David William Haxton Girdwood PhD Thesis - University of St Andrews

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Transcriptional Regulation Mediated through the Conjugation
and Deconjugation of the Small Ubiquitin-Like Modifiers
SUMO-1, SUMO-2, and SUMO-3
David William Haxton Girdwood
A Thesis Presented for the Degree of
Doctor of Philosophy
School of Biological and Medical Sciences
University of St. Andrews
June 2004
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DECLARATION
I, David William Haxton Girdwood, hereby certify that this thesis, which is
approximately 40,000 words in length, has been written by me, that it is a
record of work carried out by me and that it has not been submitted in any
previous application for a higher degree.
Date
\\ý
Signature of candidat
I was admitted as a research student in September 2000 and as a
candidate for the degree of Doctor of Philosophy in September 2000: the
higher study for which this record was carried out in the faculty of Sciences of
low
the University of St. Andrews between 2000 and 2003.
Date Signature of candidate
I hereby certify that the candidate has fulfilled the conditions of the
Resolution and Regulations appropriate for the degree of Doctor of Philosophy
in the University of St. Andrews and that the candidate is qualified to submit
this thesis in application for that degree.
}..... ý'
Date
.
Signature of supervisor .................................
2

In submitting this thesis to the University of St. Andrews I understand
that I am giving permission for it to be made available for use in accordance
with the regulations of the University Library for the time being in force,
subject to any copyright vested in the work not being affected thereby. I also
understand that the title and abstract will be published, and that a copy of the
work may be made and supplied to any bona fide library or research worker.
Date Signature of candidate
3

DECLARATION
CONTENTS
CONTENTS
....................................................................................................................... 2
............................................................................................................................... 4
List of Figures ............................................................................................................................. 8
List of Tables ............................................................................................................................... 12
List of Abbreviations ................................................................................................................... 13
Abbreviations for Amino Acids and Side-chains ........................................................................ 16
Genetic Code ............................................................................................................................... 17
ACKNOWLEDGEMENTS
18
.....................................................................................................
ABSTRACT 19
...............................................................................................................................
1. INTRODUCTION 20
................................................................................................................
1.1. GENE EXPRESSION
21
.......................................................................................................
1.1.1. Post-translational modification ................................................................................. 21
1.2. UBIQUITIN-LIKE PROTEIN MODIFIERS .................................................................... 24
1.2.1 Ubiquitin .................................................................................................................... 30
. 1.2.1.1. The Proteasome ..................................................................................................... 36
1.2.2. NEDD8 ......................................................................................................................... 38
1.2.3. ISG15 39
.................................................................................................................
1.2.4. FAT10 39
................................................................................................................
1.2.5. Atg12 & Atg8 40
.....................................................................................................
1.2.6. URM1 ............................................................................................................................ 41
1.2.7. HUB1 ............................................................................................................................. 42
1.2.8. UFM1 ............................................................................................................................ 42
1.3 SUMO .................................................................................................................................. 42
1.3.1. Localisation & Nuclear domains ................................................................................. 49
1.3.2. PML bodies ................................................................................................................... 49
1.3.3. SUMO and genome integrity ........................................................................................ 56
1.3.4. SUMO and transcriptional regulation ......................................................................... 58
1.3.5. SUMO and neurodegenerative diseases ...................................................................... 59
4

1.4. UBIQUITIN AND UBIQUITIN-LIKE PROTEASES: - CONTROL OVER
CONJUGATION ......................................................................................................................... 60
1.4.1. SUMO specific proteases and PML bodies ................................................................. 63
1.5. COMPETION FOR LYSINE RESIDUES ........................................................................ 65
1.6. THE UBL CONJUGATION PATHWAY ......................................................................... 67
1.6.1. The El superfamily 66
.......................................................................................................
1.6.2. The E2 superfamily 74
.......................................................................................................
1.6.3. E3 ligases 77
.......................................................................................................................
1.6.3.1. Ubiquitin E3s 77
...........................................................................................................
1.6.3.2. SUMO E3s 79
................................................................................................................
1.7. p300/CB P 84
............................................................................................................................
1.7.1. p300/CBP and growth control and signal transduction .............................................. 85
1.7.2. p300? CBP and disease ................................................................................................. 89
1.7.3. p300/CBP and chromatin remodelling ........................................................................ 90
1.7.4. An alternate role for p300 as an E3 ligase ................................................................... 91
1.7.5 p300/CBP and transcriptional repression .................................................................... 95
1.8. AIMS OF THE PROJECT
95
..................................................................................................
2. MATERIALS & METHODS 97
.............................................................................................
2.1. MATERIALS 98
......................................................................................................................
2.2. ANTIBODIES 98
.......................................................................................................:.............
2.3. BACTERIAL STRAINS
............................................................................. I......................
98
2.4. DNA PREPARATION 99
.......................................................................................................
2.4.1. cDNA cloning ................................................................................................................ 99
2.4.1.1. Preparation of thermocompetent bacteria .............................................................. 99
2.4.1.2. Transformation of thermocompetent bacteria ........................................................ 100
2.4.1.3. Generated plasmid constructs & siRNA oligonucleotides ..................................... 100
2.4.1.3.1. Cullin-2 .............................................................................................................. 101
2.4.1.3.2. Synthetic siRNA oligonucleotides targeting SUMO-1/-2/-3 ............................ 101
2.4.1.3.3. pSUPER retro siRNA expression plasmids ....................................................... 101
2.4.2. DNA sequencing
102
............................................................................................................
2.5. EXPRESSION & PURIFICATION OF UNLABELED RECOMBINANT PROTEINS
102
.....................................................................................................................................................
2.6. QUANTITATION OF PROTEIN 104
......................................................................................
5

2.7. SDS/PAGE & WESTERN BLOT ANALYSIS
................................................................ 104
2.8. STRIPPING OF WESTERN BLOTS 105
.............................................................................
2.9. IN VITRO EXPRESSION OF PROTEINS ..................................................................... 105
2.10. IN VITRO SUMO CONJUGATION ASSAY 105
.............................................................
2.11 IN VITRO SUMO DECONJUGATION ASSAY 106
..........................................................
2.12. PROTEIN INTERACTION ASSAY 106
............................................................................
2.13. MAMMALIAN CELL CULTURE TRANSIENT TRANSFECTIONS ..................... 108
2.13.1. Calcium phosphate transient transfections & reporter gene assays ..................... 108
2.14. PURIFICATION OF HIS6-TAGGED PROTEINS 109
.....................................................
2.15. INDIRECT IMMUNOFLUORESENCE ANALYSIS OF CELLS 109
.............................
2.16. LUCIFERASE ASSAY 110
.................................................................................................
3. RESULTS 111
...........................................................................................................................
3.1. BIOINFORMATICS: AN APPROACH TO IDENTIFY NOVEL MEMBERS OF THE
UBIQUITIN-LIKE PROTEIN SUPERFAMILY 112
...................................................................
3.1.1. Identification of three potential novel members of the SUMO family ...................... 112
3.1.2. SUMO-1 b/4/5 are conjugated both in vivo and in vitro ........................................... 113
3.1.3. Deconjugation of SUMO proteins via a SUMO specific protease ........................... 117
3.1.4. SUMO-1 b/4/5 may be psuedogenes
120
...........................................................................
3.1.5. Discussion ................................................................................................................... 122
3.2. SUMO MODIFICATION OF THE TRANSCRIPTION REGULATOR p300............ 124
3.2.1. p300 is modified by SUMO in vivo 124
............................................................................
3.2.2. SUMO conjugation to the N-terminus of p300 in vitro ............................................. 126
3.2.3. In vitro SUMO modification of the N-terminus of p300 does not require an E3..... 126
3.2.4. Identification of the lysine residues, within the CRDI domain of p300, that are
conjugated to by SUMO 131
........................................................................................................
3.2.5. SUMO modification is required for CRD1 activity in vivo ...................................... 137
3.2.6. SUMO modified CRDI recruits a histone deacetylase
140
.............................................
3.2.7. Conclusion
145
..................................................................................................................
3.3. TRANSCRIPTIONAL REPRESSION IS PREFERENTIALLY MEDIATED
THROUGH SUMO-2 AND SUMO-3 148
....................................................................................
3.3.1. Ablation of SUMO-1 & SUMO-2/3 expression following treatment with synthetic
siRNA oligonucleotides ........................................................................................................ . 149
6

3.3.2. Transcriptional repression is relieved preferentially-through siRNA targeting of
SUMO-2/3 .............................................................................................................................. 149
3.3.3. The nuclear localisation of p80 coilin is differentially regulated by knockdown of
SUMO-1 compared to that of SUMO-2/3 ............................................................................. 157
3.3.4. Conclusion .................................................................................................................. 161
4. Discussion
5. APPENDIX - VECTOR MAPS
6. BIBLIOGRAPHY 193
.............................................................................................................
7. PUBLICATIONS 248
..............................................................................................................
164
172
7

List of Figures
FIGURE 1. SCHEMATIC REPRESENTATION OF THE HISTONE CODE MODE ......... 23
FIGURE 2. PHYLOGENIC TREE SHOWING THE EVOLUTIONARY RELATIONSHIP
BETWEEN THE Ub-LIKE FAMILY OF PROTEINS 26
.............................................................
FIGURE 3. SCHEMATIC REPRESENTATION OF THE SUMO-1/2/3 AND UBIQUITIN
CONJUGATION PATHWAYS 27
.................................................................................................
FIGURE 4. A RIBBON REPRESENTATION OF THE 3D STRUCTURE OF
UBIQUITIN, A COMMON CHARACTERISTIC OF THE Ubls, AND A COMPARISON
OF THE PREDICTED SECONDARY STRUCTURES OF SUMO-1, UBIQUITIN,
NEDD8, AND UFM1 29
.................................................................................................................
FIGURE 5. SCHEMATIC REPRESENTATION OF UBIQUITIN, WITH THE
MODIFIABLE LYSINE RESIDUES AND THE ROLES OF THE DIFFERENTLY
LINKED POLYMERIC CHAINS 32
.............................................................................................
FIGURE 6. SCHEMATIC REPRESENTATION OF THE NF-KB PATHWAY, AND THE
ROLES PLAYED BY Ub, THROUGH ITS ABILITY TO FORM POLYMERIC CHAINS
WITH DIFFERENT INTERNAL LYSINE RESIDUES 34
..........................................................
FIGURE 7. ALIGNMENTS OF THE SEQUENCES FOR A NUMBER OF Ub-LIKE
PROTEINS, AND THE SPACEFILL 3D MODELS OF SUMO-1, UBIQUITIN, NEDD8,
AND REPRESENTATIONS OF SUMO-2 AND SUMO-3' BASED ON THE STRUCTURE
OF SUMO-1 45
................................................................................................................................
FIGURE 8. COMPOSITION AND REULATION OF PML BODIES 50
...................................
8

FIGURE 9. SCHEMATIC REPRESENTATION OF PROTEIN ASSOCIATION WITHIN
PML BODIES, WITH THE POTENTIAL MECHANISMS FOR TRANSCRIPTIONAL
REGULATION
........................................................................................................................... 54
FIGURE 10. SCHEMATIC REPRESENTATION OF THE ACTIVE SITE OF A
CYSTEINE PROTEASE, WITH A SCHEMATIC ILLUSTRATION OF A NUMBER OF
SUMO-SPECIFIC PROTEAES INDICATING THE CONSERVED CORE DOMAIN AND
RESIDUES. A SEQUENCE ALIGNMENT OF A NUMBER OF SUMO-SPECIFIC AND
SUMO-SPECIFIC LIKE PROTEASES, INDICATING THE RESIDUES INVOLVED IN
THE FORMATION OF THE PROTEASE ACTIVE SITE ...................................................... 62
FIGURE 11. SCHEMATIC REPRESENTATION OF A GENERAL UBIQUITIN-LIKE
PROTEIN CONJUGATION PATHWAY 69
.................................................................................
FIGURE 12. SCHEMATIC DIAGRAM SHOWING THE FOUR CONSERVERD
REGIONS IN Ub AND Ub-LIKE E1 ACTIVATING ENZYMES, AND A PHYLOGENIC
TREE DEPICTING THEIR EVOLUTIONARY RELATIONSHIPS 70
......................................
FIGURE 13. DIAGRAMMATIC REPRESENTATION OF THE SCF AND VHL E3
UBIQUITIN LIGASE COMPLEXES 80
.......................................................................................
FIGURE 14. DIAGRAMMATIC REPRESENTATION OF p300 AND THE SIGNALING
PATHWAYS WHICH ITS INVOLVED IN, AND THE KNOWN PROTEIN
INTERACTORS WITH p300 86
....................................................................................................
FIGURE 15. SCHEMATIC REPRESENTATION OF THE p53 FUNCTIONAL CIRCUIT,
INDICATING THE MECHANISMS INVOLVED IN THE CONTROL OVER ARF,
HDM2, E2F-1, AND p53 91
............................................................................................................
FIGURE 16. DIAGRAMMATIC REPRESENTATION OF THE CHROMOSOMAL
LOCATIONS OF THE SUMO GENES, A SUMMARY OF THEIR GENE MAKEUP,
AND AN ALIGNMENT OF THE SEQUENCES OF SUMO-1/2/3 WITH SUMO-lb/4/5
108
..................................................................................................................................................
9

FIGURE 17. RT-PCR OF SUMO-lb/4/5, AND TRANSFECTIONS OF HA-SUMOs INTO
COST CELLS, SHOWING THEIR FUNCTIONAL STATUS 110
.............................................
FIGURE 18. IN VITRO CONJUGATION OF SUMO-lb/4/5 112
..............................................
FIGURE 19. SSP3 DECONJUGATES SUMO-lb AND SUMO-5 113
.....................................
FIGURE 20. RT-PCR OF THE NOVELS SUMO GENES WITH TAQ POLYMERASE
115
................................................................................................................................................
FIGURE 21. p300 AND CBP CONTAIN SUMO CONSENSUS SITES IN A
REPRESSION DOAMAIN, AND p300 IS MODIFIED, WITHIN THIS DOMAIN IN VIVO
119
..................................................................................................................................................
FIGURE 22. p300 IS MODIFIED BY SUMO-1/2/3 IN VITRO
121
...........................................
FIGURE 23. TITRATION OF UBC9 INTO THE p300 CONJUGATION ASSAY ........... 123
FIGURE 24. ADDITION OF KNOWN SUMO E3s INTO p300 CONJUGATION ASSAY..
124
..................................................................................................................................................
FIGURE 25. IDENTIFICATION OF THE RESIDUES REQUIRED FOR SUMO
CONJUGATION 126
......................................................................................................................
FIGURE 26. SUMOs CONJUGATION CORRELATES WITH TRANSCRIPTIONAL
REPRESSION
130
..........................................................................................................................
FIGURE 27. TRANSCRIPTIONAL REPRESSION IS DEPENDENT ON SUMO
CONJUGATION 132
......................................................................................................................
FIGURE 28. CONTROL OVER SUMO MODIFICATION OF p300 134
.................................
FIGURE 29. SUMO MODIFICATION OF CRDI RECRUITS A HISTONE
DEACETYLASE 137
.....................................................................................................................
10

FIGURE 30. KNOCKDOWN OF ENDOGENOUS SUMO BY siRNA, AS SHOWN BY
WESTERN BLOT ANALYSIS 143
..............................................................................................
FIGURE 31. KNOCKDOWN OF ENDOGENOUS SUMO BY siRNA, AS SHOWN BY
IMMUNOFLUORESCENCE 144
.................................................................................................
FIGURE 32. AFFECT OF SUMO KNOCKDOWN ON p300 ACTIVITY 146
........................
FIGURE 33. AFFECT OF SUMO KNOCKDOWN ON ELK-I ACTIVITY 147
.....................
FIGURE 34. AFFECT OF SUMO KNOCKDOWN ON SP3 ACTIVITY 148
..........................
FIGURE 35. AFFECT OF SUMO KNOCKDOWN ON GAL4 ACTIVITY
FIGURE 36. SUMO-1 AND SUMO-2/3 PLAY DIFFERENT ROLES IN THE
...................... 149
LOCALISATION OF p80 COILIN 152
........................................................................................
FIGURE 37. SCHEMATIC REPRESENTATION OF A NUMBER OF TRANSCRIPTION
CO/FACTORS INDICATING DOMAINS AND SUMO CONJUGATION SITES ........... 158
FIGURE 38. MODEL FOR TRANSCRIPTIONAL REPRESSION BY SUMO ................ 161
11

LIST OF TABLES
TABLE 1. THE DIFFERENT UBIQUITIN-LIKE PROTEINS IN SACCHAROMYCES
CEREVISIAE AND HOMO SAPIENS ...................................................................................... 25
TABLE 2. SUMMARY OF THE KNOWN SUBSTRATES FOR SUMO MODIFICATION
46
.....................................................................................................................................................
TABLE 3. SUMMARY OF THE RELATIONSHIP BETWEEN SUMO CONJUGATION
AND TRANSCRIPTIONAL REPRESSION 130
.........................................................................
TABLE 4. LYSINE RESIDUES WITHIN A SUMO CONSENSUS MOTIF THAT ARE
MODIFIED BY UBIQUITN IN YEAST 163
...............................................................................
12

List of Abbreviations
Ac Acetylation
ADP Adenosine diphosphate
AMP Adenosine monophosphate
APL Acute promyelocytic leukaemia
APP-BPI Amyloid precursor protein-binding protein 1
Atg Autophagy
ATP Adenosine triphosphate
BLAST Basic local alignment search tool
bp Base pairs
BPV Bovine papilloma virus
BSA Bovine serum albumen
CDK Cyclin depedent Kinase
CBP CREB-binding protein
cDNA Complementary DNA
CMV Cytomegalo virus
DeUb De-ubiquitination
D-MEM Dulbecco's modified Eagle's medium
DNA Deoxyribonucleic acid
DTT Dithiothrietol
DUB Deubiquitination enzymes
E. coli
Eschericia coli
El Activating enzyme
E2 Conjugating enzyme
E3 Ligase enzyme
E6-AP E6 activating enzyme
ECL Enhanced chemiluminesence
EDTA Ethylenediaminetetracetic acid
ERK Extracellular signal-regulated kinase
FAT10 Diubiquitin
FCS Foetal calf serum
GST Glutathione S-transferase
13

h Hours
.
HA Haemagglutinin
HAT Histone acetyl-transferase
HCl Hydrochloric acid
HDAC Histone deacetylase
HDM2 Human double minute 2
HECT Homology to E6-AP C-terminus
HIPK Homeodomain-interacting protein kinase
HLH Helix-loop-helix
HPV Human papilloma virus
HUB I Homologous to ubiquitin 1
Ig Immunglobulin
IKB Inhibitor kappa B
IPTG Isopropyl-ß-D-thiogalactopyranose
ISG15 Interferon induced gene of 15 kDa
IVTT In vitro transcription and translation
KCl Potassium chloride
kDa Kilo Dalton
LB I Luria-Bertani broth
MDM2 Mouse double minute 2
Me Methylation
min minute
MonoUb Mono-ubiquitination
MW Molecular weight
NBs PML nuclear bodies
NEDD8 Neuronal precursor cell expressed developmentally
Down regulated protein 8
NES Nuclear export signal
NF-KB Nuclear Factor-Kappa B
NLS Nuclear localisation signal
NP-40 Nonidet P-40
NPC Nuclear pore complex
PAGE Polyacrylamide gel electrophoresis
PBS Phosphate buffered saline
PCR Polymerase chain reaction
Pi Phosphate
PIAS Protein inhibitor of activated STAT
14

PML Promyelocytic leukaemia protein
PODS PML oncogenic domains
PolyUb Poly-ubiquitination
PVDF Polyvinylidene difluoride
RanGAP Ran GTPase activating protein
RanBP Ran binding protein
RAR Retinoic acid receptor
RNA Ribonucleic acid
RT-PCR Reverse transcriptase PCR
Rubl Related to ubiquitin
SAE1 SUMO activating enzyme subunit 1
SAE2 SUMO activating enzyme subunit 2
S. cerevisiae Saccharomyces cerevisiae
SCF Skpl, cullin, F-box
SENP Sentrin-specific protease
SSP SUMO-specific protease
STAT Signal transducer and activator of transcription
SUMO Small ubiquitin-like modifier
SUSP SUMO-specific protease
SV40. Simian virus type 40
3D Three-dimensional
Tris 2-amino-2-(hydroxymethyl)propane-1,3-diol
Ub Ubiquitin
UBA1 Ubiquitin activating enzyme
Ubc Ubiquitin conjugating enzyme
UBL Ubiquitin-like modifiers
UBP Ubiquitin-specific processing proteases
UCH Ubiquitin C-terminal hydrolase
UD Ubiquitin domain
UDP Ubiquitin-domain protein
UFM 1 Ubiquitin fold modifier 1
ULP Ubiquitin-like protein
Ulp Ubiquitin-like protease
UV Ultra violet
URM 1 Ubiquitin related modifier 1
VCB VHL-Elongin C/ Elongin B
VHL Von Hippel-Lindau
Wrn Werner's disease protein
WT Wild-type
15

Abbreviations for Amino Acids and Side-chains
Alanine ala A
Arginine arg R
Asparagine asn N
Aspartic acid asp D
Cysteine cys C
Glutamine gln Q
Glutaminc acid glu
Glycine gly G
Histidine his H
Isoleucine He I
Leucine leu L
Lysine lys K
E
-CH3
-(CH2)3-NH-CNH-NH2
-CH2-CONH2
-CH2-000H
-CH2-SH
-CH2-CH-CONH2
-CH2-CH2-000H
-H
-CH2-imidazole
-CH(CH3)-CH2-CH3
-CH2-CH(CH3)2
-(CH2)4-NH2
Methionine met M -CH2-CH2-S-CH3
Phenylalanine phe F
-CH2-phi
Proline pro P -[N]-(CH2)3-[CH]
Serine ser S -CH2-OH
Threonine thr T -CH-(CH3)-OH
Tryptophan try w -CHZ-indole
Tyrosine tyr Y -CH2-phi-OH
Valine val v -CH-(CH3)2
16

Genetic Code
TTT'phe F TCT ser S TAT tyr Y TGT cys C
TTC phe F TCC ser S TAC tyr Y TGC cys C
TTA leu L TCA ser S TAA OCH Z TGA OPA Z
TTG leu L TCG ser S TAG AMB Z TGG try W
CTT leu L CCT pro P CAT his H CGT arg R
CTC leu L CCC pro P CAC his H CGC arg R
CTA leu L CCA pro P CAA gln Q CGA arg R
CTG leu L CCA pro P CAG gln Q CGG arg R
ATT ile I ACT thr T AAT asn N AGT ser S
ATC ile I ACC thr T AAC asn N AGC ser S
ATA ile I ACA thr T AAA lys K AGA arg R
ATG met M ACG thr T AAG lys K AGG arg R
GTT val V GCT ala A GAT asp D GGT gly G
GTC val V GCC ala A GAC asp D GGC gly G
GTA val V GCA ala A GGA glu E GGA gly G
GTG val V GCG ala A GAG glu E GGG gly G
17

ACKNOWLEDGEMENTS
I would like to thank my supervisor, Ron Hay, for sharing with me his
enthusiasm for the SUMO field, and providing me with opportunities,
encouragement, and support throughout my PhD, both with my work and in
developing my own ideas and interests. The work' within the thesis benefited
greatly from the excellent help and assistance freely given by members of the
lab and the BMS building as a whole, both past and present, and I gratefully
acknowledge their help and assistance throughout my time here. I would
additionally like both Graham Kemp and Rick Randall for their time and help
in the lab both before and during my PhD.
To my family and friends, special thanks, for your support and advice
throughout, it made a real difference.
Finally I would like to thank Stephen and Charlotte, their cats Haggis
and Pepper, for both letting me stay in their home, and for making me feel so
welcome, especially as the "just for a short while" turned into months.........
This work was supported by the Biotechnology and Biological Sciences
Research Council (BBSRC).
18

ABSTRACT
SUMO-1/2/3 are members of the ubiquitin-like family of protein
modifiers. These proteins are covalently attached to numerous proteins in a
directed and controlled manner. SUMO conjugation primarily occurs to
proteins containing an exposed SUMO conjugation motif, (I, V, L, F)KxE,
where x represents any amino acid. SUMO conjugation is controlled by key
enzymes, a SUMO activating enzyme, SAE1/2 and a SUMO conjugating
enzyme, Ubc9, which is responsible for substrate recognition, and the
efficiency of this pathway can be increased by one of many SUMO ligase
enzymes. This modification alters the substrate's characteristics and results in
a change of state, such as stability, localisation, or activity.
p300, a transcriptional co-activator, contains an evolutionary
conserved tandem SUMO modification motif, located in a transcriptional
repression domain. p300 was efficiently conjugated, both in vitro and in vivo,
by SUMO-1/2/3, within this repression domain to both SUMO conjugation
motifs. The SUMO conjugation to p300 correlated with p300 ability to repress
transcription, requiring both SUMO conjugation motifs for full transcription
repression activity. This repression activity was mediated through SUMO
recruitment of histone deacetylase 6. Repression could be alleviated through
co-expression of a SUMO-specific protease thereby suggesting a potential
regulatory mechanism for transcription control of SUMO modified substrates.
Despite utilising the same conjugation machinery, there remained the
potential for distinct roles for the SUMO isoforms. SUMO -2/3, which form a
distinct group from SUMO-1, were shown to preferentially mediate the
transcription repression abilities of a number of known SUMO modifiable
substrates: p300, Elk-1, and SP3. Further differences were observed in the
ability of SUMO-1 and SUMO-2/3 to influence the nuclear organisation of
p80 coilin.
19

1. INTRODUCTION
20

1.1 Gene Expression
1.1.1 Post-translational modification
The specialization of an organism's cells depends upon the ability to
control the expression of particular genes. Gene expression is controlled at six
different levels: transcription; RNA processing; RNA transport; translation;
mRNA turnover; and protein turnover. At the first level of control, that of gene
transcription, determines how frequently a particular gene is transcribed and as
such dictates the level of primary RNA transcript produced. The last level of
control, achieved through the modification of a protein, can determine the
consequences of gene expression. The gene product can be selectively activated,
inactivated, degraded, or compartmentalised following its translation. This
regulation will affect a proteins capacity to interact with other proteins or DNA,
and as such the proteins function. A protein, for instance, that is rapidly
degraded may have little cellular impact although its gene may be expressed.
This form of control, known as post-translational modification, can be achieved
in numerous ways, phosphorylation, hydroxylation, glycosylation, and
acetylation amongst others. Addition of these chemical groups to a target protein,
is achieved through the activity of specific enzymes while removal of the
chemical groups is mediated by other distinct enzymes. This represents a
common method of controlling a proteins function. A more advanced system of
post-translational modification utilises the attachment of one protein to another,
via an enzymatic cascade. Post-translational modification via the attachment of
other proteins provides unique mechanisms of controlling protein function.
Ünlike chemical modification such as phosphorylation, adenylation, or
glycosylation, the size of the protein attached can exert a direct influence on the
proteins immediate environment, ubiquitin being approximately one hundred
times bigger than a phosphate group. An example is histone ubiquitinylation
(Histone code hypothesis) and this size also creates the addition of a more
chemically complex surface, creating the possibility of further interactions with
additional proteins. Histones are the fundamental constituents of the
nucleosome. Nucleosomes consist of genomic DNA wrapped around an'octamer
of the conserved histone proteins H2A, H2B, H3, and H4. H1 functions as linker
histones, located between nucleosomes, which serve to promote the overall
21

I
folding of the chromatin fibre. Histones consist of a globular domain, a short C-
terminal extension, and an extended N-terminal-tail (Figure l. i. ). Post-
translational modifications of the tail domains of histones modulate chromatin
structure and gene expression (Naar et al., 2001; Berger et al., -2002). This is
achieved through the control of the accessibility ' of the gene expression
machinery to the DNA. DNA is compacted within the nucleus of a eukaryotic
cell, due to spatial limitations and as a means of protecting DNA (reviewed in
Felsenfeld and
Groudine, 2003).
Histone acetylation is the best characterised post-translational
modification of the histone tails. Histone acetyltransferases (HAT) acetylate
specific lysine residues present in the tail regions, associated with transcription
activation, conversely histone deacetylases (HDAC) reverse the acetylation of
lysine residues, and are associated with transcription repression. These distinct
histone modifications can act sequentially or in combination- the histone code
(reviewed in Strahl and Allis, 2000).
The histone-code hypothesis proposes that post-translational
modifications in histone tails are `read' by other histones and/or proteins, and are
translated into silencing or activation of gene transcription (Jenuwein et al.,
2001). For example, the phosphorylation of SerlO (S10) of histone H3
antagonizes methylation of K9 and/or K14 (Figure l. ii. ). The modifications of
the histone tails will, under the hypothesis, regulate the interactions of proteins,
such as Chromo and Bromo domain containing proteins, that specifically bind to
methylated or acetylated lysine residues, respectively, in histones (Sun et al.,
2002).
Research into histone ubiquitination has provided much evidence in
support of the histone-code hypothesis. Ubiquitination of K123 of H2B has been
demonstrated to be a prerequisite for H3 methylation of K4 in yeast. H3
methylation of K4 is a key event in the regulation of gene expression in yeast
(Dover et al., 2002). The mutation of K123 of H2B results in the disruption of
gene silencing, indicating the links between the ubiquitination of K123, mediated
by Rad6, K4 methylation of H3 by Setl, and transcriptional repression (Sun et
al., 2002; Dover et al., 2002). Rad6 mediated ubiquitination of K123 on H2B
has further been demonstrated to be a requirement for K79 methylation of H3,
22

I.
Histone tail
-K9S1O-K14-R17-K18-K23-K27-K3
Radio
- ------------------
Brei
H2B B
Globular
domain
Mb
H2B K 123 ý`ý
Figure 1. The histone-code model. In this model the histone tails extend to the left of the
globular domain, ubiquitin (ub) is indicated in red. (i) Mono-ubiquitination of histone H2B is
mediated by the E2 ubiquitin conjugating enzyme Rad6 and the ubiquitin protein ligase Brel. (ii)
Cis- and trans-regulation of histone-tail modifications as part of the histone code on histories H2B
and H3. Modified residues are indicated. Histone modifications include ubiquitination,
methylation, phosphorylation, and acetylation. Cis regulation: phosphorylation of S10 on
histone H3 negatively affects (indicated by red double blocked line) methylation of K9, whereas
acetylation of K9 and/or K14 positively regulated (indicated by double pointed arrows) by S10
phosphorylation (and vice versa). Both events take place on the same histone tail. Trans-
regulation: methylation of K4 and K79 on histone H3 are dependent on the mono-ubiquitination
of K123 on histone H2B (broken arrows).
Abbreviations: Ac, acetylation; K, lysine; Me, methylation; P, phosphorylation; S, serine; R,
arginine. (Adapted from Bach and Ostendorff, 2003).
H3

providing a further example of trans-histone regulation (Briggs et al., 2002).
Although the methylation of K4 of H3 had been implicated in gene silencing,
there is also evidence for K4 methylation of H3 in gene activation. K4 of histone
H3 exists in a di- or tri-methylated state in transcriptionally active chromosomal
(euchromatin) regions, but not in inactive (heterochromatin) regions (Santos-
Rosa et al., 2002). The role of H2B ubiquitination in this context remains
ambiguous, although a general role of the ubiquitination of histone residues
serving as key regulators of subsequent histone modifications (Figure 1. ) is
strongly supported. Thus, the modification of histone tails will influence gene
expression by controlling the accessibility of both RNA polymerases and
transcription factors to DNA.
1.2 Ubiquitin-like protein modifiers
Ubiquitin was originally isolated by Goldstein and co-workers from the
thymus and was thought to be a thymic hormone (Goldstein et al., 1975).
Ubiquitin was subsequently independently identified by two further groups, as an
'ATP-dependent proteolysis factor 1' (Ciehanover et al., 1978) and as attached to
histone H2A (Goldknopf and Busch, 1975, Goldknopf and Busch, 1977).
Subsequent work showed that ubiquitin was found in all tissues and all
eukaryotic organisms, and this ubiquitous distribution gave the protein its name.
Indeed ubiquitin has been shown to be one of the most phylogenetically well-
conserved of all proteins in eukaryotes, although there are no known homologues
in either archea or eubacterial organisms. Ubiquitins' identification was the first
example of a polypeptide modifier, and as such lends its name to the family of
polypeptide protein modifiers, the ubiquitin-like modifiers. Since the discovery
of ubiquitin, further ubiquitin-like protein modifiers
have been identified:
SUMO-1/-2/-3 (Boddy et al., 1996, Mannen et al., 1996, Matunis et al., 1996,
Okura et al., 1996, Shen et al., 1996, Kamitani et al., 1997, Lapenta et al., 1997,
Mahajan et al., 1997, Tsytsykova et al., 1998, Kamitani et al., 1998), NEDD8
(Kamitani et al., 1997), ISG15 (Haas et al., 1987), FAT10 (Liu et al., 1999b),
HUB 1 (Dittmar et al., 2002), An l a/An lb (Linnen et al., 1993), APG12
(Mizushima et al., 1998), FUBI (Kas et al., 1992), URM1 (Furukawa et al.,
2000), and UFM1 (Komatsu et al., 2004). Higher eukaryotic genomes contain
24

an increased number of ubiquitin-like proteins, compared to the lower eukaryotes
(Table. 1. ) and the evolution of new members of the ubiquitin-like family seem
likely to have occurred from the ancestral members (Figure 2. ).
Ubiquitin-like proteins fall into two separate classes (reviewed in Jentsch
and Pyrowolakis, 2000). The first class, those mentioned above, function as
modifiers in a similar manner to that of ubiquitin. They exist either in a free
form or attached *covalently to other proteins via their matured C-termini
(Figure. 3. ). These proteins are termed "ubiquitin-like modifiers", or type 1 Ubls.
The conjugation of the ubl is achieved via an enzymatic cascade involving an El
(activating enzyme), E2 (conjugating enzyme), and an E3 (ligase) enzyme. In all
cases to date, the conjugation occurs at a lysine residue within the substrate,
either within or without a "consensus motif", although recently p21
has been
shown to be ubiquitinated at its N-terminal methionine (Bloom et al., 2003). The
second class of proteins contain ubiquitin related domains but also domains with
no homology between. one another. Members of this second group are not
conjugated to other proteins. Collectively these proteins are termed type-2 UBLs
(also called UDPs, ubiquitin domain proteins), which contain ubiquitin-like
structure embedded in a variety of different classes of large proteins with
apparently distinct functions, such as Rad23, Elongin B, Scythe, Parkin, and
HOIL-1 (Tanaka et al., 1998; Jentsch and Pyrowolakis, 2000; Yeh et al., 2000;
Schwartz and Hochstrasser, 2003).
The similarities between the different ubiquitin-like proteins and their
component enzymes are as striking as their differences. Despite the lack of
sequence homology between the various ubiquitin-like modifiers, to date all
share a common tertiary structure, the ubiquitin fold (Figure 4. i. ). The body of
each adopts this, ßßaßßaß fold (Figure 4. ii. ), resulting in a stable globular
structure that is highly resistant to a range of denaturants (Vierstra and Callis,
1999, Vijay-kumar et al., 1987, Melchior, 2000). Despite the similar structure
there are significant differences in the electrostatic potentials of the various
ubiquitin and ubiquitin-like proteins and unlike most ubiquitin-like modifiers, the
members of the SUMO family possess a flexible N-terminal extension.
25

Table 1. Identifed Ubls in Saccharomyces cerevisiae and Homo Sapiens. ;
Saccharomyces cerevisiae Homo Sapiens
Ubiquitin Ubiquitin
ISG15
FAT 10
FUB1
RUB! NEDD8
SMT3 SUMO-1
SUMO-2
SUMO-3
Atg8 GATE-16
LC3
GABARAP
Atg l2 Atg l2
Urm 1
Ufml
GenBank accession numbers: ScUbiquitin P04838; hUbiquitin AAA36789; ISG15
P0161; FAT10 NP_001988; FUB1 NP001988; RUBI NP_009209; NEDD8
NP_006147; SMT3 Q12306; SUMO-1 ACAA67898; SUMO-2 CAA67896; SUMO-3
P55855; Atg8 NP_009475; GATE-16 P60520; LC3 NP_852610; GABARAP
NP_009209; ScAtg12 NP_009776; hAtgl2 NP_004698; Ufml B0005193; Urml
NP_012258
AtgS
GATE-16
GARARAP
MAP-113
UFM1
Atg12p
Atg12
MOCO1-A
MoaD. E. coG
Urml
ThiS. E. coli
FUBI
Anla
Anlb
" Ubiquitin
YeastUb
- NED08
Rubl
tSG15
FAT10
- 50140-1
- SUMO-2
-S0140-3
Sm13
Figure 2. Phylogram of all known Ub-lke proteins and Ubl-like bacterial proteins. Phylogram
was constructed via the ClustalW alignment program (www. ebi. ac. uk/clustalw) and was
generated from full length sequences.

