Transcriptional regulation of meiosis in budding yeast
Transcriptional regulation of meiosis in budding yeast
Transcriptional regulation of meiosis in budding yeast
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<strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> <strong>meiosis</strong><br />
<strong>in</strong> budd<strong>in</strong>g <strong>yeast</strong><br />
Yona Kassir 1* , Noam Adir 2 , Elisabeth Boger-Nadjar 3 , Noga Guttmann Raviv 1 , Ifat Rub<strong>in</strong>-<br />
Bejerano 4 , Shira Sagee 1 , and Galit Shenhar 5 .<br />
1. Department <strong>of</strong> Biology, Technion Haifa Israel; 2. Department <strong>of</strong> Chemistry, Technion Haifa<br />
Israel; 3. Food Eng<strong>in</strong>eer<strong>in</strong>g and Biotechnology, Technion Haifa Israel; 4. Whitehead Institute for<br />
Biomedical Research, Cambride USA. 5. Department <strong>of</strong> Molecular Cell Biology at Weizmann.<br />
Institute, Rehovot, Israel.<br />
*correspond<strong>in</strong>g author. Tel: 972-4-8294214, Fax: 972-4-8225153, e-mail:<br />
ykassir@tx.technion.ac.il<br />
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I. Introduction<br />
II. <strong>Transcriptional</strong> cascade governs <strong>in</strong>itiation <strong>of</strong> <strong>meiosis</strong>.<br />
III. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> IME1.<br />
A. The MAT signal<br />
1. Genes whose products transmit the MAT signal to IME1 and <strong>meiosis</strong><br />
1.1. RME1.<br />
1.2. IME4.<br />
1.3. RES1.<br />
B. The nitrogen signal<br />
C. The glucose Signal<br />
1. The cAMP signal-transduction pathway.<br />
1.1. The IREu element.<br />
1.1.1. Msn2 and Msn4.<br />
1.1.2. Sok2.<br />
1.2. Rim15<br />
2. The Snf1 signal-transduction pathway<br />
3. Respiration and the transcription <strong>of</strong> IME1.<br />
D. Additional prote<strong>in</strong>s regulat<strong>in</strong>g the transcription <strong>of</strong> IME1<br />
1. Mck1<br />
2. Tup1/Ssn6<br />
3. Yhp1<br />
4. The Swi/Snf chromat<strong>in</strong> remodel<strong>in</strong>g complex<br />
E. Positive and negative feedback <strong>regulation</strong>.<br />
F. Summary.<br />
IV. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> early <strong>meiosis</strong>-specific genes<br />
A. Silenc<strong>in</strong>g <strong>of</strong> early <strong>meiosis</strong>-specific genes <strong>in</strong> vegetative growth.<br />
1. The WTM genes.<br />
2. The UME2 gene.<br />
3. The UME3 and UME5 genes.<br />
4. The SIN3, UME6, and RPD3 genes.<br />
5. The ISW2 gene.<br />
B. Expression <strong>of</strong> early <strong>meiosis</strong>-specific genes under meiotic conditions.<br />
1. Ume6 is also positive regulator.<br />
2. The function <strong>of</strong> Ime1.<br />
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3. Prote<strong>in</strong>s required for the association <strong>of</strong> Ime1 with Ume6.<br />
3.1. The function <strong>of</strong> Rim11 and its homologs Mck1 and Mrk1.<br />
3.2. The function <strong>of</strong> Rim15.<br />
4. The function <strong>of</strong> Gcn5.<br />
5. The transcription <strong>of</strong> early <strong>meiosis</strong>-specific genes is also regulated by positive<br />
elements.<br />
6. Additional positive regulators.<br />
7. The role <strong>of</strong> premeiotic DNA replication and/or recomb<strong>in</strong>ation <strong>in</strong> controll<strong>in</strong>g early<br />
<strong>meiosis</strong>-specific genes expression.<br />
8. The choice between silenc<strong>in</strong>g and expression <strong>of</strong> early <strong>meiosis</strong>-specific genes.<br />
V. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> middle <strong>meiosis</strong>-specific genes<br />
A. Positive regulators <strong>of</strong> middle <strong>meiosis</strong>-specific genes<br />
1. Ndt80<br />
2. Ime2<br />
3. S<strong>in</strong>3 and Rpd3<br />
B. Negative regulators <strong>of</strong> middle <strong>meiosis</strong>-specific genes<br />
C. The recomb<strong>in</strong>ation checkpo<strong>in</strong>t<br />
VI. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> late <strong>meiosis</strong>-specific genes<br />
A. Early late genes.<br />
B. Mid late genes<br />
C. Late genes<br />
VII. A feedback loop controll<strong>in</strong>g <strong>meiosis</strong>.<br />
VIII. Conclud<strong>in</strong>g remark. The choice between developmental pathways<br />
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Abstract<br />
Initiation <strong>of</strong> <strong>meiosis</strong> <strong>in</strong> Saccharomyces cerevisiae is regulated by mat<strong>in</strong>g type and nutritional<br />
conditions that restrict <strong>meiosis</strong> to diploid cells grown under starvation conditions. Specifically,<br />
<strong>meiosis</strong> occurs <strong>in</strong> MATa/MATα cells shifted to nitrogen depletion media <strong>in</strong> the absence <strong>of</strong> glucose<br />
and the presence <strong>of</strong> a non-fermentable carbon source. These conditions lead to the expression and<br />
activation <strong>of</strong> Ime1, the master regulator <strong>of</strong> <strong>meiosis</strong>. IME1 encodes a transcriptional activator<br />
recruited to promoters <strong>of</strong> early <strong>meiosis</strong>-specific genes by association with the DNA b<strong>in</strong>d<strong>in</strong>g<br />
prote<strong>in</strong>, Ume6. Under vegetative growth conditions these genes are silent due to recruitment <strong>of</strong><br />
the S<strong>in</strong>3/Rpd3 histone deacetylase and Isw2 chromat<strong>in</strong> remodel<strong>in</strong>g complexes by Ume6.<br />
Transcription <strong>of</strong> these meiotic genes occurs follow<strong>in</strong>g histone acetylation by Gcn5. Expression <strong>of</strong><br />
the early genes promote entry <strong>in</strong>to the meiotic cycle, s<strong>in</strong>ce they <strong>in</strong>clude genes required for<br />
premeiotic DNA synthesis, synapsis <strong>of</strong> homologous chromosomes, and meiotic recomb<strong>in</strong>ation.<br />
Two <strong>of</strong> the early <strong>meiosis</strong> specific genes, a transcriptional activator, Ndt80, and a CDK2 homolog,<br />
Ime2, are required for the transcription <strong>of</strong> middle <strong>meiosis</strong>-specific genes that are <strong>in</strong>volved with<br />
nuclear division and spore formation. Spore maturation depends on late genes whose expression<br />
is <strong>in</strong>directly dependent on Ime1, Ime2 and Ndt80. F<strong>in</strong>ally, phosphorylation <strong>of</strong> Ime1 by Ime2,<br />
leads to its degradation, and consequently to shutt<strong>in</strong>g down <strong>of</strong> the meiotic transcriptional cascade.<br />
This Review is focus<strong>in</strong>g on the <strong>regulation</strong> <strong>of</strong> gene expression govern<strong>in</strong>g <strong>in</strong>itiation and progression<br />
through <strong>meiosis</strong>.<br />
Key Word: <strong>meiosis</strong>, Saccharomyces cerevisiae, transcriptional repression and silenc<strong>in</strong>g,<br />
transcriptional activation, glucose, nitrogen, signal transduction pathways.<br />
List <strong>of</strong> abbreviations: ad – activation doma<strong>in</strong>; bd- DNA b<strong>in</strong>d<strong>in</strong>g doma<strong>in</strong>; CDK – cycl<strong>in</strong><br />
dependent k<strong>in</strong>ase; EMG – early <strong>meiosis</strong>-specific genes; id – <strong>in</strong>teraction doma<strong>in</strong>; LMG – late<br />
<strong>meiosis</strong>-specific genes; MMG – middle <strong>meiosis</strong>-specific genes; MSE - middle sporulation<br />
element; MAPK – Mitogen activated k<strong>in</strong>ase; NIS – Nuclear import sequence; NLS - Nuclear<br />
localization sequence; PKA – cAMP-dependent prote<strong>in</strong> k<strong>in</strong>ase H; SA – synthetic growth media<br />
with acetate as the sole carbon source; SCB - Swi4/6 cell cycle box; SD – synthetic growth media<br />
with glucose as the sole carbon source; SPM – sporulation media; STRE-stress response element;<br />
UAS - Upstream activation sequence; UCS - Upstream controll<strong>in</strong>g sequence; URS - Upstream<br />
repression sequence.<br />
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I. Introduction<br />
The budd<strong>in</strong>g <strong>yeast</strong> Saccharomyces cerevisiae is a simple unicellular eukaryote exhibit<strong>in</strong>g several<br />
optional developmental pathways. Fig. 1 schematically illustrates the developmental options <strong>of</strong><br />
MATa/MATα diploid cells. In the presence <strong>of</strong> both carbon and nitrogen sources both haploid and<br />
diploid cells adopt the <strong>yeast</strong> form morphology; upon nitrogen limitation, and <strong>in</strong> the presence <strong>of</strong><br />
high levels <strong>of</strong> glucose, a dimorphic transition to a filamentous growth takes places [For recent<br />
reviews see (Gancedo, 2001; Lengeler et al., 2000; Pan et al., 2000)]. Haploid as well as diploid<br />
cells manifest<strong>in</strong>g these forms propagate by the mitotic cell cycle. Upon nitrogen depletion the<br />
developmental decision made by the cells depends on the presence or absence <strong>of</strong> a fermentable<br />
carbon source such as glucose, as well as on the <strong>in</strong>formation at the MAT locus. In the presence <strong>of</strong><br />
functional MATa1 and MATα2 alleles, regardless <strong>of</strong> ploidy, i.e. MATa/MATα diploids,<br />
MATa/MATα disomic cells, and haploid cells express<strong>in</strong>g both MAT alleles, cells enter the meiotic<br />
cycle (Kassir and Simchen, 1976; Kassir and Simchen, 1985; R<strong>in</strong>e and Herskowitz, 1987; Roman<br />
and Sands, 1953; Roth and Fogel, 1971). In the presence <strong>of</strong> only a s<strong>in</strong>gle functional allele, i.e.<br />
MATa and MATα haploids or MATa/MATa and MATα/MATα diploids, depletion <strong>of</strong> nitrogen<br />
leads to a cell cycle arrest at G1 (Kassir and Simchen, 1976; Roman and Sands, 1953; Strathern et<br />
al., 1981). The last developmental option S. cerevisiae cells possess is that <strong>of</strong> mat<strong>in</strong>g, cells<br />
carry<strong>in</strong>g only one <strong>of</strong> the two MAT alleles can respond to the pheromone secreted by cells<br />
express<strong>in</strong>g the other MAT allele by temporal arrest <strong>in</strong> G1, mate, and then resume cell growth<br />
[reviews on the mat<strong>in</strong>g process as well as on how the MAT alleles control cell type are (Fields,<br />
1990; Herskowitz, 1995; Sprague, 1991)].<br />
In this review we focus on how the meiotic signals, namely, the presence <strong>of</strong> the MATa and<br />
MATα alleles, the presence <strong>of</strong> a non-fermentable carbon source, the absence <strong>of</strong> glucose and<br />
nitrogen, control <strong>in</strong>itiation and progression through the meiotic cycle. We focus on transcriptional<br />
<strong>regulation</strong> rather than the function <strong>of</strong> the prote<strong>in</strong>s <strong>in</strong>volved with the specific meiotic events [for<br />
reviews on these aspects <strong>of</strong> <strong>meiosis</strong> see (Kupiec et al., 1997; Roeder, 1997)].<br />
II. <strong>Transcriptional</strong> cascade governs <strong>in</strong>itiation <strong>of</strong> <strong>meiosis</strong>.<br />
Entry and progression through the meiotic cycle depends on the expression and activity <strong>of</strong> many<br />
genes that can be roughly divided <strong>in</strong>to 3 groups: <strong>meiosis</strong>-specific, cell-division cycle (CDC) and<br />
radiation sensitive (RAD) genes. The <strong>meiosis</strong>-specific genes are expressed only under meiotic<br />
conditions, and their function is required only <strong>in</strong> <strong>meiosis</strong>. The cell-division cycle genes that are<br />
4
<strong>in</strong>volved with the nuclear cell cycle are required for <strong>meiosis</strong> (Simchen, 1974), and are expressed<br />
<strong>in</strong> both the mitotic and meiotic cycles. The RAD genes are <strong>in</strong>volved with DNA repair as well as <strong>in</strong><br />
checkpo<strong>in</strong>t surveillance, and <strong>in</strong> <strong>meiosis</strong> are required for meiotic recomb<strong>in</strong>ation and surveillance<br />
mechanisms.<br />
Initiation and progression through the meiotic cycle is regulated by a transcriptional cascade<br />
consist<strong>in</strong>g <strong>of</strong> a temporal and programmed expression <strong>of</strong> 6 classes <strong>of</strong> <strong>meiosis</strong>-specific genes (early<br />
I, early II, early middle, middle, mid-late, and late) (Chu et al., 1998; Primig et al., 2000). Fig. 2<br />
shows a schematic draw<strong>in</strong>g <strong>of</strong> this transcriptional cascade, the tim<strong>in</strong>g <strong>of</strong> meiotic events, and the<br />
<strong>in</strong>put <strong>of</strong> the meiotic signals. IME1 is the master regulator gene absolutely required for entry <strong>in</strong>to<br />
the meiotic cycle and the transcription <strong>of</strong> all <strong>meiosis</strong>-specific genes (Kassir et al., 1988; Smith<br />
and Mitchell, 1989). Briefly, <strong>in</strong> the mitotic cell cycle IME1 is silent, as its transcription is<br />
regulated by the three meiotic signals (Kassir et al., 1988). The translation and activity <strong>of</strong> Ime1 is<br />
also regulated by nutrients (Rub<strong>in</strong>-Bejerano et al., 1996; Sherman et al., 1993). IME1 encodes a<br />
transcriptional activator that is directly required for the transcription <strong>of</strong> early <strong>meiosis</strong>-specific<br />
genes (EMG) (Mandel et al., 1994; Smith et al., 1993). The transcription <strong>of</strong> the middle genes<br />
(MMG) depends on the transcription factor, Ndt80, and on the ser<strong>in</strong>e/threon<strong>in</strong>e prote<strong>in</strong> k<strong>in</strong>ase,<br />
Ime2, two early <strong>meiosis</strong>-specific genes whose transcription depends on Ime1 (Chu and<br />
Herskowitz, 1998; Hepworth et al., 1998; Mitchell et al., 1990; Yoshida et al., 1990). In addition,<br />
the function <strong>of</strong> Ime2 is directly regulated by nutrients (Donzeau and Bandlow, 1999; Mitchell et<br />
al., 1990; Yoshida et al., 1990). <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> mid-late and late genes (LMG) is<br />
less clear, however, their regulated expression depends on the upstream regulators, Ime1, Ime2,<br />
and Ndt80, as well as on nitrogen depletion (Friesen et al., 1997; Kihara et al., 1991).<br />
There is a good correlation between time <strong>of</strong> transcription and meiotic function (Chu et al.,<br />
1998; Primig et al., 2000). The transcription <strong>of</strong> EMG is <strong>in</strong>duced prior to premeiotic DNA<br />
replication, and it <strong>in</strong>cludes genes required for pair<strong>in</strong>g <strong>of</strong> homologous chromosomes (i.e. HOP1),<br />
and meiotic recomb<strong>in</strong>ation (i.e. DMC1). Furthermore, the transcription <strong>of</strong> CDC genes required for<br />
premeiotic DNA replication is <strong>in</strong>duced at this time (i.e. POL1) (Johnston et al., 1986). Middle<br />
genes are <strong>in</strong>duced follow<strong>in</strong>g completion <strong>of</strong> DNA replication and prior to the first nuclear division.<br />
These genes are <strong>in</strong>volved with nuclear division (i.e. CLB1,3,4, CDC26). The late genes are<br />
expressed follow<strong>in</strong>g completion <strong>of</strong> nuclear divisions, and are <strong>in</strong>volved with spore formation and<br />
its maturation (i.e. DIT1, SPS100) (Chu et al., 1998). However, some genes don’t follow this rule.<br />
For example, CLB5,6 are middle genes (Chu et al., 1998) that are required for premeiotic DNA<br />
replication (Dirick et al., 1998; Stuart and Wittenberg, 1998). In the mitotic cell cycle Clb5 is also<br />
required for sp<strong>in</strong>dle orientation, a process occurr<strong>in</strong>g concomitantly with DNA replication (Segal<br />
5
et al., 2000). On the other hand, <strong>in</strong> the meiotic cycle, separation <strong>of</strong> the duplicated Sp<strong>in</strong>dle-Pole<br />
Bodies and sp<strong>in</strong>dle formation occur follow<strong>in</strong>g completion <strong>of</strong> DNA replication (Goetsch and<br />
Byers, 1982). It seems, therefore, that the transcription <strong>of</strong> CLB5/6 correlates with their second<br />
putative meiotic function.<br />
III. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> IME1.<br />
The different signal pathways that lead to <strong>meiosis</strong> converge at IME1, promot<strong>in</strong>g its transcription<br />
(Kassir et al., 1988). In vegetative growth media with glucose as the sole carbon source (SD -<br />
synthetic dextrose) IME1 is not transcribed. A low basal level <strong>of</strong> IME1 mRNA is present <strong>in</strong><br />
growth media when acetate is the sole carbon source (SA - synthetic acetate) (Kassir et al., 1988).<br />
