CST Guide:

01
Section I: Research Areas
Gene Expression, Epigenetics,
and Nuclear Function
Chromatin/Epigenetic Regulation
The nucleosome, made up of four histone proteins (H2A, H2B, H3, and H4) and 147 bp of DNA, is
the primary building block of chromatin. In addition to the core histone proteins, a number of histone
variants exist (H2AX, H2AZ, MacroH2A, H3.3, and CENP-A) that confer different structural properties
to nucleosomes and function in DNA repair, chromosome segregation during mitosis, and regulation
of transcription. Histones were originally thought to function as a static scaffold for DNA packaging;
however, they are now known to be dynamic proteins, undergoing multiple types of post-translational
modifications (PTMs) and interacting with regulatory proteins to control gene expression in a highly
regulated manner.
Chromatin Modifying Enzymes
Histone Acetylases and Deacetylases
Protein acetylation plays a crucial role in regulating chromatin structure and transcriptional activity.
Acetylation marks occur on lysine residues and are applied by histone acetyltransferases (HATs) and
removed by histone deacetylases (HDACs). Acetylation within the histone tail weakens histone-DNA
and histone-histone interactions and creates an open chromatin conformation that increases transcription
factor access to DNA, while histone deacetylation limits gene activity by creating a closed
conformation that limits transcription factor binding. In addition, acetylation creates binding sites for
bromodomain-containing chromatin regulatory proteins. For example, the bromodomain-containing
protein 4 (BRD4) is a chromatin-binding protein with a preference for acetyl-lysine 14 on histone H3 as
well as acetyl-lysine 5 and acetyl-lysine 12 on histone H4.
BRD4 binds to acetylated lysine residues within active chromatin regions.
BRD4 (E2A7X) Rabbit mAb #13440: Chromatin IPs were performed with
cross-linked chromatin from 4 x 10 6 MV-4-11 cells and either 10 µl of
#13440 or 2 µl of Normal Rabbit IgG #2729, using SimpleChIP ® Enzymatic
Chromatin IP Kit (Magnetic Beads) #9003. The enriched DNA was quantified
by real-time PCR using SimpleChIP ® Human Bcl-2 Promoter Primers #12924,
human c-Myc intron 1 primers, and SimpleChIP ® Human α Satellite Repeat
Primers #4486. The amount of immunoprecipitated DNA in each sample is
represented as a percent of the total input chromatin.
BRD4 (E2A7X)
Rabbit mAb #13440
Normal Rabbit
IgG #2729
SirT6, a class III histone deacetylase, is
expressed in wild-type but not knock-out MEFs.
SirT6 (D8D12) Rabbit mAb #12486: WB analysis of extracts from SirT6 wild-type
(WT) and knockout (KO) mouse embryonic fibroblasts (MEF) using #12486 (upper)
or β-Actin (D6A8) Rabbit mAb #8457 (lower). SirT6 WT and KO MEF were kindly
provided by Dr. David Lombard, University of Michigan.
% of total input chromatin
1.2
1.0
0.8
0.6
0.4
0.2
0
Bcl-2
Lanes
1. SirT6 WT MEF
2. SirT6 KO MEF
c-Myc
kDa
60
50
40
30
α Satellite
1 2
SirT6
chapter 01: GENE EXPRESSION, EPIGENETICS, AND NUCLEAR FUNCTION
Histone Methyltransferases and Demethylases
The histone methylation state determines active and inactive regions of the genome and is crucial for
proper developmental programming. Methylation marks occur at lysine and arginine residues and are
carried out by histone methyltransferases, including lysine methyltransferases EZH2, G9a, SUV39H1,
and arginine methyltransferases PRMT1, PRMT4/CARM1, and PRMT5. Methylated histone residues
are found within all four core histone proteins and are implicated in both transcriptional activation and
silencing. Methylation facilitates binding of chromatin regulatory proteins (readers) that contain various
methyl-lysine or methyl-arginine binding domains (PHD, chromo, WD40, Tudor, MBT, Ankyrin repeats,
PWWP domains). Methylation marks are removed by demethylases such as Jumonji C domain family
proteins (JARID, JMJD, UTX, UTY, FBXL10, FBXL11) and LSD1.