Figure 3. Schematic representation of the SUMO-1/-2J-3 (Su) and ubiquitin(Ub)
conjugation pathways. Both pathways can be divided into five key stages. The first
stage in the conjugation process is the maturation of the SUMO/ubiquitin translation
products. Prior to conjugation a C-terminal inhibitory tag is required to be removed,
to expose the di-glycine residues that allow conjugation. This maturation is
catalysed by specific proteases; ubiquitin C-terminal hydrolayse (UCH) and SUMO
specific protease(SSP), for ubiquitin and SUMO respectively. The processed
modifier can then be attached to substrates via a three step enzymatic cascade. In the
first step specific activating enzymes (Els) form ATP-dependent thioester bonds
between internal cysteine residues and the conserved C-terminal glycine of the
modifiers. The El for SUMO is a heterodimer, SAE1/SAE2, whilst the El for
ubiquitin is monomeric, UBAI. In the second step specific conjugating enzymes
(E2s) accept the activated modifier via a transesterification reaction to an internal
cysteine residue. Many E2s are known to exist for ubiquitin (Ubcl-8, UbclO-11, and
Ubcl3), whilst only a single E2 has been isolated for SUMO (Ubc9). The final step
involves the formation of an isopeptide bond between the E-amino group of the
substrate target lysine and the terminal glycine of the modifier. For the ubiquitin
pathway the E3 activity is an absolute requirement for substrate modification, whilst
in the SUMO pathway, E3 activity is not normally required for conjugation, but may
serve to enhance the efficiency of the process. The modifiers can subsequently be
removed from substrates via the actions of the SUMO-specific protease (SSP) or an
ubiquitin isopeptidase. Following their deconjugation the protein modifiers are
recycled and are available for conjugation, existing in a free "pool" of modifier.
I

Ubiquitination
E3 cycle
5. Deconjugatio? 0 -
4. Ligation
LEL1
Ubi
-, C
Maturation
`Activation
J3.
ransestenfication

(ii)
SUMO-1
(1
1)
MSDQEAKYSTEDLGDKKEGEYIKL MGQDSSEIHFKVRMfTHLKKLKESYCQRQGVPMNSLRFLFEGQRIADNHTPKELQbIEEEDVIEVYQEQTGGHSTV
Ubiquitin
NEDD8
Ufm l
Key:
I strand
MQIrVKTLTGKTITLXVEPSDTIiM%IAXIQDKZOIDPDQQRLIFAG QLIDGRTLBDYNIQXZSTLHLVLRLRGG
NQ, IRNETLTG EIEIDIZPTDEVERIEERVEEEEGIPPQQQRLITSGIIQMNDERTARDSEILGGSVLHLVLRLRGGGGM. RQ
! (BEVBFRITLTBDPRLPTRVLBVPEBTPFTAVLEFAAEEFEVPAIºTSM IT$DGIOINPAQTAGNVFLRRGBELRI IPRDRVOBC
J; )
helix coil
Figure 4. i. The "ubiquitin fold". A representation of the tertiary structure of ubiquitin.
The adoption of this fold results in a stable globular structure, and is a common
characteristic of all Uhl proteins. ii. Comparison of predicted secondary structures of
SUMO-1, Ubiquitin, NEDD8, and Ufml. All four of these proteins, despite having
dissimilar amino acid sequences have all been reported to posses the "ubiquitin fold".
Predicted secondary structures were generated through the secondary structure prediction
software Jpred (www. compbio. dundee. ac. uk/-wwwjpred)

The majority of Ubls are expressed as inactive precursors, which are
made initially as fusions with C-terminal extensions, which prevent conjugation.
These inhibitory tails, which can range from a single amino acid in length to a
polypeptide, are specifically cleaved via the activity of proteases, resulting in the
release of the active Ubl and its tail.
1.2.1 Ubiquitin
Despite the similarities in conjugation pathways of the ubl proteins, once
conjugated their effects are distinctly unique. Ubiquitin was the first of the
superfamily of protein tags to be identified, and for many years was thought to be
unique in its ability to be attached to other proteins. The effects of ubiquitin
conjugation on a substrate are varied and depend primarily on how many
ubiquitin moieties are bound. The attachment of ubiquitin, in most cases, is not
to a specific lysine residue that serves as an acceptor nor are they part of a
recognition motif. There are exceptions to this rule such as in the case of IKBa,
where lysine residues 21 and 22 are unique sites of ubiquitin ligation (Scherer et
al., 1995; Baldi et al., 1996; Rodriguez et al., 1996). The conjugation of a single
ubiquitin protein, monoubiquitination, is quite distinct from having multiple
ubiquitin molecules conjugated, which is termed polyubiquitination. Ubiquitin
chains can be formed in distinct linkages, due to the presence of lysine residues
in ubiquitin molecules. The chain types and their roles will be discussed in detail
later. Ubiquitin was first isolated bound to histone H2A, indeed in vertebrates up
to 15% of histone H2A and around 5% of H2B, as well as the linker histone H1,
are ubiquitinated, predominantly in the monoubiquitinated form (Rechsteiner ed,
1988). Monoubiquitination has been shown to be involved in at least three
distinct biological functions, these being histone regulation, endocytosis, and the
budding of retroviruses from the plasma membrane (reviewed in Hicke, 2001).
The function of histone modification by ubiquitin has been demonstrated to be
important in gene expression, as previously mentioned in the histone code. The
importance of histone ubiquitination can be illustrated in yeast cells, where H2B
mutants, lacking the ubiquitinylation site do, not sporulate indicating a role in
meiosis (Robzyk et al., 2000). It is well known that post-translational
30

modifications of histone tail domains modulate chromatin structure and gene
expression, although the role of ubiquitin in this process is poorly understood.
Deubiquitination of monoubiquitinated histones is closely associated with
mitotic chromatin condensation, and consequently transcriptional repression.
The ubiquitin status of the histones is in a dynamic equilibrium, where ubiquitin
is freely exchanged with intact nucleosomes (Seale, 1981), although it has been
shown that apoptotic stimuli lead to a caspase-dependent deubiquitination of
monoubiquitinated histone H2A (Mimnaugh et al., 2001). Ubiquitination of
histone H2B in yeast (Saccharomyces cerevisiae) is required for the methylation
of histone H3 (Sun and Allis, 2002) and as such shows the involvement of
ubiquitin as a determinant of the histone code hypothesis (reviewed in Strahl and
Allis, 2000). Monoubiquitination is required as an internalisation signal on
endocytic cargo at the plasma membrane and may play a role in the control of the
activity of the endocytic machinery (reviewed in Hicke, 2001). Similarly the
budding of enveloped viruses, such as Ebola, from the plasma membrane of
infected cells, requires monoubiquitination, although this process occurs in the
opposite direction to that of vesicles in the process of endocytosis (Garoff et al.,
1998).
Ubiquitin is also capable of forming polyubiquitin chains, through the
attachment to internal lysine residues at positions 6,11,29,48, and 63 (Figure
5). The functions of Lys ll and Lys29-linked chains are unknown. The
functions of Lys63-linked chains have a range of fates: DNA repair, translation,
IKB kinase activation, and endocytosis. The most abundant ubiquitin substrate in
Saccharomyces cerevisiae is L28, a component of the large ribosomal subunit,
which forms polymeric chains through Lys63 (Spence et al., 2000). The best
documented role for polyubiquitination, is targeting of proteins for proteosomal
degradation. In this case chains of four or more in length are linked via Lys48.
Once bound to the 26S proteasome the protein to be degraded is unfolded and the
ubiquitin chain deconjugates and is recycled.
Ubiquitin is encoded for in the human genome both as a monoubiquitin
form and as a polyubiquitin (Özkaynäk et al., 1984, Wiborg et al., 1985). In
yeast, UB14 encodes a polyubiquitin gene, consisting of five consecutive
ubiquitin-coding repeats in a spacerless head-to-tail arrangement. UB14 has been
31

Ubiquitin
Lys 6 11 9 3
/
/ I
10. pIl
E2 ? ? Many Ub Ub c 13 Ubc l, Ubc4, UbcS
EZs
E3 B Many Ra RAF6 Rsp5
Eis Rad 1 8
Substrates BRCA1/ Many Su bstrates? L28 ? Membrane
BARD I substrates proteins
Process DNA repair Proteasome DNA repair Trans lation IKB kinase Endocytosis
/replication mediated activation and transport
degradation
Figure 5. Different functions for different ubiquitin linkages. Schematic representation of
ubiquitin with the modifiable lysines and roles of different linkages. Ubiquitin contains
several lysine residues and, in vivo, can form multi-ubiquitin chains linked through positions
6,11,29,48, and 63. The functions of Lysl I and Lys29-linked chains are unknown. Lys6-
linked chains are formed by the autoubiquitination of BRCAI/BARD] during DNA repair
and replication, Lys48-linked chains target substrates to the proteasome, but might have other
functions, and Lys63-linked chains have a range of fates. Figure adapted in part from
Weissman, 2001.
C

shown to be essential in providing ubiquitin to cells under stress, and that
ubiquitin is in itself is an essential component of the stress response system
(Finley et al., 1987). Transcription of the polyubiquitin gene would allow cells
to maintain the levels of free ubiquitin against the increased rate at which free
ubiquitin is depleted through the formation of ubiquitin-protein conjugates.
Following the attachment of a polyubiquitin chain, a substrate is most commonly
degraded by the 26S proteasome, although ubiquitinated cell surface exposed
membrane proteins, are targeted to the lysosome following ligand binding
(reviewed in Hicke, 1997). Ubiquitin conjugation can also play important non-
destructive functions, such as those involved in the NF-KB signalling pathway.
Ubiquitin plays multiple roles in the NF-KB signalling pathway that ultimately
leads to NF-KB translocation to the nucleus and gene expression (Figure 6). The
family of NF-KB transcription factors are a group of structurally related and
evolutionarily conserved proteins, which form either homodimers or
heterodimers (Ghosh and Karin, 2002). In vertebrates, they comprise five
subunits, p65 (ReIA), Re1B, c-Rel, and two which are transcribed and translated
as precursors p100 and p105. Proteolytic processing of p100 and p105 generate
p52 and p50 respectively. Both precursors possesss large C-terminal regions
containing ankyrin repeat domains (ARD), resulting in their cytoplasmic
retention. The ubiquitination of p105 leads to the removal of the C-terminus,
exposure of its nuclear localisation sequence, and subsequent nuclear localisation
(Palombella et al., 1994). Both p52 and p50 are only active as heterodimers,
with p60, RelB, or c-Rel. The most abundant form of NF-KB dimer is the
p5O/p65 heterodimer, which is found in most cell types, bound to the inhibitory
protein, IKB in the cytoplasm. The second point at which ubiquitin is involved is
in the proteolysis of IKB. IKBct is ubiquitinated on lysines 21 and 22, and this
polyubiquitination, following its phosphorylation, results in its degradation and
release of NF-KB, which subsequently translocates to the nucleus and activates
transcription (Figure 6). Interestingly SUMO, an ubiquitin-like modifier
competes with ubiquitin for conjugation of IKB, creating a privileged pool of NF-
KB-IKB, held in the cytoplasm (Figure 6) (Desterro et al., 1998). The trigger for
ubiquitination of IKB is its phosphorylation by the IKB kinase complex, and this
phosphorylation
33

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is also an ubiquitin-mediated event. The binding of interleukin-1 to the
interleukin-1 receptor results in the association of adaptor proteins with the
intracellular region of the receptor, and subsequent recruitment of TRAF6. The
TRAF6 molecules are multiply ubiquitinated, forming chains via the Lys63
residue in ubiquitin. By mechanisms as yet unclear this results in the activation
of the TAK1 kinase, which phosphorylates IKK (Figure 6) (Wang et al., 2001,
reviewed in Finley, 2001). Evidence has begun to emerge suggesting links
between ubiquitin-dependent proteolysis and transcriptional activation. The
most straightforward link between degradation and transcription is by controlling
the levels of transcription factors, which is true for p53 (Carr, 2000), ß-catenin
(Hart et al., 1999), and Rpn4 (Xie and Varshavsky, 2001). A slightly more
sophisticated relationship between the two is shown by the observation that
sequences within activators that specify proteolysis commonly overlap with the
transcriptional activation domains (Salghetti et al., 2000), and that components
of the transcription machinery recruited by activation domains can lead to the
degradation of activators (Chi et al., 2001, Nelson et al., 2003). Recently it has
been shown that ubiquitin-mediated degradation stimulates transcription
promoted by the oestrogen receptor (Reid et al., 2003) and that the function of
the MYC proto-oncoprotein is also modulated via the ubiquitin pathway (von der
Lehr et al., 2003, Kim et al., 2003), and there is compelling evidence that the
proteasome itself plays a direct role in transcriptional regulation.
1.2.1.1 The Proteasome
The 26S Proteasome is the predominant, 2.5 MDa enzyme, that degrades
proteins conjugated to ubiquitin, and is composed of two major subcomplexes,
the 670 kDa proteolytic core particle (CP; also known as 20S proteasome) and
the 900 kDa regulatory particle (RP; also known as PA700 and the 19S
complex). The RP particle, consisting of six ATPases (Rptl-Rpt6) and at least
twelve other non-ATPase subunits (Rpnl-12) is further subdivided into the base
and lid, with all ATPases being located in the base (Glickman et al., 1998). The
CP subunits are arranged into four seven-membered rings, the outer rings are
formed by the a subunits, whilst the inner rings are formed by the proteolytically
active ß subunits (Groll et al., 1997). These subunits can possess chymotrypsin-
36

like activity, which cleaves after hydrophobic residues, a trypsin-like activity,
which cleaves after basic residues, and a peptidylglutamyl peptidase that cleaves
after acidic residues and are responsible for the disassembly of the substrate
protein. (Baumeister et al., 1998). Peptidase activity is not limited to the CP of
the proteasome however. The Rnp11 subunit of RP lid has been shown to
contain a metalloprotease motif (Verma et al., 2002, Yao et al., 2002). The
proposed role for this subunit is in the removal of the multiubiquitin chain, which
is essential for the efficient translocation of target protein into the CP of the
proteasome (reviewed in Wilkinson, 2002, Hochstrasser, 2002). The RP has
been demonstrated to have multiple functions, such as regulating the entry to the
sealed internal chamber of the CP. The axial channel of the CP is gated by the
Rpt2 ATPase and has been shown to control both substrate entry and product
release (Köhler et al., 2001). The base of the lid has also been shown, through
the Rpn1 subunit, to bind ubiquitin-like protein domains (Elsasser and Gali et al.,
2002) and can recruit multi-ubiquitin chains through their association with
ubiquitin-associated (UBA) domains with proteins also possesssing ubiquitin-
like domains (Wilkinson et al., 2001). Interestingly, Gonzalez et al. (2002), have
shown that the ATPases of the RP, and not the proteases of the CP, become
associated with genes that are being transcribed. The ATPases not only did not
interact with the CP when bound to DNA, they did not interact with the lid of the
RP, the exact role of this association has yet to be elucidated, and it has yet to be
determined whether the RP base plays a direct role, enzymatically, in
transcriptional activation or is limited to transcription factor degradation
(reviewed in Ottosen et al., 2002).
Multiple associated proteins regulate proteasome structure and function.
The most notable include Ubp6, a deubiquitinating enzyme, Hu15, an ubiquitin-
ligase, Ecm29, which tethers the CP to the RP (Leggett et al., 2002), and the 11S
complex. The i IS complex is composed of alpha, beta, and gamma subunits and
is capable of associating with the 20S proteasome, and replaces the 19S
proteasome on at least one side. 11 S is interferon inducible, as are three subunits
from the 20S proteasome, ß1i, ß5i, and ß2i, and unlike the 19S complex, 11S
play roles in antigen processing (Groettrup et al., 1995). The 11S complex is
found in mature PML nuclear bodies, nuclear bodies which are dependent upon
37

SUMO for their maturation, and are further recruited following exposure to
arsenic trioxide, and interferon gamma (Fabunmi et al., 2000), which has
implications in promyelocytic leukemia (Lallemand-Breitenbach et al., 2001).
Thus the proteasome plays important roles, not only in protein
degradation, antigen presentation, the recycling of ubiquitin, but also in
transcriptional regulation. Despite these pivotal roles some proteasome
inhibitors are already in clinical trials, showing particular promise in treating
cancers of blood cells such as multiple myeloma (Adams, 2002).
1.2.2 NEDD8
NEDD8 (Neural precursor cell-Expressed Developmentally down-
regulated) was first reported following the identification of a set of genes with
developmentally down-regulated expression in the mouse brain (Kumar et al.,
1992). Subsequently NEDD8 was shown to function in an analogous manner to
that of ubiquitin and SUMO, and covalently conjugate to substrate proteins
(Kamitani et al., 1997). NEDD8 substrates were subsequently identified as
belonging to the Cullin family (Cullin-1, -2, -3, -4a, -4b, -5), a family of proteins,
which bear significant sequence similarity to the yeast Cdc53 protein (Osaka et
al., 1998, Wada et al., 1999, Hori et al., 1999). In yeast, Cdc53 forms part of a
protein complex, with SKIP1 and Cdc4, that functions as an ubiquitin E3 ligase
to target the yeast CDK inhibitor, p40sIC1, for ubiquitin-dependent degradation
during the G1/S transition. Thus it was likely, and subsequently shown, that
NEDD8 would be involved in the degradation pathway. This hypothesis was
substantiated by the finding that NEDD8 modification of Cullin-1 activates the
ubiquitin-ligase, SCF (beta-TrCP), and was required for IKBa degradation via
the ubiquitin/proteasome pathway (Read et al., 2000). NEDD8 modification of
Cullin-1 was shown to enhance the recruitment of Ubc4, an ubiquitin E2, to the
SCF complex (Kawakami et al., 2001). Recently NEDD8 was demonstrated to
conjugate to pVHL, where it was required for fibronectin matrix assembly and
suppression of tumour development (Stickle et al., 2004). Surprisingly, NEDD8
modification of pVHL played no role in the ubiquitination of
hypoxia inducible
factor-1 (HIF-1) a known substrate for the VCB E3 ligase complex (Stickle et
38

al., 2004). Two reports show that another protein
CAND1 (Cullin-associated
NEDD8-deassociated 1) negatively modulates, the interaction of the E3 subunits,
and selectively binds cullin-1, which has not been NEDD8 modified. The
conjugation of NEDD8 to cullin leads to the dissociation of CAND1; and
subsequent formation of the E3 complex (Liu et al., 2002; Zheng et al., 2002).
NEDD8 conjugation is apparently modulated in a number of ways: the binding
of Butl and But2 (proteins that bind to Uba three) to Uba3 (Yashiruda et al.,
2003); and by NUB I. The association of Butl and But2 to the NEDD8 El
component, Uba3 restricts NEDD8 conjugation by inhibiting the El. NUB1
(NEDD8 Ultimate Buster-1) overexpression results in a severe reduction of
NEDD8 levels (Kito et al., 2001). NUB1 interacts with S5a subunit of the RP of
the proteasome, and proteasome inhibitors were shown to completely block
NUB 1-mediated down-regulation of NEDD8 expression, which suggests that
NUB I recruits NEDD8 and its conjugates to the proteasome where they are
subsequently degraded (Kamitani et al., 2001).
The COPS signalosome is an evolutionarily conserved multiprotein
complex that possess similarities with the lid of the proteasome, RP (reviewed in
Schwechheimer and Deng, 2001). Interestingly the COPS signalosome was
demonstrated to remove NEDD8 from cullins, thus modulating the activities of
E3 ligases (Lyapina et al., 2001). The removal of NEDD8 is achieved via a
metalloenzyme in the Jabl/Csn5 subunit of the complex. The Jabl/Csn5 appears
to remove NEDD8 from cullins (Cope et al., 2002) in a similar fashion to the
deubiquitinating metalloprotease of the RP. To date only one NEDD8 specific
protease has been reported. NEDP1/DEN1 is a cysteine protease, which is
homologous to the SUMO specific proteases (Mendoza et al., 2003; Gan-Erdene
et al., 2003).
1.2.3 ISG15
ISG15 (interferon induced gene of 15 kDa), also known as UCRP
(ubiquitin cross-reactive protein) was originally identified as a member of the
ubiquitin-like proteins, which was induced upon exposure of cells to type I
interferon (IFN), viral infection (Haas et al., 1987), and lipopolysaccharide
(LPS) (Malakhov et al., 2003b). ISG15 is a 17 kDa protein comprised of two
39

domains, each of which bears a regular pattern of homology to ubiquitin. ISG15
shares a similar conjugation pathway but has its own unique
conjugation/deconjugation enzymes that would appear to have evolved from the
ubiquitin pathway enzymes (Narasimhan et al., 1996, Potter et al., 1999, Yuan
and Krug, 2001). ISG15 has been shown to conjugate to Serpin 2a (Hamerman
et al., 2002) as well as key regulators of signal transduction: PLCy1, Jakl,
STAT1, and ERK1 (Malakhov et al., 2003a, Malakhov et al., 2003b). Loss of
UBP43, an ISG15 specific protease, in mice results in a decreased life span,
brain cell injury, and hypersensitivity to interferon stimulation (Malakhov et al.,
2002; reviewed in Kim and Zhang, 2003). Recently ISG15 has been shown to
have functions in neutrophil-mediated immune mechanisms, as it has been
shown to be a novel neutrophil chemotactic factor (Owhashi et al., 2003).
1.2.4 FAT10
FAT10 is similar to, but distinct from ISG15, and contains two ubiquitin-
like domains seperated by a short linker region (Fan et al., 1996, Bates et al.,
1997, Raasi et al., 1999). FAT10, formerly known as diubiquitin, has an
exposed diglycine motif at its C-terminus, and is capable of being conjugated to
as yet unidentified target protein(s). It induces apoptosis (Raasi et al., 2001), and
is highly upregulated in hepatocellular carcinoma and other gastrointestinal and
gynaecological cancers (Lee et al., 2003).
1.2.5 Atgl2 and Atg8
In yeast, Atg12 and Atg8 are ubiquitin-like proteins involved in the other
pathway of protein breakdown, autophagy. The ubiquitin/proteasome is involved
with, amongst other things, the breakdown of short-lived proteins. Its is reported
that the half-lives of the majority of cellular proteins range from a few minutes to
more than ten days, with the definition of a long-lived protein being one with a
half-life of more than five hours. By this definition more than 99% of cellular
proteins have relatively long lives (Ohsumi, 2001). The route for degradation of
these proteins lies in their targeting to the lysosome, which is equivalent to the
vacuole in yeast. The interior of this organelle is acidic and contains multiple
enzymes to breakdown cellular compounds. This process degrades aged proteins
40

from the cytoplasm and can remove entire organelles under stress conditions,
such as starvation (Ohsumi, 2001). Both Atgl2 and Atg8 are required for this
process.
Atg12 is a 186-amino acid hydrophilic protein, which possess a C-
terminal glycine that, through a conjugation pathway analogous to that of
ubiquitin, is attached to a lysine residue in Atg5 (Mizushima et al., 1998) in
yeast. The El enzyme in this pathway is Atg7 (Tanida et al., 1999), and the E2
is AtglO (Shintani et al., 1999), although AtglO bears no homology with any
known E2 protein. The sole substrate for this pathway seems to be Atg5, to
which Atg12 is conjugated immediately after its translation. Apparently little or
no free Atg12 exists in cells, with the majority conjugated to Atg5. The Atgl2-
Atg5 complex then recruits Atgl6 to form a 350 kDa multimeric complex that is
essential for autophagy (Mizushima et al., 1999, reviewed in Jentsch and Ulrich,
1998, Ohsumi, 2001).
Atg8 was the second ubiquitin-like protein shown to be involved in
autophagy in yeast. Atg8 is processed by Atg4, a cysteine protease, removing a
single amino acid to reveal a C-terminal glycine residue. Atg8 and Atg12 are
activated by the same El enzyme, Atg7 but once activated are passed on to
distinct E2 enzymes, Atg3, in the case of Atg8 (Ichimura et al., 2000). Uniquely
Atg8 has been shown to conjugate to the amino group of
phosphatidylethanolamine (PE), and as such mediates protein lipidation
(Ichimura et al., 2000, reviewed in Ohsumi, 2001). In mammals there are three
ubls with homology to Atg8; GATE-16, GABARAP, and MAP-LC3.
1.2.6 Urml
Urml, ubiquitin-related modifier, is a ninety-nine amino acid protein that
terminates in a diglycine motif, that is absent from higher eukaryoyic genomes.
Urml is conjugated to substrate proteins, though these have yet to be identified,
through the activation of Uba4, an El-like enzyme. Urml and Uba4 show
sequence homology with the prokaryotic proteins essential for molybdopterin
and thiamin biosynthesis (Furukawa et al., 2000), providing insights into the
evolution of the ubiquitin-like proteins and their pathways. In Saccharomyces
cerevisiae loss of Urml has been reported to enhance post-translational
41

modification/proteolysis of Elongator subunit Totlp (Elplp) and abrogates its
TOT (toxin target) function. This loss of function prevents Totip mediating cell
cycle arrest in G1 (Fichtner et al., 2003).
1.2.7 HUB1
HUB1 (homologous to ubiquitin 1) is unique amongst ubiquitin-like
modifiers, possesssing a dityrosine carboxyl-terminus, which is exposed
following the processing of a single inhibitory amino acid residue. The
dityrosine motif has been shown to conjugate to cell polarity factors Sphl and
Hbtl in Saccharomyces cerevisiae (Dittmar et al., 2002). A subsequent
investigation into HUB 1, suggests that although HUB I can indeed form SDS-
resistant complexes with cellular proteins, these interactions are not through
covalent conjugation with the dityrosine motif (Luders et al., 2003). As such
HUB 1 may very well not be a true ubl, but may be a member of the type II
category of Ubiquitin-like proteins.
1.2.8 UFM1
The newest member of the ubl family is the ubiquitin-fold modifier 1 (UFM1).
Komatsu et al., whilst trying to identify GATE-16 interacting proteins, isolated a
protein Uba5, which was similar to many of the previously identified El
enzymes. Further investigation showed that Uba5 was indeed an El-like
enzyme, and was the El enzyme for UFM1 (Komatsu et al., 2004). UFM1 was
shown to be a true ubl as high molecular weight species were detected, indicating
that UFM1 was capable of conjugating to target substrates. Ufml has no
sequence homology with ubiquitin, yet still possess the characteristic ubiquitin
fold as determined by computer modelling ((MOE program (2003.02; Chemical
Computing Group Inc., Montreal, Quebec, Canada)) (Komatsu et al., 2004).
1.3 SUMO
The Schizosaccharomyces
pombe and Saccharomyces cerevisiae yeasts
contain a single gene encoding a SUMO homologue, Pmt3 and Smt3
42

espectively, whilst in higher eukaryotes there are three distinct isoforms;
SUMO-1, SUMO-2, and SUMO-3.
SMT3 was originally isolated in a screen for high copy suppressors of
mutations in the centromere binding protein MIF2 (see GenBank Accession No.
U33057). Mif2 protein is a yeast centromere protein with homology to the
mammalian centromere protein, CENP-C (Brown et al., 1995; Meluh and
Koshland, 1995). Studies using temperature-sensitive mutants showed that the
loss of yeast Mif2p function results in chromosome missegregation, mitotic
delay, and aberrant microtubule morphologies (Brown et al.,
1993). Pmt3-null
cells show various phenotypes, including aberrant mitosis, sensitivity to various
DNA damaging reagents, and high-frequency loss of minichromosomes.
Furthermore it was shown that loss of Pmt3 function resulted in a striking
increase in telomere length (Tanaka et al., 1999). In contrast, deletion of SMT3
in S. cerevisiae is lethal and yeast with mutations in UBC9 accumulate at the G2-
M boundary of the cell cycle. Mutations in the Pmt3 UBC9 equivalent, hus5, or
an E1 subunit, rad31, result in mitotic defects and impaired survival after ultra-
violet radiation (Ho and Watts, 2003; reviewed in Hay, 2001). Genetic studies in
mice have yet to be reported although RNA interference studies in
Caenorhabditis elegans indicate that the knockdown of SUMO resulted in a high
incidence of embryonic lethality, with the surviving progeny having high
incidences of a protruding vulva phenotype (Fraser et al., 2000).
Although Smt3/Pmt3 is attached to many yeast proteins, only six have
been characterised to date. In Saccharomyces cerevisiae, where Smt3
conjugation is reported to be essential for viability, three members of the septin
family of proteins are Smt3 conjugated (Johnson et al., 1999). Septins are
components of a belt of 10-nm filaments that encircle the yeast bud neck; Smt3
is attached to the septins Cdc3, Cdcll, and Shsl/Sep7, specifically during
mitosis. The same is not true, however, for Pmt3 in Schizosaccharomyces
pombe. Whereas the staining pattern of Smt3 at the mother/bud neck is observed
at mitosis in Saccharomyces cerevisiae, no similar pattern is observed at the
mother/bud neck suggesting that the septins may not be major Pmt3-modified
targets in Schizosaccharomyces pombe (Tanaka et al., 1999). Pds5 is another
43

Smt3 substrate that is maximally conjugated during mitosis, where Pds5 Smt3-
conjugation promotes the dissolution of cohesion (Stead et al., 2003).
Topoisomerase II is similarly involved in controlling centromeric cohesion,
regulating either a component of chromatin structure or topology required for
centromeric cohesion, and like Pds5, this process can be regulated by Ulp2
(Bachant et al., 2002).
In vertebrates SUMO are a family of ubiquitin-like modifiers, comprising
three members. SUMO-1 is approximately 45% identical to either SUMO-2 or
SUMO-3, following processing, whilst SUMO-2 and SUMO-3 are highly
homologous, being 96% identical to each other following processing (Figure 7).
The SUMO proteins have been characterised as covalently modifying numerous
cellular and viral proteins (Table 2) with substrate dependent consequences.
Functional differences also exist between the various isoforms, with SUMO-2
and SUMO-3 being capable of forming polymeric chains (Tatham et al., 2001).
The specific roles of SUMO-1, SUMO-2, and SUMO-3 have yet to be fully
elucidated, partially due to research focussing on SUMO-1. Recent work on
topoisomerase II during mitosis highlighted the need for care. Azuma et al.,
showed that although exogenous SUMO-1 was capable of conjugating to
topoisomerase II, none was detected when endogenous material was analysed. It
was further shown that topoisomerase II was specifically modified by SUMO-2/-
3 during mitosis, and that SUMO-2/-3 conjugation was dramatically reduced
following the addition of exogenous SUMO-1. Given all three isoforms share
the same conjugation enzymes it is therefore quite conceivable that exogenous
SUMO-1 competes with SUMO-2/-3 for the enzymes and as a result inhibits
their conjugation (Azuma et al., 2003). This is unlikely to represent the only
case where exogenous expression does not represent the endogenous situation,
and it is probable that some of the reported SUMO-1 substrates may in fact turn
out to be specifically modified by SUMO-2, SUMO-3, or both, although how
this is achieved as yet remains unsolved.
The three SUMO isoforms all conjugate to the same conserved motif,
'KxE, where IF represents a large hydrophobic amino acid and x represents any
amino acid. The sequence requirements for the SUMO consensus binding site
were investigated, both in vitro and in vivo. W could
be leucine, isoleucine,
44

hSUMO-2
hSUMO-3
ScSmt3
hSUMO-1
hUbiquitin
ScUbiquitln
ScRubl
WK x
Figure 7. (i) Primary sequence alignment of the human (h) and S. cerevisiae (Sc)
homologues of Ubiquitin, SUMO, and NEDD8. Similar residues are shown in blue.
Position of the SUMO modification motifs present in SUMO-2/3 is indicated by pKxE.
Scissors denote site of protease activity. (ii) Space-fill 3D models of SUMO-1,
Ubiquitin, NEDD8, and representations of SUMO-2 and SUMO-3. Models for SUMO-2
and SUMO-3 were predicted by submission of primary sequences with the PDB co-
ordinates for the 3D structure of SUMO-1 (Bayer et al., 1998) to the ExPASy proteomics
server of the Swiss Institute of Bioinformatics `Swiss Model' (Guex and Peitsch, 1997;
Peitsch et al., 1996). Electrostatic calculations are based upon a dielectric constants of
80.0 (solvent) and 40.0 (protein) at physiological pH. Blue indicates negative surface
potentials whilst red indicates positive surface potentials. Images generated using Swiss
PDB viewer v3.6b (Guex et al., 1999)