Upon nitrogen depletion and the presence <strong>of</strong> acetate (SPM - sporulation media), the level <strong>of</strong> IME1<br />
mRNA is transiently <strong>in</strong>creased <strong>in</strong> MATa/MATα diploids, but not <strong>in</strong> cells carry<strong>in</strong>g only a s<strong>in</strong>gle<br />
active MAT allele (Kassir et al., 1988). In addition, IME1 is subject to both positive and negative<br />
feedback <strong>regulation</strong> (Shefer-Vaida et al., 1995). An extremely large region, about 2.1 kb long,<br />
consist<strong>in</strong>g <strong>of</strong> dist<strong>in</strong>ct positive and negative elements mediates the regulated transcription <strong>of</strong> IME1<br />
(Granot et al., 1989; Sagee et al., 1998; Sherman et al., 1993; Smith et al., 1990). By deletion<br />
analysis and <strong>in</strong>sertion <strong>of</strong> specific elements <strong>of</strong> IME1 <strong>in</strong> heterologous reporter genes, the meiotic<br />
signals affect<strong>in</strong>g discrete elements were identified (Fig. 3) (Covitz and Mitchell, 1993; Sagee et<br />
al., 1998; Sherman et al., 1993; Smith et al., 1990). Below is a detailed review on how the MAT,<br />
Glucose, and Nitrogen signals are transmitted to IME1.<br />
A. The MAT signal<br />
The MAT signal is transmitted through two dist<strong>in</strong>ct elements, UCS3 and UCS4, which do not<br />
share any sequence homology (Covitz and Mitchell, 1993; Sagee et al., 1998). Deletion <strong>of</strong> any <strong>of</strong><br />
these elements leads to partial derepression, whereas deletion <strong>of</strong> both elements results <strong>in</strong> full<br />
expression <strong>of</strong> IME1 <strong>in</strong> MATa/MATa cells (Sagee et al., 1998). In addition, UCS3 is apparently<br />
required for complete relief <strong>of</strong> repression <strong>of</strong> UCS4. Nested deletion <strong>of</strong> UCS3 leads to a 2-fold<br />
reduction <strong>in</strong> the expression <strong>of</strong> ime1-lacZ <strong>in</strong> MATa/MATα cells <strong>in</strong>cubated under meiotic<br />
conditions, whereas <strong>in</strong> concomitant deletion <strong>of</strong> UCS4 and the entire upstream region, the<br />
expression <strong>of</strong> ime1-lacZ is complete (Fig. 4, compare l<strong>in</strong>e B to l<strong>in</strong>es A and C). The prote<strong>in</strong>s<br />
through which MAT <strong>regulation</strong> is transmitted to UCS3 are not known. As discussed below, Rme1<br />
transmits the MAT signal to UCS4 (Covitz and Mitchell, 1993) (see section IIIA1.1).<br />
6
Full expression <strong>of</strong> Ime1 <strong>in</strong> S288C stra<strong>in</strong>s deleted for RME1 or UCS3 and UCS4 does not lead<br />
to complete sporulation (Kassir et al., 1988; Sagee et al., 1998), suggest<strong>in</strong>g that the Mata1 Matα2<br />
prote<strong>in</strong>s might be required for a downstream meiotic event. On the other hand, <strong>in</strong> SK1<br />
background, this is not the case, and the MATa1 and MATα2 alleles are required only for the<br />
transcription <strong>of</strong> IME1 (Covitz and Mitchell, 1993). There are many reported cases <strong>of</strong> phenotypic<br />
differences between SK1 and other stra<strong>in</strong>s, i.e. S288C or W303. Genomic DNA hybridization<br />
reveals the presence <strong>of</strong> many deletions and polymorphisms between these stra<strong>in</strong>s (Primig et al.,<br />
2000). Thus, the discrepancy between results is attributed to the absence <strong>of</strong> some functions <strong>in</strong> the<br />
different stra<strong>in</strong>s.<br />
1. Genes whose products transmit the MAT signal to IME1 and <strong>meiosis</strong>. Three<br />
genes are known to transmit the MAT signal to IME1 and <strong>meiosis</strong>: RME1, IME4, and RES1.<br />
1.1. RME1. RME1 encodes a Z<strong>in</strong>c-f<strong>in</strong>ger DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> (Covitz et al., 1991;<br />
Shimizu et al., 1997a) that is a negative regulator for the transcription <strong>of</strong> IME1 and <strong>meiosis</strong> <strong>in</strong><br />
cells carry<strong>in</strong>g only one <strong>of</strong> the MAT alleles (Kassir et al., 1988; Kassir and Simchen, 1976;<br />
Mitchell and Herskowitz, 1986; R<strong>in</strong>e et al., 1981). Recessive mutations <strong>in</strong> RME1 and deletion <strong>of</strong><br />
RME1 lead to complete expression <strong>of</strong> IME1 and sporulation <strong>in</strong> mata1/MATα, MATa/MATa and<br />
MATα/MATα stra<strong>in</strong>s (Kassir et al., 1988; Kassir and Simchen, 1976; Mitchell and Herskowitz,<br />
1986). Haploid cells carry<strong>in</strong>g the rme1-1 mutation complete premeiotic DNA replication, but<br />
arrest as mono nucleate cells without loss <strong>of</strong> viability (Kassir and Simchen, 1976). This is most<br />
probably due to the pachytene checkpo<strong>in</strong>t mechanism that monitors lack <strong>of</strong> synapsis and/or<br />
recomb<strong>in</strong>ation [for review on this checkpo<strong>in</strong>t see (Roeder and Bailis, 2000)]. In MATa/MATα<br />
stra<strong>in</strong>s relief <strong>of</strong> repression is due to the substantial reduction (10 to 20-fold) <strong>in</strong> the level <strong>of</strong> RME1<br />
mRNA and prote<strong>in</strong> [(Mitchell and Herskowitz, 1986), and L. Johnston, personal communication].<br />
The Mata1/Matα2 complex b<strong>in</strong>ds to a specific element <strong>in</strong> the promoter <strong>of</strong> RME1 repress<strong>in</strong>g its<br />
transcription (Goutte and Johnson, 1988; Johnson and Herskowitz, 1985; Li et al., 1995; Stark<br />
and Johnson, 1994).<br />
Rme1 b<strong>in</strong>ds the IME1 5’ region to two sites localized with<strong>in</strong> UCS4, at -2040 to –2030 and –<br />
1959 to –1949 (Shimizu et al., 1998). The consensus sequence for b<strong>in</strong>d<strong>in</strong>g is GWACCTCAARA<br />
(Shimizu et al., 1998). The presence <strong>of</strong> these two b<strong>in</strong>d<strong>in</strong>g sites; def<strong>in</strong>ed as the RRE and the<br />
modulation element (Covitz and Mitchell, 1993), is required to repress the transcription <strong>of</strong> IME1.<br />
Deletion or mutations <strong>in</strong> a s<strong>in</strong>gle site cause only partial derepression (Shimizu et al., 1998).<br />
Moreover, deletion <strong>of</strong> both sites does not lead to complete relief <strong>of</strong> repression, (Shimizu et al.,<br />
1998), probably due to the repression activity <strong>of</strong> the UCS3 site (Sagee et al., 1998). Insertion <strong>of</strong><br />
7
the UCS4 element upstream <strong>of</strong> the CYC1 UAS results <strong>in</strong> 17-fold repression, depend<strong>in</strong>g on overexpression<br />
<strong>of</strong> Rme1, as well as on the presence <strong>of</strong> both the RRE and the modulation sites (Covitz<br />
and Mitchell, 1993). Unlike the <strong>in</strong>tact IME1 gene, repression dependent on over-expression <strong>of</strong><br />
Rme1 and dependence on MAT was not reported. It is not surpris<strong>in</strong>g, therefore, that <strong>in</strong> this<br />
heterologous system repression is accomplished by sequester<strong>in</strong>g the b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> a transcriptional<br />
activator (Shimizu et al., 1997b). The mode by which Rme1 represses transcription <strong>of</strong> IME1 is<br />
not known, however, it repression activity depends on S<strong>in</strong>4 and Rgr1 (Covitz et al., 1994), two<br />
components <strong>of</strong> the RNA polymerase SRB mediator complex (Myer and Young, 1998). These<br />
prote<strong>in</strong>s are not required for the stability <strong>of</strong> Rme1 (Covitz et al., 1994), or it’s b<strong>in</strong>d<strong>in</strong>g to DNA<br />
(Shimizu et al., 1998).<br />
Rme1 also functions as a transcriptional activator; it activates the transcription <strong>of</strong> CLN2 whose<br />
promoter <strong>in</strong>cludes Rme1’s b<strong>in</strong>d<strong>in</strong>g site (Toone et al., 1995). Accord<strong>in</strong>gly, Rme1 also activates the<br />
transcription <strong>of</strong> CYC1 and HIS3 when artificially tethered to their promoters (Covitz and<br />
Mitchell, 1993; Covitz et al., 1994). The transcription <strong>of</strong> RME1 is ma<strong>in</strong>ly <strong>in</strong>duced <strong>in</strong> G1 (Frenz et<br />
al., 2001), suggest<strong>in</strong>g that although this gene is non-essential, it might contribute to the high level<br />
expression <strong>of</strong> Cln2 at this stage <strong>in</strong> the cell cycle. <strong>Transcriptional</strong> activation is <strong>in</strong>dependent <strong>of</strong> Rgr1<br />
or S<strong>in</strong>4 (Blumental-Perry et al., 2002), although the function <strong>of</strong> Rme1 as an activator or repressor<br />
is mediated through the same doma<strong>in</strong> (Blumental-Perry et al., 2002). These results suggest that<br />
the function <strong>of</strong> Rme1 is determ<strong>in</strong>ed by <strong>in</strong>teract<strong>in</strong>g prote<strong>in</strong>s b<strong>in</strong>d<strong>in</strong>g to nearby DNA sites.<br />
The effect <strong>of</strong> MAT on the transcription <strong>of</strong> IME1 is evident only upon nitrogen depletion; In SA<br />
media the level <strong>of</strong> IME1 mRNA is low and identical <strong>in</strong> haploid and MATa/MATα diploid cells<br />
(Kassir et al., 1988), and only SPM and <strong>in</strong> MATa/MATα that IME1 is fully <strong>in</strong>duced. Moreover,<br />
s<strong>in</strong>ce nitrogen depletion <strong>in</strong>duces the transcription <strong>of</strong> RME1 (Frenz et al., 2001), it is possible that<br />
Rme1 represses transcription only <strong>in</strong> the absence <strong>of</strong> nitrogen. This hypothesis predicts that <strong>in</strong> the<br />
absence <strong>of</strong> Rme1, <strong>in</strong>itiation <strong>of</strong> <strong>meiosis</strong> will not depend on nitrogen depletion. However, cells<br />
where either RME1 or UCS3 along with UCS4 (the sites mediat<strong>in</strong>g the MAT signal) have been<br />
deleted, <strong>in</strong>duce <strong>meiosis</strong> only upon nitrogen depletion (Kassir and Simchen, 1976; Sagee et al.,<br />
1998). The effect <strong>of</strong> Rme1 is mediated through one <strong>of</strong> two mechanisms: by direct b<strong>in</strong>d<strong>in</strong>g and<br />
repression <strong>of</strong> IME1, and by activation <strong>of</strong> CLN2 transcription (Frenz et al., 2001; Toone et al.,<br />
1995). The <strong>in</strong>crease <strong>in</strong> activity <strong>of</strong> the Cln/Cdc28 complex could result <strong>in</strong> an <strong>in</strong>crease <strong>in</strong><br />
phosphorylation <strong>of</strong> Ime1, and its sequester<strong>in</strong>g from the nucleus (Colom<strong>in</strong>a et al., 1999).<br />
Consequently, the reduction <strong>in</strong> positive auto<strong>regulation</strong>, would lead to a decrease <strong>in</strong> the level <strong>of</strong><br />
IME1 mRNA.<br />
8
1.2. IME4. IME4 encodes a positive regulator absolutely required for the transcription <strong>of</strong><br />
IME1 (Shah and Clancy, 1992). The transcription <strong>of</strong> IME4 is regulated by the meiotic signals; it<br />
requires the presence <strong>of</strong> the MATa1 and MATα2 gene products and nitrogen depletion. Rme1<br />
does not transmit the MAT signal to IME4 (Shah and Clancy, 1992), suggest<strong>in</strong>g that Rme1 and<br />
Ime4 function <strong>in</strong> two dist<strong>in</strong>ct signal pathways. The region with<strong>in</strong> IME1 respond<strong>in</strong>g to Ime4, and<br />
the mode by which Ime4 activates the transcription <strong>of</strong> IME1 is not known. Over expression <strong>of</strong><br />
Ime1 partially suppresses ime4∆, suggest<strong>in</strong>g that Ime4 have an additional role <strong>in</strong> <strong>meiosis</strong> (Shah<br />
and Clancy, 1992). Ime4 associates with Mum2/SpoT-8 (Uetz et al., 2000) that is required for<br />
premeiotic DNA replication (Engebrecht et al., 1998; Tsuboi, 1983). It is possible that the second<br />
meiotic function <strong>of</strong> Ime4 is to regulate the function <strong>of</strong> this prote<strong>in</strong>.<br />
1.3. RES1. The dom<strong>in</strong>ant mutation Res1-1 bypasses the requirement for the presence <strong>of</strong><br />
both Mata1 and Matα2 for the expression <strong>of</strong> IME1 and <strong>meiosis</strong> (Kao et al., 1990). Res1-1 is not<br />
allelic to RME1, IME1 or IME4 (Kao et al., 1990; Shah and Clancy, 1992), neither is Res1 <strong>in</strong> the<br />
Ime4 or Rme1 signal pathway (Kao et al., 1990; Shah and Clancy, 1992). This gene has not been<br />
cloned, and its normal function is not known.<br />
B. The nitrogen signal<br />
The nitrogen signal is transmitted to IME1 through the UCS1 element (Fig. 3). Nested deletion <strong>of</strong><br />
this region leads to a 5 and 15-fold <strong>in</strong>crease <strong>in</strong> the expression <strong>of</strong> ime1-lacZ <strong>in</strong> vegetative growth<br />
media with either glucose (SD) or acetate (SA) as the sole carbon source, respectively [Fig. 4,<br />
compare l<strong>in</strong>es D and E, (Sagee et al., 1998)]. These results suggest that UCS1 functions as a<br />
negative element <strong>in</strong> the presence <strong>of</strong> glucose and nitrogen. By itself UCS1 has no UAS activity, it<br />
cannot promote expression <strong>of</strong> a his4-lacZ reporter gene lack<strong>in</strong>g its own UAS (Nadjar-Boger,<br />
2000). Insertion <strong>of</strong> UCS1 between the HIS4 UAS and TATA box <strong>in</strong> the HIS4UAS-HIS4TATA-lacZ<br />
reporter gene leads to a substantial reduction <strong>of</strong> expression <strong>in</strong> SD and SA [Fig. 5, (Sagee et al.,<br />
1998)]. Level <strong>of</strong> repression is 2 to 3-fold higher <strong>in</strong> SD <strong>in</strong> comparison to SA, confirm<strong>in</strong>g that the<br />
activity <strong>of</strong> UCS1 is partially regulated by glucose. Upon nitrogen depletion (SPM) a substantial<br />
<strong>in</strong>crease <strong>in</strong> expression is observed [Fig. 5 and (Sagee et al., 1998)], suggest<strong>in</strong>g that nitrogen is the<br />
major signal regulat<strong>in</strong>g the function <strong>of</strong> UCS1.<br />
The cAMP/PKA pathway transmits a nitrogen signal to IME1 and <strong>meiosis</strong> (Matsumoto et al.,<br />
1983; Matsuura et al., 1990). Mutations that cause low or no activity <strong>of</strong> PKA, such as cyr1, ras2,<br />
and cdc25 lead to the expression <strong>of</strong> IME1 and spore formation <strong>in</strong> the presence <strong>of</strong> nitrogen<br />
(Matsumoto et al., 1983; Matsuura et al., 1990; Shilo et al., 1978; Smith and Mitchell, 1989)<br />
[CYR1 encodes adenylate cyclase, RAS2 encodes a small G prote<strong>in</strong> that functions as a positive<br />
9
egulator <strong>of</strong> Cyr1, CDC25 encodes the Ras GDP/GTP exchange factor, which serves as a positive<br />
regulator <strong>of</strong> adenylate cyclase (Broach, 1991; Broek et al., 1987; Toda et al., 1985)]. On the other<br />
hand, mutations that cause constitutive PKA activity, such as RAS2-val19 (activated Ras) and<br />
bcy1 [the regulatory subunit <strong>of</strong> PKA (Broach, 1991)] are sporulation deficient, and are suppressed<br />
by over-expression <strong>of</strong> IME1 (Matsuura et al., 1990). The repression activity <strong>of</strong> UCS1 is reduced<br />
<strong>in</strong> vegetatively grown cdc25-5 cells <strong>in</strong>cubated at the non-permissive temperature (Fig. 5). Upon<br />
nitrogen depletion (SPM), this phenomenon is not observed; the same levels <strong>of</strong> expression are<br />
observed <strong>in</strong> cells <strong>in</strong>cubated at the permissive or restrictive temperature (Fig. 5). These results<br />
suggest that Cdc25 transmits the nitrogen signal to UCS1. Cdc25 is a positive regulator <strong>of</strong> both<br />
the, cAMP/PKA (Broek et al., 1987) and MAPK (Shenhar, 2001) signal transduction pathways.<br />
Therefore, further experiments are required to determ<strong>in</strong>e if this effect <strong>of</strong> Cdc25 is mediated<br />
through PKA, MAPK or a different signal pathway.<br />
MDS3 and its homologue, PMD1, are negative regulators <strong>of</strong> IME1 transcription <strong>in</strong> vegetative<br />
growth media with acetate as the sole carbon source. Deletion <strong>of</strong> these genes results <strong>in</strong> an<br />
<strong>in</strong>crease <strong>in</strong> the transcription <strong>of</strong> IME1 and the EMG IME2 and HOP1 (Benni and Neigeborn,<br />
1997). In addition, when stationary phase mds3∆ pmd1∆ diploid cells are shifted from SD to SA<br />
media, growth is ceased and about 10% <strong>of</strong> the cells enter and complete the meiotic cycle. These<br />
results suggest that Mds3/Pmd1 function upstream <strong>of</strong> Ime1, <strong>in</strong> prevent<strong>in</strong>g cell cycle arrest <strong>in</strong> the<br />