A
kDa
200
140
100
80
60
50
40
30
20
1
2 3
Ezh2
B
C
1.6
1.4
1.2
1.0
0.8
0.6
0.4
0.2
0
Ezh2 (D2C9) XP ® Rabbit mAb #5246: WB analysis of extracts from MCF7, Neuro-2a, and COS-7 cell lines (A) using #5246. IHC analysis
of paraffin-embedded human lymphoma (B) using #5246. Chromatin IPs were performed with cross-linked chromatin from 4 x 10 6 NCCIT
cells (C) and either 5 µl of #5246 or 2 µl of Normal Rabbit IgG #2729 using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads)
#9003. The enriched DNA was quantified by real-time PCR using SimpleChIP ® Human HoxA1 Intron 1 Primers #7707, SimpleChIP ® Human
HoxA2 Promoter Primers #5517, and SimpleChIP ® Human α Satellite Repeat Primers #4486. The amount of immunoprecipitated DNA in
each sample is represented as a percent of the total input chromatin.
% of total input chromatin
HoxA1
HoxA2
α Satellite
Other Modifications: Phosphorylation,
Ubiquitination, DNA Methylation
Chromatin structure is also modulated through other PTMs such as phosphorylation and ubiquitination
of histone proteins, which affect association with DNA-interacting proteins and have been recently
identified to play a role in coordinating other histone modifications. For example, histone H2B is
ubiquitinated at Lys120, which is associated with the transcribed region of active genes. Ubiquitination
of H2B at Lys120 is essential for subsequent methylation of histone H3 Lys4 and 79, two additional
histone modifications that regulate transcriptional initiation and elongation.
In addition, methylation of DNA at cytosine residues affects chromatin folding by recruiting methyl-DNA
binding proteins (MeCP2, MBD), which recruit additional chromatin modifying complexes and inhibit
DNA binding of methylation-sensitive transcriptional activators. DNA methylation is critical for proper
regulation of gene silencing, genomic imprinting, and development. Two families of mammalian DNA
methyltransferases have been identified, DNMT1 and DNMT3, which play distinct roles in embryonic
stem cells and adult somatic cells. Activity of these enzymes is regulated by accessory proteins such
as DMAP1 and UHRF1, which target the associated DNMT to mediate proper methylation patterns to
newly synthesized DNA during replication. DNA methylation changes are frequently associated with
cancer; general hypomethylation of the genome and localized hypermethylation of CpG islands within
tumor suppressor promoter regions can both be found during various stages of cancer development.
Ezh2, a member of
the Polycomb Group
proteins, is expressed
in various cell lines and
cancers, and associates
with HoxA genes.
Lanes
1. MCF7
2. Neuro-2a
3. COS-7
Ezh2 (D2C9) XP ®
Rabbit mAb #5246
Normal Rabbit
IgG #2729
Chromatin/Epigenetics Resources
Please visit our website for additional resources and products relating to the study of Chromatin/Epigenetics.
www.cellsignal.com/cstchromatin
60
50
40
30
β-Actin
Ubiquityl-Histone H2B (Lys120) (D11) XP ® Rabbit mAb #5546:
Chromatin IPs were performed with cross-linked chromatin from 4 x 10 6
HeLa cells and either 10 μl of #5546 or 2 μl of Normal Rabbit IgG #2729
using SimpleChIP ® Enzymatic Chromatin IP Kit (Magnetic Beads) #9003.
The enriched DNA was quantified by real-time PCR using SimpleChIP ®
Human γ-Actin Promoter Primers #5037, SimpleChIP ® Human γ-Actin Intron
3 Primers #5047, SimpleChIP ® Human GAPDH Promoter Primers #4471,
and SimpleChIP ® Human GAPDH Intron 2 Primers #4478. The amount of
immunoprecipitated DNA in each sample is represented as a percent of the
total input chromatin.
Ubiquityl-Histone H2B (Lys120)
(D11) XP ® Rabbit mAb #5546
Normal Rabbit
IgG #2729
% of total input chromatin
1.2
1.0
0.8
0.6
0.4
0.2
0
γ-Actin
Promoter
γ-Actin
Intron 3
GAPDH
Promoter
GAPDH
Intron 2
Ubiquitination of
histone H2B at Lys120
is associated with the
transcribed region of
active genes.
20 For Research Use Only. Not For Use in Diagnostic Procedures. See pages 302 & 303 for Pathway Diagrams, Application, and Reactivity keys.
www.cellsignal.com/cstchromatin
21