Table 2. List of SUMO modified substrates.
Transcription co-/factor Effect on transcription Reference
Tcf-4 Positive Yamamoto et at, 2003
HSF-1 Positive Hong et at, 2001
HSF-2 Positive Goodson et at, 2001
CREB Positive Comerford et at, 2003
p53 Positive(? ) Rodriguez et at, 1999; Gostissa et at, 1999
p73a Not characterised Minty et at, 2000
Dnmt3a Positive Ling et at, 2004
Dnmt3b Not characterised Kang et at, 2001
Pdxl Positive Kishi et at, 2003
APA-1 Stabilisation Benanti et al, 2002
C/EBP Negative Kim et at, 2002
ELK-1 Negative Yang et at, 2003
SREBPs Negative Hirano et at, 2003
Sp3 Negative
-
Sapetsching et at, 2002; Ross et at, 2002
IRF-1 Negative Nakagawa et at, 2002
ARNT Negative Tojo et at, 2002
AP-2 Negative Eloranta et at, 2002
Jun Negative Muller et at, 2000
c-Myb Negative Bies et at, 2002
Lef1 Negative Sachdev et at, 2001
SRF Negative Matasuzaki et at, 2003
AR Negative (? ) Poukka et at, 2000
PR Negative (? ) Takimoto et at, 2003
GR Negative (? ) Tian et at, 2002
GATA-2 Negative (? ) Chun et at, 2003
GRIP1 Negative Kotaja et at, 2002
HDAC 1 Enhanced repression David et at, 2002
HDAC4 Enhanced repression Kirsh et at, 2002
Histone H4 Negative Shiio and Eisenman, 2003
p300 Negative Girdwood et at, 2003
CtBPI Negative Kagey et at, 2003; Lin et at, 2003
PL"ZF Negative Kang et al, 2003
SOP-2 Negative Zhang et at, 2004

Nuclear body proteins
Property Reference
PML PML body formation Boddy et al, 1996
Sp100 Uncertain Sterndorf et al, 1997
HIPK2 Localisation Kim et al, 1999
TEL, TEL-AMLI Nuclear export Chakrabarti et al, 2000
Daxx Ryu et al, 2000
Genome integrity proteins
Rad52/Rad22, Rad51/Rhpl
TDG
Topo I
Topo II
WRN
BLM
PCNA
Signal transduction proteins
IKBa
CamkIl
Smad4
Axin
Mekl
Ttk69
Dorsal
FAK
MDM2
Double-strand break repair
Increase in enzymatic turnover
DNA repair
DNA repair
Not characterised
Localisation
DNA repair
Stabilisation
Regulates neuronal differentiation
Localisation/stabilisation
JNK activation
Nuclear export
Regulates neuronal differentiation
Immune response
Activation of autophosphorylation
Stabilisation
Viral proteins Property Reference
Ho et al, 2001; Shen et al, 1996
Hardeland et al, 2002
Mao et al, 2000
Mao et al, 2000
Kawabe et al, 2000
Suruki et al, 2001
Hoege et al, 2002
Desterro et al, 1998
Long et al, 2000
Lin et al, 2003
Rui et al, 2002
Sobko et al, 2002
Lehembre et al, 2000
Bhaskar et al, 2002
Kadare et al, 2003
Buschmann et al, 2001
EBV-BZLF1 PML body disruption Adamson et al, 2001
BPV-E1 Localisation Rangasamy et al, 2001
CMV-IE1/IE2 Disrupts SUMO localisation Ahn et al, 2001; Spengler et al, 2002
AdV-E1B 55K Localisation Endter et al, 2001
Neuronal proteins Property Associated disorder Reference
APP Abeta production Alzheimers Li et al, 2003
Httex 1p Stability Huntingtons Steffan et al, 2004
Neuronal tracks NIID Pountney et al, 2004
Atrophin mutant (? ) Polyglutamine tracts Terashima et al, 2002; Ueda et
Cytoplasmic
al, 2002
Yeast septins Not characterised Takahashi et al, 1999 Johnson et al, 1999
GLUT, GLUT4 Activity Giorgino et al, 2000
Nuclear pore complex
RanGAPI Localisation Matunis et al, 1996
RanBP2 Unclear, SUMO E3 Pichler et al, 1998

valine, phenyalanine, and methionine with no detrimental affects on SUMO
conjugation, whilst alanine and proline permitted weak conjugation and
tryptophan prevented conjugation. The requirement of the glutamic acid was
also shown although weak SUMO conjugation did occur when this was
substituted to an aspartic acid (Rodriguez et al., 2001). These requirements were
tested on transferable sequences containing the'PKxE motif, allowing SUMO
modification. Although the WKxE motif was demonstrated to be sufficient for
modification in vitro, modification in vivo required the additional presence of a
nuclear localization signal. The SUMO conjugation motif is an. absolute
requirement for'the vast majority of SUMO substrates but there are a number of
exceptions to this rule. APA-1, CREB, IRF-1, Tcf-4, Daxx, Axin, Hdm2,
Histones, Huntingtin, and SENP1 have all been demonstrated to be SUMO
substrates, that do not contain the consensus SUMO modification motif, with the
exceptions of SENP1, which although it contains a motif, this is not the site of
SUMO modification and Tcf-4 which is modified at a consensus site but also
possessses a second site of modification that lies out-with the consensus (Benanti
et al., 2002, Comerford et al., 2003, Nakagawa et al., 2002, Yamamoto et al.,
2003, Ryu et, 2000, Rui et al., 2002, Buschmann et al., 2001, Shiio and
Eisenman, 2003, Bailey and O'Hare, 2003). CtBP2 is also modified in vitro by
SUMO, but only in the presence of Pc2 although it lacks a consensus motif
(Kagey et al., 2003), which raises the possibility that other substrates, that lack
consensus motifs, are SUMO modified in the presence of appropriate SUMO E3
enzymes.
The effects of SUMO conjugation onto a substrate can broadly be
characterised into one of three groups: altering substrate stability, changing sub-
cellular localisation, and altering protein-protein interactions. As a large number
of SUMO substrates are either transcription factors or co-factors, SUMO
conjugation has been demonstrated to play an important role in transcriptional
regulation, exerting both positive and negative influences in a substrate
dependent manner.
The classic example of SUMO modification increasing substrate stability
is that of IiBa. SUMO conjugation to IKBa directly competes with ubiquitin,
thereby preventing IKBa degradation. This antagonism of ubiquitin is achieved
48

due to the requirements of lysine residues 21 and 22, which are blocked and
masked by SUMO attachment. Ubiquitination does not usually require specific
lysine residues for conjugation and as such SUMO conjugation to a substrate
would not prevent degradation, as ubiquitin would bind at any available lysine
residue. In the case of CREB, c-Myb, Smad4, and APA-1 the increase in
stability could be explained by a reduction in ubiquitin moieties attached
(Comerford et al., 2003, Bies et al., 2002, Benanti et al., 2002, Lin et al., 2003).
1.3.1 Localisation and Nuclear domains
The nucleus, like the cytoplasm, is compartmentalised, containing distinct
nuclear domains. Distinct nuclear domains thus far identified include, nucleoli,
Cajal bodies (CBs), gems, splicing speckles, clastosomes, paraspeckles, and
Promyelocytic leukaemia (PML) bodies (reviewed in Lamond and Sleeman,
2003). These nuclear bodies may function by accumulating factors in distinct
localisations, thereby creating concentrations of components, promoting efficient
interactions. The compartmentalisation of these components could create
domains of active/inactive factors, and as such control of their
assembly/disassembly may regulate many cellular functions (reviewed in Isogai
and Tjian, 2003).
1.3.2 PML bodies
PML bodies are known under a variety of names; PML bodies, ND 10,
kreb bodies, and PODs (PML oncogenic domains). PML bodies are in essence
an association of numerous proteins, co-localising within the nucleus. The
protein components of the PML bodies are often very transient and many of the
proteins identified as components are conditional on their expression levels
(Figure 8). Mature PML bodies, where PML forms the outer shell (Lallemand-
Breitenbach et al., 2001, LaMorte et al., 1998) are intimately linked with the
SUMO conjugation system. Much work has focused on the Promyelocytic
leukaemia (PML) gene since its integral role in Acute Promyelocytic leukaemia
was established, brought on by the t(15; 17) translocation resulting in a
PML/retinoic acid receptor (RAR) a fusion protein. In healthy cells PML
49

Figure 8. Composition and regulation of PML bodies. Table consisting of the known
components of PML bodies, indicating the conditions used to show their localisation, and
a brief summary of their function. 'Ishov et al, 1999; 2Mu et al, 1994; 3Alcalay et at,
1998; °Guo et al, 2000; 5Seeler et al, 1998; 6Lehming et a1,1998; 7Yankiwski et al, 2000;
SBoddy et al, 1996; 9Sternsdorf et al, 1997; 1°Kamitani et al, 1998; UAscoli and Maul,
1991; 12Maul et al, 1995; '3Gong et al, 1997; "Gong et at, 2000; "Everett et al, 1999;
16Seeler
et al, 2001; "Doucas et al, 1999; '8Boisvert et al, 2001; '! Everett et al, 1999;
'Maul et al, 1998; 21Fabunmi
et al, 2001; 'Anton et al, 1999; 'Cao et al, 1998; 'Carlile
et al, 1998; 'Lai and Borden, 2000; 'Ruthardt
et al, 1998; 'Goodson et al, 2001;
2'Doucas and Evans, 1999; 'Jiang et al, 1996; 30Gongora et al, 1997; 31Skinner et al,
1997; 'Klement et al, 1998; 33Trost et at, 2000; 34Desbois et al, 1996; 35 Lehembre et at,
2001; 'Vallian et al, 1998; 37Rochat-Steiner
et al, 2000; 'Maul, 1998; 'Yeager et al,
1999; 42Johnson et al, 2001; `Alcalay et al, 1998; 'Bloch et al, 1999. And a schematic
representing how the components of the PML bodies can be both recruited and released
from PML bodies following different stimuli. (Adapted in part from Negorev and Maul,
2001).

(i)
Proteins present at PML bodies at endogenous levels of expression
PML Fssential in PML, body formation; in SUMO-modified form recruits Daxx; interacts with CBP, pRb, p. 53; repressor;
considered a co-
''.
SplOO
Interacts with HPI; PML bodies are limited in their capacity to recruit Spl005.1,.
Daxx Recruited by PMLSUMO; binds to an increasing number of proteins.
BLM
Low relative amount of BLM is stationed in PML bodies".
SUMO
NDP55
Modifies PML and SpIOO covalently changing their capacity to bind other proteins"').
Uncharacterised protein(s); potentially a protein modification recognised by Mab 138; increases after heat shock; present in
all vertebrate species tested"'2.
Proteins conditionally present in or at PML bodies
TRF1l2
NBS 1
P95/nibrin
Mrel 1
Present in a subset of PMI, bodies containing telomere repeats in cells depending on an alternative telomere maintenance
RAD-51 mechanism. These proteins colocalise with PML in RAD52
Repl. Factor A
WRN
telomerase-negative immortalised cells during the late SIG,
-phase.
SENPI
HPI
SpIOOC
HMG2
CBP/p30p
pRb
USP7/HAUSP
pA28
proteosome
p$p
GAPDH
eIF-4
PLZF
HSF2
Tax
p53
ISO-20
Mutant ataxin
PKM
Int6
PAX3
Spl
FISTIHIPK3
hGCNP
Sp140
BRCA 1
(ii)
Removes St IMO from PMI. ".
Interacts with Sp10 but is present in PML bodies in variable amounts depending on the cell cycle Phase 5,6.1.5
.
Present in some PML bodies".
Interacts with SplOOB6.
Interacts with PMI. "; found in some cell types but not other'.
Phosporylated form found by some groups but not others; possibly cell cycle related colocalisation'.
At or beside some PML bodies; probably cell cycle dependent"-'
Proteosomal activator; increased recruitment to PML bodies by interferon y exposure".
Only after application of proteosome inhibitors=' or after interferon y as immunoproteosomes'".
Interacts with PML and associates with a subset of PML bodies'.
Interaction with PML is RNA dependent.
Interaction with PML23.
No interaction with PML but side by side localisation in nucleus".
Heat shock transcription factor 2 after overexpression in low number of cells, mostly cytoplasmic2.
Recruited to PML bodies when PML is overexpressee.
In specific cells, after overexpression or proteosome inhibition° or beside PML bodies with T-ag down regulations.
After overexpression of a fragment".
PML but not the other PML body proteins are located in ataxin- l aggregates after polyglutamine tract extension3`32
Mx interaction kinase after transient expression°.
In some cells possibly by binding to overexpressed Daxxx.
Interacts with PML'.
After overexpression interacts with Daxe.
Beside PML bodies upon transient overexpressio& .
After overexpression with some PML bodies4'.
Breast cancer protein I beside PML bodies upon transient overexpression.
Recruitment
IFN UP motion Recycling
PML
SUMO Ubc9
Davy
Release
Viral Proteins
E4 ORF3
ICPO
IEI
External Insults
Heat Shock
I
MMS
Cd'
Uv
40"AO-Daxx SENP-1 SITMO
PML body PML
pl W'mo-HP1
nax, i

functions as the organiser of the PML bodies, and is essential for the recruitment
of other PML body associated proteins, a function lost by the PML/retinoic acid
receptor (RAR) a fusion protein. PML is a common form of acute myeloid
leukaemia (AML), and it is common for patients to possess the PML/RARa
fusion protein, although four other different fusion partners have been reported to
date, these being the promyelocytic leukemia zinc finger gene (PLZF), the
nucleophosmin gene (NPM), the nuclear mitotic apparatus gene (NuMA), and
the signal transducer and activator of transcription 5b gene (STAT5b) (Lin et al.,
2001).
Cells from APL patients show a disruptedmicropunctate pattern in
place of the usual 10-20 PML bodies. SUMO modification was first suspected
of being a crucial component of PML bodies as both PML and SplOO, another
principal component of PML bodies, are SUMO substrates, as are many PML
body associated proteins. Furthermore it was demonstrated that upon exposure
to arsenic trioxide (As203) SUMO modification of PML was increased and
nucleocytoplasmic PML is transferred to the nuclear matrix, resulting in an
increase in size of the PML bodies (Muller et al., 1998). Treatment of APL cells
with retinoic acid or As203 restores normal NB organisation. As203 treatment
results in the proteasome-mediated degradation of SUMO modified PML and
PML/RARa, mediated through Lys160 (Lallemand-Breitenbach et al., 2001) and
subsequent cell apoptosis. PML/RARa strongly represses key regulatory genes
and slows down differentiation resulting in the arrest of the cells at the
promyelocyte stage (Du et al., 1999). Following PML degradation, healthy cells
will synthesise additional PML, whereas PML/RARa cells being arrested at the
promyelocyte stage will fail to. This results in the restoration of NBs localisation
and remission of PML (reviewed in Zhu et al., 2002). Although SUMO
modification is not required for either matrix targeting or formation of primary
PML bodies, it is required for secondary shell-like PML body formation
(Lallemand-Breitenbach et al., 2001). The ability of PML to function as a
scaffold protein for NB recruitment is shown by PML -/- cells which had nuclear
diffuse or mislocalised patterns of normally PML body-associated proteins
(Ishov et al., 1999, Zhong et al., 2000). PML mutants that lack SUMO
conjugation sites are defective in recruiting proteins to PML bodies, and this has
52

lead to the hypothesis that PML bodies are sites of nuclear storage of SUMO
modified substrates. The storage of these substrates can be affected by a number
of early gene products from several DNA viruses, which act to disrupt the PML
body. DNA viruses such as herpes simplex virus (HSV) and cytomegalovirus
virus (CMV), both code for proteins which disrupt the SUMO-1 modification of
PML and SplOO (Muller and Dejean, 1999; Everett, 2001) and the nuclear
accumulation of the CMV protein IE1 that mediates PML NB disruption, is
SUMO-1 modification-dependent (Muller and Dejean, 1999). These
observations suggest that DNA viruses may target PML bodies as a means of
relieving the transcriptionally repressive environment created by SUMO
modification. Additionally PML body composition is affected by external
stresses, such as heat shock and DNA damage, suggesting a role for PML bodies
in many response pathways
(Figure 8).
PML is able to regulate transcription via association with numerous
transcription factors/cofactors (Figure 9), and shows intrinsic repression activity
as a Ga14 fusion (Ahn et al., 1998; Vallian et al., 1997). Furthermore over
expression of PML leads to the repression of various promoters (Mu et al.,
1994). Other examples of SUMO modified substrates, which are associated with
PML bodies and exhibit repressive . capabilities include HIPK2, Daxx, and
Sp100. HIPK2 is a member of the homeodomain interacting protein kinases
(HIPK). SUMO modification of HIPK2 is required for its localisation to PML
bodies (Kim et al., 1999) where it subsequently forms a stable corepressor
complex with Groucho corepressor and HDAC1 (Choi et al., 1999). Daxx and
Sp100, like PML, have been demonstrated to have repressive capabilities when
fused to Ga14 DBD (Szostecki et al., 1990). Daxx accumulation in PML bodies
is dependent upon SUMO modification of PML (Duprez et al., 1999). Daxx
may potentially act as an adaptor protein, recruiting other PML body-associated
proteins that do not interact directly with PML (reviewed in Maul et al., 2000).
Daxx has the ability to recruit HDACs (Li et al., 2000), and thus likely represses
transcription by remodelling chromatin. Interestingly the repressive ability of
Daxx is lost by PML coexpression, suggesting a mechanism by which PML
sequesters Daxx, possibly by its retention to PML bodies (Lehembre et al., 2001;
53
,ý

Figure 9. PML associated proteins and transcriptional regulation. (i) Summary of
direct protein-protein interactions among PML associated proteins which are involved
in transcriptional activation (green) or repression (orange). (ii) Models for
transcriptional regulation by PML bodies: Left, PML bodies regulate transcription by
sequestering and thereby inactivating activators or repressors from the active pool in the
nucleoplasm. Middle, PML bodies serve as sites for assembly or activation of
transcription modulatory complexes by providing the post-translational modifying
enzymes and a high local concentration of the factors involved. Right, PML bodies
create a microenvironment that facilitates repression or activation in their direct
surroundings. Figure adapted in part from Lin et al, 2001.

4
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Li et al., 2000). SplOO has been shown to possesss a heterochromatin 1(HP1)
binding site (Lehming et al., 1998; Seeler et al., 1998). SUMO conjugation to
SplOO enhances its interaction with HPIcc, at least in vitro (Seeler et al., 2001),
providing an insight into how SUMO modification might regulate both PML
bodies and chromatin. SplOO is currently thought to be recruited to PML bodies
following the association of SplOO with an as yet unidentified adaptor protein.
This adaptor proteins recruits Sp l00 either by binding to the self-assembly
domain or by a conformational change in PML. (Negorev et al., 2001, reviewed
in Negorev and Maul, 2001). Sp l00, like Daxx, in general appears to be
recruited to PML bodies in order to reduce its availability in the nucleus and not
as a necessity of PML body structural formation.
In addition to the number of transcription factors and transcription co-
factors found in PML bodies, a number of DNA damage associated protein are
also found within this nuclear domain.
1.3.3 SUMO and genome integrity
The DNA damage response involves the coordination of numerous
cellular pathways, including DNA repair events and checkpoint arrest
mechanisms. Abnormalities in these processes are identified by their increased
sensitivities to DNA damaging agents, such as UV and ionising radiation, or to
the DNA synthesis inhibitor hydroxyurea (HU).
SUMO was co-discovered as an interactor with the DNA repair enzymes
Rad5l and Rad52 (Shen et al., 1996), although the authors termed their novel
gene UBL1 (ubiquitin-like 1), this was subsequently shown to be the same gene
as SUMO-1. Ubc9 has also been reported to associate with Rad51 (Kovalenko et
al., 1996). In yeast Rad5l/Rad52-dependent DNA repair pathways are involved
in DNA recombination and DNA double-strand break repair in yeast. In fission
yeast, Schizosaccharomyces pombe, Pmt3 (SUMO) has been shown to be
conjugated to both Rad22 and Rhp5 1, the fission yeast homologues of Rad51 and
Rad52 (Ho et al., 2001). Similarly, mammalian Rad5l proteins have been
demonstrated to play essential roles in DNA homologous recombination, DNA
repair, and cell proliferation. Rad5l and Rad52 have been reported to form a
complex with non-conjugated SUMO-1 (UBL1) in human cells (Li et al., 2000).
56

The function of this complex seems likely to regulate homologous
recombination, based on the observations that overexpression of SUMO-1 down
regulated DNA double-strand break-induced homologous recombination and
reduced resistance to ionizing radiation. Interestingly Rad51 foci colocalise with
a subset of PML NBs and do not form in cells that express a dominant-negative
PML form, suggesting that functional PML is necessary for their assembly
(Bischof et al., 2001). The role played by SUMO modification in recruitment of
Rad51 foci, either to PML or another as yet unidentified component,
is further
supported by the findings that other components of the Rad51 foci are indeed
SUMO modified, such as WRN (Kawabe et al., 2000). WRN localises to the
nucleolus under normal growth conditions, but following exposure to DNA-
damaging agents, relocates into Rad51 foci (reviewed in Muller et al., 2004).
Topoisomerase-mediated DNA damage represents an unique type of
DNA damage, and is of particular interest due to the effect many antibiotics,
anticancer drugs, toxins, carcinogens, and physiological stresses have on the
catalytic cycles of topoisomerases, and as such result in the formation of
topoisomerase-mediated DNA damage. Both topoisomerase I and topoisomerase
II are substrates for SUMO modification (Mao et al., 2000a, Mao et al., 2000b).
SUMO conjugation to topoisomerase is induced by the presence of camptothecin
(CPT), an anti-tumour drug. CPT was shown not to be the only drug to induce
SUMO modification of topoisomerase, ICRF-193, a drug which does not induce
topoisomerase II-mediated DNA damage, but traps topoisomerase II into a
circular clamp conformation, also resulted in SUMO conjugation. The
consequence of SUMO modification on topoisomerase I, is to enhance the
cleavable complex formation (Horie et al., 2002). Indeed sumoylation deficient
mutants of topoisomerase I, reduced the CPT-induced cleavable complexes but
had no effect on the in vitro catalytic activity.
DNA damage has also been shown to activate the NF-KB pathway via the
activation of NEMO (NF-KB essential modulator) / IKB kinase Y (IKKy) by
ubiquitin-like proteins. NEMO is held in the cytoplasm as a member of the IKB
kinase complex, consisting of NEMO and IKKI/a and IKK2/ß. Following DNA
damage NEMO is transported to the nucleus and SUMO modified (Huang et al.,
2003, reviewed in Hay, 2004). It is not yet clear whether NEMO shuttles
57

etween the nucleus and cytoplasm, in a manner analogous to that of both NF-
KB and IKBa or whether the translocation is DNA damage induced. Following
NEMO sumoylation and nuclear localisation, NEMO is desumoylated, and
phosphorylated by the ATM kinase. The ATM/ATR kinase family are often
associated with phosphorylation of substrates following DNA damage, and
signal the DNA damage event to the appropriate response enzymes.
Interestingly, following NEMO phosphorylation, NEMO is then modified by
ubiquitin, an event that seemingly occurs at the same lysine residues required for
SUMO conjugation. Following NEMOs ubiquitination, NEMO exits the
nucleus, associates with the IKK complex, and activates NF-KB.
Smt3 (SUMO) modification of the proliferating cell nuclear antigen
(PCNA), a DNA-polymerase sliding clamp involved in DNA synthesis and
repair, in Saccharomyces cerevisiae plays a crucial role in DNA repair. Hoege et
al., demonstrated that Smt3 conjugation occurred and competed with
ubiquitination of the same lysine residue (Hoege et al., 2002, reviewed in
Pickart, 2002, Stelter and Ulrich, 2003). PCNA is monoubiquitinated through
Rad6 and Radl8, and subsequently modified by lysine-63-linked multi-
ubiquitination, through Mms2, Ubc13, and Rad5. The ubiquitination of PCNA is
induced following DNA damage, and the resulting multi-K63 linked ubiquitin
chain leads to error-free DNA repair. Smt3 modification of PCNA conversely
leads to an inhibition of error-free DNA repair, via the competition for the
acceptor lysine with ubiquitin (Hoege et al., 2002). It is attractive to speculate
that the ubiquitin/SUMO switch present in Saccharomyces
cerevisiae for PCNA,
might be a prevalent regulatory mechanism, and there are a growing list of
documented cases where ubiquitin and SUMO directly compete for lysine
residues (Desterro et al., 1998; Sobko et al., 2002; Hoege et al., 2002; Huang et
al., 2003; Steffan et al., 2004). However human PCNA has yet to be shown to
be SUMO modified. Indeed the authors whilst, acknowledging the absence of
SUMO modified PCNA, also reported the detection, indeed prevalence of mono-
ubiquitinated PCNA in HeLa cells, and that of multi-ubiquitinated forms at lower
levels under normal conditions (Hoege et al., 2002).
58

1.3.4 SUMO and transcriptional regulation
The addition of a SUMO moiety to components of the transcriptional
apparatus does not have a common consequence as it can both activate and
repress transcription. Currently, a greater number of transcription factors, around
half of all known substrates, are repressed following SUMO conjugation.
A number of SUMO acceptor sites in many of the substrate transcription
factors, have been mapped to previously characterised repression domains, and
mutation of target lysine(s) leads to an increase in transcriptional activity.,
SUMO modification of transcription factors may also result in an increase in
transcriptional activity. Modification of the heat-shock transcription factors
HSF1 and HSF2 with SUMO-1 results in increased DNA-binding activity and
mutation of the acceptor lysine reduces the transcriptional activity of HSF1
(Hong et al., 2001; Goodson et al., 2001). Also the UV light stimulated SUMO
modification of p53, has been shown to result in a mild enhancement of
transcriptional and apoptotic responses (Rodriguez et al., 1999; Gostissa et al.,
1999), although, as yet, the molecular mechanisms have not been elucidated.
1.3.5 SUMO and neurodegenerative diseases
The majority of the studies exploring the links between SUMO and
neurodegeneration have focused on conditions in which there are apparent
intranuclear aggregates. Evidence linking SUMO-modified proteins, and their
accumulation in aggregates is shown in the case of neuronal intranuclear
inclusion disease (NIID). NIID is a rare multisystem neurodegenerative disease
characterised by large intranuclear inclusions in neurons of the central and
peripheral nervous systems. Intranuclear aggregates are the pathologic hallmark
of this disease, and are also a common feature of many other neurodegenerative
diseases including many CAG/polyglutamine disorders. The role of ubiquitin in
these diseases has been shown by the presence of ubiquitinated proteins
in long
glutamine tracts, representing aggregated proteins, a hallmark of many
neurodegenerative diseases (Lieberman et al., 1998). Recently these
intraneuronal aggregates, in NIID, were shown to stain with antibody
recognising SUMO-1 (Pountney et al., 2004). The role of SUMO in other
neuronal degenerative diseases is implicated by the observations that there is
59

increased SUMO modification in brain tissue samples from patients with
spinocerebellar ataxia type 3 (SCA3), dentatorubropallidoluysian atrophy
(DRPLA), and in a mouse model of spinocerebellar ataxia type 1 (Terashima et
al., 2002; Ueda et al., 2002).
The role for SUMO in neuronal degenerative disorders has yet to be
understood, although through the use of Drosophila experimental models of
polyglutamine disease, Chan et al., 2002, have demonstrated that SUMO is a
modifier of toxicity. Further clues to SUMOs role comes from investigations
showing SUMO-3 conjugation to amyloid precursor protein, and a possible role
played in its proteolytic processing (Li et al., 2003).
Recently SUMO has also been implicated in Huntington's disease (HD),
a condition characterised by the accumulation of a pathogenic protein Htt, which
contains an abnormal polyglutamine expansion. Marsh and colleges reported
that a pathogenic fragment of Htt (Httexlp) can either be modified by SUMO-1
or Ubiquitin. In a Drosophila model of HD, it was demonstrated that SUMO
modification of Httexlp exacerbates neurodegeneration, whilst ubiquitination of
Httexlp abrogates neurodegeneration (Steffan et al., 2004).
1.4 Ubiquitin and ubiquitin-like proteases: - control over
conjugation
Ubiquitin is expressed as a protein fusion or as polyubiquitin, and free
ubiquitin is generated by the actions of a family of cysteine proteases termed C-
terminal hydrolases (UCHs) revealing the Gly-Gly motif required for its
subsequent conjugation. Ubiquitin can subsequently be removed following its
covalent attachment to substrate proteins, by actions of a second group of
proteases known as Ub-specific proteases (UBPs/USPs), and recently two further
classes of ubiquitin proteases have been identified, a Ub-specific JAMM
(JAB 1/MPN/Mov34) motif containing an metalloprotease and cysteine proteases
containing an OTU (ovarian tumour) domain (Verma et al., 2002, Balakirev,
2003, Evans et al., 2003). Similarly two distinct proteases are required for the
control of ISG15. As yet no proteases have been proven to process ISG15, but
ISG15 is removed from substrates via the protease activity of UBP43. Unlike
60

oth ubiquitin and ISG15, the three SUMO ubls and NEDD8 are processed and
removed following conjugation by the same proteases.
The SUMO specific proteases are members of the cysteine protease
family, falling into the C48 class of proteases (Rawlings and Barrett, 2000), and
as such are distinct from the ubiquitin C-terminal hydrolyases and DUBs which
are in the classes C12 and C19 respectively. Cysteine proteases contain at their
active sites the amino acids Cys, His, and Asp, and this catalytic triad is used to
define the cysteine protease family (Figure 10). Members of the SUMO family
of SUMO specific proteases include proteases with no activity towards SUMO,
rather. they contain a conserved catalytic domain which is found in all members
of this group (Figure 10. ii and iii. ); furthermore the catalytic core shares a similar
tertiary structure. Initially eight SUMO specific proteases were identified due to
the presence of this core domain, termed "ULP domain" (Ubiquitin like protease)
after the yeast Smt3 protease first identified, these being SENP1,2,3,5,6,7,
and 8 (Figure 10. iii. ) (Yeh et al., 2000). The Schizosaccharomyces pombe and
Saccharomyces cerevisiae genome each contain two genes encoding distinct
Smt3/Pmt3 proteases, Ulpl and U1p2 (SMT4). Ulpl is localised to distinct
regions of S. pombe in a cell cycle dependent manner (Taylor et al., 2002).
During S and G2 phases of the cell cycle the Ulpl protein is localised to the
nuclear periphery. Although not essential for cell viability, cells lacking the Ulpi
gene display severe cell and nuclear abnormalities. Ulpl-null (Ulpl. d) cells are
sensitive to ultraviolet radiation and have similar, although less pronounced
phenotypes, to rad31. d and hus5.62. These cells contain mutations in one subunit
of the activator and the conjugator for Smt3/Pmt3 respectively (Ho and Watts,
2003). The second protease, U1p2, is localised throughout the nucleus, and the
distinct localisations of the proteases accounts, in part, for its distinct in vivo
substrate selectivity (Li et al., 2000). Ulp2-null cells, like U1p1-null cells,
display multiple defects, including temperature-sensitive growth, abnormal cell
morphology, decreased plasmid and chromosome stability, and severe
sporulation defects. U1p2-null cells are hypersensitive to DNA-damaging agents,
hydroxyurea, and benomyl. DNA damage results in an impaired ability to
recover from cell cycle arrest. The studies on the proteases in yeast also revealed
61

Figure 10. Comparison of SUMO-specific and SUMO-like cysteine proteases. (i)
Representation of the active site of the cysteine protease, and schematic of its
activation. The cysteine residue, activated by a histidine residue, plays a role in the
nucleophile that attacks the peptide carbonyl bond. (ii) Schematic illustration of two
of the six human SUMO-specific proteases (SENP1 and SENP2) and the two yeast
homologues (ScUlpI and ScUlp2). The conserved core domain is in blue, whilst the
arrows indicate the positions of conserved residues of the catalytic triad (histidine,
asparate, and cysteine) an additional glutamine residue predicted to aid in the
formation of the active site. (iii) Sequence alignment of the conserved catalytic core
domain of the SUMO specific-like protease family. Vertical blue bars indicate
highly conserved amino acids. Full triangles mark the amino acid residues of the
catalytic triad, (His, Asn/Asp/Glu, and Cys) and the Gln residue involved in the
formation of the oxyanion hole in the active site. Sequences: Human sentrin-specific
protease 1 (SENP1) (AF149770); Human SENP2 (Q9HC62); Human SENP3
(Q9H4L4); Human SENP5 (Q96HI0); Human SENP7 (Q9BQF6); Ulpl,
Saccharomyces cerevisiae Ulpl protease; Human NEDD8 protease
(NEDP1/DEN1/SENP8); African swine fever virus (ASFV) protease (Q00946);
Human adenovirus 2 protease (ADE2) (P03252).