presence <strong>of</strong> nitrogen (Benni and Neigeborn, 1997). Growth arrest and sporulation are suppressed<br />
<strong>in</strong> cells carry<strong>in</strong>g the RAS2-val19 mutation, suggest<strong>in</strong>g that these genes might be upstream<br />
activators <strong>of</strong> the Ras pathway (Benni and Neigeborn, 1997). The region <strong>in</strong> IME1 that is regulated<br />
by these genes is not known. It will be <strong>in</strong>terest<strong>in</strong>g to determ<strong>in</strong>e if their effect is mediated through<br />
the UCS1 element.<br />
As discussed above, the Mata1 and Matα2 gene products are required to <strong>in</strong>duce expression <strong>in</strong><br />
the absence <strong>of</strong> nitrogen, suggest<strong>in</strong>g that UCS1 might also mediate the MAT signal. However, the<br />
repression activity <strong>of</strong> UCS1 and its relief (<strong>in</strong> a UASHIS4-UCS1-his4-lacZ reporter) is identical <strong>in</strong><br />
isogenic haploid and diploid cells (Boger-Nadjar, 2000), <strong>in</strong>dicat<strong>in</strong>g that the activity <strong>of</strong> UCS1 is<br />
not regulated by MAT. However, it is still possible that with<strong>in</strong> the context <strong>of</strong> IME1 promoter,<br />
UCS1 might <strong>in</strong>teract with the UCS3 and/or UCS4 elements that mediate MAT <strong>regulation</strong>.<br />
C. The glucose Signal<br />
The UAS activity <strong>of</strong> IME1 is conf<strong>in</strong>ed to UCS2, a region that conta<strong>in</strong>s alternate positive and<br />
negative elements [Fig. 3, (Sagee et al., 1998)]. Nested deletions <strong>of</strong> either one <strong>of</strong> its three positive<br />
elements, UASru, IREu, and UASrm lead to a reduction <strong>in</strong> expression <strong>of</strong> ime1-lacZ <strong>in</strong> SPM (Fig.<br />
10
4, l<strong>in</strong>es F,G,H), confirm<strong>in</strong>g their function as UAS elements (Sagee et al., 1998; Shenhar and<br />
Kassir, 2001). In addition, <strong>in</strong> the presence <strong>of</strong> glucose UASru and IREu function as repression<br />
elements [Fig. 4, l<strong>in</strong>es F,G and (Shenhar and Kassir, 2001)]. Deletion <strong>of</strong> UASru <strong>in</strong>creases<br />
expression <strong>in</strong> SD, but <strong>in</strong> SA or SPM expression is absent (Fig. 4, l<strong>in</strong>e F), suggest<strong>in</strong>g that UASru<br />
functions as a URS element <strong>in</strong> the presence <strong>of</strong> glucose and as a UAS element <strong>in</strong> the absence <strong>of</strong><br />
glucose and/or the presence <strong>of</strong> acetate. Nested deletion <strong>of</strong> IREu leads to the same levels <strong>of</strong><br />
expression <strong>of</strong> ime1-lacZ <strong>in</strong> both SD and SA media [Fig. 4, l<strong>in</strong>e G, (Sagee et al., 1998)],<br />
suggest<strong>in</strong>g that its URS activity is regulated by either the carbon or nitrogen source. These<br />
UASru, IREu and UASrm element function as a carbon source regulated UAS when <strong>in</strong>serted<br />
upstream <strong>of</strong> a his4-lacZ reporter gene (Fig. 6). Decreased levels <strong>of</strong> expression are observed <strong>in</strong> SD,<br />
and <strong>in</strong>creased expression is observed <strong>in</strong> SA, while <strong>in</strong> SPM there is a slight <strong>in</strong>crease <strong>in</strong> activity, but<br />
only <strong>in</strong> the presence <strong>of</strong> UASru [Fig. 6 and (Sagee et al., 1998)]. These results imply that IREu and<br />
UASrm are regulated only by the glucose signal, and that UASru is ma<strong>in</strong>ly regulated by glucose<br />
but nitrogen has also a partial effect on its activity.<br />
1. The cAMP/PKA signal-transduction pathway. Genetic analysis suggests that the<br />
cAMP/PKA signal pathway transmits a nitrogen signal [see section IIIB and (Gancedo, 2001; Pan<br />
et al., 2000)]. However, biochemical and genetic analysis demonstrate that this pathway is one <strong>of</strong><br />
the major signal transduction pathways transmitt<strong>in</strong>g a glucose signal to <strong>yeast</strong> cells. This is an<br />
essential pathway that transiently <strong>in</strong>creases the level <strong>of</strong> cAMP <strong>in</strong> response to glucose addition [for<br />
reviews see (Broach, 1991; Thevele<strong>in</strong> and de W<strong>in</strong>de, 1999)]. Addition <strong>of</strong> 10mM cAMP to diploid<br />
cells <strong>in</strong>cubated <strong>in</strong> sporulation conditions leads to a small but significant reduction <strong>in</strong> the<br />
expression <strong>of</strong> IME1 and the level <strong>of</strong> sporulation (Fig. 7). The expression <strong>of</strong> either UASrm-his4lacZ<br />
or UASru-his4-lacZ is slightly <strong>in</strong>creased by addition <strong>of</strong> cAMP. However, about 1.6-fold<br />
reduction <strong>in</strong> the expression <strong>of</strong> IREu-his4-lacZ is observed, similar to the effect cAMP has on<br />
sporulation and IME1 expression, suggest<strong>in</strong>g that the activity <strong>of</strong> IREu is negatively regulated by<br />
cAMP. The signal transduction pathways that transmit the glucose signal to UASru and UASrm<br />
are not known.<br />
1.1. The IREu element. The sequence <strong>of</strong> IREu (-1153 to –1121) reveals that it <strong>in</strong>cludes<br />
two known positive elements, a stress response element, STRE, and the Swi4/Swi6 cell cycle<br />
box, SCB (Fig. 8). An almost identical element, IREd, is present at –788 to –756 <strong>of</strong> IME1 (Fig.<br />
3). IREd differs from IREu <strong>in</strong> two residues localized to the STRE and SCB elements (Fig. 8). An<br />
ime1-lacZ chimeras whose 5’ end are term<strong>in</strong>ated downstream or upstream <strong>of</strong> either IREu or IREd<br />
show that IREu functions as a positive element, whereas IREd as a negative element (Sagee et al.,<br />
11
1998). Furthermore, unlike IREu, IREd promotes only low UAS activity when <strong>in</strong>serted upstream<br />
<strong>of</strong> his4-lacZ (Fig. 6), suggest<strong>in</strong>g that the STRE and/or the SCB elements are the transcriptional<br />
activation sequences with<strong>in</strong> IREu. The cAMP/PKA pathway negatively regulates IREu,<br />
promot<strong>in</strong>g its URS activity and prevent<strong>in</strong>g its UAS activity <strong>in</strong> the presence <strong>of</strong> glucose (Sagee et<br />
al., 1998; Shenhar and Kassir, 2001). Deletion <strong>of</strong> BCY1- the regulatory subunit <strong>of</strong> PKA (Toda et<br />
al., 1987a) leads to no expression <strong>of</strong> IREu-his4-lacZ, whereas a temperature sensitive mutations<br />
<strong>in</strong> CDC25 leads to a substantial <strong>in</strong>crease <strong>in</strong> the activity <strong>of</strong> IREu <strong>in</strong> the presence <strong>of</strong> either glucose<br />
or acetate as the sole carbon source (Sagee et al., 1998). Transcription factors that regulate the<br />
activity <strong>of</strong> IREu are the two-homologous DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>s, Msn2 and Msn4, Ime1 and<br />
Sok2. Msn2 and Msn4 are absolutely required for the activity <strong>of</strong> IREu, Ime1 is required for the<br />
complete UAS activity <strong>of</strong> IREu, and Sok2 is a negative regulator for IREu activity (Sagee et al.,<br />
1998; Shenhar and Kassir, 2001).<br />
1.1.1. Msn2 and Msn4. These C2H2 Z<strong>in</strong>c-f<strong>in</strong>ger prote<strong>in</strong>s b<strong>in</strong>d the STRE site present <strong>in</strong><br />
many stress-<strong>in</strong>duced genes (Mart<strong>in</strong>ez-Pastor et al., 1996; Schmitt and McEntee, 1996), <strong>in</strong>clud<strong>in</strong>g<br />
the IREu and IREd elements <strong>in</strong> IME1 (Sagee et al., 1998). Competition experiments reveal that<br />
IREu is a better competitor than IREd (Sagee et al., 1998), suggest<strong>in</strong>g that Msn2 and Msn4 b<strong>in</strong>d<br />
to the STRE element <strong>in</strong> IREu. In vitro transcribed and translated Msn2 can b<strong>in</strong>d the STRE<br />
sequence and the IREu element (Mart<strong>in</strong>ez-Pastor et al., 1996; Shenhar, 2001), suggest<strong>in</strong>g that<br />
b<strong>in</strong>d<strong>in</strong>g is <strong>in</strong>dependent <strong>of</strong> post-translational modifications, and/or the presence <strong>of</strong> additional<br />
prote<strong>in</strong>s. However, two l<strong>in</strong>es <strong>of</strong> evidence suggest that Msn2 forms a heterodimer with Sok2 (see<br />
section IIIC1.1.2 below): i. Msn2 physically associates with Sok2, and ii. Deletion <strong>of</strong> the<br />
postulated Sok2 b<strong>in</strong>d<strong>in</strong>g site with<strong>in</strong> IREu (the SCB element) abolishes the activity <strong>of</strong> IREu<br />
(Shenhar and Kassir, 2001). These results suggest that <strong>in</strong> vivo, Msn2 forms a heterodimer with<br />
Sok2, and that Sok2 facilitates its b<strong>in</strong>d<strong>in</strong>g to the DNA. In the absence <strong>of</strong> Sok2, an imposter<br />
prote<strong>in</strong> can promote the b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> Msn2/4 to STRE (Fig. 8) (Shenhar and Kassir, 2001). Msn2/4<br />
are apparent targets <strong>of</strong> the cAMP/PKA pathway. This is concluded from the follow<strong>in</strong>g<br />
observations: i. Deletion <strong>of</strong> both MSN2 and MSN4 suppresses the lethality <strong>of</strong> a stra<strong>in</strong> deleted for<br />
the three TPK genes [TPK1-3 are the three homologous genes encod<strong>in</strong>g the catalytic activity <strong>of</strong><br />
PKA (Smith et al., 1998; Toda et al., 1987b)], ii. Msn2 <strong>in</strong>cludes sites required for its nuclear<br />
import (NLS) as well as for its export (NIS), whose functions are regulated by glucose through<br />
the cAMP/PKA pathway (Gorner et al., 2002). Glucose starvation and <strong>in</strong>activation <strong>of</strong> the<br />
cAMP/PKA pathway through mutations, leads to nuclear localization, whereas addition <strong>of</strong><br />
glucose or cAMP leads to cytoplasmic localization (Gorner et al., 1998; Gorner et al., 2002).<br />
Interest<strong>in</strong>gly, the NIS element is also regulated by the TOR signal<strong>in</strong>g pathway. When this signal<br />
12
pathway is activated, the b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> Msn2/4 to Bmh2, the 14-3-3 adaptor, sequesters it from the<br />
nuclei (Beck and Hall, 1999). 3. The NLS site <strong>of</strong> Msn2 conta<strong>in</strong>s four PKA consensus sites. These<br />
residues are phosphorylated <strong>in</strong> vivo <strong>in</strong> response to glucose addition, depend<strong>in</strong>g on the presence <strong>of</strong><br />
the three TPK genes. Ser<strong>in</strong>e to alan<strong>in</strong>e mutations <strong>of</strong> all these sites lead to constitutive nuclear<br />
localization <strong>of</strong> Msn2, whereas ser<strong>in</strong>e to aspartic acid, mimick<strong>in</strong>g a phosphorylated ser<strong>in</strong>e residue,<br />
leads to its cytoplasmic localization (Gorner et al., 2002).<br />
1.1.2. Sok2. SOK2 encodes a DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> that is highly homologous to the<br />
DNA-b<strong>in</strong>d<strong>in</strong>g doma<strong>in</strong> <strong>of</strong> Swi4 and Phd1 prote<strong>in</strong>s that are known to b<strong>in</strong>d the SCB consensus<br />
sequence. This homology suggests that Sok2 might also b<strong>in</strong>d the same sequence. Direct b<strong>in</strong>d<strong>in</strong>g<br />
<strong>of</strong> Sok2 to IREu was not reported, however, the follow<strong>in</strong>g genetic evidence suggests that Sok2<br />
b<strong>in</strong>ds IREu under all growth conditions. The IREu-his4-lacZ reporter shows a 10-fold <strong>in</strong>crease <strong>in</strong><br />
expression <strong>in</strong> the triple mutant sok2∆ msn2∆ msn4∆ <strong>in</strong> comparison to the double mutant msn2∆<br />
msn4∆. On the other hand, IREu-his4-lacZ is not expressed <strong>in</strong> the sok2T598A msn2∆ msn4∆<br />
triple mutant. The sok2T598A allele, similar to the SOK2 deletion allele is defective <strong>in</strong><br />
repression, but unlike the latter, the defective Sok2 prote<strong>in</strong> is present <strong>in</strong> the cells (Shenhar and<br />
Kassir, 2001). These results imply that Sok2 b<strong>in</strong>ds IREu under all growth conditions, and that <strong>in</strong><br />
its physical absence an imposter prote<strong>in</strong> can b<strong>in</strong>d and activate transcription. Sok2 functions as a<br />
transcriptional repressor for several PKA target genes such as GAC1, SSA3, SWI5 and IME1 (Pan<br />
and Heitman, 2000; Shenhar and Kassir, 2001; Ward et al., 1995). Furthermore, it functions as a<br />
negative regulator <strong>of</strong> pseudohyphal growth (Ward et al., 1995) and <strong>meiosis</strong> (Shenhar and Kassir,<br />
2001), and as a positive regulator <strong>in</strong> the mitotic cell cycle (Ward et al., 1995). Glucose regulates<br />
both the transcription <strong>of</strong> SOK2 and the repression activity <strong>of</strong> Sok2 prote<strong>in</strong>. The latter is concluded<br />
from the observation that a Sok2-Gal4(bd), expressed from the ADH1 promoter, represses the<br />
transcription <strong>of</strong> UASGAL1-UASHIS4-his4-lacZ, but only <strong>in</strong> the presence <strong>of</strong> glucose (SD media)<br />
(Shenhar and Kassir, 2001). The signal pathway regulat<strong>in</strong>g the transcription <strong>of</strong> SOK2 is not<br />
known (Shenhar and Kassir, 2001). However, the signal pathway transmitt<strong>in</strong>g the glucose signal<br />
regulat<strong>in</strong>g Sok2 repression activity is the cAMP/PKA pathway. This is evident from the<br />
follow<strong>in</strong>g observations: i. Over-expression <strong>of</strong> SOK2 suppresses the temperature sensitive growth<br />
defect <strong>of</strong> a tpk1∆ tpk2-ts tpk3∆ mutant (Ward et al., 1995); ii. Repression <strong>of</strong> IREu-his4-lacZ and<br />
UASgal1-UAShis4-his4-lacZ by either Sok2 or Gal4(bd)-Sok2, respectively, is relieved <strong>in</strong> cdc25-<br />
5 cells <strong>in</strong>cubated at the non-permissive temperature, as well as by a threon<strong>in</strong>e to alan<strong>in</strong>e mutation<br />
<strong>in</strong> a PKA consensus site <strong>in</strong> Sok2 (Shenhar and Kassir, 2001). Furthermore, Sok2 is a<br />
phosphoprote<strong>in</strong> whose phosphorylation depends on this same threon<strong>in</strong>e (T598) residue (Shenhar<br />
and Kassir, 2001).<br />
13
In the absence <strong>of</strong> glucose Sok2 does not repress transcription. Relief <strong>of</strong> repression <strong>in</strong> the<br />
absence <strong>of</strong> glucose depends on its N-term<strong>in</strong>al doma<strong>in</strong>, am<strong>in</strong>o acids 1-247 as well as on Ime1 [see<br />
section IIIE and (Shenhar and Kassir, 2001)]. The mode by which Sok2 represses transcription<br />
and how Ime1 relieves repression is not known.<br />
1.2. Rim15. Rim15 encodes a ser<strong>in</strong>e/threon<strong>in</strong>e prote<strong>in</strong> k<strong>in</strong>ase whose activity <strong>in</strong> glucose<br />
growth media is <strong>in</strong>hibited due to its phosphorylation by PKA (Re<strong>in</strong>ders et al., 1998). In addition,<br />
the transcription <strong>of</strong> RIM15, and consequently the steady state level <strong>of</strong> Rim15, is <strong>in</strong>creased <strong>in</strong><br />
acetate growth media (Vidan and Mitchell, 1997). Diploid cells deleted for RIM15 or carry<strong>in</strong>g a<br />
k<strong>in</strong>ase-dead po<strong>in</strong>t mutation are sporulation deficient (Vidan and Mitchell, 1997). Deletion <strong>of</strong><br />
RIM15 causes a drastic reduction <strong>in</strong> the transcription <strong>of</strong> IME1. The mode <strong>of</strong> action <strong>of</strong> Rim15, and<br />
the IME1 element regulated by it are not known.<br />
2. The Snf1 signal-transduction pathway. SNF1 encodes a prote<strong>in</strong> k<strong>in</strong>ase whose activity as a<br />
k<strong>in</strong>ase is <strong>in</strong>hibited <strong>in</strong> the presence <strong>of</strong> high levels <strong>of</strong> glucose (Jiang and Carlson, 1996). Snf1<br />
function is required for the expression <strong>of</strong> glucose-repressed genes (Lesage et al., 1996). Snf1<br />
phosphorylates the DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>, Mig1 (Treitel et al., 1998) that recruits the Tup1/Ssn6<br />
repression complex to the glucose-repressed genes to whom it b<strong>in</strong>ds (Nehl<strong>in</strong> et al., 1991). Snf1 is<br />
required for the high level <strong>in</strong>duction <strong>in</strong> the transcription <strong>of</strong> IME1 <strong>in</strong> sporulation conditions, and<br />
diploid cells deleted for SNF1 arrest prior to premeiotic DNA replication, meiotic recomb<strong>in</strong>ation,<br />