(i)
(ii)
(iii)
SENP1
SENP2
SENP3
SENP7
Ulpl
NEDP1
ASFV
PDE2
SENP1
SENP3
SENP5
SENP7
Ulpi
NEDP1
ASFV
ADE2
ýZ
ISO&
ý' R
ScUlp 1 (621 aa)
SENPI (643 aa)
SENP2 (1112 aa)
ScUlp2 (1034 aa)

the presence of a possible feedback mechanism. Bylebyl et al (2003), showed
that overproduction of a catalytically inactive version of Ulpl can substantially
overcome the U1p2-null defects, a result that was shown to be reciprocal. The
double protease mutant accumulates Smt3-protein conjugates to a much lesser
extent than either single mutant, suggesting a mechanism that limits Smt3-
protein ligation when Smt3 deconjugation by both Ulpl and Ulp2 are
compromised.
Ulpl is the major Smt3 precursor-processing enzyme in yeast, and
whenpresent at endogenous levels is capable of cleaving Smt3 conjugates that
accumulate in Ulp2-null cells. In contrast Ulp2, possessses a more limited ability
to cleave Ulpl cell-specific Smt3 conjugates (Li et al., 2000). Ulp2 has been
demonstrated to cleave the Smt3-Smt3 chains, formed via covalent attachment to
the lysine residue of the conjugation motif in the N-terminus of Smt3 (Bylebyl et
al., 2003), although despite relieving several of the Ulp2-null phenotypes, the
polymeric chains were shown not to be essential in yeast. Wild type strains
lacking any of the nine lysines in SUMO were viable, had no obvious growth
defects or stress sensitivities, and showed similar patterns of SUMO conjugation
as the wild type strain (Bylebyl et al., 2003). The specificity of the proteases
towards substrates is controlled in part by the non-essential amino-terminal
region of Ulpl. The Ulpl, Ulp domain (UD), a 200-residue segment, conserved
among Ulps, which includes the core cysteine protease domain, is capable of
supporting wild-type growth and is capable of cleaving Smt3 from substrates in
vitro (Li et al., 2003). Interestingly, the yeast strain containing the amino-
terminally deleted Ulpl protease was capable of suppressing defects associated
with cells lacking the Ulp2 protease, whilst wild-type Ulpl proteases are not.
Additionally it was observed that many Smt3 conjugates accumulated to high
levels in cells lacking the Ulp2 protease. These results showed that, although the
catalytic domain was capable of processing Ulp2 protease substrates, the amino-
terminal domain of Ulpl restricted protease activity towards certain substrates
whilst enabling the cleavage of other substrates.
In vertebrates, only three of the seven potential SUMO proteases, SENP1,
SENP2, and SENP6 have been shown, to have SUMO processing and
deconjugating activity in vitro (Gong et al., 2000; Hang and Dasso, 2002; Kim et
al., 2000; Zhang et al., 2002). SENP8, as mentioned earlier, is a NEDD8
64

specific protease, and it is possible that other members of this protease group will
turn out to have specificity towards other Ubl proteins. The gene products from
several animal viruses, including the adenovirus L3 protease, the I7 products of
fowlpox and vaccinia virus, and openreading frame (ORF) S273R of African
swine fever virus (ASFV) (Li and Hochstrasser, 1999) also possesss this Ulp
domain, the "core Ulp domain" of -90 amino acids, containing the conserved
catalytic cysteine and histidine residues. Andres and colleagues subsequently
investigated this observation and demonstrated that the ASFV protease was
indeed capable of processing at Gly-Gly-Xaa sites, and was involved in the
processing of the viral polyproteins pp62 and pp220 (Andres et al., 2000). The
adenovirus protease has been well characterized, and is similarly capable of
processing at Gly-Gly-Xaa (Webster et al., 1993; Mangel et al., 1996; Ding et
al., 1996).
1.4.1 SUMO specific proteases and PML bodies
Given the fact that SUMO modification of PML is required for both NB
formation and PML recruitment of proteins such as, Daxx, CBP it was likely that
the role of the family of SUMO specific proteases would prove important in both
the regulation of PML body formation and regulation of transcriptional activity.
Repression mediated through SUMO conjugation to Sp3 has been shown to be
relieved via the expression of SUMO specific proteases (Ross et al., 2002),
thereby providing a mechanism for controlling transcription. Work on a murine
SENP2 isoform, SuPr-1, provided further detail into the workings of the
proteases. Best and colleagues demonstrated that PML is required for SuPr-1
activity and enhances SuPr-1 action, presumably by having SUMO substrates in
the PML body, which can subsequently be removed by the protease. A
catalytically inactive form of the protease was capable of binding to SUMO
modified PML, whereas the wild type was not, and it was further noticed that the
inactive mutant was capable of activating c-Jun dependent transcription just as
well as the wild type (Best et al., 2002). Recently it has been demonstrated that
SENP1, is capable of being a target for SUMO modification in itself, and this
modification is enhanced in catalytically inactive forms of the protease (Bailey
and
O'Hare, 2003).
65

1.5 Competition for lysine residues
SUMO is one member of the protein tags, which is capable of modifying
substrates on their lysine residues, yet a further number of post-translational
modifiers also attach via a substrates lysine residue, most notably acetylation.
To date the number of identified substrates, where ubiquitin and SUMO compete
for the same lysine residues is small. The first such substrate identified was
IKBa (Desterro et al., 1999). As previously mentioned, Hay and colleagues
demonstrated that the conjugation of SUMO to IKBa directly inhibited the
conjugation of ubiquitin, thereby creating a "privileged pool" of IKBa resistant
to degradation. NEMO is also modified by SUMO and ubiquitin on identical
lysine residues. SUMO modification of NEMO results following DNA damage
and nuclear localisation. Subsequently following SUMO deconjugation, NEMO
is ubiquitinated and exported to the cytoplasm (Huang et al., 2003, reviewed in
Hay, 2004). Furthermore it was demonstrated that PCNA, in yeast, was also
modified by SUMO and ubiquitin, and again there was competition for the same
lysine residue and controlled PCNAs DNA repair process (Hoege et al., 2002).
The ubiquitination of PCNA is induced following DNA damage, and the
resulting multi-K63 linked ubiquitin chain leads to error-free DNA repair.
SUMO modification of PCNA conversely leads to an inhibition of error-free
DNA repair, via the competition for the acceptor lysine with ubiquitin. A further
example of competition is that involving MEK1, in Dictyostelium. SUMO
conjugation of MEK1 occurs in response to elevated levels of cAMP that are a
consequence of nutrient starvation. Under these conditions nuclear MEK1 is
SUMO modified and transported to the cytoplasmic cortex. Interestingly the
lysine residue for SUMO modification is also the target for ubiquitination, and
following desumoylation, MEK1 translocates back to the nucleus where it is
ubiquitinated and degraded (Sobko et al., 2002). Competition by ubiquitin and
SUMO for the same lysine residue also occurs on the Huntingtin (Htt) protein, as
previously mentioned. Interestingly Htt does not contain a consensus SUMO
modification motif, and raises the likelihood of further examples of substrates
that are modified by both SUMO and ubiquitin on identical lysine residues.
66

Since modification by all members of the ubiquitin-like family of protein
modifiers, and acetylation can all occur on lysine residues, transcription factors
can potentially undergo a cascade of modifications that modulate their functions.
An example of this is shown in the case of Sp3, where the major site of SUMO
modification is identical to the major, site of acetylation (Braun et al., 2001).
SUMO modification of Sp3 results in transcriptional repression (Sapetschnig et
al., 2002; Ross et al., 2002), whilst the role played by Sp3 acetylation has yet to
be determined (Braun et al., 2001). In addition to SUMO preventing
ubiquitination of IKBa, the acetylation of Smad7 has been shown to protect
Smad7 from ubiquitin-mediated degradation, suggesting competition between
acetylation and Ubiquitin at critical lysine residues (Gronroos et al., 2002). Thus
the complex relationship between the various post-translational modifiers can
potentially enhance or repress the functional potential of various substrates. This
modulation of protein function will provide an important mechanism to help
control the diverse arrays of gene expression programs that are required for the
lives of a complex organism (reviewed in Freiman and Tjian, 2003).
1.6 The Ubl Conjugation Pathway
The Ubl modifier is first activated by its cognate El enzyme, that uses ATP to
adenylate the exposed C-terminal glycine residue of the Ubl (2) (Figure 11,
numbers in brackets refer to position in schematic), following which a thioester
bond is formed between the C-terminus of the Ubl and a cysteine residue in the
El, releasing AMP (3). The Ubl is transferred from the El to the E2, in a
transesterification reaction (4). The thioester linked E2-Ubl, either with or
without the help of an E3 ligase activity, forms an isopeptide linkage between the
C-terminus of the Ubl and an E-amino group of a lysine or N-terminal residue of
a substrate protein (5).
1.6.1 The El super family
The common feature of all the known ubiquitin-like protein modification
systems is in the activation of the enzymatic cascade. This is achieved via a
unique El, activating enzyme, for each of the conjugation pathways. Currently
El enzymes have been found for ubiquitin, ISG15, NEDD8, APG12, SUMO,
67
{°

URM1, and UFM1 conjugation pathways (Figure 12). The ubiquitin-activating
enzyme exists as two isoforms, Ela (117 kDa) and Elb (110 kDa). Ela is
phosphorylated and localised to the nucleus in a cell cycle-dependent manner
(Stephen et al., 1996), due to the presence of a nuclear localisation sequence
(SPLSKKRR), which is essential for both Ela phosphorylation and nuclear
localisation (Stephen et al., 1997). In contrast Elb is predominantly cytoplasmic
and is not phosphorylated (Stephen et al., 1996). Whilst the El's for ubiquitin,
ISG15, APG12, and URM1 are monomeric, the El enzymes for NEDD8 and
SUMO are heterodimeric, being composed of APPBPI/UBA3 (Gong et al.,
1999), and SAE1/SAE2 (Desterro et al., 1999) respectively (Figure 12). UBA3
and SAE2 are homologous to the C-terminal of the ubiquitin El, and contain
both the nucleotide-binding motif involved in adenylation and the catalytic
cysteine involved in the thioester intermediate. SAE1 and APPBP1 conversely,
share homology with the N-terminal segment of the ubiquitin-activating enzyme.
Although no ubiquitin-like modifiers exist in bacteria, there are members of the
El super family present. There are distinct parallels between ubiquitin-like
activation and the biosynthesis of several sulphur-containing enzyme cofactors
(Furukawa et al., 2000). The microbial synthesis of thiamine and molybdopterin
each requires a specific sulphurtransferase, ThiF or MoeB, respectively. All the
El proteins identified to date share, along with ThiF and MoeB, conserved
motifs and the same structural folds. In both cases the sulphur chemistry of both
involves the addition of a sulphur atom to the carboxyl-terminal carboxyl group
of short polypeptides, ThiS or MoaD, for ThiF and MoeB respectively, each of
which terminates in a glycine-glycine dipeptide. Similar to El enzymes, MoeB
activates the C terminus of MoaD to form an acyl-adenylate. Subsequently, a
sulphurtransferase converts the MoaD acyl-adenylate to a thiocarboxylate that
acts as the sulphur
donor during Molybdenum (Moco) biosynthesis (Leimkuhler
et al., 2001 and Pitterle et al., 1993). The structures of these bacterial members
of the El super family have been resolved (Wang et al., 2001 and Rudolph et al.,
2001) including the MoeB-MoaD complex (Lake et al., 2001). The similarities
in both structure and function of ubiquitin and the ubls, together with their
activating enzymes, and those of the ThiS-ThiF and MoeB-MoaD pathways
68
4'

O
EI-Sll EI-SII E2-Sll ýý
Ubl/JýbFi ATP Ub MP Ubl/\S-E1 -Ubl/\S-E2 t~ tc Ub NH-Protein
(2) (3) (4) (5)
Figure 11. Schematic representation of a Ubl protein conjugation. The Ubl modifier is
first activated by its cognate E1 enzyme, in an ATP dependent reaction, to adenylate the
exposed C-terminal glycine residue of the Ubl (2), following which a thioester bond is
formed between the C-terminus of the Ubl and a cysteine residue in the El, releasing
AMP (3). The Ubl is transferred from the El to the E2, in a transesterification reaction
(4). The thioester lonked E2-Ubl, either with or without the help of an E3 ligase, forms
an isopeptide linkage between the C-terminus of the Ubl and an amino group of a lysine
residue of a substratre protein (5).

Figure 12. Schematic diagram showing the four conserved regions found in Ub and Ub-
like protein, El activating proteins, and -a phylogram showing the evolutionary
relationship of the El proteins. (i) The, monomeric El enzymes for the activation of Ub
in both humans- and S. cerevisiae (hUBA1 and scUBA! ), and the heterodimeric El
enzymes for NEDD8, its yeast homologue, Rubl; SUMO and its yeast homologue Smt3
are shown. Four conserved regions are found in the majority of all Ub and Ub-like
protein El activating enzymes, indicated I to IV, as defined by (Johnson et al., 1997).
Recently an El described for the Ufml Ubl has been characterised,
that is significaantly
smaller than the other El proteins and contains only one of the conserved domains.
Adapted in part from (Desterro et al., 1999). (ii) Phylogram of various El proteins and
bacterial El-like proteins. Phylogram was constructed via the ClustaiW alignment
program (www. ebi. ac. uk/clustalw) and was generated with full length sequences.

CFs 632
hUbiquitin hUBAI IV
. 4-197 301-438 4-50406 608-664 926-913
0 6(X)
ScUbiquitin ScUBA I IV
(ii)
Rub]
NEDD8
S mB p
Ula lp
18-162 322-40.; 416-574 576-632 793-995 1024
(168
148
UBA3p . 391-402 IV
APP-BPI
hUBA3
14-160 451 5341
1-138
Aos lp cys 177
UBA2p
SUMO-1
SAE]
SUMO-2
15-161 26(-347
2(X)-276 299
c ý. s 213
IV
49-1£ 3 245-321 441
in
I IV
3-151 153-209 303-395 636,
(y' 1 - 1-3
SUMO-3 SAE2 18-)(4 263-_346; IV
Uba5
Move
THF
9-147 149-3(9 ? li-*X (AO
Moee-like
('ý s 250
72-229 404
IJba4
SAE2
- USE I
UBE1L
- UbaS
SAE1
X97
1058
APPBP1
1 -. 9

make it likely that these eukaryotic protein post-translational modification
pathways evolved from a similar protein-based sulphide donor system in
bacteria.
The initial catalytic step in ubl activation involves the formation of a
high-energy thiol-ester linkage between the El and the carboxyl-terminal glycine
of the ubiquitin-like protein. This process is ATP dependent and results in the
production of AMP and PPi. The mechanism for this energy dependent
activation was described some twenty-one years ago (Haas et al., 1982). The
crystallization of the El heterodimer for NEDD8, APPBPI-UBA3, provides
insight into functionality of the El (Walden et al., 2003). The El displays a
large active site groove that is divided into two distinct clefts, with ATP being
bound in one cleft whilst NEDD8 is bound in the other. In the NEDD8 El, there
is a distance of some 30A between the adenylation site and the catalytic cysteine,
Cys216. In order for NEDD8 (Ubl) to transfer from the adenylate, a-significant
conformational change is required, as the catalytic cysteine is required to move
by at least 10A, to allow the formation of the thioester bond (reviewed in
VanDemark et al., 2003). The crystal structure also revealed that UBA3, which
shares sequence similarity with the C-terminal half of the ubiquitin El, adopts an
ubiquitin-like fold. The ubiquitin-like fold of UBA3 sits primarily in cleft one,
and its deletion reduces the binding of the E2 enzyme (Walden et al., 2003).
Thus El functions in a two-step process.
Biochemical analysis of point mutants of ubiquitin had previously
identified the important roles of arginine 42 and 72 in the interaction with
ubiquitin-activating enzyme (Burch et al., 1994). The corresponding residues in
SUMO family members is either glutamate or glutamine and alanine in NEDD8.
The crystal structure of the NEDD8 El enzyme, indicates that Ala 72 of NEDD8
interacts with the hydrophobic Leu 206 and Tyr 207 residues of UBA3. The
orientation of the residues corresponding to Leu 206 and Tyr 207 in other El
enzymes correlates with the nature of the side chain at position 72 in the other
Ubls (Walden et al., 2003). Mutation of these two key residues of UBA3 to
aspartate, reduces NEDD8 adenylation, but was shown not to be sufficient for
72

APPBPI-UBA3 to promote adenylation of ubiquitin, showing that specificity is
regulated by other conformation restraints (Walden et al., 2003).
The El enzyme for the ISG15 ubl, UBE1L, was originally identified as
being reduced in expression in lung cancers (Kok et al., 1993 and McLaughlin et
al., 2000), but it was not shown to be the El enzyme for ISG15 until later (Yuan
et al., 2001). Although pharmacological targeting of a Ubls El enzyme may be
unlikely to be of therapeutic interest due to lack of specificity, viral proteins do
target El function. Influenza B virus strongly induces the ISG15 protein during
infection via the interferon response but the virus has evolved ways to counteract
this response. Influenza B virus NS1 protein, is capable of preventing ISG15
from being activated by UBE1L (Yuan et al., 2001), thereby preventing ISG15
conjugation to substrates. El activity can also be regulated by cellular stress,
such as oxidative stress. The activity of the ubiquitin-activating enzyme
increases 2-6.7 fold, as determined by thiol ester formation, during recovery
from oxidation (Shang et al., 1997).
Several chemicals have also been reported which act to inhibit the ubiquitin-
activating enzyme. Panepophenanthrin, from the mushroom strain Panus rudis,
was isolated during screening trials for proteasome inhibitors (Sekizawa et al.,
2002). Following the discovery of this natural product, Lei and co-workers,
reported on the total synthesis of (+)-panepophenanthrin (Lei et al., 2003).
However although both report the inhibition of the El-ubiquitin complex there is
no evidence as to the specificity of this mechanism, as to whether it functions as
a general E1 inhibitor or an ubiquitin specific inhibitor..
Interestingly there is an example of a protein with both E1/E2 activities,
TAF, 1250 in Drosophila (Pharr et al., 2000). TAFI, 250 is the central subunit
within the general transcription factor TFIID, which was shown by Pham and
colleagues to mediate monoubiquitination of histone 1.
73

1.6.2 The E2 super family
The second enzyme in the cascade, known as E2, forms a thioester with
its cognate Ubl, following their maturation and subsequent activation by the El.
Ubcs (ubiquitin conjugating enzymes) can be placed into one of
four broad
groups, by comparison of their amino acid sequences (Jentsch, 1992). Class I
enzymes possess a conserved catalytic core domain of about 150 residues with a
minimum of 25% sequence identity. Class II enzymes have an extra C-terminal
extension attached to the core domain, whilst Class III enzymes have an N-
terminal extension to the core domain. Class IV enzymes possess both the N-
and C-terminal extensions. Specificity for both Ubl and substrate recognition is
in part determined by these extensions to the core domain, whilst amino acid
insertions and other sequence and structural heterogeneity will undoubtedly play
important roles.
As it was not initially recognised that Ubls other than ubiquitin had
distinct E2's, and as they are highly homlogous, the E2's are known as Ubcs.
Indeed the E2 for SUMO, Ubc9, when initially discovered was thought to act in
the ubiquitin system (Yasugi and Howley, 1996), whilst the E2 for the NEDD8
pathway is named Ubcl2 (Gong et al., 1999). Subsequently Ubc9 was shown
not be involved in the ubiquitin pathway, but the SUMO conjugation pathway
(Desterro et al., 1997 and Schwartz et al., 1998).
The Ubcs from the SUMO and NEDD8 pathways are incapable of
forming thioesters with either ubiquitin or any other Ubl (Desterro et al., 1997,
Schwartz et al., 1998, and Gong et al., 1999), demonstrating the specificity for
its own unique conjugation pathway. The ubiquitin pathway is reported to
consist of eleven Ubc isoforms, and these isoforms have different substrate
specificities. Ubcl, Ubc4, and Ubc5 would appear to be involved in general
ubiquitination pathways which lead to the degradation of abnormal proteins
(Seufert et al., 1990a, Seufert et al., 1990b). Ubc2 (Rad6) is involved in DNA
damage repair and sporulation (Finley and Chau, 1991). Ubc3 (cdc34) is linked
to the control of cyclin stability (Finley and Chau, 1991). Ubc6 has been
demonstrated to be essential for protein translocation through the membrane
(Sommer and Jentsch, 1993). UbclO is involved in the biogenesis of
74

peroxisomes (Wiebel and Kunau, 1992). Ubc13 forms a heterodimeric complex
with mms2, to promote Lys63 ubiquitin chain formation. This heteromeric
complex is largely cytosolic, but relocates to the nucleus following DNA
damage, where they are subsequently recruited to chromatin (Ulrich et al., 2000).
Thus despite belonging to the same class of enzyme and possesssing homologous
amino acid sequences, there is distinct, and specific, functional versatility, such
as the promotion of different ubiquitin chain linkages. Modification of the
ubiquitin-conjugating enzyme, to create chimeric proteins containing appropriate
protein-binding peptides fused to their C-termini, redirects the specificity of
ubiquitination (Sullivan and Vierstra, 1991, Gosink and Vierstra, 1995).
The crystal structures for both Ubc9 and Ubc9 bound to a substrate
RanGAPI have been resolved (Tong et al., 1997; Giraud et al., 1998; Bernier-
Villamor et al., 2002) and along with mutagenesis and biochemical studies has
provided a working hypothesis for the role of Ubc9 in SUMO transfer. A
catalytic cysteine residue (C93 in Ubc9) is required to accept the transfer of the
Ubl from the El to the E2. The Ubcs also possess a conserved asparagine
residue, Asp85 in Ubc9, which whilst being dispensable for both E2 folding and
E2-Ub thiol ester formation is required for E2-catalysed Ubl conjugation (Wu et
al., 2003). Mutational analysis of residues in Ubc9, which are not conserved
between Ubcs, AsplOO and LyslOl, which are located close to the active site
cysteine in the tertiary structure of Ubc9, inhibit both the transesterification
reaction from SAE1/SAE2 and substrate recognition (Tatham et al., 2003).
NMR experiments had previously shown that SUMO interacted with Ubc9
through its ubiquitin domain and that Ubc9 interacted with SUMO through a
structurally conserved region (Liu et al., 1999). The elucidation of the crystal
structure for Ubc9-RanGAPI revealed extensive RanGAPI interactions with the
surface of Ubc9 and provided the mechanism for interaction between the SUMO
modification motif WKXE/D and Ubc9 recognition. Conserved residues on the
surface of Ubc9 create shallow grooves, complementary electrostatic, and
hydrogen bonding interactions form a platform into which the IFKXE/D motif
can specifically dock, resulting in the proper orientation and coordination of the
acceptor lysine (Bernier-Villamor et al., 2002). This was consistent with earlier
work, which had previously identified a substrate recognition site on Ubc9 by
75

NMR (Lin et al., 2002). It is important to remember though, that not all of the
substrates for SUMO modification possess the motif. The N-terminus of Ubc9,
and by analogy other E2 enzymes due to its conservation, also plays important
regulatory roles. R 13A/K 14A and R 17A/K 18A mutations in Ubc9 were shown
to disrupt the interaction with SUMO, whilst retaining some degree of interaction
with the El enzyme (Tatham et al., 2003). Furthermore, although the ability of
the mutant to transfer SUMO-1 from El to E2 was significantly disrupted, the
transfer of SUMO-1 to substrate was unaffected. Thus, although distant from the
active site these conserved residues within the N-terminus, and hence the N-
terminus still plays a modulatory role.
Work on Ubc9p, the Ubc9 from Saccharomyces cerevisiae, identified
regions for Smt3p-Smt3p chain formation and thioester bond formation.
Bencsath and co-workers showed that the surfaces involved in thioester bond
formation map to the surface of the El binding site, and that the same surface
binds Smt3p. Furthermore, it was demonstrated that a mutation in Ubc9p that
disrupts El binding, but not Smt3p binding, impairs thioester bond formation,
which suggests that it is the El interaction at this site that is crucial (Bencsath et
al., 2002). The authors raise the point that as other E2s utilise this surface for
binding to ubls, E3s, and other proteins and as such creating a multipurpose
protein scafold, this could suggest that the entire El-E2-E3 conjugation pathway
has coevolved for a given ubiquitin-like protein.
As in the case of the El enzyme, certain viruses have evolved with these
pathways. The African swine fever virus genome encodes its own viral
ubiquitin-conjugating enzyme, UBCvI (Hingamp et al., 1992, Rodriguez et al.,
1992). In vitro recombinant UBCvI can self-ubiquitinate, as well as ubiquitinate
histones, and the ASFV virion protein, PIG 1 (Hingamp et al., 1992, Hingamp et
al., 1995). In vivo evidence indicates that a DNA binding protein, SMCp is a
substrate for the virally subverted ubiquitin pathway (Bulimo et al., 2000).
76

1.6.3 E3 ligases
1.6.3.1 Ubiquitin E3s
The high degree of sequence and functional similarity of the various E1
and E2 enzymes of the Ubl pathways is not found to the same extent for the final
enzymes, the ligases, or E3 protein. The most studied E3s belong to the
ubiquitin pathway although, due to the large number of E3s of this pathway (they
number into the hundreds) there still remains a considerable amount of effort
before they are fully understood. In general the E3 reaction involves at least two
distinct steps, beginning with the binding to the substrate via the ubiquitination
signal, followed by the covalent ligation of one or more ubiquitin proteins to the
substrate, and as such ubiquitin ligases are defined as "enzymes that bind,
directly or indirectly, specific protein substrates and promote the transfer of
ubiquitin, directly or indirectly, from a thioester intermediate to amide linkages
with proteins or polyubiquitin chains". Despite the large number of ubiquitin
ligases, all known E3s feature one of two structural elements, with the exception
of p300, which will be discussed later. The first of these ligases are known as
HECT domain E3s. Work on the human papiloma virus (HPV) showed that the
ubiquitin dependent degradation of p53 depended on the HPV E6 gene product
and a -100 kDa cellular protein named E6-AP (E6-associated protein). E6 and
E6-AP form a complex that functions as a p53-specific E3. The final third of the
E6-AP sequence is 35-45% identical to distinct domains in a large number of
identified proteins. These domains are collectively known as the HECT domain
(homologous to E6-AP carboxyl terminus). The HECT domain contains a
strictly conserved cysteine residue positioned -35 residues upstream of the C-
terminus. The conserved cysteine of E6-AP is required for p53 ubiquitination, as
it acts as a site of thiol ester formation with ubiquitin. There are numerous lines
of evidence to indicate that all HECT domain E3s use a similar mechanism of
covalent catalysis, often with UbcH7 or UbcH8 as their E2. The construction of
HECT E3s is modular, the unique N-terminus of each family member interacts
with specific substrate(s), whilst the HECT domain mediates E2 binding. The
second ubiquitin ligase types, are known as RING E3s. ' Many eukaryotic
proteins display a series of histidine and cysteine residues with a characteristic
spacing that allows for the coordination of two zinc ions in a cross-brace
77

structure called the Really Interesting New Gene (RING) finger. RING finger
proteins number in the hundreds and have been implicated in a wide range of
cellular functions. The conservation in the RING fingers is not in the sequence,
but rather the spacing of the zinc ligands. As the zinc ions and their ligands are
catalytically inert, it suggests that RING fingers do not function as chemical
catalysts, but rather as molecular scaffolds that function to bring other proteins
together. There are two varieties of ubiquitin RING E3s, those that function as
single-subunit and those that form part of a multi-subunit complex (reviewed in
Deshaies, 1999; Borden, 2000; Joazeiro and Weissman, 2000). That the single-
subunit E3s function alone has yet to be proven categorically, as many can form
complexes with proteins besides their substrate, but so far only the E2 has been
shown as a requirement for substrate ubiquitination. Following the binding of
their E2, single-subunit RING E3s have the ability to catalyse their own
ubiquitination in vitro. This autoubiquitination, in some circumstances, is due to
ubiquitin attachment to the RING protein, or fusion protein, and as such
represents. an in vitro artefact. However, in other situations the RING E3
autoubiquitination has been demonstrated in vivo, and as such has the potential to
represent a physiological mechanism of E3 down-regulation, as is the case for
both c-Cbl and Mdm2. c-Cbl is involved in the ubiquitin-dependent down-
regulation of activated receptor protein tyrosine kinases (RTKs) (Joazeiro et al.,
1999; Waterman et al., 1999; Joazeiro and Weissman, 2000). Mdm2
ubiquitinates itself and the tumour suppressor protein, p53, and this relationship
will be discussed in detail later. The second type of RING E3 requires that it is
incorporated into a multiprotein complex to display ligase activity. There are
three types of known multisubunit E3s, in which a small RING finger protein is
an essential component. The three E3s are the SCF E3s (Skp1-Cullin-F-box
protein), the APC (Anaphase-Promoting Complex), and the VCB (VHL-Elongin
C/ Elongin B) E3s (reviewed in Tyers and Willems, 1999). These multisubunit
E3s possesss a -100-residue RING finger protein, Rbxl (Hrtl/Rocl) that
functions as an organiser, suportive of the scaffold hypothesis. Rbxl interacts
strongly with the cullin protein family; cullinl in SCF and culling in VCB
complexes. The assembly of the appropriate cullin, Rbxl, and specific E2, at
least in vitro, results in an active ligase that will either autoubiquitinate the E2 or
78

produce free polyubiquitin chains. The APC E3 contains Apc 11, a RING finger
protein, amongst its many subunits.
Single-subunit RING E3s directly recognise the ubiquitination signals of
their substrates, through domains that are distinct from their RING finger. In
multisubunit RING Eis, the substrate recognition is mediated through a separate
subunit. The Skpl protein of the SCF complex, for instance, recognises the
substrate-specific F-box proteins through their F-box motif (Figure 13), whilst
the recognition of the VCB E3 ligase is mediated through the pVHL, the product
of the Von Hippel-Lindau tumour suppressor gene. The pVHL protein is
recruited to the complex through interactions with the heterodimeric adaptor
proteins Elongin B and Elongin C. Mutations of pVHL are often found in a
subset of cancers, and these mutations are predicted to destabilise the interaction
of pVHL with Elongin B/C (Figure 13).
1.6.3.2 SUMO E3s
Since the initial discovery of the SUMO conjugation system, an
intriguing question was whether or not an E3 ligase activity was required for
SUMO modification. Studies had shown that there was no necessity for an
SUMO E3-like enzyme for modification in vitro, and unlike the ubiquitin
pathway, the SUMO E2, Ubc9 directly bound to a substrate through the WKXE
motif. This however, did not rule out the possibility of an E3-like activity in
vivo, being required for the conjugation of those substrates that lack the WKXE
motif. This question remained unanswered for some five years until the
discovery of Sizl a yeast member of the mammalian protein inhibitor of
activated STAT (PIAS) family, as a potential SUMO E3-like protein (Takahashi
et al., 2001, Sachdev et al., 2001, Johnson and Gupta, 2001).
PIAS proteins, as their name would suggest, are involved in the STAT
signalling transduction pathway. The STAT signal transduction pathway is
activated following the binding of cytokines to the cell surface receptors, and
induces a variety of cellular responses. Janus (JAK) kinases are constitutively
79

ED,
0
SCF (Skipl-Cullin-F-box protein)
F-Box > Substrate
-0 -ý
1i2 c'dc ýn
P 13
ß-TrCP 1KB, ß--catcnin
VHL (VHL-Elongin C/Elongin B)
w
ffli
F-Box Substrate
pVHL
HIFIu
Figure 13. E, 3 ubiquitin ligase complexes. Similar overall architecture between the SCF
and VHL complexes which participate in the ubiquitin-dependent degradation of many
cellular proteins. Each complex is composed of a set of adapter proteins that recruit
different binding partners through specific protein-protein interactions. Complex subunits
that share sequence similarity are shown in the same colour, and their respective substrates
are indicated below. Adapted in part from Desterro et al, 2000.

associated with the cytoplasmic domain of cytokine receptors. Binding of the
ligand to its receptor induces dimerisation of the receptor chains, bringing
together two JAK kinases that are activated by transphosphorylation. Activated
JAKs phosphorylate various substrates in the cell, including the receptor itself
and STAT transcription factors. Phosphorylated STATs then dimerise and
migrate to the nucleus where they activate the transcription of genes, which
mediate the cytokine-induced biological response.
PIAS family of proteins were identified as STAT interacting proteins
using yeast two-hybrid screens. There are four PIAS proteins in vertebrates;
PIAS1, PIAS3, PIASx, and PIASy (Liu etal., 1998). PIAS1 was identified as a
specific interaction partner for STAT1, and PIAS3 was subsequently cloned on
the basis of homology to PIAS 1. PIAS 1 and PIAS3 inhibit DNA binding of the
STAT1 and STAT3 factors, thereby inhibiting signal transduction (Chung et al.,
1997, Liu et al., 1998), whilst PIASy represses STAT1 and the activity of the
androgen receptor without affecting DNA binding (Gross et al., 2001). The role
of PIAS/Siz as SUMO E3s further increases the important roles played by these
proteins.
Interestingly Sizl/PIAS members contain a conserved domain with a
similarity to a zinc-binding RING-domain, which is often found in ubiquitin
ligases. Ubiquitin RING domain E3 ligases do not possess intrinsic enzymatic
activity, rather they function as adapters that bring together the E2 and the
substrate, and this was shown to be true for the PIAS family of SUMO E3s.
Both Siz1 and PIASy were shown to physically bind Ubc9 and substrate, which
were septins (Takahashi et al., 2001, Johnson and Gupta, 2001) and LEF1, in the
initial examples (Sachdev et al., 2001). Sizl was shown to be essential for
septin-Smt3 conjugation in vivo. While Sizl was not required for conjugation in
vitro, the presence of an E3 greatly enhanced the efficiency of the reaction.
In yeast there are only two known SUMO ligases, Sizl and Siz2.
However sizl-siz2 double mutants do not show mitotic arrest suggesting that
other E3 ligases remain to be discovered. In higher eukaryotes two other
families of SUMO E3 ligases, RanBP2 (Nup358) (Pichler et al., 2002) and Pc2
(Kagey et al., 2003), have been identified.
81