and spore formation (Honigberg and Lee, 1998). Over expression <strong>of</strong> either Msn2 or Msn4<br />
suppresses a temperature sensitive allele <strong>of</strong> SNF1 (Estruch and Carlson, 1993), suggest<strong>in</strong>g that<br />
Snf1 effect on IME1 might be mediated through Msn2/4. It is not known, though, whether<br />
Msn2/4 are direct targets <strong>of</strong> Snf1. Genetic evidence suggests that Snf1 is <strong>in</strong> a separate pathway<br />
than the cAMP/PKA pathway (Thompson-Jaeger et al., 1991), and both regulate Msn2/4. The<br />
sequence <strong>of</strong> IME1 shows no homology to the Mig1 b<strong>in</strong>d<strong>in</strong>g site, and the region <strong>in</strong> IME1 regulated<br />
by Snf1 is not known.<br />
In the meiotic cycle Snf1 is also required follow<strong>in</strong>g the transcription <strong>of</strong> IME1. When IME1 is<br />
placed on a multi-copy plasmid it is highly expressed <strong>in</strong> both wild type and snf1∆ diploid cells,<br />
nevertheless, <strong>in</strong> the SNF1 deleted stra<strong>in</strong> over express<strong>in</strong>g Ime1, the EMG IME2 is only partially<br />
expressed, 10-15% <strong>of</strong> the cells complete premeiotic DNA replication and meiotic recomb<strong>in</strong>ation,<br />
but spores are not formed (Honigberg and Lee, 1998). It was suggested that Snf1 is required <strong>in</strong><br />
the meiotic cycle at three po<strong>in</strong>ts, for the transcription <strong>of</strong> Ime1, for the transcription <strong>of</strong> Ime2, and<br />
for spore formation (Honigberg and Lee, 1998).<br />
14
3. Respiration and the transcription <strong>of</strong> IME1. The presence <strong>of</strong> glucose prevents the activity<br />
<strong>of</strong> the mitochondria whose function is required for sporulation (Berger and Yaffe, 2000). The<br />
transcription <strong>of</strong> IME1 is absent <strong>in</strong> diploid cells without mitochondria – petites, or <strong>in</strong> cells treated<br />
with oligomyc<strong>in</strong> - an <strong>in</strong>hibitor <strong>of</strong> the mitochondrial ATPase (Tre<strong>in</strong><strong>in</strong> and Simchen, 1993). It is not<br />
known whether mitochondrial function is also required at a stage preced<strong>in</strong>g the transcription <strong>of</strong><br />
IME1. The site with<strong>in</strong> IME1 that responds to mitochondria function is not known, nor is it known<br />
how the respiration signal is transmitted. The connection between mitochondria function and<br />
IME1 transcription is also evident from the identification <strong>of</strong> the Rim1 (Rim101) Rim8, Rim9,<br />
Rim13 and Rim20 prote<strong>in</strong>s as positive regulators <strong>of</strong> IME1 (Li and Mitchell, 1997; Su and<br />
Mitchell, 1993a). RIM1 encodes a C2H2 z<strong>in</strong>c f<strong>in</strong>ger prote<strong>in</strong> that is required for high-level<br />
expression <strong>of</strong> IME1 as well as for efficient sporulation (Su and Mitchell, 1993b). Activation <strong>of</strong><br />
Rim1 requires its cleavage by the products <strong>of</strong> Rim8, Rim9 and Rim13 (Li and Mitchell, 1997), as<br />
well as the activity <strong>of</strong> Rim20 that might be required for present<strong>in</strong>g Rim1 to Rim13 (Xu and<br />
Mitchell, 2001). Rim1 is localized to mitochondria, and is required for mitochondria DNA<br />
replication, and consequently for the ma<strong>in</strong>tenance <strong>of</strong> the mitochondria (Berger and Yaffe, 2000;<br />
Van Dyck et al., 1992). Thus, the effect <strong>of</strong> these RIM1,8,9,13,20 genes on the transcription <strong>of</strong><br />
IME1 may be <strong>in</strong>direct, through their effect on the ma<strong>in</strong>tenance <strong>of</strong> the mitochondria.<br />
D. Additional prote<strong>in</strong>s regulat<strong>in</strong>g the transcription <strong>of</strong> IME1.<br />
1. Mck1. MCK1 encodes a prote<strong>in</strong> k<strong>in</strong>ase required for efficient transcription <strong>of</strong> IME1, for<br />
expression <strong>of</strong> early and middle genes such as IME2 and SPS1,2 respectively, and for spore<br />
maturation (Neigeborn and Mitchell, 1991). Expression <strong>of</strong> IME1 from the hetrologous GAL1<br />
promoter suppresses the requirement <strong>of</strong> Mck1 for the transcription <strong>of</strong> EMG and MMG, but spore<br />
maturation rema<strong>in</strong>s defective, suggest<strong>in</strong>g that Mck1 directly regulate IME1 transcription and<br />
spore maturation (Neigeborn and Mitchell, 1991). MCK1 is one <strong>of</strong> 4 <strong>yeast</strong> genes show<strong>in</strong>g<br />
homology to mammalian glycogen synthase k<strong>in</strong>ase 3 (GSK3-β), RIM11, YOL128c, and MRK1.<br />
These homologs are not required for the transcription <strong>of</strong> IME1 (Hajji et al., 1999; Mandel et al.,<br />
1994; Rabitsch et al., 2001), suggest<strong>in</strong>g that the transcription <strong>of</strong> IME1 is only partially dependent<br />
on Mck1.<br />
Mck1 <strong>in</strong>hibits the k<strong>in</strong>ase activity <strong>of</strong> PKA both <strong>in</strong> vitro and <strong>in</strong> vivo, through a mechanism that<br />
does not depend on Mck1 k<strong>in</strong>ase activity (Rayner et al., 2002). This result implies that deletion <strong>of</strong><br />
MCK1 might <strong>in</strong>crease rather than decrease the transcription <strong>of</strong> IME1. Therefore, the effect <strong>of</strong><br />
Mck1 on the transcription <strong>of</strong> IME1 is most probably not mediated by PKA. Mck1 is subject to<br />
15
autophosphorylation, <strong>in</strong>clud<strong>in</strong>g on a conserved tyros<strong>in</strong>e (Y199) (Rayner et al., 2002).<br />
Phosphorylation <strong>of</strong> this tyros<strong>in</strong>e residue <strong>in</strong> Mck1, similar to other GSK3β homologs, is required<br />
for its k<strong>in</strong>ase activity on exogenous substrates, but not for autophosphorylation (Rayner et al.,<br />
2002). The Ptp2 and Ptp3 tyros<strong>in</strong>e phosphatases are required for Mck1 dephosphorylation (Zhan<br />
et al., 2000). Deletion <strong>of</strong> either PTP2 or PTP3 has no effect on <strong>meiosis</strong>, however, the double<br />
mutant ptp2∆ ptp3∆ is sporulation deficient, it arrests <strong>in</strong> <strong>meiosis</strong> prior to premeiotic DNA<br />
replication, with a significant reduction <strong>in</strong> the expression <strong>of</strong> IME1 and IME2, and no expression<br />
<strong>of</strong> middle or late <strong>meiosis</strong>-specific genes (Zhan et al., 2000). A third tyros<strong>in</strong>e phosphatase, Yvh1,<br />
might contribute to this <strong>regulation</strong> (Guan et al., 1992). Deletion <strong>of</strong> YVH1 leads to m<strong>in</strong>or effects on<br />
the transcription <strong>of</strong> IME1, a reduced transcription <strong>of</strong> IME2, and a reduction <strong>in</strong> the efficiency <strong>of</strong><br />
asci formation (Park et al., 1996), but <strong>in</strong> the double mutant yvh1∆ ptp2∆ sporulation is almost<br />
dim<strong>in</strong>ished (Park et al., 1996). Thus, the effect <strong>of</strong> these tyros<strong>in</strong>e phosphatases on <strong>meiosis</strong> may be<br />
partially mediated through Mck1. The transcription <strong>of</strong> YVH1 is <strong>in</strong>duced upon nitrogen depletion,<br />
suggest<strong>in</strong>g that it might be <strong>in</strong>volved with transmitt<strong>in</strong>g the nitrogen signal (Park et al., 1996).<br />
Over expression <strong>of</strong> Mck1 suppresses the sporulation defect <strong>of</strong> cells deleted for TPS1 (De Silva-<br />
Udawatta and Cannon, 2001). TPS1 encodes one <strong>of</strong> the subunits <strong>of</strong> the trehalose phosphate<br />
synthase complex (Re<strong>in</strong>ders et al., 1997). Thus, Tps1 may either regulate Mck1, or suppression <strong>of</strong><br />
mck1∆ by Tps1 is due to unrelated <strong>in</strong>crease <strong>in</strong> the level <strong>of</strong> Ime1. The region <strong>in</strong> IME1 regulated by<br />
Mck1 and its mode <strong>of</strong> function is not known. However, the additive effects <strong>of</strong> mutations <strong>in</strong><br />
MCK1, MDS3, PMD1, RIM1 and IME4 suggest that these genes affect different regulatory<br />
elements <strong>in</strong> the IME1 promoter (Su and Mitchell, 1993b).<br />
2. Tup1/Ssn6. The Tup1/Ssn6 complex functions as a general transcriptional repressor upon<br />
recruitment by specific DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>s (Keleher et al., 1992). These prote<strong>in</strong>s are also<br />
negative regulators for the transcription <strong>of</strong> IME1 (Mizuno et al., 1998), but the specific DNAb<strong>in</strong>d<strong>in</strong>g<br />
prote<strong>in</strong> that tethers them to IME1 is not known. Two regions <strong>in</strong> IME1 promoter respond<br />
to Tup1, the first one is from -914 to – 621 and the second from -1215 to –915 (Mizuno et al.,<br />
1998), function<strong>in</strong>g as URS <strong>in</strong> the presence <strong>of</strong> Tup1, and UAS <strong>in</strong> the absence <strong>of</strong> Tup1,<br />
respectively (Mizuno et al., 1998). S<strong>in</strong>ce these regions carry the IREd and IREu repeated<br />
elements (Fig. 3) that function as repression and activation sequences, respectively, it should be<br />
<strong>in</strong>terest<strong>in</strong>g to determ<strong>in</strong>e if the Tup1/Ssn6 complex mediates its effect through these elements, and<br />
whether Sok2 is the DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> that recruits them to these elements.<br />
16
3. Yhp1. YHP1 encodes a homeodoma<strong>in</strong> prote<strong>in</strong> that b<strong>in</strong>ds to the UASv element <strong>in</strong> IME1<br />
promoter, between -702 to –675 (Fig. 3) (Kunoh et al., 2000). This 28 bp element by itself does<br />
not function as a UAS, but when tethered to heterologous reporter genes, it causes a 2-fold<br />
reduction <strong>in</strong> expression, suggest<strong>in</strong>g that Yhp1 functions as a repressor (Kunoh et al., 2000). The<br />
mode by which Yhp1 represses transcription is not known, but its effect is <strong>in</strong>dependent <strong>of</strong> the<br />
Tup1/Ssn6 repression complex (Kunoh et al., 2000). The transcription <strong>of</strong> YHP1 is reduced <strong>in</strong><br />
vegetative cells grown <strong>in</strong> the presence <strong>of</strong> acetate as the sole carbon source <strong>in</strong> comparison to<br />
glucose (Kunoh et al., 2000), imply<strong>in</strong>g that it might transmit a glucose signal. However, deletion<br />
<strong>of</strong> YHP1 has no effect on either the transcription <strong>of</strong> IME1, or on the ability <strong>of</strong> cells to enter and<br />
complete <strong>meiosis</strong> and sporulation (Kunoh et al., 2000).<br />
4. The Swi/Snf chromat<strong>in</strong> remodel<strong>in</strong>g complex. Two components <strong>of</strong> the Swi/Snf<br />
complex, Snf2 and Swi1 are required for high-level expression <strong>of</strong> IME1 (Yoshimoto et al., 1993).<br />
This complex is <strong>in</strong>volved with transcriptional activation <strong>of</strong> genes to whom it is tethered through<br />
<strong>in</strong>teraction with specific activation doma<strong>in</strong>s [for review see (Carlson and Laurent, 1994)].<br />
E. Positive and Negative feedback <strong>regulation</strong>.<br />
The transcription <strong>of</strong> IME1 is subject to positive <strong>regulation</strong>. Over expression <strong>of</strong> Ime1 leads to<br />
<strong>in</strong>crease <strong>in</strong> the level <strong>of</strong> ime1-lacZ, through its effect on the function <strong>of</strong> IREu (Shenhar and Kassir,<br />
2001). The effect <strong>of</strong> Ime1 on additional elements has not yet been determ<strong>in</strong>ed. This<br />
auto<strong>regulation</strong> is required to relieve repression by Sok2. This is deduced from the follow<strong>in</strong>g<br />
results: i. Expression <strong>of</strong> IREu-his4-lacZ is substantially reduced <strong>in</strong> diploid cells deleted for IME1<br />
(Shenhar and Kassir, 2001), ii. Gal4(bd)-Sok2 represses the transcription <strong>of</strong> GAL1UAS-HIS4 UAS -<br />
his4-lacZ <strong>in</strong> SD but has no repression activity <strong>in</strong> SA (Shenhar and Kassir, 2001). However, <strong>in</strong><br />
diploid cells deleted for IME1 the transcription <strong>of</strong> the reporter gene is repressed <strong>in</strong> both SD and<br />
SA media (Shenhar and Kassir, 2001). S<strong>in</strong>ce <strong>in</strong> the presence <strong>of</strong> glucose Ime1 is not expressed, it<br />
is not surpris<strong>in</strong>g that the effect <strong>of</strong> Ime1 is observed only <strong>in</strong> SA. As will be detailed <strong>in</strong> section<br />
IVB2, Ime1 does not b<strong>in</strong>d directly to DNA, and is recruited to the DNA by <strong>in</strong>teract<strong>in</strong>g with a<br />
DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>. S<strong>in</strong>ce the N-term<strong>in</strong>al doma<strong>in</strong> <strong>of</strong> Sok2 is also required to relieve repression<br />
<strong>in</strong> SA, it is tempt<strong>in</strong>g to suggest that Ime1 associates with the N-term<strong>in</strong>al doma<strong>in</strong> <strong>of</strong> Sok2. No<br />
<strong>in</strong>formation on possible associations between these two prote<strong>in</strong>s has yet been reported.<br />
The transcription <strong>of</strong> IME1 as well as the steady-state level <strong>of</strong> Ime1 prote<strong>in</strong> <strong>in</strong> meiotic<br />
conditions is transient, when <strong>in</strong>duced upon a shift to SPM, the level <strong>of</strong> IME1 peaks at about 6-8<br />
17
hours <strong>in</strong> SPM, followed by a decl<strong>in</strong>e (Guttmann-Raviv and Kassir, 2002; Kassir et al., 1988;<br />
Shefer-Vaida et al., 1995). This transient transcription depends on both Ime1 and Ime2<br />
(Guttmann-Raviv and Kassir, 2002; Mitchell et al., 1990; Shefer-Vaida et al., 1995; Smith and<br />
Mitchell, 1989; Yoshida et al., 1990). This negative feedback effect is on transcription <strong>of</strong> IME1<br />
rather than the stability <strong>of</strong> the mRNA, s<strong>in</strong>ce <strong>in</strong>sertions <strong>of</strong> two separate regions, -621 to –924 and –<br />
924 to –1368 (Fig. 3) upstream <strong>of</strong> cyc1-lacZ exhibit the same feedback <strong>regulation</strong> (Shefer-Vaida<br />
et al., 1995). Ime1 is a non-stable prote<strong>in</strong> that is degraded by the proteasome and whose half-life<br />
depends on Ime2 (Guttmann-Raviv and Kassir, 2002). IME2 encodes a <strong>meiosis</strong>-specific prote<strong>in</strong><br />
k<strong>in</strong>ase that <strong>in</strong>teracts with Ime1 and phosphorylates it <strong>in</strong> vitro (Guttmann-Raviv and Kassir, 2002).<br />
We suggest that the effect <strong>of</strong> Ime2 on shutt<strong>in</strong>g down the transcription <strong>of</strong> IME1 is due to its effect<br />
on the stability <strong>of</strong> Ime1 prote<strong>in</strong>. In the presence <strong>of</strong> Ime2, degradation <strong>of</strong> Ime1 would lead to the<br />
establishment <strong>of</strong> Sok2 repression and silenc<strong>in</strong>g. On the other hand, <strong>in</strong> cells deleted for IME2,<br />
accumulation <strong>of</strong> Ime1 leads to cont<strong>in</strong>ues positive auto<strong>regulation</strong>, and <strong>in</strong>crease <strong>in</strong> its transcription,<br />
and consequently the availability <strong>of</strong> Ime1 prote<strong>in</strong> (Guttmann-Raviv and Kassir, 2002).<br />
F. Summary.<br />
IME1 encodes the master regulator required to open a developmental pathway, that <strong>of</strong> <strong>meiosis</strong>, <strong>in</strong><br />
S. cerevisiae. It is not surpris<strong>in</strong>g, therefore, that its transcription is regulated by an unusual large<br />
5’ region that is subject to multiple <strong>regulation</strong>s. Fig. 9 is a summary <strong>of</strong> the results described<br />
above, on the current knowledge on how the meiotic signals are transmitted to IME1. A<br />
comb<strong>in</strong>atorial effect <strong>of</strong> at least 10 dist<strong>in</strong>ct elements ensures that IME1 will be transcribed only<br />
under the appropriate conditions, namely <strong>in</strong> MATa/MATα diploids, the absence <strong>of</strong> glucose, the<br />
presence <strong>of</strong> acetate, and nitrogen depletion. Two negative elements, UCS3 and UCS4 restrict<br />
elevated transcription <strong>of</strong> IME1 to cells express<strong>in</strong>g Mata1 and Matα2 peptides. In vegetative<br />
growth media with glucose as the sole carbon source IME1 is silent. This <strong>regulation</strong> is<br />
accomplished by us<strong>in</strong>g 4 dist<strong>in</strong>ct elements, UCS1 IREu UASru, and UASv, whose function as<br />