RanBP2(Nup358) is a giant nucleoporin, comprising an amino-terminal
700-residue leucine-rich region, four RanBP1-homologous domains, eight zinc-
finger motifs, and a carboxyl terminus with high homology to cyclophilin
(Yokoyama et al., 1995). RanBP2 is one of the key constituents of the
RanGTPase system, a system that is integral for nuclear transport, serving as a
docking factor for import complexes on their way to the nucleus. The other
constituents of this system are the guanine nucleotide exchange factor RCC1
(Ohtsubo et al., 1987); the RanGTPase-activating protein RanGAP 1 (Bischoff et
al., 1994); the Ran-binding protein RanBP1 (Coutavas et al., 1993); NTF2
(Moore and Blobel, 1994); and the Impß-related transport receptors. The
evolution of the distinct nuclear and cytoplasmic compartments that characterise
eukaryotic cells, requires a means of separating the two compartments whilst
allowing exchange of factors between the two. This is achieved by the nuclear
envelope (NE), that is penetrated by nucleopore complexes (NPCs), thus
allowing the exchange of macromolecules between the compartments. RanBP2
has recently been shown to be involved in nuclear mRNA export (Forler et al.,
2004). The compartmentalisation of eukaryotic cells, although considerably
energy intensive, allows the regulation of key cellular events, such as the control
of access of transcriptional regulators to chromatin. The nuclear envelope is
spanned in places by the nuclear pore complexes (NPCs). The number of NPCs
in a cell will depend on the demand for nuclear transport-and will vary depending
on cell size, synthetic, and proliferative activity, with about 3000-5000 NPCs in a
proliferating human cell (Gorlich and Kutay, 1999). Metazoan nuclear
envelopes contain an additional structural element, called lamina, which is
associated with the nuclear face of the inner nuclear membrane.
RanBP2(Nup358) is a component of the short (100nm) filaments that extend
from the cytoplasmic face of the NPC during interphase, which relocates to both
spindle microtubules and kinetochores (Joseph et al., 2002). The relocation of
RanBP2 occurs in association with RanGAP I. RanBP2 has been demonstrated
to play an important role in nuclear envelope breakdown. Higher eukaryotes
have an open mitosis, requiring that once per. cell cycle the nuclear envelope
breaks, and the nuclear and cytoplasmic compartments mix. This involves the
disassembly of all major structural components of the nuclear envelope,
including the nuclear pore complex. RanBP2 is essential for kinetochore
82

function, shown by the defects of chromosome congression and segregration in
the absence of RanBP2, and aberrant kinetochore formation, due to the inhibition
of the assembly of kinetochore components (Salina et al., 2003). The
implication of this is that RanBP2 plays an essential role in integrating nuclear
envelope breakdown. with kinetochore maturation and function (Salina et al.,
2003).
RanBP2 also possessses SUMO E3 ligase activity. It was shown that
RanBP2 interacted directly with Ubc9 and promoted the SUMO-1 modification
of Sp100, by stimulating transfer of SUMO-1 from Ubc9 to Sp100 (Pichler et al.,
2002). Although possesssing a RING finger domain, it was shown not to be
required for SUMO E3 ligase ability, and both RanBP2-DRING and point
mutations of the cysteine residues still possesssed E3 ability. Furthermore the
treatment of recombinant protein with alkylating agents did not prevent RanBP2
E3 activity towards Sp100, suggesting that thioester bond formation is not
essential for this activity. RanBP2, as with the Siz/PIAS family of SUMO Eis,
was capable of stimulating the formation of SUMO chains on itself, although the
physiological relevance of this still remains to be elucidated.
The third known SUMO E3 ligase identified to date, is a member of the
Polycomb group of proteins. Polycomb group (PcG) proteins form large
multimeric complexes (PcG bodies) and are associated with the stable repression
of gene expression. PcG proteins were originally identified in Drosophila as
repressors of homeotic gene expression (Kennison et al., 1995; Simon et al.,
1995). Flies containing mutations in PcG genes showed aberrant expression of
homeotic genes, outside their normal segment boundaries, leading to the
alteration of body part identity (Simon et al., 1995; Schumacher and Magnuson,
1997). Many homologs of the Drosophila PcG proteins have been identified in
mammals (Brock and van Lohuizen, 2001), and multiple core PcG complexes are
found in a variety of cell types (Seawalt et al., 1998; Francis et al., 2001; Gunster
et al., 2001). Although PcG complexes are associated with DNA, individual
PcG proteins do not seem capable of directly binding DNA. (Francis et al.,
2001). It is thought that the recruitment of PcG complexes to DNA, is mediated
by sequence specific DNA binding proteins, such as, YY1, and E217 (Dahiya et
al., 2001; Satijin et al., 2001; Ogawa et at., 2002). PcG complexes form
83

discrete foci within the nucleus, termed PcG bodies (Gerasimova and Corces,
1998; Saurin et al., 1998), and these bodies are often found near centromeres.
The SUMO E3 ligase ability of a Polycomb protein, Pc2, was found, in the
context of the SUMO modification of the transcriptional corepressor carboxyl-
terminus binding proteins (CtBP), CtBP1 and CtBP2 (Kagey et al., 2003). CtBP
was originally identified by its interaction with the adenovirus E1A protein,
which was required for E1A transcriptional repression (Boyd et al., 1993;
Schaeper et al., 1999). CtBP interacts with Pc2, leading to its recruitment in PcG
bodies (Sewalt et al., 1999). Pc2 has recently been shown to be able to recruit
Ubc9 to PcG bodies (Kagey et al., 2003), and could provide the first indication
that PcG bodies, similar to PML bodies, may function as centres of SUMO
modification within the nucleus. It was also noted that Pc2 could stimulate
SUMO modification of CtBP2, both in vivo and in vitro, despite the absence of a
consensus SUMO conjugation motif. This suggests that other potential SUMO
substrates, which do not possessses a SUMO conjugation motif, could be
modified in the presence of the correct E3.
1.7 p300/CBP
p300 and its paralogue CREB-binding protein (CBP) are large multi-domain
proteins that play key roles in transcriptional regulation, signal transduction
pathways, and protein stability. p300/CBP contain distinct, conserved, domains:
a bromodomain, three cysteine-histidine (CH)-rich domains (CHI, CH2, and
CH3), a KIK domain. The CBP binds to CREB via the KIK domain as well as
binding to transactivation domains of other nuclear factors including Myb and
Jun. Additionally p300/CBP possess an ADA2-homology domain; which shows
homology to Ada2p, a yeast transcriptional coactivator (Figure 14) (Chan and La
Thangue, 2001). Both p300 and CBP interact with numerous proteins and are
components of many signalling pathways (Figure 14). p300 and CBP were both
initially identified in protein interaction assays, p300 through its interaction with
the adenoviral-transforming E1A protein (Stein et al., 1990; Eckner et al., 1994)
and CBP through its association with the transcription factor CREB (Chrivia et
al., 1993). CBP can specifically interact with the protein protein kinase A-
phosphorylated, activated form of CREB, and in transfection experiments
84

enhances the CREB"mediated activation of reporter constructs in vivo in a
manner that depends on the integrity and function of the protein kinase A
phosphorylation site (Kwok et al, 1994). E1A is a transforming viral protein that
forces quiescent cells into S phase. E! A binds to the CH3 domain through the
EIA amino-terminus and CR1 (conserved region 1) domains, and represses p300
transcriptional activity. EIA also targets retinoblastoma protein (Rb), which is
essential for the regulation of GI/S transition by controlling the transcription
factor E2F. E2F activates a subset of genes whose products are involved in
DNA synthesis or in cell cycle control. p300/CBP possessses intrinsic histone
acetyltransferasc (tiAT) activity and this activity is regulated in a cell cycle
dependent manner and shows a peak at the G1/S transition (Ait-Si-Ali et a!.,
1998; Ait-Si-Ali et at., 2000). The intrinsic HAT activity of p300/CBP provides
a means of influencing chromatin activity by modifying nucleosomal histones.
p300/CBP is required for cell proliferation, performing an important function
during the G 1/S transition, by influencing the activities of the genes required for
cell cycle progression. Additionally p300 has a cell cycle inhibitory function,
and is likely to regulate the promoters for genes required for arrest in GOIG 1.
7.1.1 p300/CUP and growth control and signal transduction
p300/CBP do not solely associate with Ela and E2F, rather there is a
diverse and constantly increasing number of transcription factors, that have been
shown to form stable physical complexes with, and respond to, the coactiviating
properties of p300/CBP. Many insights into the functioning of p300/CBP come
from investigating the role Ela plays in compromising the functions of
p300/CBP. Proto-oncogenes, such as c-Afyb, utilise CBP as a co-activator,
unless compromised by Ela. Much thought has gone into understanding why a
viral oncoprotcin would compromise mitogenic transcription factors. Theories
put forward include the possibility p300/CBP may allow for a stronger
coactivator to play a role in transcriptional activation. Alternatively, p300/CBP
may be involved with regulating transcription of a subgroup of AP-1 or c-Myb-
responsive genes that antagonise proliferation. It has been speculated, therefore,
that some transcription factors use p300/CBP preferentially for regulating a
category of genes or, alternatively, for responding to a particular transcriptional
85

stimulus that, if inactivated, would be advantageous to the ultimate aims of a
viral oncoprotein (Shikama et al., 1997).
Ela can also cause an increase in the overall transcriptional activity of
some transcription factors, such as YY1. YY1 has transcriptional repression
properties in the absence of Ela (Shi et al., 1991). Although YY1-binding sites
are frequently present in transcriptional control regions, some sites function to
repress transcription, and, in these cases, p300 is believed to be responsible for
the repressive activity (Lee et al., 1995). This repression can be relieved by Ela
binding, an effect dependent upon the N-terminal p300/CBP-binding domain in
Eta. YY1 and Eta associate in vivo with p300 through distinct C-terminal
domains, and it is thought that both proteins can associate with p300 at the same
time (Shikama et al., 1997).
p300/CBP are also connected to a further class of sequence-specific
transcription factors, the ligand-dependent nuclear receptors. The ligand-binding
domains of multiple nuclear receptors, including the retinoic acid (RAR and
RXR), oestrogen (ER), progesterone (PR), thyroid hormone (TR), and
glucocorticoid (GR) receptors, interact with the N-terminal region of p300/CBP
(Figure 14). Several other coactivators are implicated in the hormone-dependent
activation of nuclear receptors which can associate with receptor bound to
p300/CBP.
Signal transduction mediated by the JAK-STAT pathway also involves
p300/CBP (Bhattacharya et al., 1996). Interferon a (IFN-a) induces the
transcription of a variety of genes required to produce antiviral effects, many of
which require the ISGF3 transcription factor (Darnell et al., 1994). ISGF3 is a
multicomponent activity composed of three subunits, STAT1 and STAT2,
together with a weak DNA-binding component known as p48. Adenovirus Ela
impedes transcriptional activation mediated by IFN-a by inactivating p300/CBP,
which binds through its N-terminal region to STAT2. The compromisation of
the IFN signalling pathway is likely to have beneficial effects for viral
replication.
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7.1.2 p300/CBP and disease
The essential role of p300/CBP is further shown by genetic studies.
Inactivation of a single allele of cbp leads to bone malformations and
monoallelic deletion of p300 results in a partially penetrant neural tube closure
defect. In humans haploinsufficiency of cbp leads to Rubinstein-Taybi
Syndrome (RTS) (Petri] et al., 1995; Kalkhoven et al., 2003), a congenital
disease characterised by malformation of facial bones and digits as well as
increased incidence of heart defects. Studies in mice show that CBP loss and
histone deacetylation were observed in two separate pathological contexts, both
in amyloid precursor protein-dependent signalling and amyotrophic lateral
sclerosis, indicating that these modifications may well have roles in
neurodegenerative diseases. It has been demonstrated that p300 and CBP play
essential roles in neuronal apoptosis. p300/CBP also binds phospho-CREB, a
transcription factor shown to be involved in neuroprotection (Lonze and Ginty,
2002). p300/CBP degradation normally occurs through the proteasome pathway
although p300/CBP can also be degraded by Caspase-6 (Rouaux et al., 2003).
The controlled degradation of p300/CBP and resulting decrease in HAT activity
is critical in neuronal apoptosis.
A role for the gene encoding p300 as a tumour suppressor is suggested
from studies where the gene is mutated in human tumour cells. Specifically,
missense mutations occur in both colorectal and gastric carcinomas. The
mutations identified alter residues 1399 and 1680, located within the C-terminal
C/H-rich region. Further observations for p300/CBP roles as a tumour
suppressor is provided from the studies in which the oncogenic capacity of Ela is
compromised by coexpressing p300 (Smits et al., 1996). Further evidence
suggesting a role for CBP in tumourigenesis, is provided by CBP translocation
events that occur during myeloid leukaemia. In myeloid leukaemia, a large
proportion of the MOZ gene is translocated to the gene encoding CBP, resulting
in the fusion of the MOZ acetyltransferase domain to the largely intact CBP
(Muraoka et al., 1996). The physiological significance of this translocation
remains to be elucidated, although it is possible that the generation of this fusion
protein will the acetyltransferase to be targeted constitutively to particular groups
of p300/CBP-regulated genes.
89
F, ,

Despite the many functional similarities between p300 and CBP, they are
not functionally interchangeable, as there are subtle differences in the expression
of p300 and CBP during development, although mouse embryos nullizygous for
either p300 or CBP die at midgestation, heterozygosity of cbp causes distinct
haematological defects and an increased risk of developing cancer, an effect not
seen' in mice with a single allele of p300 (Kung et al., 2000; reviewed in
Goodman and Smolik, 2000). Further differences include the observations that
fibroblasts derived from homozygous p300 knockouts are defective for retinoic
acid receptor but not CREB signalling (Yao et al., 1998; Kawasaki et al., 1998).
Full activation of CREB-dependent transcription by CBP depends on CBP
phosphorylation at Ser301, an event that does not occur for p300 (Impey et al.,
2002). p300 also plays a role in the apoptotic response to DNA damage
following ionizing damage (IR) whilst CBP does not (Yuan et al., 1999).
Additionally the Karposi sarcoma-associated virus protein vIRF - has been
reported to be stimulated by CBP whilst repressed by p300 (Jayachandra et al.,
1999).
7.1.3 p300/CBP and chromatin remodeling
Although p300/CBP possessses intrinsic HAT activity, in many
circumstances, it appears to function as part of larger complexes. Indeed
p300/CBP can bind to other acetyltransferases such as GCN5 and PCAF
(p300/CBP-associated factor), resulting in the assembly of protein complexes
consisting of two or more acetyltransferases (Yang et al., 1996). Acetylation of
multiple sites in the core histone tails is commonly associated with
transcriptional activity, whilst hypo-acetylation is associated with transcriptional
repression. The consequences of acetylation of lysine residues, within the N-
terminal tails of histones can be varied, and forms part of the histone code, as
described earlier. The acetylation of histones by p300/CBP, directed by the
histone chaperone RbAp48 (Zhang et al., 2000), helps the transfer of H2B-H2B
dimers from nucleosomes to NAP-1, a chaperone protein (Ito et al., 2000).
Histones H2A and H2B are known to be absent from transcriptionally active
chromatin and is readily exchanged out of transcribed chromatin in vivo.
However increased histone acetylation alone is usually insufficient for
90

transcriptional activation (Hebbes et al., 1994; Gregory et al.,
1999). Efficient
gene expression requires cooperation between acetyltransferases and other
chromatin modifying activities such as ATP-dependent remodelling complexes
(Hassan et al., 2001; Narlikar et al., 2002). Potentially as important may be the
acetylation of transcription factors through factor acetyl-transferase activities
(FAT). This activity was first determined in the context of the tumour
suppressor p53 (Gu and Roeder, 1997), but there is growing evidence that
acetylation of transcription factors, is a common feature in gene expression.
p300/CBP form complexes with proteins other than acetyltransferases and can
function as protein bridges, connecting different DNA sequence-specific
transcription factors to the transcriptional apparatus (reviewed in Chan and La
Thangue, 2001). The interactions of p300/CBP with components of the general
transcriptional machinery, such as TFIID, TFIIB, and the RNA polymerase II
holoenzyme (RNAPII) suggests that these interactions are likely to contribute to
p300/CBP function (Kwok et al., 1994; Kee et al., 1996; Yuan et al., 1996;
Mikecz et al., 2000). The simultaneous interaction of multiple transcription
factors with p300/CBP could contribute to transcriptional synergy, where the
interaction of two or more transcription factors has a combined effect greater
than the sum of the individual transcription factors. Conversely, the competition
for the binding to p300/CBP may mediate signal induced repression.
Furthermore it has been rationalised that given the limited repertoire of
activators, co-activators, and cofactors that respond to diverse regulatory cues,
cells may use cooperativity and transcriptional synergy so that a combination of
the few available ubiquitous, signal- and tissue-specific activators can create a
potentially very large number of regulatory complexes.
1.7.4 An alternate role for p300 as an E3 ligase
The relationship between p300 and p53 is complex, with seemingly '
contradictory reports of effects of p300 on the levels of p53 (Li et al., 1997;
Kawai et al., 2001; Livengood et al., 2002). The p53 gene is a tumour
suppressor gene that plays a critical role in safeguarding the integrity of the
genome. p53 is mutated or part of its regulatory circuit is functionally inactivated
in almost all cancers, which highlights its importance in preventing
91

tumourigenesis. p53 is regulated by two interactive feedback loops. p53 and
Hdm2 (Mdm2 in the mouse), form one of these feedback loops, in which p53
positively regulates Hdm2 by activating HDM2 transcription, and Hdm2
negatively regulates p53 by promoting p53 ubiquitination and subsequent
degradation via the 26S proteasome, by functioning as an E3 ligase (Figure 15)
(Sherr, 1998; Zhang and Xiong, 2001). E2F-1 and p14M (p19"' in the mouse)
form an additional feedback loop, in which E2F-1 activates ARF transcription,
and p14"RF promotes the ubiquitin dependent degradation of E2F-l. Furthermore
p14 "RF interacts with Hdm2, inhibiting Hdm2 E3 ligase function, which will
stabilise p53 (Bates et al., 1998; Zhang et al., 1998; Stott et al., 1998)(Fig. 15. ).
Additionally p53 represses the transcription of the ARF gene (Fig. 15). SUMO
modification adds an extra layer of complexity to this regulatory mechanism.
Hdm2 and p53 are both modified by SUMO (Gostissa et al., 1999; Rodriguez et
al., 1999; Muller et al., 2002; Xirodimas et -al., 2002). HDM2 and ARF
expression has been shown to increase the level of SUMO modified p53 (Chen
and Chen, 2003). This increase in SUMO modification was shown to be
regulated by Hdm2 and Arf mediated nucleolar targeting (Chen and Chen, 2003).
The acetylation, of three specific lysine residues in the C-terminus of
p53 increases the DNA binding capacity of p53, possibility due to a
conformational change of an inhibitory regulatory domain. Ionizing radiation
promotes the N-terminal phosphorylation of p53, and as a consequence increases
the affinity between p53 and p300/CBP and results in the stimulation of p53
DNA binding activity following its acetylation. However it has been reported
that although acetylated p53 binds to short fragments of DNA with a greater
affinity than that of non-acetylated, acetylated p53 does not appear to
significantly affect p53 DNA binding activity on chromatin templates (Espinosa
and Emerson, 2001). As such the growth suppression and apoptotic promoting
functions of p300/CBP are mediated, in part, by their interaction with p53.
CBP has been shown to act as a p53 coactivator that potentiates p53
transcriptional activity. p53 interacts via its N-terminus activation domain with
several domains within the C-terminus of p300/CBP, which can up-regulate
expression of the Mdm2 gene. Mdm2 is the negative regulator of p53, acting as
an ubiquitin ligase, and as such, increasing levels of Hdm2 lead to decreased
92

(i) Transcriptional control.
(ii) Post-translational control.
do ý-.
p53
Proteosomal degradation
\ýý
Nucleolar targeting
-
p14ARF
E2F-1
Proteosomal degradation
Figure 15. The p_53 functional circuit. (i)Transcriptional regulation of the core circuit.
p53 positively regulates the expression of the HDM2 gene, whilst repressing expression
of ARF. E2F-1 positively regulates ARF expression. (ii) Post-translational regulation
of the core circuit. p14ARF promotes the SUMO modification of Hdm2 and p53, when
in a complex with Hdm2. Hdm2 also undergoes autoubiquitination, thereby regulating
its own stability. p14ARF further promotes the ubiquitination of E2F-1, whilst
repressing Hdm2 ability to ubiquitinate p53. Effects on protein levels are indicated by (-
), reduction, and (+), increase.

levels of p53. Decreasing levels of p53 will in turn lead to decreased expression
of Hdm2, as previously mentioned. This auto-inhibitory feed back loop is
subverted by viral protein such as E1A, leading to decreased levels of p53, and
allows viral replication. p53 is the most commonly mutated tumour suppressor
in the majority of cancers investigated to date. The Tax (human T-cell leukemia
virus (HTLV-1)) protein is also capable of binding to p300/CBP, and directly
competes for the binding of p53 at the CR2 (conserved region 2) domain of
p300/CBP. The mechanism by how E1A subverted the feedback loop, was only
recently proposed. The N-terminal of p300 shows partial sequence homology to
the E6-AP, an ubiquitin ligase and is found mutated in Angelman's syndrome.
Angelman's syndrome is a neurological disorder, characterised by
neurodevelopmental impairments. E6-AP when bound to E6 was also capable
of targeting p53 for ubiquitination and this induces its degradation. Purified
p300 showed ubiquitin ligase activity, in vitro, an activity that was abolished in
the presence of E1A (Grossman et al., 2003). The ubiquitin ligase activity was
further mapped to the N-terminal region of p300. In vitro experiments showed
that p300 and Mdm2 were required for promoting polyubiquitination of p53
(Grossman et al., 2003). In the absence of p300, only single ubiquitins were
conjugated to p53. Expression of E1A caused a decrease in polyubiquitinated
forms of p53, but not monoubiquitinated forms (Grossman et al., 2003). A
caveat of this work was that p300 was only able to polyubiquitinate
monoubiquitinated p53, and thus, may be functioning as an E4, promoting rather
than controlling p53 polyubiquitination.
However the intrinsic ubiquitin ligase
ability of p300 could explain how it can control both p53 activation and its
destruction. As p300 regulates a range of short-lived transcription factors, it is
possible that it may act as an ubiquitin ligase in other cases, although this is yet
to be proven. However there are conflicting reports on this as Li et al., have
shown that p53 is monoubiquitinated and localised in the cytoplasm when Mdm2
is expressed at low levels, whereas p53 is polyubiquitinated and subsequently
degraded when Mdm2 is expressed at high levels (Li et al., 2003).
94

1.7.5 p300 and transcriptional repression
p300/CBP can also act as a transcriptional repressor via the modulation of
adaptor proteins, such as the proliferating cellular nuclear antigen. (PCNA). The
PCNA protein can associate with p300 (Hasan et al., 2001), an association that
potently inhibits both the acetyltransferase activity and transcriptional activation
properties of p300 (Hong and Chakravarti, 2003). p300/CBP also contains a
conserved transcriptional repression domain (Snowden et al., 2000). This
repression domain was located in the N-terminal half of p300/CBP upstream
from the bromodomain, termed cell cycle regulatory domain 1 (CRDI).
Repression mediated by this domain could be relieved via coexpression of
p21"'af/`ipl a cyclin-dependent kinase inhibitor (Snowden et al., 2000). This
stimulatory effect of p21waflcipl, in the context of p300 coactivation, was first
reported for the p65 NF-xB subunit, resulting in the stimulation of KB-dependent
gene expression (Perkins et al., 1997). Of special interest was the observation
that the repression domain of p300 contained two contiguous "classic" SUMO
consensus modification motifs.
1.8. Aims of the project
The aims of the work described herein were to investigate whether p300
was a bona fide SUMO substrate, identify whether the tandem SUMO
modification motifs were the sites of SUMO conjugation and elucidate the
biological significance of the potential modification. SUMO modification of
p300 was to be investigated through use of a permanent cell line expressing
6HIS-tag SUMO, allowing the pull-down of any associated proteins, under
conditions that inactivate the SUMO-specific proteases, preventing substrate
deconjugation. The identification of the SUMO conjugation site was to be
investigated through the use of p300 mutants lacking the predicted conjugation
domain in vivo, and subsequently in vitro using site-directed mutagenesis and in
vitro SUMO conjugation assays.
The occurrence of the tandem SUMO modification motifs in a previously
identified transcriptional repression domain, CRD1, of p300 suggested a role for
SUMO in the regulation of p300 transcriptional capacity. The relationship
95

etween that of SUMO conjugation and transcriptional activity of p300 was
investigated, through use of a Ga14 luciferase reporter system, with SUMO
competent and deficient versions of'p300.
This relationship could
be further
investigated through the control of the SUMO modification pathway, by either a
dominant negative version of Ubc9 or over-expression of a SUMO-specific
protease.
Finally through use of emerging siRNA technology the potential of
functional differences between the three SUMO paralogues were to be
researched. This work was to focus on the potential roles played in
transcriptional regulation and localisation by the various SUMO isoforms.
96

2. MATERIALS & METHODS
97

2.1. Materials
All materials not produced in house were purchased from Sigma unless
otherwise stated.
2.2. Antibodies
HA-SUMO-1, HA-SUMO-2, and HA-SUMO-3 were detected in
Western-blot experiments using mAb 12CA5 (at 1: 5000 dilution), which
recognises YPYDVPDYA from influenza HA, obtained from BabCO.
Ubc9 was detected using an affinity purified sheep pAB (in house) at a
1: 1000 dilution.
SUMO-1 was detected using the mouse anti-GMP1 mAb (1: 1000)
(Zymed), and SUMO-2 and SUMO-3 were detected using a rabbit anti-Sentrin2
pAb (1: 2500) (Zymed). The SUMO-1 and SUMNO-2/3 antibodies were both
used at 1: 300 dilutions for visualisations in immunofluorescence.
p300 and CBP were detected by anti-p300 mAb (Pharmingen) and anti-
CBP pAb (Santa Cruz) respecfuly.
Sheep anti-mouse (Amersham), donkey anti-sheep (Jackson
ImmunoResearch) and donkey anti-rabbit (Amersham) horseradish-peroxidase
conjugated IgGs were used to detect the primary antibodies at 1: 2500 dilution in
Western-blot experiments. The secondary Immunoflurescence antibodies; goat
anti-mouse FITC conjugate, and goat anti-rabbit Texas Red conjugate (Oxford
Biotechnology Ltd. ) were used at a concentration of
2.3. Bacterial Strains
1: 250.
E. coli DH5a (genotype: ý80dlacZAM15, rec Al, end Al, gyr A96, thi-
1, hsd R17 (rk-, mk+), sup E44, rel Al, deoR, D(lacZYA-argF) U169) was used
for routine DNA preparation. E. coli B834 (F-, ompT, hsdSB, (rB-, m8-) dcm,
gal) was used for all protein expression unless otherwise stated.
98

2.4. DNA preparation
The DNA preparations (maxi-preps, mini-preps, and gel extractions) used
for cloning and transfections were prepared with Qiagen`"' kits, in accordance
with the manufactures instructions. DNA restriction enzymes were obtained
from both Promega ` "' and New England Biolabs' ' (NEB). The Vent polymerase
used in polymerase chain reactions (PCR) was obtained from NEB. A
Boeringher "TitanTm one tube RT-PCR System" was used for reverse
transcription followed by PCR amplification (RT-PCR). The quality and
quantity of DNA were analysed by spectrophotometric readings at 260 nm and
280 nm and by electrophoresis in an agarose gel in the presence of ethidium
bromide, followed by U. V. measurement.,
2.4.1. cDNA cloning
2.4.1.1. Preparation of thermocompetent bacteria
A single colony of the appropriate bacterial strains were used to inoculate
5 mis of L-broth medium overnight, which was subsequently used to inoculate 5
L of L-broth medium. The bacteria cultures were allowed to grow at room
temperature until the optical density at 600 nm were in the 0.5-0.8 region. The
cultures were subsequently divided into pre-chilled 50 ml sterile centrifuge tubes
and kept on ice for thirty minutes. The cultures were centrifuged at 3600 rpm,
for five minutes at 4°C, the supernatants were removed and 20 mls of both pre-
chilled 100 mM CaC12 and 40 mM MgSO4 were used to resuspend the bacterial
pellet, and subsequently left to incubate on ice for a further thirty minutes.
Following the incubation, the bacterial solution was re-centrifuged at 3600 rpm
for five minutes at 4°C. The supernatant was subsequently removed and the
pellet resuspended in 2.5 mis of CaCl2 and 2.5 mis of MgSO4. Glycerol was
added to 10%, the cells were aliquoted as desired, snap-frozen and stored at
-70°C.
99

2.4.1.2. Transformation of thermocompetent bacteria
Approximately 10 ng of DNA was added to 100 µl of bacteria, and left on ice for
thirty minutes, following which the bacteria and DNA mixture was heat-shocked
by being left at 42°C for between two and four minutes, before returned to ice for
a further twenty minutes. Subsequently 1 ml of LB was added and the mixture
was incubated at 37°C for between thirty and sixty minutes. Following this
incubation the mixture was centrifuged for one minute at 13000 rpm, the pellet
was subsequently resuspended in 100 µl of the supernatant and plated out onto
L-agar plates, containing the appropriate selection antibiotic, and left at 37°C for
twelve hours.
2.4.1.3 Generated plasmid constructs and siRNA oligonucleotides
siRNA
Oigonucleotide sequences were designed against 21 nucleotide
(nt) long
sequences that terminated in double thymidine nucleotide (nt). Subsequently the
21-nt was blasted to ensure the 21-nt was unique to the gene of interest.
Synthetic oligonucleotides take advantage of a naturally occurring RNA
interference pathway. The siRNA oligonucleotide mimics the naturally forming
siRNA duplex created by the double stranded RNA cleavege mediated through
the Rnase III family member, Dicer. The siRNA oligonucleotides are
subsequently incorporated into the RNA-inducing silencing complex (RISC).
Following' the unwinding of the of the siRNA duplex in an ATP dependent
manner, the single-stranded antisense strand guides RISC to messenger RNA that
has a complementary sequence which results in the endonucleolytic cleavage off
the target mRNA.
An alternative to the synthetic oligonucleotides, is plasmid based siRNA
vectors. These vectors are considerably cheaper than the synthetic
oligonucleotides, and can be efficiently co-transfected along with other plasmids.
Mammalian expression vectors have been generated that direct the synthesis of
siRNA-like transcripts (pSUPER: supression of endogenous RNA). PSUPER
possess a polymerase-Ill H1-RNA gene promoter, and it produces a small RNA
transcript lacking a polyadenosine tail and a well defined start of transcription
100

and termination signal consisting of five thymidines in a row. Importantly, the
cleavage of the transcript at the termination site is after the second uridine
yielding a transcript resembling the end of the synthetic siRNAs. The pSUPER,
and other shRNA vectors, contains a 19-nt sequence derived from the target
transcript, separated by a short spacer from the reverse complement of the same
19-nt sequence. The resulting RNA hairpin can be cleaved by Dicer into siRNA,
and subsequently mediated mRNA destruction.
2.4.1.3.1 Cullin-2
The full length open reading frame of human Cullin-2 cDNA was obtained via
RT-PCR from HeLa cell mRNA (kind gift from Dr. David C. Hay), with the
primers Cul-2F (5'-CGAAGGATCCATGTCTTTGAAACCAAGAGTAGTAG-
3') and Cul-2R (5'-TCAGGAATTCACGCGACGTAGCTGTATTC-3').
2.4.1.3.2. Synthetic siRNA oligonucleotides targeting the SUMO isoforms
Synthetic siRNA oligonucleotides were created against the following target
sequences; SUMO-1, AACUGGGAAUGGAGGAAGAAG; SUMO-2,
AAGAUCAAGAGGCACACGUCG; SUMO-3,
AACAGACACACCUGCACAGUU.
2.4.1.3.3. pSUPER retro siRNA expression plasmids
The siRNA expression plasmids were designed as described previously
(Brummelkamp et al, 2002), were inserted into the pSUPER retro plasmid
(oligoengine). The siRNA oligonucleotides created were (upper strand only):
SUMO-1,
GATCCCCCTGGGAATGGAGGAAGAAGTTCAAGAGACTTCTTCCTCCA
TTCCCAGTITFI GGAAA;
SUMO-2,
GATCCCCGATCAAGAGGCACACGTCGTTCAAGAGACGACGTGTGCCT
CTTGATCTTTTTGGAAA;
SUMO-3,
GATCCCCCGACAGGGATTGTCAATGATTCAAGAGATCATTGACAATC
CCTGTCGT =GGAAA.
101