repression elements is mediated through dist<strong>in</strong>ct signal transduction pathways. In acetate growth<br />
media a low level <strong>of</strong> transcription is accomplished by the positive activity <strong>of</strong> UASru, IREu,<br />
UASrm and UASv that is opposed by negative <strong>regulation</strong> from UCS1 as well as from three<br />
constitutive URS elements, URSu, URSd and IREd. Upon nitrogen depletion, relief <strong>of</strong> UCS1<br />
repression promotes an <strong>in</strong>crease <strong>in</strong> transcription.<br />
18
IV. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> early <strong>meiosis</strong>-specific genes (EMG)<br />
A. Silenc<strong>in</strong>g <strong>of</strong> early <strong>meiosis</strong>-specific genes <strong>in</strong> vegetative growth.<br />
In vegetative growth conditions with either glucose or acetate as the sole carbon source <strong>meiosis</strong>specific<br />
genes are silent. Silenc<strong>in</strong>g <strong>of</strong> EMG is not only due to the absence <strong>of</strong> their ma<strong>in</strong><br />
transcriptional activator, Ime1, but is rather due to active repression. The genes required to<br />
promote this silenc<strong>in</strong>g are WTM3, UME2, UME3, SIN3, RPD3, UME5, UME6, and ISW2<br />
(Goldmark et al., 2000; Strich et al., 1989; Strich et al., 1994; Vidal and Gaber, 1991). Except for<br />
Ume6 and Isw2, these genes are not essential for the expression <strong>of</strong> EMG under meiotic conditions<br />
(Goldmark et al., 2000; Strich et al., 1989).<br />
1. The WTM genes. UME1 (WTM3) and its two homologs WTM1 and WTM2 repress the<br />
transcription <strong>of</strong> the EMG IME2 synergistically (Pemberton and Blobel, 1997). These prote<strong>in</strong>s also<br />
function as transcriptional repressors <strong>of</strong> the silent HMR cassette, and when tethered artificially to<br />
heterologous genes. These genes are non-essential; the wtm1 wtm2 wmt3 triple mutant is viable<br />
(Pemberton and Blobel, 1997). The Wtm prote<strong>in</strong>s are expressed constitutively <strong>in</strong> both mitotic and<br />
meiotic conditions, and are localized to the nucleus (Pemberton and Blobel, 1997). The mode by<br />
which they cause repression is not known.<br />
2. The UME2 gene. UME2 (SRB9, SSN2) is a component <strong>of</strong> the SRB subcomplex <strong>of</strong> RNA<br />
polymerase II holoenzyme (Kornberg, 1999). Although it is a negative regulator <strong>of</strong> EMG under<br />
vegetative growth conditions (Strich et al., 1989), when fused to lexA it activates transcription<br />
(Song and Carlson, 1998).<br />
3. The UME3 and UME5 genes. The UME3 (SRB11, SSN8) and UME5 (SRB10, SSN3)<br />
encode <strong>in</strong>tegral components <strong>of</strong> the SRB subcomplex <strong>of</strong> RNA polymerase II holoenzyme (Cooper<br />
and Strich, 1998; Kornberg, 1999). UME3 encodes a cycl<strong>in</strong> C homolog that associates with the<br />
cycl<strong>in</strong> dependent k<strong>in</strong>ase, Ume5 (Cooper et al., 1997; Cooper and Strich, 1998). Ume5<br />
phosphorylates the C-term<strong>in</strong>al CTD repeats <strong>of</strong> RNA polymerase II, but this event is <strong>in</strong>dependent<br />
<strong>of</strong> its repression activity (Cooper and Strich, 1998), thus, it is not know how Ume5 represses the<br />
transcription <strong>of</strong> EMG. Ume3 is degraded <strong>in</strong> meiotic conditions, and this degradation, that is<br />
<strong>in</strong>dependent <strong>of</strong> Ume5, is required for complete relief <strong>of</strong> repression <strong>of</strong> EMG (Cooper et al., 1997).<br />
19
4. The SIN3, UME6, and RPD3 genes. UME6 encodes a C6Zn2 prote<strong>in</strong> that b<strong>in</strong>ds the<br />
URS1 sequence present <strong>in</strong> many genes <strong>in</strong>clud<strong>in</strong>g EMG (Anderson et al., 1995; Strich et al.,<br />
1994). The C6Zn2 doma<strong>in</strong> is sufficient for b<strong>in</strong>d<strong>in</strong>g, and is required for its repression activity<br />
(Anderson et al., 1995; Strich et al., 1994). Deletion <strong>of</strong> UME6 causes high expression <strong>of</strong> EMG, as<br />
well as additional non-meiotic genes carry<strong>in</strong>g the URS1 element, <strong>in</strong> vegetative growth conditions<br />
(Bowdish and Mitchell, 1993; Lopes et al., 1993; Park et al., 1992; Strich et al., 1994). In<br />
addition, Ume6 functions as a transcriptional repressor when tethered to heterologous genes<br />
follow<strong>in</strong>g its fusion to lexA (Kadosh and Struhl, 1997). This repression activity <strong>of</strong> Ume6 depends<br />
on the availability <strong>of</strong> S<strong>in</strong>3 (Ume4, Rpd1) and Rpd3 (Kadosh and Struhl, 1997).<br />
Coimmunoprecipitation and two-hybrid assays demonstrate physical association between<br />
Ume6(515-530) and S<strong>in</strong>3(426-472) (the PAH2 doma<strong>in</strong>), as well as between S<strong>in</strong>3 and Rpd3<br />
(Kadosh and Struhl, 1997; Kasten et al., 1997; Washburn and Esposito, 2001).<br />
S<strong>in</strong>3 functions as a transcriptional repressor when tethered to heterologous genes follow<strong>in</strong>g its<br />
fusion to lexA (Wang and Stillman, 1993). The repression activity <strong>of</strong> S<strong>in</strong>3 depends on Rpd3<br />
(Kadosh and Struhl, 1997; Kasten et al., 1997). In accord, the double mutant s<strong>in</strong>3 rpd3 have the<br />
same phenotype as either s<strong>in</strong>gle mutant (Vidal and Gaber, 1991).<br />
RPD3 encodes histone deacetylase whose deletion, similar to SIN3 deletion, causes an <strong>in</strong>crease<br />
<strong>in</strong> acetylation <strong>of</strong> lys<strong>in</strong>es 5 and 12 <strong>in</strong> histone H4 <strong>in</strong> various genes carry<strong>in</strong>g the URS1 element<br />
(Burgess et al., 1999; Rundlett et al., 1998). Recruitment <strong>of</strong> the S<strong>in</strong>3/Rpd3 complex by Ume6 to<br />
DNA results <strong>in</strong> decreased acetylation <strong>of</strong> histone H3 and H4 <strong>in</strong> a restricted region, up to two<br />
nucleosomes from the b<strong>in</strong>d<strong>in</strong>g site <strong>of</strong> Ume6 (Kadosh and Struhl, 1998).<br />
5. The ISW2 gene. Isw2 <strong>in</strong> a complex with Itc1 functions as an ATP dependent chromat<strong>in</strong>remodel<strong>in</strong>g<br />
factor that is required for the repression <strong>of</strong> EMG under vegetative growth conditions<br />
(Goldmark et al., 2000). Isw2 functions <strong>in</strong> parallel to the S<strong>in</strong>3/Rpd3 histone deacetylase complex,<br />
as deduced from the observation that the level <strong>of</strong> expression <strong>of</strong> EMG are dramatically <strong>in</strong>creased<br />
<strong>in</strong> the s<strong>in</strong>3 isw2 double mutant <strong>in</strong> comparison to the s<strong>in</strong>gle mutants (Goldmark et al., 2000). Isw2<br />
physically associates with Ume6, and s<strong>in</strong>ce it’s b<strong>in</strong>d<strong>in</strong>g to the URS1 element depends on Ume6,<br />
it was concluded that Ume6 recruits the Isw2 complex to URS1 (Goldmark et al., 2000). DNaseI<br />
and micrococcal nuclease digestion <strong>of</strong> the chromat<strong>in</strong> near the EMG REC104 show dependence on<br />
Isw2, suggest<strong>in</strong>g that the Isw2 complex forms an <strong>in</strong>accessible chromat<strong>in</strong> structure near the URS1<br />
element (Goldmark et al., 2000). These results suggest that Isw2 would be localized to the<br />
nucleus <strong>in</strong> vegetative growth media. However, this is not the case, Isw2 is localized to the<br />
cytoplasm <strong>in</strong> vegetative growth cultures, and to the nucleus, the cytoplasmic and sp<strong>in</strong>dle<br />
microtubuli, <strong>in</strong> meiotic cultures (Trachtulcova et al., 2000). There is no good explanation to<br />
20
clarify these contradict<strong>in</strong>g results. Isw2 is required for <strong>meiosis</strong>, cells deleted for ISW2 arrest under<br />
meiotic conditions prior to premeiotic DNA replication, and asci are not formed (Trachtulcova et<br />
al., 2000). It is not known whether Isw2 is required for the transcription <strong>of</strong> EMG under meiotic<br />
conditions, nor why cells deleted for ISW2 are sporulation deficient.<br />
B. Expression <strong>of</strong> early MSG under meiotic conditions.<br />
1. Ume6 is also a positive regulator. Ume6 is required for the transcription <strong>of</strong> EMG under<br />
meiotic conditions (Bowdish and Mitchell, 1993; Bowdish et al., 1995; Steber and Esposito,<br />
1995; Strich et al., 1994; Szent-Gyorgyi, 1995). In addition, S<strong>in</strong>3 and Rpd3 are required for their<br />
high level <strong>of</strong> expression (Lamb and Mitchell, 2001). Ume6, S<strong>in</strong>3 and Rpd3 are present and<br />
physically associate <strong>in</strong> both vegetative growth and meiotic conditions (Lamb and Mitchell, 2001).<br />
When tethered to heterologous genes, Ume6 can serve as either a transcriptional repressor or<br />
activator, depend<strong>in</strong>g on S<strong>in</strong>3/Rpd3 and Ime1, respectively (Bowdish et al., 1995; Kadosh and<br />
Struhl, 1997). Different doma<strong>in</strong>s <strong>in</strong> Ume6 are required for transcriptional repression and<br />
activation (Bowdish et al., 1995; Washburn and Esposito, 2001). The Ume6T99N mutant prote<strong>in</strong><br />
is competent <strong>in</strong> repression <strong>of</strong> EMG <strong>in</strong> growth conditions, but is defective <strong>in</strong> their expression<br />
under meiotic conditions (Bowdish et al., 1995). On the other hand, the M530T mutation <strong>in</strong><br />
UME6 has a defect only <strong>in</strong> repression (Washburn and Esposito, 2001). In agreement, the<br />
transcriptional activator, Ime1 associates with Ume6(1-232) (Rub<strong>in</strong>-Bejerano et al., 1996), while<br />
the transcriptional repressor, S<strong>in</strong>3, with Ume6(515-530) (Kadosh and Struhl, 1997; Kasten et al.,<br />
1997; Washburn and Esposito, 2001). The positive and negative roles <strong>of</strong> Ume6 are mediated<br />
through its b<strong>in</strong>d<strong>in</strong>g to the URS1 element whose presence is required for complete expression <strong>of</strong><br />
EMG <strong>in</strong> starved cells (Bowdish et al., 1995).<br />
2. The function <strong>of</strong> Ime1. IME1 encodes as positive regulator <strong>of</strong> <strong>meiosis</strong>. Over-expression<br />
<strong>of</strong> Ime1 bypasses the requirement for MATa1 and MATα2 gene products for sporulation (Kassir<br />
et al., 1988). Diploid cells deleted for IME1 arrest under meiotic conditions <strong>in</strong> G1, as unbudded<br />
cells (Foiani et al., 1996; Kassir et al., 1988), prior to execution <strong>of</strong> any meiotic event, namely,<br />
expression <strong>of</strong> <strong>meiosis</strong>-specific genes, premeiotic DNA replication, commitment to meiotic<br />
recomb<strong>in</strong>ation, meiotic divisions, and spore formation (Foiani et al., 1996; Kassir et al., 1988;<br />
Mitchell et al., 1990; Smith and Mitchell, 1989). The requirement <strong>of</strong> IME1 for transcription<br />
suggests that it might encode a transcription factor. However, its predicted am<strong>in</strong>o acid sequence<br />
does not show any homology to known DNA-b<strong>in</strong>d<strong>in</strong>g motifs (Sherman et al., 1993; Smith et al.,<br />
21
1993). Nevertheless, when Ime1 is tethered to heterologous genes it functions as a potent<br />
transcriptional activator (Mandel et al., 1994; Smith et al., 1993).<br />
As described above, the transcription <strong>of</strong> IME1 is subject to extensive <strong>regulation</strong> by the MAT<br />
alleles and nutrients. On top <strong>of</strong> it, translation <strong>of</strong> IME1 mRNA is regulated by nutrients (Sherman<br />
et al., 1993). In vegetative growth media with acetate as the sole carbon source low but<br />
substantial levels <strong>of</strong> IME1 or ime1-lacZ mRNAs are observed, but Ime1-lacZ prote<strong>in</strong> is not<br />
detected. On the other hand, upon nitrogen depletion, the level <strong>of</strong> IME1 mRNA is not <strong>in</strong>duced <strong>in</strong><br />
MATa/MATa cells, but Ime1-lacZ prote<strong>in</strong> is readily observed (Sherman et al., 1993). Furthermore,<br />
α-factor and heat-shock treatment <strong>in</strong>creases the transcription <strong>of</strong> IME1, but translation occurs only<br />
<strong>in</strong> cells arrested <strong>in</strong> G1 by either α-factor, or the CDC mutations cdc28-4 and cdc4-3 (Sherman et<br />
al., 1993). These results suggest that nitrogen and/or cell cycle progression <strong>in</strong>hibits translation <strong>of</strong><br />
IME1 mRNA. S<strong>in</strong>ce nitrogen depletion leads to a G1 arrest, it is possible that the effect <strong>of</strong><br />
nitrogen is <strong>in</strong>direct, and that a G1 arrest is a prerequisite for efficient translation. IME1 has an<br />
atypical 5 UTR (untranslated region), 229 bp long (Sherman et al., 1993), which might mediate<br />
this <strong>regulation</strong>. Sequence analysis reveals that this RNA region can form stem-and-loop<br />
secondary structure that might <strong>in</strong>hibit translation. However, deletion <strong>of</strong> this region has no effect<br />
on the translation <strong>of</strong> IME1 mRNA (Ben- Dov, 1994). Thus, it is not known how nitrogen and/or<br />
G1 phase controls the efficiency <strong>of</strong> translation <strong>of</strong> IME1 mRNA.<br />
Over expression <strong>of</strong> Ime1 <strong>in</strong> acetate growth media does not <strong>in</strong>duce <strong>meiosis</strong> and sporulation <strong>in</strong><br />
logarithmic cultures <strong>of</strong> wild-type diploids (Colom<strong>in</strong>a et al., 1999; Sherman et al., 1993). On the<br />
other hand, <strong>in</strong> diploid cells arrested <strong>in</strong> G1 by recessive temperature sensitive mutations <strong>in</strong><br />
CDC28, CDC4, CDC25, CDC35 (CYR1), or concomitant deletion <strong>of</strong> the three CLN genes, high<br />
percentages <strong>of</strong> asci are formed (Colom<strong>in</strong>a et al., 1999; Sherman et al., 1993; Shilo et al., 1978),<br />
suggest<strong>in</strong>g that post-translational modification <strong>of</strong> Ime1 or another factor required for <strong>meiosis</strong><br />
depends on lack <strong>of</strong> function <strong>of</strong> these prote<strong>in</strong>s. Indeed, <strong>in</strong> vegetative cultures, depend<strong>in</strong>g on the<br />
Cdc28/Cln function, Ime1 is phosphorylated and sequestered from the nucleus (Colom<strong>in</strong>a et al.,<br />
1999). The localization <strong>of</strong> Ime1 <strong>in</strong> the cytoplasm prevents its transcriptional activation function,<br />
and entry <strong>in</strong>to <strong>meiosis</strong>. Cdc4 is an F-box prote<strong>in</strong> [for review see (Patton et al., 1998)] required for<br />
degradation <strong>of</strong> specific targets <strong>in</strong> G1. Interest<strong>in</strong>gly, one <strong>of</strong> its substrates is Far1, an <strong>in</strong>hibitor <strong>of</strong><br />
Cln/Cdc28 function (Henchoz et al., 1997). Thus, the effect <strong>of</strong> Cdc4 on sporulation may be<br />
mediated through Cdc28/Cln function. The role <strong>of</strong> Cdc25 and Cdc35, the two positive regulators<br />
<strong>of</strong> PKA on the function <strong>of</strong> Ime1 is most probably through their effect on the association <strong>of</strong> Ime1<br />
with Ume6 (see below).<br />
22
It is not clear if the activity <strong>of</strong> Ime1 as a transcriptional activator is subject to <strong>regulation</strong>. Us<strong>in</strong>g<br />
the SK1 stra<strong>in</strong> and a LexA-Ime1 fusion, it was shown that transcriptional activation <strong>of</strong> the<br />
reporter gene lexAop-lacZ depends on nitrogen depletion and the presence <strong>of</strong> a prote<strong>in</strong> k<strong>in</strong>ase,<br />
Rim11 (Smith et al., 1993). Genetic analysis showed that <strong>in</strong> this system Rim11 was required to<br />
relieve a repression activity <strong>of</strong> Ime1 that was modulated by the C-term<strong>in</strong>al doma<strong>in</strong> <strong>of</strong> Ime1. This<br />
conclusion was based on the follow<strong>in</strong>g results: i. A lexA-Ime1 fusion truncated for the C-term<strong>in</strong>al<br />
66 am<strong>in</strong>o acids activated transcription <strong>of</strong> lexAop-lacZ <strong>in</strong> rim11∆ cells (Smith et al., 1993). ii.<br />
LexA-Ime1L321F mutant prote<strong>in</strong> is impaired <strong>in</strong> both association with Rim11 and transcriptional<br />
activation (Malathi et al., 1997). On the other hand, us<strong>in</strong>g the S288C stra<strong>in</strong> and a Gal4(bd)-Ime1<br />
fusion, it was shown that transcriptional activation <strong>of</strong> the reporter gene gal1-lacZ is <strong>in</strong>dependent<br />