Expression plasmids for HDAC1, HDAC4, and HDAC6 were kind gifts
from T. Kouzarides (Cambridge, UK). SSP3/SENP2, wild type and mutant
DNA were generous gifts from Barbara Ink (GalaxoWellcome), Heidi Mendoza,
and Owen A. Vaughan (University of St. Andrews). The dominat negative Ubc9
mutant plasmid was kindly provided by Joanna M. P. Desterro. The expression
plasmids for HA-SUMO-1, HA-SUMO-2, HA-SUMO-3, GST-SUMO-1 GG,
GST-SUMO-2 GG, and GST-SUMO-3 GG were provided by Michael H.
Tatham (University of St. Andrews). Purified SAE1/2, Ubc9, RanBP2, and all
PIAS proteins were generously provided by Ellis Jaffray. The expression
plasmids for His-p300 CRD+, His-p300 CRD-, Ga14 N-, Ga14 N+, and Ga14
(Figure 16a) were provided as part of collaboration by Neil D. Perkins
(University of Dundee). The Ga14 fusions both wild-type and sumoylation
deficient for Sp3 and Elk-1 (Figure 16a) were provided by Grace Gill (Harvard
University) and Andrew Sharrocks (Manchester University) respectively.
2.4.2. DNA sequencing
All constructs were verified by automated DNA sequencing on an ABI
PRISMT' 377 DNA sequencer (St. Andrews University DNA sequencing unit).
2.5. Expression and Purification of unlabeled Recombinant Proteins
GST-SUMO-1-GG, GST-SUMO-2-GG, GST-SUMO-3-GG, GST-
SAE1/2, and all p300 CRD1 domain mutants were expressed from E. coli strain
B834 as described previously (Jaffray et al, 1995). All GST-SUMO proteins
were cleaved by thrombin while bound to glutathione-agarose beads. For freeze-
drying, proteins were dialysed against 20 mM ammonium bicarbonate, 1 mm
dithiothrietol (DTT), before concentration calculations. Lyophilised protein
samples were taken up in 50 mM Tris/HCL pH - 7.5,1 mM DTT to a
concentration of 10 mg. ml-'.
The CRD 1 domain proteins were purified on glutathione-agarose beads
(GGA) equilibrated with PBS + 0.5M NaCl. The sonicated supernatant was
filter sterilised and mixed with the GGA beads for 10-20 minutes under agitation
102

p300 N+
p300 N-
107IKDE10 538IKEE-"
Sp3 wt AD AD ID
Sp3 mut
Elk-1 wt
Elk-1
Ga14 AdMLP reporter
229LKSE23'
LRSE232
Ga14 bs AdMLP Luciferase
Figure 16a. Schematic representation of the Ga14 fusion proteins, as indicated, and Ga14
AdMLP reporter construct. Plasmids were originally described in Girdwood et al., 2003
(p300); Ross et al., 2002 (Sp3), and Yang et al., 2003 (Elk-1).
1004-1044
.. LKTEIKEE..

on a roller. The bead/supernatant mix was added to a plastic column and the
flow-through was re-circulated three times. The column was washed with four
column volumes of PBS + 0.5M NaCl, one volume at a time. The GST fusion
protein was eluted by the addition of glutathione buffer, and 0.5 column volume
fractions were collected. The fractions were analysed by running on 10% SDS-
PAGE, and the fractions containing protein were pooled and dialysed overnight
at 4°C to remove glutathione. The proteins were subsequently concentrated
through use of a centrifugal device (MicroconTM)
2.6. Quantitation of protein
Protein concentrations were " determined using Bradford's method
(Bradford, 1976). Protein samples were mixed with Bradford's (BioradTM) and
the absorbance at 595 nm was measured on a spectrophotometer (Hitachi U-
1100). Protein absorbencies were converted to mg/ml concentrations using a
standard curve constructed by measuring the absorbencies of a range of bovine
serum albumin (BSA) concentrations.
2.7. SDS/PAGE and Western Blot analysis
Protein samples were resuspended in disruption buffer (1X: 20mM
Tris/HCL pH 6.8,2% SDS, 5% f -mercaptoethanol, glycerol and 2.5%
bromophenol blue) and denatured at 100°C for five minutes, before loading on
6%-15% SDS-polyacrylamide gel (acrylamide percentage appropriate for the
size of proteins to be separated), or following lysis SDS sample buffer, samples
were diluted 1: 3 in RIPA buffer (25mM Tris-Hcl pH8.2,50mM NaCl, 0.5% NP-
40,0.5% Deoxycholate, 0.1% Azide) containing protease inhibitor tablets. Bio-
Rad' mini-gel equipment was used in accordance with the manufactures
instructions. New England Biolabs' prestained molecular weight markers were
used as standards to establish the apparent molecular weights of proteins
resolved on SDS-polyacrylamide gels. Separated polypeptides were either
stained with Coomassie Brillant Blue (0.2% Coomassie brilliant blue R250; 50%
methanol; 10% acetic acid) for thirty minutes and then destained (20% methanol;
10% acetic acid) or transferred to a polyvinylidene diflouride mebrane (PVDF)
(Sigma) using a wet blotter (Bio-Rad Systems). The membranes were blocked
104

with PBS containing 5% skimmed milk powder (Tesco) and 0.1% Tween 20, and
subsequently incubated with monoclonal or polyclonal antibodies diluted in
blocking buffer. Horseradish peroxidase conjugated anti-mouse IgG, anti-rabbit
IgG (Amersham) and anti-sheep IgG were used as' secondary antibodies.
Western blotting was performed using ECL detection system.
2.8. Stripping of P. V. D. F. membranes
After ECL, the membranes were washed in PBS for 20 minutes before
being transferred into stripping buffer (142 µl ß-mercaptoethanol, 2 ml 20%
SDS, 1.25 ml Tris pH 6.7 and 16.6 ml dH2O) and incubated for 30 minutes in a
hybridisation oven at 72°C. The membranes were subsequently washed twice in
250 ml PBS containing 0.1% Tween 20. The membranes were blocked for 1
hour in PBS containing 5% skimmed milk powder and 0.1% Tween 20 and then
probed as in the Western blot procedure.
2.9. In vitro Expression of Proteins
In vitro transcription/translation of proteins was performed using 1 µg of
plasmid DNA and a wheat germ-coupled transcription/translation system
according to the manufacturer's instructions (Promega). 35S-methionine
(Amersham Pharmacia Biotech) was used in the reactions to generate
radiolabelled protein.
2.10. In vitro SUMO conjugation assay
SUMO conjugation assays were performed in 10 µl volumes containing
0.84 µ1 of 1251-labeled
SUMO-1 or 1 µg of SUMO-1, SUMO-2, SUMO-3, GST-
SUMO-1, GST-SUMO-2, and GST-SUMO-3 (as indicated), an ATP
regenerating system and buffer (50 mM Tris pH 7.5,5 MM MgC12,2 mM ATP,
10 mM creatine phosphate, 3.5 U. ml'' creatine kinase) and 0.6 U. ml"' IPP with
either 1µ1 of 35S-methionine labelled substrate (5'-p300) or varying
concentrations of unlabeled GST-p300 fragments (as indicated). Assays
contained 120 ng (1.1 pmoles) purified recombinant SAE1/SAE2 and 650 ng
(35.9 pmoles) Ubc9, unless otherwise shown. Reactions were incubated at 37°C
for 4 h. Reactions were stopped following the addition of SDS sample buffer,
105

eaction products were fractionated by electrophoresis in polyacrylamide gels (6-
10%) containing SDS, stained, destained and dried, before analysis by
phosphorimaging (Fujix BAS 1500, MacBAS software).
2.11. In vitro SUMO Deconjugation Assay
Recombinant GST-PML substrate was conjugated with SUMO as
described previously. Conjugation was terminated by the addition of
iodoacetamide to 10mM and incubated at 20°C for 30 minutes. lodoacetamide
was quenched by the addition of ß-mercaptoethanol to 15mM and incubated at
20°C for a further 15 minutes.
Deconjugation of SUMO-GST-PML was performed in a total volume of 10µl;
containing 2µg of GST-PML and the indicated amounts of SSP3 (provided by H.
Mendoza)(0-243ng) in deconjugation buffer (50mM Tris pH7.5,2mM MgC12,
and 5mM ß-mercaptoethanol. Reactions were incubated at 37°C for 3 hours,
before being terminated with SDS sample buffer, reaction products were
fractionated by electrophoresis in polyacrylamide gels (10%) containing SDS,
stained, and destained.
4F
2.12. Protein Interaction Assays
GST, unmodified GST-p300-CRD1 and GST-p30085210719
or SUMO-
modified GST-p300-CRD1 and GST-p300852_1071 (Figure 16b) were purified from
reaction components on glutathione agarose. HDAC4 and HDAC6 were 15S_
labeled by in vitro translation in a wheat germ extract and diluted 10-fold in 150
mM NaCl, 50 mM Tris (pH 7.5), 1 mM DTT, and 0.1% NP40 before
clarification by centrifugation. The soluble fraction (100 µl) was added to the
loaded glutathione agarose beads (10 µl packed volume) and incubated for 2 hr
at 37°C. Beads were collected by centrifugation, washed extensively with the
above buffer and bound proteins fractionated by SDS PAGE, and analysed by
phosphorimaging.
106

(1)
C/H1 CRDI Bromo C/H2 C/H3
PMO wt III on IIIII
p300 i-1255
p300 ss2-ion
p3001017-1029
p300 CRD1
p300 192-1044
(ii)
p300 Aloo4-1o44
RanBP2 2532-2767
1 1255
0-91
852 CRD 1 1071
1017 029
192
1044
2532 2633 2685 2710 2761 2767
Figure 16b. Fusion constructs. (i) Schematic representation of p300 fusion constructs,
as described in material and methods. (ii) Schematic representation of a fragment
from the SUMO E3, RanBP2, that possess the E3 ligase activity.

2.13. Mammalian cell culture transient transfections
HeLa His6-SUMO-1 stable cell line was grown in DMEM (Gibco)
supplemented with 10% FCS and puromycin (2µg/ml). HeLa His6-SUMO-2 and
HeLa His6-SUMO-3 stable cell lines were grown in DMEM supplemented with
10% FCS and puromycin (0.5µg/ml). COST and HeLa cells were maintained in
exponential growth in Dulbecco's modified Eagle's medium, containing 10%
foetal bovine serum. For transfections, a total of 12µg of plasmid DNAs were
transfected for 10 hours in subconfluent 75cm3 flasks using LipofectamineTM
according to manufacturers instructions (Invitrogen). After 16 hours of
expression, cells were washed in PBS and cellular extracts were either added to
1X disruption buffer or 6M guanidinium-HCL.
2.13.1. Calcium phosphate transient transfections and reporter gene assays.
Cells growing in log phase were plated into individual wells of a six well
plate at 40% confluency, 1 to 2h prior to transfection to allow the cells to
adhere. All transfection solutions were equilibrated to room temperature. siRNA
encoding plasmids (1µg) were co-transfected into U2-OS cells along with Ga14-
p300, Gal4-Sp3, and Gal4-Elk-1 expression plasmids (0.5 ng) and 2 µg of Ga14
AdML luciferase reporter plasmids. Synthetic siRNA oligos were similarly
transfected at 100 nM each. The reporter plasmid DNAs along with the
pSUPER retro plasmid DNA or synthetic siRNA oligos was diluted into 438 µl
of water containing 61 pl of 2M CaC12
and added in drops with gentle agitation
to 500 gl of 2X HEPES-buffered saline (0.274 M NaCl, 1.5 mM Na2HPO4,
54.6 mM HEPES [pH 7.1]). The DNA, CaC12, or synthetic siRNA oligo, and
HEPES-buffered saline were mixed by pipetting and, split three ways, then
immediately sprinkled onto the cells, in three wells of a six well plate, ensuring
that all the cells were covered. Following a 24-h incubation, the precipitate was
removed and fresh complete medium was added to the cells, and the cells left for
a further 24-h, before being subjected to SDS-page and western blotting or
harvested and luciferase activity determined as appropriate.
108

2.14. Purification of His6-tagged proteins
After twenty-six hours the transfected cells were washed with PBS and
resuspended in 10 ml of PBS. 1 ml was removed, pelleted and lysed in SDS
sample buffer (5% SDS, 0.15 M Tris-HCI pH 6.7,30% glycerol, 0.72 M ß-
mercaptoethanol) diluted 1: 3. The remaining 9 ml was pelleted and lysed in 5 ml
6M guanidinium-HCI, 0.1 M Na2HPO4/NaH2PO4,0.01 M Tris-HC1 pH 8.0 plus
5 mM imidazole and 10mM ß-mercaptoethanol. per 75 cm3 flask. Endogenous
material were directly lysed in 5m1 6M guanidinium-HCI, 0.1 M
Na2HPO4/NaHZPO4,0.01 M Tris-HC1 pH 8.0 plus 5 mM imidazole and 10mM ß-
mercaptoethanol. per 75 cm3 flask. Both transfected and endogenous cell lysates
were sonicated, to reduce viscosity, the lysates were mixed with 50µ1 of
Nie+NTA-agarose beads prewashed with lysis buffer and incubated for 3h at
room temperature. The beads were subsequently washed twice with 6M
guanidinium-HCI, 0.1 M Na2HPO4/NaHZPO4,0.01 M Tris-HC1 pH 8.0,10mM f-
mercaptoethanol; three times with 8M urea, 0.1 M Na2HPO4/NaH2PO4,0.01 M
Tris-HC1 pH 8,10 mM ß-mercaptoethanol; twice with 8M urea, 0.1
Na2HPO4/NaHZPO4,0.01 M Tris-HC1 pH 6.3,10 mM ß-mercaptoethanol (buffer
A); buffer A plus 0.2% Triton X-100; buffer A; buffer A plus 0.1% Triton X-
100, and then three times with buffer A. The beads were finally eluted with 200
mM imidazole in 5% SDS, 0.15 M Tris-HC1 pH 6.7,30% glycerol, 0.72 M ß-
mercaptoethanol. The eluates were subjected to SDS-PAGE (6-10%) and the
proteins transferred to a PVDF membrane (Sigma). Western blotting was
performed with either pAb against p300 (Santa Cruz) or mAb specific to the HA-
epitope (Babco).
2.15. Indirect immunotluorescence analysis of cells
Cells were cultured on 13 mm glass coverslips (Scientific Laboratory
Supplies Ltd. ) in 6 well plates and subjected to transient transfections as
necessary. On completion of transfection, cells were washed twice in PBS
containing 1 mM MgCl2 and 1 mM CaCl2. Cells were subsequently fixed with
warm 3% Paraformaldehyde/PBS for 10 minutes, washed in PBS three times,
and fixation quenched by two 10 minute 0.1 M glycine/PBS incubations. After a
5 minute PBS wash, cells were permeabilised for 10 minutes in 0.1% Triton-X-
109
4

100/PBS. Following three further 5 minute PBS washes, the cells were blocked
in 0.2%/PBS BSA for 10 minutes. Immunostaining with the relevant
concentration of primary antibodies was performed for 2 hours at room
temperature, in 0.2% BSA/PBS before being subjected to three 5 minute 0.2%
BSA/PBS washes. The cells were then incubated for a further 30 minutes with
the corresponding fluorescently labelled secondary antibodies diluted in 0.2%
BSA/PBS at 1: 250 dilution. The cells were given a final three 5 minute PBS
washes before the coverslips were gently mounted onto slides using Movial
(Calbiochem) and stored overnight at 4oC. Fluorescence microscopy was carried
out with a DeltaVision microscope (Olympus IX70) with a 1.40 NA 100X
objective and Photometric CH300 CCD camera. 10X 0.2 µm sections were
taken. Images were deconvoluted using SoftWorx (Applied Precision) software.
2.16. Luciferase Assay
Transfections to be assayed for luciferase activity were stopped by
washing cells twice with PBS, and then subsequently lysed in 200 µl of
luciferase lysis buffer ( 25 mM Tris-phosphate pH 7.8,8 MM MgC12,1 mM
DTT, 1% Triton X-100 and 15% glycerol; made up in dH2O). 50 µl of the lysate
- was injected with luciferase assay buffer (0.25mM luciferin, 1mM ATP, and 1%
BSA diluted in the lysis buffer) into a luminometer (Berthold 'Sirius
Luminometer) and the luciferase activity was recorded. The activity was stated
in relative light units (R. L. U. ).
110

3. RESULTS & DISCUSSIONS
111

3.1 Bioinformatics: an approach to identify novel members of the
ubiquitin-like protein superfamily
Previous bioinformatics within the group had focused primarily on
database searching, of submitted proteins, to identify those proteins containing
the reported SUMO modification motif. This approached allowed the generation
of a list of putative SUMO modified substrates. The completion of many
genomes potentially allowed the generation of a comprehensive list of potential
SUMO substrates. The draft human genome sequence, as accessible through the
Sanger Institute (httl2: //www. satiger. ac. uk/HGP/) enables us to search for similar
nucleotide sequences or "blasting" (Basic Local Alignment Search Tool) of
proteins of interest against the draft human genome sequence. Thus to utilise the
completion of the draft version of the human genome, the protein sequences of
all known ubiquitin-like proteins were "blasted" against the draft sequence in an
attempt to identify novel members of the ubiquitin-like protein modifier
superfamily.
3.1.1 Identification of three possible new members of the SUMO
family
The database searching of the draft sequence of the human genome
revealed the presence of an additional three members of the SUMO family,
located on chromosomes six, seven, and twenty (Figure 17). The sequences for
all three novel members had previously been submitted to GenBank
(Accession numbers XP_066029, CAA20019, and XP_168354 respectively).
These genes were annotated by the Ensembl automatic analysis pipeline using
either a GeneWise model from a human/vertebrate protein, a set of aligned
human cDNAs followed
. by GenomeWise for open reading frame (ORF)
prediction or from Genscan exons supported by protein, cDNA and EST
evidence. GeneWise models are further combined with available aligned cDNAs
to annotate UTRs. All three annotated genes possesssed ORFs and contained a
112

di-glycine motif towards the end of their C-terminal region. Of these three
potential new genes, one had high homology to SUMO-1, that on chromosome
20, termed SUMO-lb, and two possesssed high homology to SUMO-2/-3, those
on chromosomes six and seven, termed SUMO-4 and SUMO respectively.
Interestingly, all three contained an internal SUMO modification motif (Figure
16a), the new genes however, possesssed no introns unlike the previous three
members.
3.1.2 SUMO-lb/4/5 are conjugated both in vitro and in vivo
To clone the genes, mRNA was extracted from HeLa cells (mRNA
Isolation system, Promega), which included a DNase treatment step to digest any
contaminating genomic DNA. Subsequently RT-PCR was preformed to amplify
the genes from the mRNA (Figure. 18. ). The' DNA products were then cloned
into HA-pcDNA3 and pGEX-4T vectors, and the correct sequences were verified
by DNA sequencing.
All members of the SUMO family are capable of being covalently bound
to substrates via lysine residues, following their processing by the proteases, and
conjugation through the enzymatic cascade involving SAE1/2, Ubc9, SUMO
ligase. Due to the sequence similarities of the novel members with SUMO-1/-2/-
3, it was likely that these novel members of the SUMO gene family would be
functionally similar. To test this hypothesis HA-SUMOs la/lb/2/3/4/5 and
empty pcDNA3 vectors were transfected into COST cells. Following
transfection cells were lysed in RIPA buffer and subjected to Western blot
analysis with anti-HA antibody (Figure. 18. ). All three potential new members of
the SUMO family were shown to be capable of covalently modifying a range of
cellular substrates, as indicated by the analogous pattern of detection. Western
blot analysis indicated that the levels of SUMO-lb were greatly elevated,
compared to the other SUMO isoforms, there being approximately 100 times
more SUMO-lb conjugated than SUMO-la (Figure 18). The differences in
levels of conjugation of SUMO-lb, suggest that SUMO-lb has greater stability
than the other SUMO isoforms, or is more resistant to removal via the actions of
the SUMO specific proteases.
113

Figure 17(i). Diagrammatic representation of the human chromosomes. The
chromosomal locations of both the SUMO gene members, and potential new SUMO gene
members are shown (indicated by red arrow). Locations were identified through the blast
software service option of the human genome sequencing project, through the sanger
centre (www. sanger. ac. uk/HGP). (ii) Table providing GenBank accession numbers,
chromosome location, number of exons, and number of pseudogenes for the SUMO gene
family. (iii) Sequence alignment of SUMO-1/-2/-3 with the predicted sequences for the
potential new members of the SUMO family.

(I)
(ii)
123456789 10 ii 12
13 14 15 16 17 18 19 20 21 22 XY
Genes GenBank Accession number Chromosome Exons Pseudogenes
SUMO-1 CAA67898 2 5 8
SUMO-2 CAA67896 21 4 0
SUMO-3 P55855 17 4 23
SUMO-Ih XP_066029 20 1
SUMO-4 CAA2(X)19 6 1
SUMO-5 XP 168354 7 1
(iii)
Sabo-2
SW®0-3
SUND-5
SUMO-4
SUM-2
SWF-3
SUMO-5
SUMO-4
SU-1 s T$DLGDKPMGZY
SUND-lb ruimZD sn lJAII'
IB
ýA'! ý
SIDE)-1 sIýý
sý------sasa-ý
s xa ,4 VW z srv-------
-
-

(t An
z
(iii q rý
x
175
83
62
m
OO o o
vý ýn
ý' x xx x x
a t t ý ..
HA-SUMO
conjugates
Figure 18. (i) RT-PCR
was performed on HeLa
SUMO DNA poly(A)+ RNA.
from RT-PCR Amplification
reaction. (ii) Conjugation of
proteins in vivo. COS7 of transfected SUMO
cells were transfected
la/lb/2/3/4/5-GG.
with either
Total pcDNA3 or HA-SUMOcell
lysates
were analyzed by
anti-HA Western
mAb. Molecular blotting
mass standards using
are expressed in kilodaltons.
6

To investigate the mechanisms of their conjugation, a purified GST-PML
construct (Tatham et al., 2001) or 35S-Met-labelled Sp100, generated by in vitro
transcription/translation, was incubated with recombinant Ubc9, SAE1/2, and
" either SUMO-la, SUMO-lb, SUMO-2, SUMO-3, GST-SUMO-4, or SUMO-5
(as described in material and methods). The incubation of the substrates, with
the components of the conjugation pathway, resulted in the appearance of slower
migrating species, with all of the SUMO forms tested (Fig. 18i. ). The specificity
of the reactions was demonstrated by the absence of any other forms of GST-
PML or 35S-SplOO when incubated in the absence of purified SUMO, Ubc9, or
SAE1/2, furthermore substitution of SUMO for a tagged GST-SUMO further
reduces the mobility of the substrate, demonstrating that the occurrence of the
slower migrating forms observed are due to the conjugation of SUMO to the
substrates. In the reactions lacking substrate, the formation of poly(SUMO)
chains was observed, indicating that the all three potential new members,
functioned in a manner analogous to that of SUMO-2/-3 are were capable of
forming the SUMO chains (Fig. 19. ii. ).
3.1.3 Deconjugation of SUMO proteins via a SUMO specific
protease
To test whether the new SUMO family members were deconjugated in a
similar manner to those already identified, deconjugation assays were set up.
SUMO modification of substrates is controlled on many levels, including their
deconjugation from their substratcWsing the GST-PML substrate that had
previously been demonstrated to be modified by all SUMO species, the
deconjugating ability of the SUMO specific protease, available within the group,
SSP3/SENP2 was investigated. Recombinant GST-PML was independently
modified by both SUMO-lb and SUMO-5 in the presence of recombinant
SAE1/2 and Ubc9; and subsequently conjugation was terminated by the addition
of iodoacetamide. After quenching of the iodoacetamide with ß-
mercaptoethanol the reaction products were used as substrates for a range of
SSP3/SENP2 concentrations with a three hour incubation at 37°C (Figure 20)(as
117

(i)
(ii)
SUMO-1aSUMO-2 SUMO-lb SUMO-4 SUMO-5
SUMO - ++ - ++ +- ++++- ++++ -
GST-SUMO - -- --- ----- + +- ++ ++ "
- +
SAE1l2 + ++ +++ - ++- +++ -++- ++ ++ +
-++-
Ubc9 +++ +++ -+++-++ - ++ + -+
++++ +
GST-PML ++- ++- -++++-+ -+++ +- -++++-
175
83
63
47
32
25 r `" ý. ý" W- go or
16
SUMO-1a
SUMO-lb
SUMO -+-+++-
GST-SUMO -+
SAE1! 2
Ubc9
++++-++
+++++-+
175-
83-
62-
!! '
....
w.. * w.,.
. ý.
-
wow
SUMO-4
SUMO-5
+
sumo
mº conjugates
ow
- - - - + + + -
- + + + - - - - +
+ + - + + + - + +
+ + + - + + + - +
a
06
SUMO
conjugates
-44
+ SUMO
Figure 19. SUMO-1b/4/5 are capable of conjugation to target substrates
and to a similar extent. (i) GST-PML or (ii) 35S Sp1OO. In the absence of
substrate a reduction in monomeric SUMO levels were observed, and high
weight molecular species were observed, consistent with the formation of
poly(SUMO) chains. Due to the presence of an internal thrombin site
SUMO-4 was left as a GST fusion, GST-SUMO-4-GG. Reaction products
were fractionated by SDS-PAGE before the gels were either (i) stained
with coomassie brilliant blue or (ii) dried and analysed by
phosphorimaging. The positions of conjugated SUMO are indicated.

+I SUMO- I blo.
GST-PML º,,
SUMO-lb
+1 SUMO-5
0'
GST-PML ºr
SUMO-5 º
SSP3/SENP2
039 27 81 243 (ng)
.,.,,
ý
q... , new __ ý
SSP3/SENP2
039 27 81 243 (ng)
Figure 20. SUMO modification of GST-PML is removed following the
addition of a SUMO specific protease. Following a3h incubation in
deconjugation buffer, with varying amounts of SSP3/SENP2 present (as
indicated) at 37°C, the SUMO modified substrate was not seen. With no
SSP3/SENP2 added or at very low concentrations, SUMO modification was
left intact. Reaction products were fractionated by SDS-PAGE before the gels
werevisualised via staining with coomassie
brilliant blue.

detailed in materials and methods). As indicated by Figure 20, assays that
contained no or very low concentrations of SSP3/SENP2 did not affect the
SUMOylation status of the substrate. The assays containing the higher
concentrations of resulted in the removal of all SUMO modified GST-PML
forms and increase in the available pool of unconjugated "free" SUMO.
3.1.4 SUMO-lb, SUMO-4, and SUMO-5 may be psuedogenes
Data obtained from the human genome is analysed against a range of
critera, to check for validity, before being submitted to the accessible databases.
To confirm that the newly identify genes are expressed and that the original RT-
PCR constructs generated were amplified directly from mRNA and not
contaminating DNA, RT-PCR reactions were repeated in duplicate, one set
containing the reverse transcriptase DNA polymerase and the second set had the
reverse transcriptase enzyme substituted by Taq polymerase. The resulting PCR
products demonstrated that there was possible DNA contamination (Figure 21).
Thus there was the possibility that the newly identified "novel" SUMO genes
may in fact represent pseudogenes. Pseudogenes are not true genes, as although
their DNA sequence is found within the genome, they are not transcribed, and
there are many pseudogenes within the human genome.
120

+ Reverse transcriptase +++---
+ Taq polymerase ---+++
SUMO lb 45 lb 45
Figure 21. Contamination of mRNA preparation with genomic DNA. The
RT-PCR for the amplification of the potential new SUMO genes was
repeated under standard conditions with the reverse transcriptase enzyme
included. To exclude artefacts from DNA targets like residual genomic DNA
contaminations from RNA preparations, a control reaction was set up where
the RT was not preformed. This was achieved by the addition of Taq DNA
polymerase to the RT-PCR mixture instead of the normal enzyme mix.
4-

3.1.5 Conclusion
That the RNA used for the RT-PCR was potentially contaminated with
DNA did not directly rule out that these potentially novel genes were expressed.
However the fact that none of these potential new genes contained any introns
unlike SUMO-l/-2/-3, was suggestive that they were in fact pseudogenes.
Further evidence was provided by bioinformatics analysis that indicated that
there was no similarity between the mouse and human promoter regions and that
represented they were not in EST databases. (C. W. Crumbley, Glaxo Smithkline,
personal communication). With the combination of evidence suggesting that the
genes were not expressed, the decision was taken not to follow up this line of
study, although one possibility was to clone the gene from a cDNA library.
Recently, however Owerback and colleagues reported the expression of SUMO-
4, in the liver (Bohren et al., 2004). SUMO-4 is polymorphic, the predominant
form having a methionine at amino acid position 55, whilst a second form exists
with a valine residue in place of the methionine. This finding was similar to the
findings from the potential SUMO-4 gene identified through the human genome,
which was submitted as having a valine at amino acid 55, but following cloning
and sequencing from HeLa mRNA, all clones contained a methionine at this
residue. Subsequently it was confirmed that the reported SUMO-4 gene was the
same gene (NCBI accession number: CAA20019) as the potential SUMO-4 gene
identified by our bioinformatics search. Owerback and colleagues demonstrated
that SUMO-4 is expressed by performing control RT-PCR reactions. The reverse
transcriptase step of the RT-PCR is omitted, and no PCR product is generated,
thereby showing that the positive reaction was not due to DNA contamination.
As this would seem to indicate that SUMO-4 is a bona fide gene, and novel
member of the SUMO family, the same may yet be true for SUMO-lb and
SUMO-5. Interestingly the SUMO-4 gene that we identified through the human
genome database searching was preformed in 2002, and in the intervening two
years, the draft version of the human genome has undergone many revisions, and
currently the potential SUMO-4 gene has been reannotated as being a
pseudogene. As such there remains the possibility that the SUMO-4 sequence
identified independently both by our group and by Bohren et al., may yet turn
122

out to be a pseudogene. Conversely there remains the possibility that SUMO-lb
and SUMO-5 may be expressed and as such novel members of the SUMO
family.
123
t

3.2 SUMO modification of the transcriptional regulator p300
p300 and CREB binding protein can both activate and repress
transcription. Contained within the previously identified cell cycle regulatory
domain (CRD1), a transcriptional repression domain of p300 that lies between
residues 1017 to 1029, we identified two copies of the SUMO modification
sequence, iKxE (Figure 22. i. ). Mutations within the conjugation motif, which
reduced SUMO modification, resulted in an increase in p300-mediated
transcriptional activity. Expression of a SUMO-specific protease or a
catalytically inactive Ubc9 relieved repression, demonstrating that the repressive
effect of p300 CRD 1 domain was dependent on the conjugation of SUMO.
SUMO-modified CRD1 domain bound HDAC6 in vitro, and the ability of
SUMO conjugation to cause transcriptional repression was relieved both by
histone deacetylase inhibitors and siRNA targeting of HDAC6. Thus the ability
of p300 to act as a transcriptional repressor is dependent on the conjugation of
SUMO to the CRD 1 repression domain, and suggests that SUMO-dependent
repression is mediated by recruitment of HDAC6.
3.2.1 p300 is modified by SUMO in vivo
To demonstrate directly that p300 was modified by SUMO in vivo, a
stable cell line expressing 6His SUMO-1 was lysed directly with guanidine
hydrochloride and 6His proteins isolated on Ni-NTA agarose. Western blotting
analysis of the eluted proteins revealed that endogenous p300 was modified by
6His SUMO-1 in vivo (Figure 22. ii. ). The specificity of this procedure was
demonstrated by the simultaneous analysis of the parental HeLa cells, which
lacked 6His-SUMO-1 (Figure 22. ii. ).
To establish that the tandem modification motif within the CRD1 domain
was indeed required for SUMO modification and to determine if p300 could also
be modified by SUMO-2 and SUMO-3, COS7 cells were cotransfected with
6His p300, residues 192-1044 containing the CRD1 domain, or a version that
124
1, Iý

(i)
p300
WKxEWKxE
y9ISESKVEDCKMESTETEERSTELKTEIKEEEDQPSTSATQSSPAPGQSPý
1035
CBP KEETDTTEQKSEPMEVEEKKPEVKVEAKEEEEENSSNDTASQSTSPSQP082
CRD I domain
HA-SUMO-1 HA-SUMO-2 HA-SUMO-3
NaNN
Cl
C, a
6His oö
s°
(ii) HeLa SUMO-1 (iii)
Q z° Z ä° x
p300 4050
4175
1175
Ni-beads
Anti-HA
extract
Anti-HA
extract wrr"+
Anti-p300 I
Figure 22. SUMO modifies p300 and CBP. (i) p300 and CBP possess a tandem SUMO
modification motif (W)KxE) within the p21-inducible transcriptional repression domain, CRD I.
(ii) Endogenous p300 and CBP are modified by SUMO-1. The SUMO-1 modification of p300
is occurs at the CRDI domain. HeLa His6-SUMO-1 stable cell line and parental HeLa cell line
were lysed in a buffer containing 6M Gaunadine Hydrochloride and purified on Ni' heads as
described in materials and methods. Eluates were subjected to Western blotting with either
p300 or CBP specific antibodies. (iii) COST cells were transfected with His6 p300192-1044 or
His6 p300l92-1(X)4 and Ha-SUMO. Following transfection, samples were taken and crude cell
extracts were prepared for Western blotting and probed with antibodies against Ha and p300, to
test for transfection efficiency. The reaming cellular extracts were lysed in 6M Guanadine
Hydrochloride and purified on Ni' beads. Eluates were Western blotted and probed with anti-
Ha antibody.
4175
/ 16.5
4175

lacks the CRD1 domain (01004-1045), and HA versions of SUMO. CRD1
containing p300 is modified by SUMO-1, SUMO-2, and SUMO-3, whereas
mutant p300 that has the CRD1 domain removed is not modified (Figure 22. iii. ).
To monitor expression of HA-SUMO and p300 a fraction of the lysate, prior to
Ni-NTA chromatography, was analysed by Western blotting using the HA-
specific mAb 12CA5 and p300 specific mAb. Thus the observed differences
were not due to differences in the levels of expression of either HA-SUMO or
p300 (Figure 22. iii. ). SUMO modification of endogenous CBP was also
detected, albeit to a lesser extent (Figure 22 (ii), bottom panel).
3.2.2 SUMO conjugation to the N-terminus of p300 in vitro
modification,
To confirm that the N terminus of p300 is an in vitro substrate for SUMO
"S-Met-labelled
p300 (1-1255) was generated by in vitro
transcription/translation, and incubated with recombinant SUMO, Ubc9, and
SAE1/2. Incubation of p300 (1-1255) with either SUMO-1, SUMO-2, or
SUMO-3, and the purified components of the SUMO conjugation pathway,
resulted in the appearance of a more slowly (labelled) migrating species that was
consistent with SUMO modification (Figure 23). To demonstrate that the more
slowly migrating species of p300 was indeed SUMO modified, SUMO, Ubc9, or
SAE1/2 were omitted from the in vitro reactions. Under these conditions
appearance of the more slowly migrating species was abolished. Substitution of
SUMO with a tagged GST-SUMO further reduced the mobility of p300 (1-1255)
confirming that the occurrence of the slower migrating forms observed are due to
the conjugation of SUMO to the N terminus of p300 (1-1255).
3.2.3 In vitro SUMO modification of N-terminus of p300 does not
require an E3
A major difference between the ubiquitin and SUMO conjugation
pathways, is that SUMO conjugation in vitro does not require the presence of an
E3 protein. Rather, the addition of an E3-like protein can enhance the in vitro
modification of target substrates. These E3 ligases include RanBP2, and
126

Assay mix
CD
2
O
SUMO
SUMO-1 -p300
SUMO-2
SUMO-3
bpw ' mw s A! WOM **4r* 44- p3001_,,
5;
ow, 44
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1ý
k
A_
w.
SUMO
-p300
r* rº 4- p300 1-1255
_, ., _ f-p300
SUMO
-p300
i-1255
Figure 23. p300 is modified by SUMO-l/-2/-3 in vitro. The N-terminal half of p300,
as 1-1255, containing the CRD 1 domain, was 35S-labeled by in vitro translation. The
35S_
labeled p3001 1255 was incubated in the assay for SUMO conjugation or in assay
mixtures missing elements necessary for SUMO conjugation, as
indicated. Reaction
products were fractionated by SDS-PAGE before the gels were dried and analysed by
phosphorimaging. The positions of the free and conjugated forms of p3001_1255 are
indicated.

members of the PIAS family; PIAS 1, PIASy, and PIASxa. Theses four SUMO
E3 were available within the group, and were available for study. E3 activity can
be tested in vitro by determining the lowest concentration of Ubc9 that results in
substrate concentration and then testing a range of E3 concentrations. If there is
an E3-like affect, the amount of substrate concentration will increase as the E3
concentration increases. Initially a range of recombinant Ubc9 concentrations
were titrated into the in vitro modification assays, whilst keeping all other
variables as standard (Figure 24), using both recombinant SUMO-1 or the mutant
SUMO-2 (KI IR) protein that is incapable of forming polymeric chains with
itself under the in vitro conditions. This is due to the mutation of the lysine
residue within the SUMO consensus motif located within SUMO-2/3. At Ubc9
concentration of 25ng a very weak modification was observed, and the band
intensity increased at concentrations of Ubc9 above this (Figure 24). In vitro
SUMO modification assays were performed using 25ng of Ubc9 and titrating in
SUMO E3 ligases over a range of concentrations. Recombinant RanBP22532-2767
fragment (provided by Ellis Jaffray, Figure 16b), which contains the SUMO E3
ligases catalytic domain or members of the PIAS family (provided by Ellis
Jaffray) were titrated as indicated (Figure 25), with either SUMO-1 or SUMO-2
(KIIR) recombinant proteins as the modifiers. Enhancement of SUMO
modification of the N terminus of p300 was not observed with any of the
concentrations of RanBP225322767
or PIAS proteins, with either SUMO-1 or
SUMO-2 (KIIR) (Figure 25). As a positive control, to show the assay was
functional SplOO was used as a substrate to show the effects of an E3 ligase on
substrate modification. SUMO modification of SplOO has been reported to be
enhanced by the RanBP2 E3 (Pichler et al., 2002). Modification of SplOO by
SUMO-2 (KIIR) was enhanced by RanBP22532
2767, although at higher E3
concentrations there was an inhibitory effect. Modification of Sp100 by SUMO-1
was also observed, albeit to a lesser extent than SUMO-2 (KIIR), and the same
inhibitory effect was noticed at high E3 concentrations (Figure 25). A caveat to
these experiments was the possibility that full length p300 may be required for an
interaction with an E3, and thus function. Although as yet no SUMO E3 ligases
have been shown to bind directly to their substrates
128

Ubc9(ng) 05 10 15 20 25 50 100 200 375
SUMO- I
SUMO-2 (KIIR)
z WW son Awa W= olm sI
VMw
I
-7
]+ SUMOs
4 p3001-1255
J+ SUMOs
4 p3001-i,; 5
Figure 24. Titration of Ubc9 into SUMO conjugation assay. The 35S_
labeled
p300, _, 255 was incubated in the assay for SUMO conjugation with increasing
amounts of Ubc9, to determine the minimal amount of Ubc9 required for
SUMO conjugation. Assays were carried out in the presence of SUMO-1
and SUMO-2 (K 11 R) as indicated. Reaction products were fractionated by
SDS-PAGE before the gels were dried and analysed by phosphorimaging.
The positions of the free and conjugated forms of p300, _, 255 are indicated.

(i)
(iii)
, ý.. - .ý
SUMO-1
RanBP2,,,,,
, r, w+ . "A" -ý -'ý+ --~ wý+ý" tai
PIAS I
,, ý,, l. º
- +1 SUMO
' Sp l 00
(ii)
SUMO-2(KIIR)
RanBP2,,,,,
767
p3001-1255 00 sw +.....,, .. º ývr ,sw. r. -... ,. ", "., r, 'ww
(x)
RanBP225I'1767
mWI! --
Figure 25. RanBP2 and the PIAS family; PIASI, y, and xa, have all been reported as
SUMO E3's. Neither RanBP2 nor the PIAS family of SUMO E3 ligases have ligase
activity towards p300 modification in vitro. SUMO modification assays were set up,
with increasing amounts of E3 ligase present. (i-iv)RanBP2 and PIAS I were added at 0,
1,2,4,8,16,25,50, and 100ng, whilst (v-viii) PIASy and PIASxa were added at 0,2,4,
6,25,50,100ng. (ix, x) 35S-labeled SplOO was included as a positive control, RanBP2 is
known to enhance its SUMO modification, showed the reported preference
for SUMO-2
modification in the presence of RanBP2. At high concentrations of RanBP2, SUMO
modification of Sp l00 was inhibited. Reaction products were fractionated by SDS-
PAGE before the gels were dried and analysed by phosphorimaging. The positions of
the free and conjugated forms of p300i_i255 and Sp100 are indicated.

3.2.4 Identification of the lysine residues, within the CRD1
domain of p300, that are conjugated to by SUMO
Modification by SUMO usually occurs at the lysine residue contained within the
iKxE conserved motif, requiring both a large hydrophobic and glutamic acid for
efficient conjugation (Rodriguez et al, 2001). To investigate the sequence
requirement for SUMO modification of p300, p300 sequences 1017-1029,
comprising the CRD1 repression domain, were fused to GST and produced as
recombinant protein, and sequenced to check for correct sequences (Figure 16b).
The purity of the GST-CRD1 proteins was determined by SDS-PAGE analysis
following staining with Coomassie brilliant blue R250 and subsequent de-
staining (Fig. 26. see Appendix). The purified GST-CRD1 was incubated with the
required components of the SUMO conjugation assay, containing "I-labelled
SUMO-1. Under these experimental conditions, two species of GST-p300
mCRD1 were generated that corresponded to single and
double SUMO-1
modified forms. The conjugation was particularly efficient at low concentrations
of GST-p300 CRD 1 substrate (Figure 25. iii). To determine the sites at which
p300 is modified by SUMO, alanine scanning mutagenesis of the 1017-1029
region was performed in the context of the GST-p300 CRD1 fusion protein.
CRD1 mutants were created by the hybridisation of oligonucleotides containing
the appropriate mutations to encode an alanine residue, as indicated (Fig. 26. i. ).
Annealed oligonucleotides were subsequently cloned into a modified GST
expression vector, and transformed into the E. coli strain B834. Proteins were
expressed and purified by GST affinity chromatography. The purity of the
expressed proteins was analysed on SDS-PAGE, following staining with
Coomassie brilliant blue R250 and subsequent de-staining, which indicated that
fusion proteins were not contaminated by any additional proteins (Fig. 26. ii).
These purified proteins were subsequently used as substrates
for SUMO
modification in vitro (Figure 26. iii. ). Mutation of single residues in the iKxE
SUMO modification motif, L1019A, K1020A, E1022A, 11023A, K1024A, and
K1026A abolished the presence of the higher molecular weight band,
representing that of the double SUMO modified form of the CRD 1 peptide. The
131

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mutants were still capable of being modified by a single SUMO protein, as a
higher molecular weight form was still observed (Figure 26. iv. ). A further set of
mutants, changing both the SUMO acceptor lysines to either alanine or arginine
was generated. When used as substrate in the in vitro SUMO conjugations, the
K1020A/K1024A and K1020R/K1024R mutants failed to show any
modification, as indicated by the absence of any higher molecular weight species
(Figure 26. iv. ). The fused SUMO modification sites from PML and adenovirus
E1B55K were also modified in vitro, although the extent of modification to the
tandem motif was less than that of the GST-p300 CRD1 (Figure 26. iv. ).
As part of the collaborative effort with Neil D. Pekins (University of
Dundee) to investigate the SUMO modification of p300, the same alanine
mutants- of the CRD 1 domain, within a Ga14 reporter construct, were used to
investigate the relationship between SUMO conjugation and transcriptional
activity (Fig. 27. ). The results, originally preformed by D. Bumpass and
reproduced with kind permission, indicated that there was a strong correlation
between the ability of SUMO to conjugate to the CRD 1 domain, and its ability to
mediate transcriptional repression in vivo (Table. 3. ).
3.2.5 SUMO modification is required for CRD1 activity in vivo
The construction of the Lys to Ala/Arg mutants of the CRD1 domain
showed that the SUMO modification motif was required for SUMO conjugation,
and that mutations affecting SUMO conjugation also affected p300
transcriptional repression capabilities. To provide direct evidence that SUMO is
required for p300 repressive effect on transcription, and rule out the possibility of
modification by other posttranslational modifiers that act through the CRD 1
domain, further experiments were undertaken. These experiments involved
SUMO-specific proteases, which would remove any SUMO proteins bound to
CRD1 and a dominant negative Ubc9 mutant of the SUMO conjugation pathway,
which would inhibit SUMO conjugation. These were utilised to show the role of
SUMO in p300 mediated repression.
To measure the repressive effect of SUMO modified CRD1, as indicated
through luciferase activity, Gal4-p300 N+, containing the CRD 1 domain or
Ga14-p300 N-, which lacks the CRD1 domain (Figure 16a), were cotransfected
137

into U-2 OS cells along with expression plasmids for SUMO-specific protease
(SSP3/SENP2), catalytically inactive form of the protease C548A SSP3/SENP2,
and catalytically inactive SUMO E2, C93S Ubc9, that acts a dominant negative
mutant, as indicated (Figure 28). The SSP3/SENP2 protease was utilised due to
its availability within the lab, and its nuclear localisation (O. A. Vaughan,
personal communication).
Cotransfections with pcDNA3 empty vector and
Ga14-p300 N-, increased expression by almost 300-fold when compared to Ga14
expression plasmid (Figure 28). In comparison cotransfection with Gal4-p300
N+, which contains CRD1, lead to a much smaller increase of expression by
around 11-fold (Figure 28). Reporter activity is therefore repressed by 24-fold,
when CRD 1 is expressed in the presence of pcDNA3 empty vector.
Cotransfection of a plasmid expressing SSP3/SENP2 with
Ga14-p300 N-, had
little influence on reporter activity, however when SSP3/SENP2 was
coexpressed with Gal4-p300 N+ plasmid, a dramatic increase of reporter activity
was observed (Figure 28). Indeed under these conditions only residual
repressive activity, 1.4-fold, was observed (Figure 28). The relief of repression
was not observed when Ga14-p300 N+ was cotransfected with the plasmid
encoding the catalytically inactive C548A SSP3/SENP2 (Figure 28). Titration of
SSP3 and C548A SSP3/SENP2 revealed that SSP3/SENP2 relieved CRD1-
mediated repression even at very low levels of cotransfected DNA. At these
levels of cotransfected DNA, C548A SSP3/SENP2 did not alter reporter activity,
but higher levels of C548A SSP3/SENP2 do appear to relieve repression. This
may be due to a transdominant effect on the processing of SUMO (Fig. 28. ii. ).
The results clearly demonstrate that SUMO deconjugation by SSP3/SENP2
protease activity relieves p300 CRD1-mediated transcriptional repression.
Further evidence to support SUMO modified CRD1 role in transcriptional
repression came via use of C93S Ubc9, a catalytically inactive form of the
SUMO conjugating enzyme Ubc9, which functions as a dominant-negative
mutant. The dominant negative Ubc9 mutant, lacking the catalytic cysteine
residue (C93S), is not able to accept the transfer of SUMO from SAE1/2 to
Ubc9. The Ubc9 mutant will form an oxy-ester with SUMO, preventing SUMO
transfer and inhibiting SUMO conjugation.
138

(Iý
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4001
300
2 00 1
100
(ii) 20
c
ca
U
fC
a8
ö
16
12
4
Ga14 p300 N+
(1005-1044)
Ga14 p300 N
Ga14
0 0 20 40 60 80
cotransfected DNA (ng)
SP3/SENP2
Figure 2tß. SUMO modification of the CRD domain is required for repression.
(i) SUMO-specific protease 3 (SSP3) relieves p300-mediated repression. Expression
plasmids encoding the indicated Ga14 or Ga14-p30() fusion proteins (511o) were
cotransfected into U-2 OS cells with Gal4 AdMLP luciferase reporter plasmid (2p, g) and
either pcDNA3 or pcDNA expression constructs for SSP3 or C548A SSP3 (50ng).
(ii) Dose response of SSP3 action. An expression plasmid encoding
1044 fusion protein (5ng) was cotransfected into U-2 OS cells with a
Gal4-p300 N+l005-
Ga14 AdMLP
luciferase reporter plasmid (2µg)and increasing amounts of pcDNA3 expression
constructs for either SSP3 or C548A SSP3. Results are displayed as fold activation
compared to transtection with Ga14 lueiferase and the pcDNA3 empty. Results shown in
(i), and (ii) are the means of three separate experiments, and standard deviations are
shown.

The effects of SSP3/SENP2 and C93S Ubc9 on SUMO conjugation to
p300 were directly demonstrated by expressing HA-SUMO-1 and 6His-p300
with either SSP3/SENP2 or C93S Ubc9.6His-tagged p300 was isolated on Ni-
NTA agarose and eluted proteins analysed by Western blotting with antibody
specific to the HA-tag. Analysis of the Western blot indicated that there was no
SUMO conjugation to p300 in the presence of SSP3/SENP2, whilst SUMO
conjugation to p300 was severely reduced by expression of
C93S Ubc9 (Figure
29. i). p21 has been demonstrated to relieve p300 transcriptional repressive
capabilities (Snowden et al., 2000). Thus there was the possibility that p21
expression may result in a decrease of SUMO conjugation to p300. Although
p21 does not directly bind to p300 itself, and functions through an as yet
unidentified adaptor protein(s). p21 expression was shown not influence the
level of SUMO conjugation to the CRD1 domain (Figure 29. ii), showing that
p21's repressional relieving capabilities is not mediated through the removal of
SUMO or direct competition. A control reaction consisted of cotransfecting
alcohol dehydrogenase (ADH), which was cloned into the same expression
vector as p21, RSV (Figure 29. ii).
3.2.6 SUMO-Modified CRD1 recruits a Histone Deacetylase
SUMO modification of the CRD 1 domain could lead to transcriptional
repression by a variety of mechanisms. A common feature of transcriptional
repression in higher eukaryotes is the recruitment of histone deacetylases
(HDACs). To examine the possibility that HDAC activity was
involved in
SUMO-dependent, CRD1-mediated repression, we employed the HDAC
inhibitor Trichostatin A. The addition of Trichostatin A had no effect on reporter
activity mediated by Ga14 alone, it dramatically increased transcription mediated
by a Ga14 fusion protein containing the CRD 1 domain (Figure 30. i. ) These
results were originally preformed by D. Bumpass and reproduced with kind
permission. Trichostatin A had only a marginal effect on transcription mediated
by a Ga14-p300 fusion protein lacking the CRD1 domain (Figure 30. i. ). To
identify the HDAC responsible for the interaction with SUMO modified CRD1
domain, an in vitro binding assay was established. This consisted of GST fusion
140

ýiý
Ni-beads
Anti-HA
extract
Anti-HA
extract
Anti-p300
extract
Anti-SSP3
extract
Anti-Ubc9
Q
Z
da
HA-SUMO-1
6His p30O
(ii)
u N
M
c
-fl
Z.
++
a vD -d
qw
Ni-beads
Anti-HA
extract
HA-SUMO- I
6His p300
Q
Q
4 175
Anti-HA 4 175
extract
Anti-p300 4 175
Figure 29. Control over SUMO conjugation to p300, SSP3 and do Ubc9 prevent
SUMO modification of p300, whilst p2l has no direct control over conjugation. (i)
COST cells were transfected with HA-SUMO-1,6His-p300, and either pcDNA3, SSP3,
or the dominant negative Ubc9, as indicated. Following transfection crude samples
were taken to test for transfection efficiency, whilst the remaining cell lysate were lysed
in 6M Guanadine HCI and purified on Ni-beads. (ii) To investigate whether p21
competed with SUMO for binding to the CRDI domain p21 and control vector ADH,
were co-transfected with HA-SUMO-1 and 6His-p300, and analysed as previously
described.

construct, possessing either the minimal CRD 1 domain, or in case, both SUMO
conjugation sites and the surrounding p300 sequence was required for binding, a
larger construct possessing amino acids 852-1071 were used (Figure 16b). As
such recombinant GST fusion constructs, containing either an extended 220
amino acid fragment of p300, that includes the CRD1 domain (residues 852-
1071) or the CRD1 domain (residues 1018-1027) were used as substrates in
SUMO conjugation assays (Figure 16b). Following incubation with individual
HDACs, generated by in vitro transcription/ translation in the presence of 3SS-
Met, GST fusion proteins were recovered on glutathione agarose and their
binding capacity for individual HDACs was determined. Both SUMO modified
p300 fragments (p3008521071
and p3001005-1o) bound to HDAC6, whereas
unmodified fragments and that of GST were unable to bind to HDAC6 (Figure
29. ii. ). The interaction was specific to HDAC6 as no other HDAC was seen to
interact with the SUMO modified fragments (Figure 30. ii. ).
To establish the importance of HDAC6 in the regulation of p300 activity,
endogenous HDAC6 expression was ablated using siRNA. Plasmids expressing
HDAC6-specific siRNA or a scrambled siRNA were cotransfected with Gal4-
p300 N- or Gal4-p300 N+ (containing CRD1) and a Ga14 luciferase reporter
plasmid. Gal4-p300 N-, which cannot be SUMO modified, drives high levels of
reporter activity which are unaffected by plasmids expressing siRNA to HDAC6.
In contrast, transcriptional repression mediated by Gal4-p300 N+CRD1 is
relieved by siRNA to HDAC6 but not by the control scrambled siRNA (Figure
30. iii. ) These experiments were originally preformed by D. Bumpass, and are
reproduced with kind permision.
142

Figure 30. SUMO-mediated p300 repression is mediated by a Histone Deacetylase
(i)Trichostatin A alleviates CRDl-dependent p300 repression. HEK 293 cells were
transfected with 5 µg Ga14 E1B CAT reporter together with 10 ng of Ga14,10 ng of Ga14-
p300 (192-1044), or 0.2 ng of Ga14-p300 (192-1004) expression plasmids. Trichostatin A
was added to the cells after 4 hr and processesed for CAT assay 20 hr later(ii) GST-p300-
CRD1 or GST-p300852.1071
(Figure 16b) were modified by SUMO-1 in vitro and the
modified species recovered on glutathione agarose. 35S-labeled in vitro-translated HDAC4
and HDAC6 were incubated with glutathione agarose bound GST, unmodified GST-p300
proteins, or the SUMO modified GST-p300 proteins as indicated. After extensive washing,
bound proteins were analysed by SDS PAGE and phosphorimaging. 10% of the 35S-labeled
HDAC4 and HDAC6 input was also analysed. (iii) Ga14-p300 N- and Ga14-p300 N+mCRD1
expression plasmids (0.5ng) were cotransfected into U-2 OS cells together with a Ga14-
dependent luciferase reporter (1.5 µg) and 2 µg of either the scrambled siRNA plasmid or a
pool of the three HDAC6 siRNA expression plasmids as indicated. Plotted results presented
are the means of three separate experiments, and standard deviations are shown. The results
shown in Figure. 29i and iii are taken from work carried out by D. Bumpass et al and are
reproduced with kind permission.

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3.2.7 Conclusion
Through the generation of p300 CRD 1 mutants the minimal repression
domain within p300 was mapped; residues 1017-1029. Occuring within this
domain are two copies of the iKxE which represent the consensus modification
site for the small ubiquitin-like protein SUMO. Here, we demonstrate that
endogenous p300 is modified by SUMO in vivo and the isolated sequence is
modified by SUMO in vitro in the presence of SUMO-activating enzyme and
Ubc9. We also demonstrate that endogenous CBP is SUMO modified, although
to a lesser extent than p300, although no further characterisation of SUMO
modification of CBP was preformed. Although Best et al, do report a
relocalisation of CBP following overexpression of a SUMO protease (Best et al.,
2002). Mutagenesis reveals a strict correlation between SUMO modification and
repression (Figure 25 and 26). Each SUMO modification motif appears to
function independently, and both motifs are required for full repression activity
(Figure, 26). The CRD 1 SUMO modification sequence is also present in the
equivalent repression domain of CBP (Snowden et al., 2000), is highly
conserved in human, mouse, and Xenopus versions of the proteins, and is also
found in Drosophila CBP. Not only is the double SUMO modification motif
conserved, but its location, just to the amino terminal side of the bromodomain,
is also conserved.
SUMO is conjugated to a numerous cellular substrates via an enzymatic
pathway, analogous to, but distinct from that of ubiquitin conjugation (Hay,
2001; Pichler et al., 2002; VanDemark and Hill, 2002) that results in the transfer
of SUMO from Ubc9 to the target protein. Substrate recognition is mediated
through acceptor lysine residues on target proteins within the consensus motif
ipKxE, and structural analysis has indicated that this sequence is contacted by
Ubc9 (Bernier-Villamor et al., 2002; Lin et al., 2002). Thus, the sequence
LKTEIKEE in p300 is likely to be directly recognised by Ubc9. Recently, it has
been shown that selection of target proteins for modification by SUMO is aided
by a number of proteins that appear to function in a fashion that is similar to the
specificity-determining E3s in the ubiqüitin system (Johnson and Gupta, 2001;
Kahyo et al., 2001; Takahashi et al., 2001; Kagey et al., 2003). We could not
145

show any E3-ligase activity mediated through members of the PIAS family or
RanBP2 towards SUMO modification of the N-terminal half of p300. This does
not rule out E3-ligase activity towards the SUMO modification of
full length
p300. A more direct role for SUMO modification is evident in the case of c-Myb
and SP3, in which SUMO modification of a negative regulatory domain mediates
transcriptional repression (Bies et al., 2002; Ross et al. 2002; Sapetsching et al.,
2002).
p300 transcriptional repression is relieved by Trichostatin A in vivo , and
the SUMO-dependent binding of HDAC6 to the CRDI domain suggests that
p300 transcriptional repression is mediated by SUMO-dependent recruitment of
HDAC6 to the CRD 1 of p300. This is supported by the observation that CRD 1-
dependent, p21-mediated relief of repression was blocked by coexpression of
HDAC6 and that siRNA-mediated ablation of endogenous HDAC6 expression
leads to CRD1-dependent derepression (Girdwood et al., 2003). That HDAC6
expression did not result in any further repression of the CRD1 domain when
expressed in the absence of p21, suggests that this domain is maximally
repressed. This is consistent with the very low basal levels of activity seen with
the Gal4-p300 N+CRD 1 vectors which (Figure 16a) display similar levels of
activity to Ga14 alone. Thus, it is likely that SUMO-modified p300 recruits
HDAC6 to a subset of promoters that are susceptible to both p300-mediated
repression and p21 inducibility (Gregory et al., 2002). Deacetylation of core
histones and other transcriptional regulators could then lead to the observed
effects on transcription. Histone deacetylase 1,4, and 6 have all been reported to
be substrates for SUMO modification (David et al., 2002; Kirsh et al., 2002;
Tatham et al., 2001), and while non-modified forms of HDACI and HDAC4
appear to be less active deacetylases, the role of SUMO modification in vivo has
yet to be established. Members of the class II HDACs (-4, -5, -6, and -7) all
appear to undergo rapid nucleo-cytoplasmic shuttling (Khochbin et al., 2001),
and recent evidence suggests that HDAC6 binds ubiquitin through its conserved
C-terminal zinc finger domain (Hook et al., 2002; Seigneurin-Berny et al.,
2001). Thus, regulated nuclear entry of HDAC6 coupled with SUMO
modification of the CRD 1 domain could serve to regulate the transcriptional
output of p300.
146

p300-mediated repression activity in vivo is relieved by SSP3 (Figure 27
and 28), a SUMO-specific protease also known as SENP2, SMT3IP2, or Axam
(Hang and Dasso, 2002; Kadoya et at., 2002; Nishida et al., 2001) which
removes SUMO from modified substrates. While this directly demonstrates that
p300 repression is mediated by SUMO and not some other ubiquitin-like protein,
it also suggests a mechanism for control of p300 repression activity. Modulation
of SSP3/SENP2 activity or subcellular localisation by signalling molecules could
allow the protease to deconjugate SUMO modified p300 and thus relieve
repression in response to various signals. It has recently been demonstrated that
Axam/SMT3IP2/SENP2/SSP3 can activate transcription in a PML-dependent
fashion (Best et al., 2002). In addition to transcriptional repression, SUMO
modification also appears to have additional diverse effects. These include
antagonism of ubiquitination, alteration of subcellular localisation, and
transcriptional activation. It is likely that the diverse biological effects of SUMO
modification, including the repression of p300-dependent transcription reported
here, are mediated by the ability of SUMO-modified proteins to selectively
recruit new protein partners that alter their properties.
147

3.3 Transcriptional repression is preferentially mediated
through SUMO-2 and SUMO-3
siRNA technology provides a useful mechanism of decreasing if not
completely ablating the levels of the targeted gene, through the endonuclolytic
cleavage of the target mRNA (as detailed in material and methods). To
investigate the potential for distinct roles of SUMO-1. and SUMO-2/3 as
transcriptional regulators, siRNA has been used to ablate expression of the
different SUMO isoforms individually. Using three previously identified
substrates repressed by SUMO modification it was demonstrated that ablation of
SUMO-2/3 substantially derepressed transcriptional activity of p300, Sp3, and
Elk-1. Ablation of SUMO-1 results in only a modest derepression of
transcription.
The conjugation of all three SUMO species to p300 occurs with equal
efficiency in the presence of El and E2 in vitro, and all three conjugate to the
same iKxE SUMO conjugation motif (Figure 23). Despite the sub-groupings of
the SUMO isoforms, there have been few reported differences in the functional
consequences regarding modification by SUMO-1 or SUMO-2/-3. There are
large pools of free SUMO-2/-3 in cells and under certain stress situations the
conjugation of SUMO-2/-3 (Saitoh and Hinchey, 2000) to substrates can be
induced, although hypoxia has been demonstrated to increase the levels of
SUMO-1 mRNA, suggesting that stress responses may not be restricted to
SUMO-2/-3 alone. Indeed SUMO-1 has been shown to modify NEMO
following genotoxic stress and result in its translocation to the nucleus (Huang et
al., 2003).
148

3.3.1 Ablation of SUMO-1 and SUMO-2/-3 expression following
treatment with synthetic siRNA oligonucleotides
Initially synthetic oligonucleotides were the only siRNA technology
available, although subsequently plasmid siRNA expression constructs were
developed. As such the initial work was preformed with synthetic
oligonucleotides, although later, due to their availability, plasmid based siRNA
technology was utilized. To check the specificity and efficiency of the siRNA
mediated silencing synthetic siRNA oligonucleotides (100nM) were transfected
by calcium phosphate as described in material and methods, and SUMO levels
were determined by both Western blotting and immunofluorescence. Western
blot analysis demonstrated that SUMO-1 levels were reduced following
transfection of SUMO-1 siRNA oligonucleotides, whilst the levels of SUMO-2/-
3 were unaffected. Experiments with siRNA oligonucleotides against SUMO-2
+3 gave the reciprocal results (Figures 31). As shown by immunofluorescence,
U2-OS cells transfected with the random sequence (RSC) oligonucleotide, cells
showed the predicted distribution with all three SUMO isoforms being
predominatly nuclear in distribution, and could be observed in discrete nuclear
foci (PML bodies). SUMO-1 levels were observed to be lower than those of
SUMO-2/-3 as determined by Western blot analysis. Following treatment of U2-
OS with the SUMO-1 specific siRNA oligonucleotide, the reduction in the levels
of SUMO-1 staining was observed, and similarly following treatment with
SUMO-2 and SUMO-3 oligonucleotides, a reduction in the amounts of SUMO-
20 was noticed (Figure 32).
3.3.2 Transcriptional repression is relieved preferentially
through shRNA targeting of SUMO-2/-3
To identify whether there are any functional differences between
conjugation by SUMO-1 and that of SUMO-2/-3, in their transcriptional
repression roles, pSUPER retro constructs (Oligoengine) were constructed as per
manufacturers instructions, for each SUMO isoform (as described in material and
149

1.
11.
Anti-SUMO-1 Anti-SUMO-2/-3
RSC S1 S2+S3 siRNA RSC Sl S2+S3
_
1Conjugated
SUMO
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Actin -º
Amount of SUMO-1 Amount of SUMO-2/3
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60
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Figure 31. i. U2-OS cells were transfected with siRNA targeting SUMO-1 or SUMO-2 and
SUMO-3, or control siRNA respectively and left for 48 hours, before whole cell extracts
were run on 10% SDS-PAGE gels and subjected to Western blotting with either antibodies
against SUMO-1 or SUMO-3, which also recognizes SUMO-2. The knocking down of
SUMO-1 mRNA was demonstrated not to affect levels of SUMO-2 or SUMO-3, and this
was demonstrated to be reciprocal. ii. The Western blots were subjected to quantification
via MacBas software and the relative amounts of the proteins, as a percentage of the control
were determined.

Figure 32. Knockdown of endogenous SUMO levels through siRNA mediated
silencing. Endogenous SUMO shows a predominantly nuclear localisation, and
accumulates into punctate nuclear dot like structures. Transfection RSC
oligonucleotides has no effect on the levels of either SUMO-1 or SUMO-2/3.
Transfection with the oligonucleotide targeting SUMO-1 specifically knockdown the
levels of SUMO-1 and does not affect the levels of SUMO-2/3. Conversly transfection
of oligonucleotides targeting SUMO-2/3 reduced SUMO-2/3 levels and did not affect
SUMO-1 levels. Synthetic siRNA oligonucleotides (100nm) were transfected by
calcium phosphate as described in material and methods. Endogenous SUMO-1 was
detected with anti-GMPI (Zymed) antibody with Texas Red as secondary, whilst
SUMO-2/-3 was detected with an anti-Sentrin-2 antibody (Zymed) with FITC as the
secondary, as indicated. White text denotes siRNA oligonucleotide transfected.
Fluorescence microscopy was carried out with a DeltaVision microscope (Olympus
IX70) with a 1.40 NA 100X objective and Photometric CH300 CCD camera. lOX 0.2
[um sections were taken. Images were deconvoluted using SoftWorx (Applied
Precision) software.

methods, and Appendix). Co-transfection of the synthetic oligonucleotides with
the appropriate reporter constructs had proved lethal to the U2-OS cells, so the
pSUPER retro plasmids were utilised. To identify differential roles played by
the two SUMO subgroups, these pSUPER retro SUMO-1 or pSUPER retro
SUMO-2 with SUMO-3 vectors were to be co-transfected with a luciferase
reporter construct and Ga14 fusions of known SUMO substrates, whose
modification has been documented to result in transcriptional repression (Figure
16a). The siRNA targeting of the appropriate SUMO or SUMOs RNA, would
result in a reduction in the amount of SUMO available for conjugation and hence
affect a substrates repressional status. Additional experiments were to be
preformed co-transfecting SUMO conjugation deficient versions of the
substrates, to act as a control, to shown that the changes in luciferase activity
were directly related to the SUMO modification status of a substrate, as there
should be no additional affect on luciferase activity. The three SUMO substrates
chosen were; p300, Sp3 (Sapetschnig et al., 2002; Ross et al., 2002), and Elk-1
(Yang et al., 2003) (Figure 16a), and the repression status of each following
siRNA targeting of the different SUMO isoforms was investigated.
, Knockdown of SUMO-2/3 resulted in a substantial relief of p300-SUMO
mediated repression, whilst the knockdown of SUMO-1 resulted in only a
modest increase in transcription (Figure 33. i and ii). The knockdown of SUMO-
2/3 also resulted in an increase in the transcriptional activity of the SUMO
conjugation deficient mutant of p300, N- (Figures 16 and 33. i and ii). The
shRNA mediated targeting of SUMO-2/3 similarly preferentially relieved Elk-1
mediated transcriptional repression, compared to the relief through SUMO-1
knockdown (Figure 34. i and ii). The Elk-1 constructs that were SUMO
conjugation deficient were shown not to be significantly effected by SUMO
knockdown. Co-expression of Ga14-Sp3 with the pSUPER retro SUMO-1 vector
resulted in a minimal relief of repression, whilst similarly to those results
obtained from Elk-l, the co-expression with the pRETRO super plasmids for
SUMO-2/3 resulted in a substantial relief of repression, but only with the SUMO
conjugation competent Sp3 construct (Figure35. i and ii).
To investigate how SUMO deficient constructs, such as p300 N-, were
152

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Effect of SUMO siRNA on p300 mediated repression.
N+ N- & Si N- & S2+S3
Figure 33. Relief of repression is preferentially mediated through the siRNA targeting of
SUMO-2 and SUMO-3.2 µg of'the luciferase reporter construct were translected along with
the indicated plasmid 5no N+/N- (Figure l6a). Additionally transfections were carried out
with either pSUPER retro, empty vector (2 ug), pSUPER retro targeting SUMO-1 (I1. tg) with
empty vector (I «g), or pSUPER retro targeting SUMO-2 (11t,, ) and SUMO-3 (l u") as
indicated. Plasmids were transfected by calcium phosphate into U2-OS cells. Results of the
lucif'erase assays are plotted both as raw R. L. U. readings and as fold activation compared to the
plasmid without shRNA coexpression, and are the means of three separate experiments, and
standard deviations are shown where applicable.