<strong>of</strong> growth conditions or Rim11 (Mandel et al., 1994; Rub<strong>in</strong>-Bejerano et al., 1996). In both stra<strong>in</strong>s<br />
Rim11 is required for the transcription <strong>of</strong> EMG and sporulation (Mandel et al., 1994; Mitchell<br />
and Bowdish, 1992). The reasons for the disparity between these reports are not known, but<br />
several possibilities can be suggested. i. The use <strong>of</strong> different stra<strong>in</strong> backgrounds, S288C and SK1.<br />
ii. The b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> the Gal4(bd) and lexA to the DNA requires its dimerization. The gal4(bd)-<br />
IME1 gene <strong>in</strong>cludes the Gal4 dimerization signal, whereas the lexA-IME1 gene might lack the<br />
lexA dimerization sequence (Golemis and Brent, 1992). Under meiotic conditions Ime1 can<br />
oligomerize, and this activity depends on Rim11 (Rub<strong>in</strong>-Bejerano et al., 1996). Therefore, it is<br />
possible that the ability <strong>of</strong> the lexA-Ime1 prote<strong>in</strong> to activate transcription only under meiotic<br />
conditions and only <strong>in</strong> the RIM11 stra<strong>in</strong> is due to the ability <strong>of</strong> the prote<strong>in</strong> to dimerize and b<strong>in</strong>d<br />
the DNA only under these conditions.<br />
Deletion and mutation analysis reveals that Ime1 is composed <strong>of</strong> at least two doma<strong>in</strong>s essential<br />
for <strong>meiosis</strong>: a transcriptional activation doma<strong>in</strong> (ad) (am<strong>in</strong>o acids 165-228), and an <strong>in</strong>teraction<br />
doma<strong>in</strong> (id) (am<strong>in</strong>o acids 270-360) (Mandel et al., 1994; Smith et al., 1993). The N-term<strong>in</strong>al 160<br />
am<strong>in</strong>o acids are not essential for <strong>meiosis</strong> (Mandel et al., 1994). However, an Ime1 prote<strong>in</strong><br />
truncated for this doma<strong>in</strong> gives rise to lower levels <strong>of</strong> asci <strong>in</strong> comparison to cells express<strong>in</strong>g this<br />
truncated prote<strong>in</strong> fused to the Gal4(bd), suggest<strong>in</strong>g that the later might either provide a nuclear<br />
localization signal or <strong>in</strong>crease the stability <strong>of</strong> the prote<strong>in</strong> (Mandel et al., 1994).<br />
Two hybrid assays reveal that Ime1 <strong>in</strong>teracts with Ume6, and that this <strong>in</strong>teraction is through<br />
am<strong>in</strong>o acids 270-360 <strong>of</strong> Ime1 [Ime1(id)] and am<strong>in</strong>o acids 1-232 <strong>of</strong> Ume6 [Ume6(id)] (Colom<strong>in</strong>a<br />
et al., 1999; Rub<strong>in</strong>-Bejerano et al., 1996; Xiao and Mitchell, 2000). The validity <strong>of</strong> this<br />
<strong>in</strong>teraction is evident from the use <strong>of</strong> different stra<strong>in</strong> backgrounds and different two-hybrid<br />
systems, namely, the Gal4(bd)-Ime1(id) with Ume6(id)-Gal4(ad) (Rub<strong>in</strong>-Bejerano et al., 1996;<br />
Xiao and Mitchell, 2000), and the tetR-Ime1(id) with Ume6(id)-VP16 (Colom<strong>in</strong>a et al., 1999).<br />
23
However, direct demonstration <strong>of</strong> physical association between Ime1 and Ume6 is still miss<strong>in</strong>g.<br />
The use <strong>of</strong> the two-hybrid assay enables the demonstration that this <strong>in</strong>teraction is negatively<br />
regulated by both glucose and nitrogen. The <strong>in</strong>teraction is absent <strong>in</strong> vegetative growth conditions<br />
with glucose as the sole carbon source, and excessive <strong>in</strong>teraction takes place under meiotic<br />
conditions (SPM media) (Colom<strong>in</strong>a et al., 1999; Rub<strong>in</strong>-Bejerano et al., 1996). Shift<strong>in</strong>g glucose<br />
grown stationary cells to acetate growth media leads to partial <strong>in</strong>teraction (Rub<strong>in</strong>-Bejerano et al.,<br />
1996), suggest<strong>in</strong>g that the <strong>in</strong>teraction <strong>of</strong> Ime1 with Ume6 depends on the absence <strong>of</strong> both glucose<br />
and nitrogen. Furthermore, this <strong>in</strong>teraction is absolutely dependent on Rim11 (Colom<strong>in</strong>a et al.,<br />
1999; Rub<strong>in</strong>-Bejerano et al., 1996), and partially dependent on Rim15 (Vidan and Mitchell,<br />
1997).<br />
Two models were suggested for the role <strong>of</strong> Ime1 Ume6 and Rim11 <strong>in</strong> the transcription <strong>of</strong><br />
EMG. I. Bowdish et al proposed that Ume6 is converted from a repressor to an activator<br />
follow<strong>in</strong>g phosphorylation by Rim11, and that Ime1, which associates with Rim11 (Bowdish et<br />
al., 1994; Rub<strong>in</strong>-Bejerano et al., 1996), is required to recruit Rim11 to Ume6 (Bowdish et al.,<br />
1995). ii. Rub<strong>in</strong>-Bejerano et al proposed that Ume6 recruits Ime1 to the URS1 element present <strong>in</strong><br />
EMG, and that this recruitment promotes the Ime1 transcriptional activation doma<strong>in</strong> to <strong>in</strong>duce the<br />
transcription <strong>of</strong> EMG. Accord<strong>in</strong>g to the later, Rim11 is required for the association between Ime1<br />
and Ume6 (Rub<strong>in</strong>-Bejerano et al., 1996). The follow<strong>in</strong>g results support the second model: i. Ime1<br />
functions as a transcriptional activator (Mandel et al., 1994; Smith et al., 1993). ii. Fusion <strong>of</strong> an<br />
heterologous transcriptional activation doma<strong>in</strong>, that <strong>of</strong> Gal4, to Ime1(id), leads to the expression<br />
<strong>of</strong> the EMG, IME2, and sporulation (Mandel et al., 1994). Moreover, this Ime1(id)-Gal4(ad)<br />
fusion prote<strong>in</strong> promotes <strong>meiosis</strong> <strong>of</strong> ime1∆ diploid cells only when galactose is added to the<br />
sporulation media (Mandel et al., 1994). S<strong>in</strong>ce galactose is required to relieve repression <strong>of</strong> Gal80<br />
on the transcriptional activation <strong>of</strong> Gal4(ad) (Johnston and Carlson, 1992), it was suggested that<br />
the normal function <strong>of</strong> Ime1 is to activate transcription (Mandel et al., 1994). iii. A Gal4(ad)-<br />
Ume6(159-836) fusion prote<strong>in</strong> that is expressed from the IME1 promoter bypasses the<br />
requirement for IME1 for the transcription <strong>of</strong> EMG and sporulation: it leads to expression <strong>of</strong> the<br />
EMG, HOP1, and to 40% sporulation (Rub<strong>in</strong>-Bejerano et al., 1996). iv. A Gal4(ad)-Ume6 fusion<br />
prote<strong>in</strong> can activate the transcription <strong>of</strong> the EMG SPO13 <strong>in</strong> SD media if it carries mutations that<br />
prevent its association with S<strong>in</strong>3 (Washburn and Esposito, 2001). In the absence <strong>of</strong> the Gal4(ad),<br />
these ume6 mutants do not promote expression <strong>of</strong> EMG (Washburn and Esposito, 2001). These<br />
results suggest that by itself Ume6 does not function as a transcriptional activator, and its<br />
conversion <strong>in</strong>to a positive regulator depends on the recruitment <strong>of</strong> the transcriptional activation<br />
doma<strong>in</strong> <strong>of</strong> Ime1 (Rub<strong>in</strong>-Bejerano et al., 1996; Washburn and Esposito, 2001). Currently, this is<br />
24
the established model, however, the ultimate pro<strong>of</strong> would be the demonstration that Ime1 is<br />
present on the complex formed on URS1, and/or that it associates with the transcription<br />
mach<strong>in</strong>ery. Both models assume that s<strong>in</strong>ce Ime1 functions through Ume6, deletions <strong>of</strong> either one<br />
<strong>of</strong> these genes would have a similar phenotype <strong>in</strong> meiotic cultures. However, ime1∆ diploids<br />
arrest <strong>in</strong> G1, whereas ume6∆ cells ma<strong>in</strong>ly arrest at G2. We assume that the expression <strong>of</strong> EMG <strong>in</strong><br />
vegetative cultures promotes the entry and progression through some meiotic events.<br />
Ime1 has at least two functions, it supplies a transcriptional activation doma<strong>in</strong>, but it is also<br />
required to relieve repression <strong>of</strong> S<strong>in</strong>3/Rpd3 (Washburn and Esposito, 2001). Diploid cells deleted<br />
for IME1 and express<strong>in</strong>g Gal4(ad)-Ume6 from the ADH1 promoter are sporulation deficient<br />
(Washburn and Esposito, 2001), but the Gal4(ad)-Ume6(M530T) mutant prote<strong>in</strong> promotes low<br />
levels <strong>of</strong> sporulation (10%). S<strong>in</strong>ce the latter prote<strong>in</strong> is defective <strong>in</strong> association with S<strong>in</strong>3, it was<br />
concluded that Ime1 is required to relieve repression mediated by the S<strong>in</strong>3/Rpd3 complex<br />
(Washburn and Esposito, 2001).<br />
The <strong>in</strong>teraction between Ime1 and Ume6 is regulated by nutrients, Rim11 and Rim15. S<strong>in</strong>ce<br />
two-hybrid assays were used to determ<strong>in</strong>e this <strong>in</strong>teraction, the regulated expression <strong>of</strong> the reporter<br />
genes might result from <strong>regulation</strong> at any <strong>of</strong> the follow<strong>in</strong>g levels: i. Regulated physical<br />
association between Ime1 and Ume6, or ii. Regulated transcriptional activation by the<br />
Ume6/Ime1 complex. The latter model assumes that Ime1 and Ume6 physically associate under<br />
all growth conditions, but <strong>in</strong> glucose growth media the result<strong>in</strong>g complex does not function as a<br />
transcriptional activator. This hypothesis is supported by the follow<strong>in</strong>g results. i. In vegetative<br />
growth media association <strong>of</strong> Ume6 with the S<strong>in</strong>3/Rpd3 complex represses transcription (Kadosh<br />
and Struhl, 1997). ii. A po<strong>in</strong>t mutation <strong>in</strong> Ume6 that prevents its association with S<strong>in</strong>3 promote<br />
transcriptional activation when the mutant prote<strong>in</strong> is fused to Gal4(ad) (Washburn and Esposito,<br />
2001). Direct demonstration <strong>of</strong> regulated (or not) physical association between Ime1 and Ume6 is<br />
required to dist<strong>in</strong>guish between these models.<br />
In accord with the role <strong>of</strong> Ime1 as a transcriptional activator, the prote<strong>in</strong> is localized to the<br />
nucleus (Colom<strong>in</strong>a et al., 1999; Rub<strong>in</strong>-Bejerano et al., 1996; Smith et al., 1993). Localization <strong>of</strong><br />
Ime1 is <strong>in</strong>dependent <strong>of</strong> Rim11 (Rub<strong>in</strong>-Bejerano et al., 1996), but as described above, it is<br />
<strong>in</strong>hibited by the Cln/Cdc28 function (Colom<strong>in</strong>a et al., 1999). Fig. 10 schematically illustrates how<br />
nutrients control the availability and activity <strong>of</strong> Ime1<br />
25
3. Prote<strong>in</strong>s required for the association <strong>of</strong> Ime1 with Ume6.<br />
3.1. The function <strong>of</strong> Rim11 and its homologs Mck1 and Mrk1. Rim11 is absolutely<br />
required for the <strong>in</strong>teraction between Ime1 and Ume6 (Colom<strong>in</strong>a et al., 1999; Rub<strong>in</strong>-Bejerano et<br />
al., 1996), the expression <strong>of</strong> EMG, and spore formation (Bowdish et al., 1994; Rub<strong>in</strong>-Bejerano et<br />
al., 1996). Rim11 associates with Ime1(id) under all growth conditions (Bowdish et al., 1994;<br />
Rub<strong>in</strong>-Bejerano et al., 1996), and <strong>in</strong> vitro it phosphorylates both Ime1 (Bowdish et al., 1994) and<br />
Ume6 (Malathi et al., 1997). The Rim11 homologs Mck1 and Mrk1 have only m<strong>in</strong>or effects on<br />
these processes (Neigeborn and Mitchell, 1991; Rabitsch et al., 2001).<br />
The transcription <strong>of</strong> RIM11 is constitutive <strong>in</strong> vegetative and meiotic conditions; however, <strong>in</strong><br />
ime1∆ cells its expression <strong>in</strong> meiotic cultures is reduced, probably reflect<strong>in</strong>g the use <strong>of</strong> the URS1<br />
like site present <strong>in</strong> its promoter (Bowdish et al., 1994). The level <strong>of</strong> Rim11 prote<strong>in</strong> is slightly<br />
reduced <strong>in</strong> glucose growth media <strong>in</strong> comparison to acetate (Bowdish et al., 1994). Rim11<br />
functions as a k<strong>in</strong>ase that shows autophosphorylation (Bowdish et al., 1994). Rim11 is<br />
phosphorylated on tyros<strong>in</strong>e 199 (Zhan et al., 2000). Tyros<strong>in</strong>e to phenylalan<strong>in</strong>e mutation <strong>in</strong> this<br />
residue results <strong>in</strong> impaired expression <strong>of</strong> ime2-lacZ <strong>in</strong> vivo, and <strong>in</strong> a defect <strong>in</strong> phosphorylation<br />
Ume6 <strong>in</strong> vitro (Zhan et al., 2000). This mutation does not impair autophosphorylation (Zhan et<br />
al., 2000), suggest<strong>in</strong>g different requirement for phosphorylation <strong>of</strong> exogenous substrates (see<br />
section IIID1).<br />
Rim11 associates with the C-term<strong>in</strong>al doma<strong>in</strong> <strong>of</strong> Ime1, am<strong>in</strong>o acids 270-360 (Malathi et al.,<br />
1999; Rub<strong>in</strong>-Bejerano et al., 1996). In vitro, Rim11 phosphorylates the C-term<strong>in</strong>al 20 am<strong>in</strong>o<br />
acids residues <strong>in</strong> this region <strong>of</strong> Ime1 (Malathi et al., 1999). Simultaneous mutations <strong>of</strong> 8 ser<strong>in</strong>e<br />
threon<strong>in</strong>e and tyros<strong>in</strong>e <strong>of</strong> these residues have the follow<strong>in</strong>g effects: i. In vitro phosphorylation <strong>of</strong><br />
Ime1 by Rim11 is absent, ii. Ime1 does not <strong>in</strong>teract with Ume6, iii. IME2 is not expressed, and iv.<br />
Asci are not formed (Malathi et al., 1999). Ime1 is detected by antibodies directed aga<strong>in</strong>st<br />
phosphotyros<strong>in</strong>es, and tyros<strong>in</strong>e to phenylalan<strong>in</strong>e mutation <strong>in</strong> this doma<strong>in</strong> results <strong>in</strong> impaired<br />
expression <strong>of</strong> ime2-lacZ <strong>in</strong> vivo, suggest<strong>in</strong>g that Rim11 phosphorylates these tyros<strong>in</strong>e resides <strong>in</strong><br />
Ime1.<br />
Rim11 controls <strong>meiosis</strong> by regulat<strong>in</strong>g not only Ime1, but also Ume6. Us<strong>in</strong>g the two-hybrid<br />
method, coIP, and <strong>in</strong> vitro k<strong>in</strong>ase assay, Malathi et al show that Rim11 physically associates with<br />
Ume6, it phosphorylates Ume6(1-232), and the association between Rim11 and Ume6 is<br />
<strong>in</strong>dependent <strong>of</strong> Ime1 (Malathi et al., 1997). In addition, the two-hybrid assay reveals <strong>in</strong>teraction<br />
between Ume6 and Mck1 (Xiao and Mitchell, 2000). In vivo, Ume6 is a phosphoprote<strong>in</strong>, whose<br />
level <strong>of</strong> phosphorylation is <strong>in</strong>creased <strong>in</strong> vegetative growth media with acetate as the sole carbon<br />
source <strong>in</strong> comparison to glucose (Xiao and Mitchell, 2000). Moreover, hyper phosphorylation is<br />
26
observed upon nitrogen starvation (Xiao and Mitchell, 2000). S<strong>in</strong>gle deletion <strong>of</strong> RIM11, MCK1 or<br />
MRK1 has no effect on the <strong>in</strong> vivo pattern <strong>of</strong> phosphorylation <strong>of</strong> Ume6 (Xiao and Mitchell, 2000),<br />
but a substantial reduction <strong>in</strong> the pattern <strong>of</strong> phosphorylation is observed for the rim11∆ mck1∆<br />
mrk1∆ triple mutant (Xiao and Mitchell, 2000). The redundant function <strong>of</strong> these GSK3-β<br />
homologs is also evident <strong>in</strong> cells carry<strong>in</strong>g the partially active rim11K68R allele. Efficient<br />
sporulation is observed for cells carry<strong>in</strong>g the MCK1 MRK1 wild type alleles, and no sporulation<br />
<strong>in</strong> either mck1∆ MRK1 or MCK1 mrk1∆ stra<strong>in</strong>s (Xiao and Mitchell, 2000). Ume6 sequence<br />
reveals several consensus sites for phosphorylation by the mammalian GSK3-β homolog.<br />
Simultaneous substitution <strong>of</strong> ser<strong>in</strong>e and threon<strong>in</strong>e residues to alan<strong>in</strong>e <strong>in</strong> one such site (T99A<br />
T103A T107) leads to a reduction <strong>in</strong> phosphorylation, and concomitantly a dramatic reduction <strong>in</strong><br />
the ability <strong>of</strong> Ume6(1-232) to <strong>in</strong>teract with Ime1, suggest<strong>in</strong>g that phosphorylation is a prerequisite<br />
for <strong>in</strong>teraction (Xiao and Mitchell, 2000).<br />
The two-hybrid method demonstrates that the <strong>in</strong>teraction between Rim11 and Ume6 is<br />
regulated by the carbon source; the <strong>in</strong>teraction is low <strong>in</strong> glucose growth media and it is <strong>in</strong>creased<br />
<strong>in</strong> acetate growth media (Malathi et al., 1997). However, coIP shows physical association<br />
between Ume6 and Rim11 that is <strong>in</strong>dependent on nutrients (Malathi et al., 1997). The authors<br />
suggest that this is due to lower levels <strong>of</strong> Galbd-Ume6 <strong>in</strong> glucose versus acetate growth media<br />
(Malathi et al., 1997). However, it is also possible that this is due to the <strong>in</strong>ability <strong>of</strong> the Gal4(bd)-<br />
Rim11/Gal4(ad)-Ume6 complex to activate transcription, due to the activity <strong>of</strong> S<strong>in</strong>3/Rpd3.<br />
Nevertheless, the ability <strong>of</strong> Rim11 to <strong>in</strong> vitro phosphorylate Ume6 is <strong>in</strong>creased when both<br />
prote<strong>in</strong>s are isolated from acetate grown cells, suggest<strong>in</strong>g that the carbon source regulates either<br />
the activity <strong>of</strong> Rim11, or the ability <strong>of</strong> its substrate, Ume6, to be phosphorylated (Malathi et al.,<br />
1997).<br />
The association <strong>of</strong> Ime1 with Ume6, and the association <strong>of</strong> Rim11 with both Ime1 and Ume6<br />
suggest that one association may serve as a scaffold for the second association. There are no<br />
direct evidence for association <strong>of</strong> Rim11 with Ime1 and Ume6 <strong>in</strong> cells deleted for UME6 or<br />
IME1, respectively. However, a Gal4ad-Ume6T99N mutant prote<strong>in</strong> shows no <strong>in</strong>teraction with<br />
Rim11 <strong>in</strong> a two-hybrid assay, and reduced <strong>in</strong>teraction with Ime1 (Malathi et al., 1997). These<br />
results suggest that either the association between Ume6 and Rim11 is required for the <strong>in</strong>teraction<br />
between Ime1 and Ume6 (Malathi et al., 1997), or that the Ume6 T99 residue is required for the<br />
association <strong>of</strong> Ume6 with both Rim11 and Ime1.<br />
3.2. The function <strong>of</strong> Rim15. Rim15 is absolutely required for the transcription <strong>of</strong> EMG<br />
such as IME2, HOP1 and SPO13 (Vidan and Mitchell, 1997). As described above (section<br />
IIIC1.2), Rim15 is required for the transcription <strong>of</strong> IME1, however, expression <strong>of</strong> IME1 from an<br />
27
heterologous promoter does not suppress this phenotype, suggest<strong>in</strong>g that Rim15 is required for an<br />
additional event. Indeed, Rim15 is required for efficient <strong>in</strong>teraction between Ime1 and Ume6, <strong>in</strong><br />
cells deleted for RIM15 5-fold reduction <strong>in</strong> their <strong>in</strong>teraction is observed (Vidan and Mitchell,<br />
1997). This effect is probably due to the effect <strong>of</strong> Rim15 on Ume6. It is required for complete <strong>in</strong><br />
vivo phosphorylation <strong>of</strong> Ume6 <strong>in</strong> SA and SPM media (Xiao and Mitchell, 2000). Rim15 is not<br />
required for the <strong>in</strong> vitro k<strong>in</strong>ase activity <strong>of</strong> Rim11 on Ime1 (Vidan and Mitchell, 1997).<br />
4. The function <strong>of</strong> Gcn5. The conversion <strong>of</strong> URS1 from a repression to activation element<br />
requires the function <strong>of</strong> the histone acetylase, Gcn5 (Burgess et al., 1999). Diploid cells carry<strong>in</strong>g<br />
a recessive mutation <strong>in</strong> GCN5 arrest under meiotic conditions prior to premeiotic DNA<br />
replication and meiotic recomb<strong>in</strong>ation (Burgess et al., 1999). These cells express IME1, but are<br />
defective <strong>in</strong> the transcription <strong>of</strong> IME2 (Burgess et al., 1999). Us<strong>in</strong>g antibodies directed aga<strong>in</strong>st<br />
acetylated histones H3 and H4, Burges et al., show that under meiotic conditions, depend<strong>in</strong>g on<br />
Gcn5, specific hyper acetylation on histone H3 is observed <strong>in</strong> the IME2 promoter (Burgess et al.,<br />
1999). Deletion <strong>of</strong> RPD3 that is required for deacetylation <strong>of</strong> Histone H4 <strong>in</strong> the IME2 promote,<br />
does not suppress gcn5, suggest<strong>in</strong>g that acetylation <strong>of</strong> histones is a prerequisite for the<br />
transcription <strong>of</strong> EMG (Burgess et al., 1999). It is not known how Gcn5 is recruited to the<br />
promoter <strong>of</strong> IME2 or additional EMG.<br />
5. The transcription <strong>of</strong> EMG is also regulated by positive elements. In addition to the<br />
negative element, URS1, the promoters <strong>of</strong> EMG carry positive elements required for their<br />
transcription under meiotic conditions: UASH element <strong>in</strong> HOP1, and the T4C sequence <strong>in</strong> IME2<br />
(Bowdish and Mitchell, 1993; Vershon et al., 1992). Homologous sequences are found <strong>in</strong><br />
additional EMG (Chu et al., 1998). Deletion or mutations <strong>of</strong> these elements prevent high-level<br />
expression <strong>of</strong> EMG (Bowdish and Mitchell, 1993; Vershon et al., 1992). Abf1 b<strong>in</strong>ds to the UASH<br />
element (Gailus-Durner et al., 1996). ABF1 encodes an essential DNA-b<strong>in</strong>d<strong>in</strong>g transcriptional<br />
activator required for the transcription <strong>of</strong> various genes as well as for DNA replication; it b<strong>in</strong>ds to<br />
orig<strong>in</strong>s <strong>of</strong> DNA replication (Svetlov and Cooper, 1995). The prote<strong>in</strong> that b<strong>in</strong>ds the T4C element <strong>in</strong><br />
IME2 is not known.<br />
6. Additional positive regulators. Ime2, Rim4, and Ndt80 are early and early late genes<br />
required for high-level transcription <strong>of</strong> EMG. Detailed description on their functions is given <strong>in</strong><br />
section VA.<br />
28
7. The role <strong>of</strong> premeiotic DNA replication and/or recomb<strong>in</strong>ation <strong>in</strong> controll<strong>in</strong>g EMG<br />
expression. Hydroxyurea (HU) is rout<strong>in</strong>ely used to <strong>in</strong>hibit DNA synthesis as it <strong>in</strong>hibits<br />
ribonucleotide reductase (Elford, 1968; Slater, 1973). Shift<strong>in</strong>g cells to meiotic conditions <strong>in</strong> the<br />
presence <strong>of</strong> 40 mM and 200 mM hydroxyurea leads to a reduction or no expression, respectively,<br />
<strong>of</strong> EMG (Davis et al., 2001; Lamb and Mitchell, 2001). It was suggested that perturbation <strong>of</strong><br />
premeiotic DNA replication <strong>in</strong>hibits the transcription <strong>of</strong> EMG. However, cells deleted for CLB5<br />
along with CLB6 or MUM2/SPOT8 arrest prior to premeiotic DNA replication, with complete<br />
expression <strong>of</strong> EMG (Davis et al., 2001; Dirick et al., 1998; Stuart and Wittenberg, 1998). These<br />
results imply that HU blocks DNA replication at different po<strong>in</strong>t than clb5 clb6 and mum2, and<br />
that only the HU block transmits a checkpo<strong>in</strong>t signal to repress transcription. However, it is also<br />
possible, that the effect <strong>of</strong> HU is not mediated through DNA replication. The latter hypothesis is<br />
strengthened by the observation that treatment <strong>of</strong> <strong>yeast</strong> cells with HU leads to a substantial<br />
decrease <strong>in</strong> RNA and prote<strong>in</strong> synthesis (Slater, 1973).<br />
The effect <strong>of</strong> HU is mediated through Rpd3 and S<strong>in</strong>3 whose presence is required for the<br />
reduction <strong>in</strong> transcription (Lamb and Mitchell, 2001). In addition, <strong>in</strong> the presence <strong>of</strong> HU there is a<br />
reduction <strong>in</strong> phosphorylation <strong>of</strong> Ume6, a phenomenon that is associated with reduction <strong>in</strong> the<br />
activity <strong>of</strong> Ume6 (Lamb and Mitchell, 2001).<br />
8. The choice between silenc<strong>in</strong>g and expression <strong>of</strong> EMG. Silenc<strong>in</strong>g and expression <strong>of</strong><br />
EMG are dependent on the URS1 element present <strong>in</strong> their promoter. URS1 serves as a silenc<strong>in</strong>g<br />
element <strong>in</strong> vegetative growth media with glucose as the sole carbon source, and is converted <strong>in</strong>to<br />
an activation element under meiotic conditions, i.e. nitrogen depletion <strong>in</strong> the presence <strong>of</strong> acetate.<br />
Fig. 11 summarizes the current knowledge and model for how this <strong>regulation</strong> is accomplished.<br />
Under all growth conditions Ume6 b<strong>in</strong>ds to URS1. However, nutrients control the prote<strong>in</strong><br />
complexes that are recruited by Ume6 to the URS1 element. In glucose growth media Ume6<br />
recruits two repression complexes, the HDAC S<strong>in</strong>3/Rpd3 that leads to deacetylation <strong>of</strong> lys<strong>in</strong>es <strong>in</strong><br />
histone H4, and the Isw2 complex that leads to chromat<strong>in</strong> remodel<strong>in</strong>g. These two activities are<br />
required for complete silenc<strong>in</strong>g <strong>of</strong> EMG. The S<strong>in</strong>3/Rpd3 complex is conserved, and functions as a<br />
transcriptional silencer <strong>in</strong> all eukaryotes (Ng and Bird, 2000; Paz<strong>in</strong> and Kadonaga, 1997).<br />
However, Ume6 is not conserved, and <strong>in</strong> mammals, it is the Mad/Max and nuclear receptors that<br />
recruit S<strong>in</strong>3/Rpd3 to the DNA (Ng and Bird, 2000; Paz<strong>in</strong> and Kadonaga, 1997). Similarly, the<br />
Isw2 complex is also conserved and functional <strong>in</strong> all eukaryotes [for review see (Cairns, 1998)].<br />
Under meiotic conditions (acetate media and nitrogen depletion) the S<strong>in</strong>3/Rpd3 and Isw2<br />
repression complexes are not functional. Relief <strong>of</strong> repression <strong>of</strong> S<strong>in</strong>3 depends on Ime1 that is<br />
29
expressed only <strong>in</strong> the absence <strong>of</strong> glucose. The mode by which Ime1 relieves repression <strong>of</strong><br />
S<strong>in</strong>3/Rpd3 and how Isw2 does not repress transcription under these conditions are not known.<br />
Relief <strong>of</strong> repression does not suffice for transcriptional activation, and requires Gcn5, the histone<br />
acetylase, as well as a transcriptional activation prote<strong>in</strong>, Ime1. Ume6 recruits Ime1 to URS1, and<br />
the <strong>in</strong>teraction between these two prote<strong>in</strong>s depends on the absence <strong>of</strong> glucose and nitrogen. These<br />
nutrient signals are transmitted to both Ime1 and Ume6, by two prote<strong>in</strong> k<strong>in</strong>ases, Rim15 and<br />
Rim11. S<strong>in</strong>ce Rim15 is required for complete phosphorylation <strong>of</strong> Ume6 <strong>in</strong> SA and SPM, and<br />
s<strong>in</strong>ce its k<strong>in</strong>ase activity is <strong>in</strong>hibited <strong>in</strong> the presence <strong>of</strong> glucose by PKA, it is assumed that the<br />
glucose signal that <strong>in</strong>hibits the association <strong>of</strong> Ime1 with Ume6 is transmitted, at least partially,<br />
through Rim15. S<strong>in</strong>ce Rim15 is only partially required for the <strong>in</strong>teraction <strong>of</strong> Ime1 with Ume6, the<br />
glucose signal must be transmitted through an additional prote<strong>in</strong>. Rim11 phosphorylates both<br />
Ime1 and Ume6, and this phosphorylation is essential for their <strong>in</strong>teraction. It is not known how<br />
nutrients regulate the function <strong>of</strong> Rim11.<br />
V. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> middle <strong>meiosis</strong>-specific genes (MMG).<br />
The regulated transcription <strong>of</strong> middle <strong>meiosis</strong>-specific genes (MMG) depends on the presence <strong>of</strong><br />
two positive elements, MSE (Middle Sporulation Element) (gNCRCAAAA/T) and an Abf1<br />
b<strong>in</strong>d<strong>in</strong>g site (Chu et al., 1998; Chu and Herskowitz, 1998; Hepworth et al., 1995; Hepworth et al.,<br />
1998; Ozsarac et al., 1995; Ozsarac et al., 1997; Pierce et al., 1998). The MSE elements present <strong>in</strong><br />
different MMG are not identical, some, for example that <strong>in</strong> CLB1, function only as positive<br />
elements, whereas others, for example that <strong>of</strong> SMK1 or NDT80 (designated <strong>in</strong>here as MSE*),<br />
function also as repression elements <strong>in</strong> vegetative growth conditions and early meiotic times<br />
(Pierce et al., 1998; Xie et al., 1999). Schematic illustration on the <strong>regulation</strong> <strong>of</strong> MMG is<br />
illustrated <strong>in</strong> Fig. 13, and discussed <strong>in</strong> the text below.<br />
A. Positive regulators <strong>of</strong> MMG.<br />
Ndt80 and Ime2 are two <strong>meiosis</strong>-specific positive regulators absolutely required for the<br />
transcription <strong>of</strong> MMG (Chu et al., 1998; Chu and Herskowitz, 1998; Hepworth et al., 1998;<br />
Ozsarac et al., 1997). The sequential transcription <strong>of</strong> MMG follow<strong>in</strong>g transcription <strong>of</strong> the early<br />
genes is due to the regulated transcription <strong>of</strong> both NDT80 and IME2 (Chu and Herskowitz, 1998;<br />
Hepworth et al., 1998).<br />
30
1. Ndt80. NDT80 encodes a transcriptional activator that b<strong>in</strong>ds to the MSE and MSE* elements<br />
and activates transcription (Chu and Herskowitz, 1998). Cells deleted for NDT80 arrest follow<strong>in</strong>g<br />
the completion <strong>of</strong> premeiotic DNA replication and meiotic recomb<strong>in</strong>ation, at pachytene, as mono<br />
nucleate cells (Chu and Herskowitz, 1998; Hepworth et al., 1998; Xu et al., 1995). Ectopic<br />
expression <strong>of</strong> Ndt80 <strong>in</strong> vegetative growth conditions is sufficient to <strong>in</strong>duce the transcription <strong>of</strong><br />
most MMG (probably the ones carry<strong>in</strong>g the MSE rather than the MSE* element) (Chu et al.,<br />
1998; Chu and Herskowitz, 1998), suggest<strong>in</strong>g that under growth conditions, the second positive<br />
regulator, Ime2, is non-essential. Ime2 is also non-essential under meiotic conditions. When<br />
Ndt80 is expressed from a heterologous, IME2 <strong>in</strong>dependent promoter, the MMG, SPS4, is<br />
expressed <strong>in</strong> both IME2 and ime2∆ cells (Pak and Segall, 2002a). Nevertheless, <strong>in</strong> the latter,<br />
expression <strong>of</strong> SPS4 is both delayed and lower than <strong>in</strong> the IME2 diploid cells, suggest<strong>in</strong>g that Ime2<br />
may be required for high and efficient activity <strong>of</strong> Ndt80 (Pak and Segall, 2002a). This hypothesis<br />
is supported by the observation that Ime2 phosphorylates Ndt80 (Benjam<strong>in</strong> et al., 2002). Ndt80 is<br />
a phosphoprote<strong>in</strong> (Benjam<strong>in</strong> et al., 2002; Tung et al., 2000), whose <strong>in</strong> vivo extent <strong>of</strong><br />
phosphorylation depends on IME2 (Benjam<strong>in</strong> et al., 2002). Furthermore, <strong>in</strong> vitro k<strong>in</strong>ase assay<br />
demonstrates that Ime2 directly phosphorylates Ndt80 (Benjam<strong>in</strong> et al., 2002).<br />
Ndt80 is also required for high levels <strong>of</strong> transcription <strong>of</strong> IME1 as well as for its transient<br />
expression (Hepworth et al., 1998). This effect might be mediated through Ime2, s<strong>in</strong>ce Ndt80<br />
regulates the transcription <strong>of</strong> IME2, through an MSE element present <strong>in</strong> its promoter (Chu and<br />
Herskowitz, 1998).<br />
NDT80 is a <strong>meiosis</strong>-specific gene whose transcription is dependent on Ime1 and Ime2 (Fig. 13)<br />
(Chu and Herskowitz, 1998; Hepworth et al., 1998; Pak and Segall, 2002a). This regulated<br />
transcription is mediated by two URS1 and two MSE elements (Chu and Herskowitz, 1998;<br />
Hepworth et al., 1998; Pak and Segall, 2002a). One MSE element functions either as a repression<br />
or activation element under growth and meiotic condition, respectively, whereas the second one<br />
functions only as an activation element (Pak and Segall, 2002a; Xie et al., 1999). Furthermore,<br />
both URS1 elements contribute to repression <strong>of</strong> NDT80 <strong>in</strong> vegetative cultures and for its high<br />
level expression under meiotic conditions [(Pak and Segall, 2002a) and Fig. 13]. The presence <strong>of</strong><br />
the MSE elements delays NDT80 transcription by the Ume6/Ime1 complex <strong>in</strong> comparison to<br />
other EMG that carry only the URS1 element (Chu and Herskowitz, 1998; Hepworth et al., 1998;<br />
Mitchell et al., 1990; Pak and Segall, 2002a; Yoshida et al., 1990).<br />
2. Ime2. IME2 encodes a <strong>meiosis</strong>-specific prote<strong>in</strong> k<strong>in</strong>ase that shows 58.9% similarity and<br />