(I)
(ii)
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1200000
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400000
200000
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Effect of SUMO siRNA on Elk-1 mediated repression.
0 F']
elk elk & S1 elk & 52+53 elk k-r
DNA
Effect of SUMO siRNA on ELk-1 Fold Activation
Liii
0 elk & Si elk & S2+S3
DNA
O00
elk k-r k-r & Si k-r & 52+S3
Figure 34. Relief of repression is preferentially mediated through the siRNA targeting of
SUMO-2 and SUMO-3.2 µg of the luciferase reporter construct were transtected along with
the indicated plasmid Elk- I/Elk- I K-R (Figure 16a). Additionally transfections were carried
out with either pSU PER retro, empty vector (2 µg), pSUPER retro targeting SUMO-1 (l p-)
with empty vector (I dug), or pSUPER retro targeting SUMO-2 (l µg) and SUMO-3 (lug) as
indicated. Plasmids were translected by calcium phosphate into U2-OS cells. Results of the
luciferase assays are plotted both as raw R. L. U. readings and as mold activation, compared to
the plasmid without shRNA coexpression, and are the means of three separate experiments, and
standard deviations are shown where applicable.
k-r & Si k-r & S2+S3

C
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20
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Effect of SUMO siRNA on Spa mediated repression.
Sp3
Effect of SUMO siRNA on Sp3 Fold Activation.
EL
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Sp3 & S1 Sp3 & S2+S3
DNA
oQ
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Figure 35. Relief of repression is preferentially mediated through the siRNA targeting of
SUMO-2 and SUMO-3.2 «g of the luciferase reporter construct were transfected along with
the indicated plasnild 200ng Sp3/Sp3 K-R (Figure 16a). Additionally transfect ions were
carried out with either pSUPER retro, empty vector (2 µg), pSUPER retro targeting SUMO-1
(l pg) with empty vector (I pg), orpSUPER retro targeting SUMO-2 (I LIg) and SUMO-3 (lug)
as indicated. Plasmids were transfected by calcium phosphate into U2-OS cells. Results of the
luciferase assays are plotted both as raw R. L. U. readings and as fold activation compared to the
plasmid without shRNA coexpression, and are the means of three separate experiments, and
standard deviations are shown where applicable.
Sp3 K-R

(1) 1
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6000
5000
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3000
2000
1000
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Effect of SUMO siRNA on basal Ga14 activity
Ga14 Ga14 & Si
DNA
Effect of SUMO siRNA on Ga14 fold activation
Ga14 Ga14 & Si
DNA
Ga14 &S2/S3
Ga14 &S2/S3
Figure 36. Ga14 is activated by siRNA targeting of SUMO-2 and SUMO-3.2 it,, of the
luciferase reporter construct were transfected with 5ng of the Ga14 plasmid. Additionally
transfections were carried out with either pSUPER retro, empty vector (2 [tg ), pSUPER retro
targeting SUMO-1 (l. t(T) with empty vector (lug), or pSUPER retro targeting SUMO-2 (I 1-)
and SUMO-3 (11.
(g) as indicated. Plasmids were transfected by calcium phosphate
into U2-OS
cells. Results of the luciferase assays are plotted both as raw R. L. U. readings and as fold
activation compared to the plasmid without shRNA coexpression and are the means of three
separate experiments, and standard deviations are shown where applicable.

eing activated following the siRNA knockdown of SUMO, Ga14 constructs
were transfected with either empty vector as a control, SUMO-1, or SUMO-2
and SUMO-3. pSUPER retro plasmids targeting SUMO-1 resulted in a modest
increase in Ga14 activation whilst those targeting SUMO-2 and SUMO-3 lead to
a more substantial increase in Ga14 activation (Figure 36. i and ii) as observed for
Ga14 p300 N-.
3.3.3 The nuclear localisation of p80 coilin is differentially
regulated by knockdown of SUMO-1 compared to that of SUMO-
2/-3
SUMO modification controls the nuclear localization of numerous
proteins, most notably recruiting many to PML bodies. To investigate the
differential roles potentially played by SUMO-1 and SUMO-2/3 in nuclear
localization of SUMO substrates siRNA targeting of the SUMO was preformed.
The effect of substrate localization following ablation of SUMO-1 or SUMO-2/3
could then be monitored through imunoflouresence.
The Cajal (coiled) body is a discrete nuclear organelle that was first
described in mammalian neurons in 1903. Because the molecular composition,
structure, and function of Cajal bodies were unknown, these enigmatic structures
were largely ignored for most of the last century. This was changed by the
discovery of p80-coilin, which can be used as a protein marker for cajal bodies.
However despite current widespread use of coilin to identify Cajal bodies in
various cell types, its structure and function are still little understood. Previous
work had identified that p80 coilin is SUMO modified in vivo (J. M. P. Desterro,
personal communication). p80 coilin could not be SUMO modified in vitro,
possibly due to the lack of a specific E3-like ligase, and no functional test
existing for p80 coilin, thereby excluding any further work into the biological
significance of this modification at that time.
To further investigate p80 coilin SUMO modification in vivo, U2-OS
cells transfected with the RSC control oligonucleotide showed coilin localized
into either cajal bodies or as nuclear aggregates. Treatment of cells with SUMO-
157

1 siRNA completely removed the appearance of coilin in cajal bodies, whilst
treatment with SUMO-2/-3 siRNA left the cajal body coilin intact but completely
removed the appearance of the aggregates (Figure 37. ii. ). PML localisation in
contrast was not affected by SUMO siRNA, presumably due to the effectiveness
of the knockdown. As SUMO levels were not completely knockout, PML,
which is efficiently modified by SUMO may out compete many of the numerous
SUMO substrates for the available SUMO (Figure 37. ii. ).
158

Figure 37. Endogenous PML and p80 coilin both localise to different nuclear dot
formations within the nucleus. The knockdown of SUMO has no effect on PML
localisation, but dramatically influences p80 coilin distribution. Synthetic siRNA
oligonucleotides (100nm) were transfected by calcium phosphate as described in
materials and methods. (i) Endogenous PML was detected with anti-PML antibody,
(ii) endogenous p80 coilin was detected with an anti-coilin antibody with FITC as the
secondary, as indicated by the coloured text. White text denotes siRNA
oligonucleotide transfected. Fluorescence microscopy was carried out with a
DeltaVision microscope (Olympus IX70) with a 1.40 NA 100X objective and
Photometric CH300 CCD camera. IOX 0.2 µm sections were taken. Images were
deconvoluted using SoftWorx (Applied Precision) software.

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3.3.4 Conclusion
In vertebrates the number of SUMO genes increases to three: SUMO-1,
SUMO-2, SUMO-3 and numerous pseudogenes, as compared to lower
eukaryotes. At the protein level the SUMO-2 and SUMO-3 are highly
homologous (Kamitani et al., 1998), being 95% identical to each other following
processing and are regarded as being functionally homologous. SUMO-1
homology is significantly reduced compared to that of
SUMO-2/-3, being
approximately 50% identical following processing. Furthermore SUMO-2/-3,
unlike SUMO-1, are capable of forming polymeric SUMO chains due to the
presence of internal SUMO modification consensus motifs iKxE (where 1 is a
large hydrophobic residue and x can be any amino acid) at their N-terminal
extensions. The consequences of these polymeric chains in vivo have yet to be
established although their existence has been determined. The conjugation of all
three SUMO species occurs with equal efficiency in the presence of El and E2 in
vitro, however in vivo there are examples of substrates that are predominantly
modified by one SUMO paralogue. RanGAP1 is preferentially modified by
SUMO-1 while Left (Sachdev et al., 2001) is preferentially modified
by SUMO-
2. As cells progress through the cell cycle, topoisomerase II, is preferentially
modified by SUMO-2/3 during mitosis (Azuma et al., 2003). It is as yet unclear
how this specificity/control of conjugation is controlled.
Targeting of SUMO-1 or SUMO-2/-3 by synthetic siRNA
oligonucleotides specifically reduces the expression of targeted SUMO species,
and does not result in the decreased levels of the SUMO species not targeted
(Figures 30 and 31). The extent of the reduction of SUMO-1 is greater than that
for the two other SUMO species SUMO-2 and SUMO-3. This result is not
unexpected in that it has been reported previously that, whilst
SUMO-1 has
greater mRNA levels compared to those of SUMO-2 and SUMO-3, there are
greater amounts of SUMO-2 and SUMO-3 at the protein level, than SUMO-1.
Due to the high degree of identity between SUMO-2 and SUMO-3,95%
following processing, the available antibody, raised against SUMO-3 (Sentrin-2)
also recognizes SUMO-2. The reduction in protein levels of SUMO-1 and
SUMO-2/-3 was also characterised by immunofluorescence in U-2 OS cells.
Targeting of SUMO-1 resulted in the disappearance of SUMO-1 punctate dots,
161

and a general reduction in SUMO-1 background staining, in the majority of cells
transfected (Figure 31). The removal of SUMO-1 punctate dots was shown not
to inhibit the formation of SUMO-2/-3 punctate dots, suggesting that SUMO-1 is
not a requirement of SUMO-2/-3 localisation or accumulation (Figure 31).
Furthermore knockdown of SUMO-1 levels did not prevent the formation of
PML bodies (Figure 37), as observed via fluorescence, although it has previously
been noted that SUMO is not required for primary PML body formation, only
secondary PML body formation (Lallemand-Breitenbach et al., 2001). The
knockdown of SUMO-2 and SUMO-3 was also observed in U-2 OS cells,
although this was achieved with a lower degree of efficiency than that of the
SUMO-1 knockdown. The reduction of SUMO-2 and SUMO-3 resulted in the
reduction in SUMO-2/-3 punctate dots, although this was not universal and
presumably depended upon the transfection efficiency. As observed with
SUMO-1, knockdown with SUMO-2 and SUMO-3 did not affect PML
localisation.
Many substrates of SUMO are either transcription factors or cofactors,
and whilst not exclusively, predominantly have been shown to mediate
transcriptional repression, following conjugation by SUMO. Three such
examples are the transcription co-activator p300 (Girdwood et al., 2003), the
ETS domain transcription factor Elk-1 (Yang et al., 2003), and the ubiquitous
transcription factor Sp3 (Sapetschnig et al., 2002; Ross et al., 2002). To further
expedite the characterisation of the roles played by SUMO-1 and those of
SUMO-2/-3 in transcriptional repression, pSUPER retro vectors were
constructed for each of the SUMO species, and were used to investigate their
roles in the repression of theses substrates. As such Ga14 fusions bearing both
SUMO modifiable p300, ELK-1, and Sp3 and mutants either lacking the required
domain or bearing lysine to alanine or arginine point mutations (as previously
described in Girdwood et al., 2003; Yang et al., 2003; Ross et al., 2002; Figure
16a) were co-transfected in the presence of empty pSUPER retro plasmid,
pSUPER retro SUMO-1, pSUPER retro SUMO-2, or pSUPER retro SUMO-3 as
indicated (Figures 33,34, and 35). The co-transfection of SUMO modifiable
substrate with the pSUPER retro SUMO-1 plasmid resulted in a modest increase
in transcription when compared to those co-transfected with only empty vector.
162

This result was repeated when the pSUPER retro SUMO-1 plasmid was co-
transfected with the SUMO modification deficient version of the Ga14 fusion
substrates. The co-transfection assays were repeated, with pSUPER retro
plasmids targeting SUMO-2 and SUMO-3, a substantial relief of repression was
observed (Figures 33,34, and 35), although it was also observed with the SUMO
modification deficient version of the Ga14 fusion substrates (Figures 32,33, and
34). As these substrates were not being modified by SUMO, the observed
increase in luciferase readings is likely due to an activation event rather than a
relief of repression. In support of this theory, when the pSUPER retro plasmids
were co-transfected with Ga14 a similar pattern of activation was noticed,
indicating that SUMO may be having a repressive effect at the promoter level of
transcription (Figure 36). Whilst this work was in progress Holmstrom et al.,
reported that SUMO-2 and SUMO-3 Ga14 fusions had a greater capacity to
repress Ga14 activity than SUMO-1, although this was only seen to a modest
degree (Holmstrom et al., 2003). Interestingly p80 coilin's nuclear localisation
was affected differently depending on whether SUMO-1 or
SUMO-2/-3 levels
were being reduced. SUMO-1 knockdown prevented the localisation of p80
coilin to cajal bodies, and instead resulted in nuclear aggregation. The
knockdown of SUMO-2/-3 had no effect on coilin cajal body localisation, and
indeed reduced the levels of the nuclear aggregates, and further suggests that
SUMO-1 and SUMO-2/-3 are involved in differential roles, outside that of
transcriptional repression.
163

A
4. DISCUSSION
164

4.1 Discussion
SUMO-1/-2/-3 are key members of the growing family of protein
modifiers, that covalently modify selected cellular substrates. The SUMO
conjugation pathway proceeds through an enzymatic cascade that is analogous,
though distinct from that of ubiquitin. These differences between the
conjugation pathways of ubiquitin and SUMO are not at the mechanistic level;
rather each pathway possessses distinct enzymes for their pathways.
While both
ubiquitin and SUMO conjugation pathways possess a sole El enzyme, the
ubiquitin pathway contains around ten E2s whereas there appears to be only a
single SUMO conjugating enzyme. There are reportedly around seven hundred
ubiquitin E3 enzymes, 'whilst only six individual E3-like enzymes have been
reported for the SUMO conjugation pathway to date. Prior to conjugation
SUMO-1/-2/-3 are processed via actions of cysteine proteases to remove an
inhibitory C-terminal tag, and exposes the characteristic di-glycine motif, and
again distinct from that of the ubiquitin the same class of protease is capable of
both the initial processing and the deconjugation of SUMO from the target
substrates. The conjugation of SUMO to a substrate usually requires the
presence of a SUMO consensus motif, although there are a growing number of
substrates that lack this consensus motif, and potentially, only through use of
first principal proteomic approaches will the comprehensive list of SUMO
substrates be identified. A large number of previously characterised repression
domains, contain SUMO modification motifs, and have subsequently been
shown to be SUMO substrates (Figure 38).
The ability of SUMO to recruit HDAC6 following its conjugation to p300
is the first proven example of the long held theory in the SUMO field, that the
conjugation of SUMO provides a novel interaction surface, one capable of
recruiting additional proteins. SUMO has been shown to enhance the
recruitment of target proteins into repressive domains, such as PML bodies. This
newly identified second mechanism for SUMO to mediate repression would
appear to be due to the ability of SUMO to impart repressive properties upon
target proteins. Following the identification of SUMO dependent recruitment of
HDAC6 to p300, a further example of SUMO dependent HDAC recruitment has
been identified. SUMO mediated repression of the Elk-1 transcription factor had
165

(i) Modification of histone deacetylases.
Catalytic domain
(ii) SUMO dependent repression.
HLH
Catalytic domain
Gln Gln C2H2
ADI ADII bZIP
ETS BRDC
C/HI CRD BROMO C/H2 C/H3
DBD LZ
hHDAC1
hHDAC4
h c-Myb
hSp3
hC/EBPa
r//, 1111A h c-Jun
hELK
hp300
KAKRVKTEDEKEK45'
EAKGVKEEVKLA482
AGVGVKGEPIESD566
LLKKIKQEVESPT530
AIDRIKEEEPDFE546
RPLVIKQEPREED166
RLQALKKEPQTVP233
EAPNLKSEELNV136
LPPEVKVEGPKE256
TELKTEIKEEEDQ'°29
Figure 38. SUMO conjugation and transcription repression. SUMO conjugation
has been
mapped to a number of previously identified repression domains, of transcription factors and
co-factors.
Solid vertical bars indicate SUMO conjugation motifs, horizontal lines indicate previously
identified repression domains. HLH, helix-loop-helix; Gln, rich in glutamine; AD, activation
domain; bZIP, basic zipper; LZ, leucine zipper C/H, cysteine-histide rich domains; Bromo,
bromodomain; ETS, B, R, D, and C represent designated regions of ELK-l; C/EBP,
CCAAT/enhancer-binding protein. h, human.

previously been attributed to SUMO conjugation to R domain of Elk-1, a domain
that contains two SUMO conjugation sites, and subsequently the repression was
shown to be by SUMO recruitment of HDAC2 (Yang and Sharrocks, 2004).
Thus the recruitment of HDACs, by SUMO-modified proteins would lead to the
alteration in the local balance between acetylases and deacetylases, increasing
the preference for the deacetylation of chromatin, associated with states of
transcriptional repression. Sharrocks and colleagues further identified that
HDAC3 also has a role in the repression of p300, suggesting that p300 may
recruit both HDACs (Yang and Sharrocks, 2004). That p300 and Elk-1 are able
to recruit different HDACs following their modification by SUMO demonstrates
that the substrate of the SUMO modification also plays a key role in determining
the specificity of HDAC recruited, presumably by the overall tertiary fold of the
substrate. The importance of the substrate in HDAC recruitment was further
demonstrated by the observation that GAL-SUMO fusion proteins activity are
not affected by the siRNA targeting of the HDACs, demonstrating that
unconjugated SUMO is unable to recruit HDACs to the promoters. Furthermore
the ability of the protease to control SUMO mediated transcription repression
also provides strong evidence that unconjugated SUMO is not able to mediate
transcriptional repression. The exact mechanisms as to how SUMO directs these
different repressive functions still remains to be elucidated. Further questions
remaining to be resolved include the mechanisms by which SUMO-2/-3
preferentially mediate transcriptional repression, although this could be
explained by the relative abundance of the SUMO-2/-3 isoforms compared to
that of SUMO-1, and that a significant proportion of SUMO-1 is conjugated to
RanGAP1, thereby limiting its availability further.
A common feature of all ubl-proteins that have been functionally
characterised to date, with the exception of Atgl2, is that only a small proportion
of a substrate is modified at any one time. Although only relatively low levels of
SUMO-modified substrates are observed, there can be dramatic consequences of
SUMO conjugation/deconjugation on the transcriptional activities of a number of
transcription factors, including those of p300, Elk-1, and Sp3. Thus far few
theories have been put forward to try and explain this apparent paradox. One
hypothesis on this matter, (Girdwood et al, 2004) draws on analogies from a
167

wide range of biological systems, in which specific proteins are required for- the
assembly, but not maintenance of multiprotein complexes. The model for
SUMO proposes that modification of a substrate may result in the recruitment of
chaperonins that assemble the modified protein into a stable complex. Once
formed, the chaperonins can dissociate and SUMO can be deconjugated leaving
the once modified protein locked into the repressed state. A good example of
this, state A to state B transition comes from the field of DNA replication. In
DNA replication, helicase loaders assemble monomeric helicase units into the
ring shaped hexameric replicative helicases that encircle the naked
DNA. The
loading of the monomeric helicase units by the helicase loaders is ATP
dependent, but once loaded they remain loaded following the hydrolysis of ATP.
Applying this model to that of SUMO conjugation and transcriptional repression,
Hay proposes that following the ATP dependent SUMO conjugation to a
substrate there follows a rapid SUMO-dependent incorporation into a repression
complex, a complex that will remain intact following the deconjugation of
SUMO, and that will remain active in transcriptional repression (Girdwood et al,
2004). The eventual break up of the complex and hence loss of transcriptional
repression will be a more gradual event, far slower than the incorporation, and
thus explain why despite the low levels of SUMO conjugated substrates are
observed, the consequences are biologically significant (Figure 39) (Girdwood et
al, 2004). Following the transition from active to repressed state, there must be
mechanisms to revert this state and relieve repression. In principle
transcriptional activation could be accomplished by signal-induced modification
of the repressed transcription factor, such as its phosphorylation or acetylation,
which in turn mediates disassembly of the complex and the release of the active
transcription factor. Variation of the activation pathway can be envisaged in
which signal-induced modification of the transcription could lead to enhanced
SUMO deconjugation and dissociation of the complex. Signal-induced relief of
repression is evident in the case of Elk-1 where signalling through the MAP
kinase pathway results in phosphorylation of Elk-1 with concomitant loss of
SUMO modification and activation of Elk-l-dependent transcription (Yang et al,
2003). In this scenario Elk-1 phosphorylation could have multiple consequences,
and could act to recruit SUMO-specific proteases and induce conformational
168

Active state
INIX NF-X
SUMOylatio
FAST
1NIF-X
i
(U-M0
SUMO-dependent
Incorporation into
Repression comply
(FAST)
i Release from
Repression comple
ý
ý (SLOW)
i
i
Repressed state
Figure 39. Model for transcriptional repression by SUMO. See text for details.
Figure courtesy of Ron Hay.
SUMO-independent'
Retention in
repression comple

changes in Elk-1 that allow release from the "repression complex".. In the case
of p300, expression of the cyclin-dependent kinase inhibitor p21 relieves p300-
dependent repression without affecting the level of SUMO-modified p300
(Figure 28). This could be explained if p21 simply induced the disassembly of
the repression complex containing predominantly unmodified p300. Indeed this
hypothetical model can be directly applied to other SUMO dependent events,
such as NEMOs recruitment to the ATM scaffold, and subsequent release
following ubiquitination (Huang et al, 2003; Hay, 2004).
v
Due to the many similarities shared between the various ubl-proteins,
research into SUMO is likely to provide insights into the functioning of the other
ubl-proteins, with the techniques developed to study SUMO being directly
applied to the other members of the superfamily of polypeptide modifiers. This
research into the field of SUMO will undoubtedly identify novel substrates for
SUMO modification, and likely provide further examples of the importance of
SUMO modification in the regulation of transcriptional activity, protein stability,
sub-nuclear domain organisation, and competition amongst the other post-
translational modifiers for lysine residues. Indeed a proteomic approach to
identify ubiquitinated substrate was recently undertaken in S. cerevisiae. Four of
the identified peptides contained an ubiquitin conjugating lysine residue, in a
classic SUMO conjugation motif (Table 4) (Peng et al, 2003). Two peptides
came from uncharacterised proteins, YGR268C and YOL109W. The remaining
two were identified in an inositol transporter protein, ITR1, and interestingly the
fourth belonged to UIP3, an Ulpl interacting protein. Although research into the
SUMO field of post-translation modification has answered many of the initial
questions regarding its function and similarity to ubiquitin, both in conjugation
and regulation. Future research will undoubtedly answer many more questions
and help determine the physiological role of SUMO and the role played by
SUMO in the development of diverse processes associated with cancer,
neurodegenerative conditions and viral infections.
170

Protein Peptide sequence
YGR268C DTHDDELPSYEDVIKEEER
YOL109W EQAEASIDNLKNEATPEAEQVK
ITR 1 VHELKYEPTQEIIDI
UIP3 MQTPSENTDVKLDTLDEPSAH
Table 4. Proteins with identified ubiquitination sites from S. cerevisiae. Table
showing the identified ubiquitin conjugated lysine residues (in bold and larger size)
and potential SUMO modification sites (underlined).

5. Appendix I Vector Maps
172

Cloning primers:
Pvul
NEOr
B
AMPr P CMV
pcDNA3 Cullin-2
7683bp
B. GH
40 on
amHl
Cu12
Tthl11I vpp- 1%
EcoRl
Smal
Upstream (BamHl): 5'-C GAA GGA TCC ATG TCT TTG AAA CCA AGA GTA GTA G-3'
Downstream (EcoRl): 5'-T CAG GAA TTC ACG CGA CGT AGC TGT ATT C-3'
CUL2:
Subunit of the pVHL ubiquitin E3 ligase complex
Organism: Homo sapiens
Coding region: 2283bp
GenBank accession number: NP_003582
Notes:
Modified by the ubl-protein NEDD8.
173

Bss
Mlu 1
Oligos annealed:
Upper (BamHl): 5'-GA TCC TCT AGA CCC GGG TCG ACT AGA TCT G-3'
Lower (EcoRl): 5-TT AAC AGA TCT AGT CGA CCC GGG TCT AGA G-3'
Notes:
Introduces Xbal and BgIII cloning sites.
Ban-Hl
Xbal
Bglll
Smal
EcoRl
thIII l
w11
174

Mlu l
Bss
Oligos annealed:
BamH 1
Xbal
Bgll1
Smal
EcoRl
l
ýat11
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA ACT GAA ATA AAA GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TIC CTC CTC TIT TAT TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEIKEEEDQ
SUMO conjugation status:
Two sites, full modification
175

Mlu l
Bss
Oligos annealed:
BamHl
Xbal
Bglll
Smal
EcoRl
t ml
kat11
Upper (Xbal): 5'-CT AGA ACT GCC TTA AAA ACT GAA ATA AAA GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC TTT TAT TTC AGT TTT TAA
GGC AGT T-3'
Amino acid Sequence:
TALKTEIKEEEDQ
SUMO conjugation status:
Two sites, full modification
176

Mlu l
Bss
Oligos annealed:
BamHl
Xbal
Bg111
Smal
EcoRl
LhIHI
Upper (Xbal): 5'-CT AGA ACT GAG GCC AAA ACT GAA ATA AAA GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC TTT TAT TTC AGT TTT GGC
CTC AGT T-3'
Amino acid Sequence:
TEAKTEIKEEEDQ
SUMO conjugation status:
One site, partial modification
w11
177

Mlul
Bss
Oligos annealed:
BamHl
Xbal
Bglll
Smal
EcoR1
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA ACT GAA ATA AAA GAG GAG GAA GAC
CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC TTT TAT TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEIKEEEDQ
SUMO conjugation status:
Two sites, full modification
thIIII
w11
178

Bss
Mlu 1
Oligos annealed:
BamHi
Xbal
Bgll1
Smal
EcoRl
thIII I
kat11
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA GCC GAA ATA AAA GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC TTT TAT TTC GGC TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKAEIKEEEDQ
SUMO conjugation status:
Two sites, full modification
179

Mlul
Bss
Oligos annealed:
BamHl
Xbal
BgIll
Smal
EcoRl
thM 1
kat11
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA ACT GCC ATA AAA GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC TTT TAT TTC GGC TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTAIKEEEDQ
SUMO conjugation status:
One site, partial modification
180

Mlul
Bss
Oligos annealed:
BamHl
Xbal
BgIll
Smal
EcoRl
thIII 1
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA ACT GAA GCC AAA GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC TTT GGC TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEAKEEEDQ
SUMO conjugation status:
One site, partial modification
Ut11
181

Mlu l
Bss
Oligos annealed:
BamH1
Xbal
Bgll1
Smal
EcoRl
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA ACT GAA ATA GCC GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC GGC TAT TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEIAEEEDQ
SUMO conjugation status:
One site, partial modification
ýl
thil
w11
182

Mlul
Bss
Oligos annealed:
BamHl
Xbal
BgIll
Smal
EcoRl
Upper (Xbal): 5'-CT AGA ACT GAG TTA AAA ACT GAA ATA AAA GCC GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC GGC TTT TAT TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEIKAEEDQ
SUMO conjugation status:
One site, partial modification
sui
w11
183

Mlu l
Bss
Oligos annealed:
BamHl
Xbal
BgIl1
Smal
EcoRl
thIII l
ýat11
Upper (XbaI): 5'-CT AGA ACT GAG TTA AAA ACT GAA ATA AAA GAG GCC GAA
GAC CAG TGA A-3'
Lower (BgIII): 5-GA TCT TCA CTG GTC TTC GGC CTC TTT TAT TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEIKEAEDQ
SUMO conjugation status:
One site, partial modification
184

Miul
ßm
Oligos annealed:
BamH 1
Xbal
Bgl ll
Smal
EcoRl
hm 1
Upper (Xba 1): 5'-Q ACA ACT GAG TTA AAA ACT GAA ATA AAA GAG GAG GCC
GACCAGTGAA-3'
Lowcr (Bg1I
I): 5-GA Jý, I' TCA CTG GTC GGC CTC CTC M TAT TTC AGT TTT TAA
CTC AGT T-3'
Amino acid Sequence:
TELKTEIKEEQDQ
SUMO conjugation status:
Two sites, full modification
, at11
185
ýý
ý,

Mlul
BssHl l
Oligos annealed:
Narl
lacIq
tacp
pGEX2T TEV K1020A/K1024A
ORI
4985bp
GST s
Ampr
BamHl
Xbal
- Bglll
Smal
EcoRl
TthIIIl
Aatl 1
Upper (Xbal): 5'-CT AGA ACT GAG TTA GCC ACT GAA ATA GCC GAG GAG GAA
GAC CAG TGA A-3'
Lower (BgIII): 5-GA TCT TCA CTG GTC TTC CTC CTC GGC TAT TTC AGT GGC TAA
CTC AGT T-3'
Amino acid Sequence:
TELATEIAEEEDQ
SUMO conjugation status:
No sites, no modification
186

Mlu l
BssHll
Oligos annealed:
Narl
lacIq
tacp
GST
pGEX2T TEV K1020R/K1024R
oRI
4985bp
Ampr
BanH l
Xbal
- Bglll
Smal
EcoR l
TthIII 1
Aatl l
Upper (Xbal): 5'-CT AGA ACT GAG TTA AGG ACT GAA ATA AGG GAG GAG GAA
GAC CAG TGA A-3'
Lower (Bg1II): 5-GA TCT TCA CTG GTC TTC CTC CTC CCT TAT TTC AGT CCTTAA
CTC AGT T-3'
Amino acid Sequence:
TELRTEIREEEDQ
SUMO conjugation status:
No sites, no modification
187

Mlul
Bss
Oligos annealed:
BamH1
Xbal
Bgll1
Smal
EcoR 1
Upper (Xbal): 5'-CT AGA CCC AGG GTG ATC AAG ATG GAG GTG AAG AGG GAG-3'
Lower (Bg1II): 5-GA TCT TCA CTC CCT CTT CAC CTC CAT CTT GAT CAC CTT CCT
GGG T-3'
Amino acid Sequence:
PRLVIKMEVKRE
SUMO conjugation status:
Two sites, full modification
aiml
Ut11
188

pUC on
B3pw III
Anip
Empty Vector
1-1 ow
$gill sbtiCI
ý1 7000
pFUP Bi. rrtm
fw/Ptrfier]
7195 bp
%RI PLW
Fpulo
Purchased from Oligoengine.
Sequencing primers:
1000
ýaooý
Prn
Dra III 9ecII BsiWI
SLANT
DgG IAwl
I-h Prams B. u
PGK Age[
s ,i
spulü21
5'-GGAAGCCTTGGCTTTTG-3' binding site: 1241-1257
5'-GATGACGTCAGCGTTCG-3' binding site: 2645-2629
I
! %II
Hindill
Dam HI
I LORI
Not
Ptnel
Nool
ßäi
189

SUMO-1
cl
pUC «i
Armp
_, -'sow
AI BM I
70W
I5
pFUPBi. wtm
/
loon
SUMO-1 2xo--
4ao
BrG ICI
51buflor
HIM ai IET BýII
BIPW III 6aoRI
, pl
CIO
Pur
PGK
Para
BM)
BSI
Dra III CI I g5iwl
siRNA oligonucleotides (upper strand only):
Axel
Bpulü2l
ii
Hinall
Bam Hl
E n'
NotI
Pmd
GATCCCCCTGGGAATGGAGGAAGAAGTTCAAGAGACTTCTTCCTCCATTCCCAGTTT
TTGGAAA
Notes:
Oligonucleotide replaces the stuffer sequence indicated in the plasmid map.
Sequencing primers:
5' -GGAAGCCTTGGCTTTTG-3'
5'-GATGACGTCAGCGTTCG-3'
binding site: 1241-1257
binding site: 2645-2629
rkl
190

SUMO-2
9acl
pJC«i
SgAl Mci
II
70M
SUMO-2 anaaýýý
oaa
BxG ICI
gel l
H r
Hl
6ccRI
1044 Not I
MPrams
Bepw ul 6MAI
$bPI PLU
PGK
Agel
I4
BMnI
Posr11 ßI
Dra III ecl I Bti l
siRNA oligonucleotides (upper strand only):
BPUIID21
\-_-PmeI
GATCCCCGATCAAGAGGCACACGTCGTTCAAGAGACGACGTGTGCCTCTTGATCTTT
TTGGAAA
Notes:
Oligonucleotide replaces the stuffer sequence indicated in the plasmid map.
Sequencing primers:
5'-GGAAGCCTTGGCTTTTG-3'
5'-GATGACGTCAGCGTTCG-3'
binding site: 1241-1257
binding site: 2645-2629
191

SUMO-3
Sgßi BE,
c i
&xGl4acl
siI sell
Hindill
Anip Bam Hl
6caRl
1o Nct I
Pmcl
pUC on
OFUPER. ICtlO Stufk! T
Naal
SUMO-3 B
aaaa
3000
Hl
MIET Bcj11
B6pw III 6aoRl
%pI Pur
PGK
Age[
Ppulo
Posrl
BMm I
gel
pra III %cl NMI
siRNA oligonucleotides (upper strand only):
Bpulü2l
GATCCCCCGACAGGGATTGTCAATGATTCAAGAGATCATTGACAATCCCTGTCGTTT
TTGGAAA.
Notes:
Oligonucleotide replaces the stuffer sequence indicated in the plasmid map.
Sequencing primers:
5' -GG AAGCCTTGGCTTTTG-3' binding site: 1241-1257
5' -GATGACGTCAGCGTTCG-3'
binding site: 2645-2629
192

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7. PUBLICATIONS
Girdwood D, Bumpass D, Vaughan OA, Thain A, Anderson LA, Snowden AW,
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Repression Is Mediated by SUMO modification. Molecular Cell 11,1043-1054.
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SUMO, Chapter 10. Van G. Wilson. (Ed) Sumoylation: Molecular Biology and
Biochemistry.
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