37% identity to the human cycl<strong>in</strong>-dependent k<strong>in</strong>ase, CDK2 (Kom<strong>in</strong>ami et al., 1993). The crystal<br />
31
structure <strong>of</strong> hCDK2 reveals that cycl<strong>in</strong> A b<strong>in</strong>ds to two regions: PSTAIRE and the T-loop (Jeffrey<br />
et al., 1995). This b<strong>in</strong>d<strong>in</strong>g is required to <strong>in</strong>duce a conformational change that promotes activation<br />
<strong>of</strong> the k<strong>in</strong>ase. Ime2 does not carry the PSTAIRE sequence, but it shows 67% similarity and<br />
52.3% identity to the T-loop region <strong>of</strong> hCDK2 (Fig. 12). This homology raises the possibility that<br />
Ime2 might be activated by b<strong>in</strong>d<strong>in</strong>g to a cycl<strong>in</strong> like molecule, and that <strong>in</strong> the meiotic cycle it<br />
might replace Cdc28, the <strong>yeast</strong> CDK that regulates <strong>in</strong>itiation and progression <strong>in</strong> the cell cycle<br />
(Lew et al., 1997). The follow<strong>in</strong>g results support the hypothesis that <strong>in</strong> the meiotic cycle Ime2<br />
might replace Cdc28: i. Similarly to Cdc28, phosphorylation by Ime2 affects stability <strong>of</strong> Ime1<br />
Cdh1 and Sic1, the latter two prote<strong>in</strong>s are known substrates <strong>of</strong> Cdc28 <strong>in</strong> the mitotic cell cycle<br />
(Bolte et al., 2002; Dirick et al., 1998; Guttmann-Raviv and Kassir, 2002). ii. Ime2 is absolutely<br />
required for premeiotic DNA replication and meiotic recomb<strong>in</strong>ation <strong>in</strong> cdc28-4 cells <strong>in</strong>cubated at<br />
the non-permissive temperature (Guttmann-Raviv et al., 2001). However, there are no reports on<br />
any <strong>yeast</strong> prote<strong>in</strong>s that b<strong>in</strong>d to the T-loop like sequence <strong>in</strong> Ime2 and are required for its k<strong>in</strong>ase<br />
activity. Moreover, a recomb<strong>in</strong>ant Ime2 prote<strong>in</strong> isolated from E. coli is active <strong>in</strong> phosphorylat<strong>in</strong>g<br />
histone H1 (Donzeau and Bandlow, 1999), suggest<strong>in</strong>g that unlike Cdc28, Ime2 is active <strong>in</strong> the<br />
absence <strong>of</strong> additional <strong>yeast</strong> prote<strong>in</strong>s. However, phosphorylation <strong>of</strong> its native substrate, Gpa2 (see<br />
below) requires the isolation <strong>of</strong> Ime2 from <strong>yeast</strong> cells (Donzeau and Bandlow, 1999), suggest<strong>in</strong>g<br />
that specificity, for at least for some substrates, might require post-translation modification or the<br />
presence <strong>of</strong> an activator.<br />
Ime2 is a positive regulator <strong>of</strong> <strong>meiosis</strong> required for multiple functions: the correct tim<strong>in</strong>g and<br />
high level transcription <strong>of</strong> early <strong>meiosis</strong>-specific genes, the transcription <strong>of</strong> middle and late genes<br />
(Mitchell et al., 1990; Yoshida et al., 1990), the correct tim<strong>in</strong>g <strong>of</strong> entry <strong>in</strong>to premeiotic DNA<br />
replication meiotic recomb<strong>in</strong>ation and nuclear division (Benjam<strong>in</strong> et al., 2002; Foiani et al.,<br />
1996), as well as for ascus formation (Benjam<strong>in</strong> et al., 2002; Foiani et al., 1996; Mitchell et al.,<br />
1990; Yoshida et al., 1990). When Ime2 is over expressed, but only <strong>in</strong> the SK1 background, it<br />
bypasses the requirement for Ime1 for the transcription <strong>of</strong> EMG and sporulation (Mitchell et al.,<br />
1990), suggest<strong>in</strong>g an Ime1 <strong>in</strong>dependent pathway for activat<strong>in</strong>g the transcription <strong>of</strong> these genes<br />
(Mitchell et al., 1990). It is not known how Ime2, that encodes a k<strong>in</strong>ase, replaces a transcriptional<br />
activator, nor if under normal conditions this pathway has any contribution to the transcription <strong>of</strong><br />
these genes.<br />
Ime2 functions also as a negative regulator for the follow<strong>in</strong>g functions: i. Phosphorylation <strong>of</strong><br />
Ime1 by Ime2 leads to its degradation by the 26S proteasome (Guttmann-Raviv and Kassir,<br />
2002). It is possible that the absence <strong>of</strong> Ime1 reestablishes repression by Sok2 and S<strong>in</strong>3 [see<br />
sections IIIC1.1.2, IVB1.2, and (Shenhar and Kassir, 2001; Washburn and Esposito, 2001)],<br />
32
esult<strong>in</strong>g <strong>in</strong> the transient transcription <strong>of</strong> IME1 and EMG. Accord<strong>in</strong>g to this model, <strong>in</strong> cells<br />
deleted for IME2, the cont<strong>in</strong>uous presence <strong>of</strong> Ime1 leads to the non-transient expression <strong>of</strong> IME1<br />
and EMG (Mitchell et al., 1990; Yoshida et al., 1990). ii. In the meiotic cycle, upon completion <strong>of</strong><br />
premeiotic DNA replication and the presence <strong>of</strong> Ime2, two subunits <strong>of</strong> the DNA polymerase αprimase<br />
complex, Pol1 and Pol12, are degraded (Foiani et al., 1996). iii. The level <strong>of</strong> the<br />
Clb/Cdc28 <strong>in</strong>hibitor, Sic1, is reduced under meiotic conditions, a process regulated by Ime2<br />
(Dirick et al., 1998). iv. When Ime2 is ectopically expressed <strong>in</strong> the cell cycle it phosphorylates<br />
Cdh1, an event caus<strong>in</strong>g dissociation <strong>of</strong> Cdh1 from APC (anaphase promot<strong>in</strong>g complex – the<br />
ubiquit<strong>in</strong> ligase required for proteolysis <strong>of</strong> specific substrates), and consequently stabilization <strong>of</strong><br />
its substrates, such as Clb2 and Cdc5 (Bolte et al., 2002). It is not known if Ime2 phosphorylate<br />
Cdh1 <strong>in</strong> the meiotic cycle, and what are the consequences <strong>of</strong> this event for entry <strong>in</strong>to the meiotic<br />
divisions. v. In the meiotic cycle Ime2 is required to limit premeiotic DNA replication and<br />
nuclear division to one and two rounds, respectively (Foiani et al., 1996). It is assumed that this<br />
phenomenon results from stabilization <strong>of</strong> specific prote<strong>in</strong>s or regulators <strong>of</strong> DNA replication and<br />
nuclear divisions.<br />
The availability and function <strong>of</strong> Ime2 is regulated <strong>in</strong> several levels:<br />
<strong>Transcriptional</strong> <strong>regulation</strong>: The transcription <strong>of</strong> IME2 is silent <strong>in</strong> vegetative growth conditions by<br />
the b<strong>in</strong>d<strong>in</strong>g and activity <strong>of</strong> the Ume6/S<strong>in</strong>3/Rpd3 and Ume6/Isw2 complexes to the URS1 element<br />
present <strong>in</strong> its promoter. Under meiotic condition, recruitment <strong>of</strong> Ime1 by Ume6 promotes the<br />
transcription <strong>of</strong> Ime2. An additional histone deacetylase activity, that <strong>of</strong> the Set3/Hos2 complex,<br />
regulates the transcription <strong>of</strong> IME2 as well as NDT80 early <strong>in</strong> <strong>meiosis</strong> (Pijnappel et al., 2001).<br />
Deletion <strong>of</strong> SET3 or HOS2 has no effect dur<strong>in</strong>g vegetative growth (although this might be due to<br />
repression by S<strong>in</strong>3/Rpd3), but it leads to <strong>in</strong>creased and advanced transcription <strong>of</strong> IME2 and<br />
NDT80, as well as moderate <strong>in</strong>crease <strong>in</strong> the transcription <strong>of</strong> IME1 (Pijnappel et al., 2001).<br />
Consequently, these cells progress faster through MI, MII and asci formation (Pijnappel et al.,<br />
2001). Ime2 is subject to positive auto<strong>regulation</strong> that is most probably mediated by the MSE<br />
element present <strong>in</strong> its promoter and Ndt80 (see above, VA1).<br />
Prote<strong>in</strong> stability: Ime2 is an extremely non-stable prote<strong>in</strong> (Bolte et al., 2002; Guttmann-Raviv and<br />
Kassir, 2002) with a half-life <strong>of</strong> about 5 m<strong>in</strong>utes (Guttmann-Raviv and Kassir, 2002). It is not<br />
known if the stability <strong>of</strong> Ime2 is subject to any <strong>regulation</strong>.<br />
Activity <strong>regulation</strong>: Ime2 is subject to phosphorylation (Benjam<strong>in</strong> et al., 2002; Guttmann-Raviv<br />
and Kassir, 2002). In vitro k<strong>in</strong>ase assays reveals autophosphorylation (Benjam<strong>in</strong> et al., 2002;<br />
Guttmann-Raviv and Kassir, 2002; Kom<strong>in</strong>ami et al., 1993), but its effect on the activity and/or<br />
stability <strong>of</strong> Ime2 is not known. The k<strong>in</strong>ase activity <strong>of</strong> Ime2 is negatively regulated by its C-<br />
33
term<strong>in</strong>al non-essential doma<strong>in</strong> (Kom<strong>in</strong>ami et al., 1993). This is deduced from the follow<strong>in</strong>g<br />
observations: i. Over expression <strong>of</strong> an Ime2 prote<strong>in</strong> truncated for this doma<strong>in</strong> promotes <strong>meiosis</strong> <strong>in</strong><br />
the presence <strong>of</strong> either nitrogen or glucose (Kom<strong>in</strong>ami et al., 1993), and ii. The <strong>in</strong> vitro-k<strong>in</strong>ase<br />
activity <strong>of</strong> Ime2 is higher for a truncated Ime2 prote<strong>in</strong> <strong>in</strong> comparison to the wt prote<strong>in</strong> (Kom<strong>in</strong>ami<br />
et al., 1993). Thus, <strong>in</strong> the presence <strong>of</strong> nutrients Ime2 is less, or non-active. The nutrient signal is<br />
transmitted to the C-term<strong>in</strong>al doma<strong>in</strong> through the Gα prote<strong>in</strong>, Gpa2 (Donzeau and Bandlow,<br />
1999). Ime2 specifically associates with the GTP bound Gpa2 (Donzeau and Bandlow, 1999), a<br />
form whose level is <strong>in</strong>creased <strong>in</strong> the presence <strong>of</strong> glucose [for review see (Versele et al., 2001)]. In<br />
addition, the association between Gpa2 and Ime2 requires the presence <strong>of</strong> nitrogen (Donzeau and<br />
Bandlow, 1999). Cells deleted for GPA2 show similar phenotypes to cells over express<strong>in</strong>g the<br />
truncated Ime2 prote<strong>in</strong>, namely, sporulation <strong>in</strong> the presence <strong>of</strong> glucose and nitrogen (Donzeau<br />
and Bandlow, 1999). Gpa2 associates with the glucose receptor, Gpr1, and transmits the glucose<br />
signal to adenylate cyclase [for review see (Pan et al., 2000)], however, the effect <strong>of</strong> Gpa2 on<br />
Ime2 is <strong>in</strong>dependent <strong>of</strong> PKA (Donzeau and Bandlow, 1999). This is deduced from the<br />
observation that the bcy1-tpk1 w1 mutation that leads to low activity <strong>of</strong> PKA does not suppress the<br />
reduction <strong>in</strong> sporulation exhibited by cells express<strong>in</strong>g the constitutive active Gpa2G132V prote<strong>in</strong><br />
(Donzeau and Bandlow, 1999). The <strong>in</strong> vitro k<strong>in</strong>ase activity <strong>of</strong> Ime2 on histone H1 is decreased by<br />
the addition <strong>of</strong> recomb<strong>in</strong>ant Gpa2γS (Donzeau and Bandlow, 1999), suggest<strong>in</strong>g that Gpa2 <strong>in</strong>hibits<br />
the k<strong>in</strong>ase activity <strong>of</strong> Ime2 (Donzeau and Bandlow, 1999). In vitro k<strong>in</strong>ase assays also demonstrate<br />
that Ime2 phosphorylates Gpa2 (Donzeau and Bandlow, 1999), but the role <strong>of</strong> this<br />
phosphorylation is not known.<br />
Ime2 k<strong>in</strong>ase activity fluctuates <strong>in</strong> the meiotic cycle, it first peaks concomitantly with<br />
premeiotic DNA replication and then, concomitantly with nuclear division (Benjam<strong>in</strong> et al.,<br />
2002). The second <strong>in</strong>crease <strong>in</strong> activity is regulated by Cdc28 (Benjam<strong>in</strong> et al., 2002).<br />
The activity <strong>of</strong> Ime2 is also regulated by Ids2 (Sia and Mitchell, 1995). Over expression <strong>of</strong><br />
Ime2 <strong>in</strong> vegetative growth media is toxic (Bolte et al., 2002; Guttmann-Raviv and Kassir, 2002;<br />
Sia and Mitchell, 1995), and this toxicity is relived <strong>in</strong> cells deleted for IDS2 (Sia and Mitchell,<br />
1995), suggest<strong>in</strong>g that Ids2 is a positive regulator <strong>of</strong> Ime2. Ids2 is not required for <strong>meiosis</strong> (Sia<br />
and Mitchell, 1995), however, <strong>in</strong> its absence over expression <strong>of</strong> Ime2 (<strong>in</strong> the SK1 stra<strong>in</strong>) leads to<br />
the transcription <strong>of</strong> only the early <strong>meiosis</strong>-specific genes, while middle and late genes rema<strong>in</strong><br />
silent and spores are not formed (Sia and Mitchell, 1995). This result suggests that the activity <strong>of</strong><br />
Ime2 is dist<strong>in</strong>ctly regulated at early and middle meiotic times (Sia and Mitchell, 1995), <strong>in</strong><br />
agreement with the f<strong>in</strong>d<strong>in</strong>g <strong>of</strong> Benjam<strong>in</strong> et al., (Benjam<strong>in</strong> et al., 2002) reported above. The effect<br />
<strong>of</strong> Ids2 is mediated through the C-term<strong>in</strong>al doma<strong>in</strong> <strong>of</strong> Ime2: Over expression <strong>of</strong> Ime2 truncated<br />
34
for this doma<strong>in</strong> suppresses ime1∆ <strong>in</strong> both wt and ids2∆ cells (Sia and Mitchell, 1995). The steadystate<br />
level <strong>of</strong> Ids2 is decreased and then disappears <strong>in</strong> cells shifted to meiotic conditions (Sia and<br />
Mitchell, 1995).<br />
RIM4 is an EMG required for high-level expression <strong>of</strong> EMG, premeiotic DNA replication,<br />
timely and efficient commitment to meiotic recomb<strong>in</strong>ation, nuclear division, and spore formation<br />
(Deng and Saunders, 2001; Soushko and Mitchell, 2000). Rim4 function is mediated through<br />
both the Ime1 and Ime2 transcriptional activation pathways (Soushko and Mitchell, 2000). rim4∆<br />
diploids over-express<strong>in</strong>g Ime2 are sporulation pr<strong>of</strong>icient, suggest<strong>in</strong>g that Rim4 functions<br />
upstream <strong>of</strong> Ime2 (Soushko and Mitchell, 2000). Rim4 carries two RNA recognition motifs that<br />
are important for its role <strong>in</strong> <strong>meiosis</strong> (Soushko and Mitchell, 2000). The mode by which Rim4<br />
affects transcription is not known, though it was proposed that it might stabilize IME2 mRNA<br />
(Deng and Saunders, 2001; Soushko and Mitchell, 2000).<br />
3. S<strong>in</strong>3 and Rpd3. Random screen<strong>in</strong>g for mutations prevent<strong>in</strong>g expression <strong>of</strong> MMG<br />
identified NDT80 and SIN3 (Hepworth et al., 1998). S<strong>in</strong>3 as well as Rpd3 are required for the<br />
transcription <strong>of</strong> MMG, suggest<strong>in</strong>g that they function also as positive regulators, or that their<br />
function as positive regulators might be due to repression <strong>of</strong> a negative regulator (Hepworth et al.,<br />
1998). Diploid cells deleted for SIN3 or RPD3 arrest at the same po<strong>in</strong>t as ndt80 cells, namely,<br />
follow<strong>in</strong>g the completion <strong>of</strong> premeiotic DNA replication, as mono nucleate cells (Hepworth et al.,<br />
1998). Po<strong>in</strong>t <strong>of</strong> arrest is not due to defects <strong>in</strong> recomb<strong>in</strong>ation, s<strong>in</strong>ce concomitant deletion <strong>of</strong> SPO13<br />
and SPO11 does not allow a bypass <strong>of</strong> the arrest, and the triple mutant cells rema<strong>in</strong> sporulation<br />
deficient (Hepworth et al., 1998; Xu et al., 1995).<br />
B. Negative regulators <strong>of</strong> MMG.<br />
SMK1 is a middle gene subject to silenc<strong>in</strong>g by b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> Sum1 to the MSE* element <strong>in</strong> its<br />
promoter (Xie et al., 1999). Sum1 also represses the transcription <strong>of</strong> NDT80, <strong>in</strong> cells deleted for<br />
SUM1 premature expression <strong>of</strong> NDT80 is observed (Pak and Segall, 2002a). Sum1 associates<br />
with Hst1, and the result<strong>in</strong>g complex, conta<strong>in</strong><strong>in</strong>g additional prote<strong>in</strong>s, exhibits NAD + dependent<br />
histone deacetylase activity (Pierce et al., 1998; Pijnappel et al., 2001; Rusche and R<strong>in</strong>e, 2001).<br />
Deletion <strong>of</strong> either SUM1 or HST1 leads to expression <strong>of</strong> SMK1 <strong>in</strong> vegetative growth conditions<br />
(Xie et al., 1999). This expression is probably <strong>in</strong>dependent <strong>of</strong> Ndt80, s<strong>in</strong>ce NDT80 rema<strong>in</strong>s silent<br />
<strong>in</strong> sum1∆ or hst1∆ cells (Xie et al., 1999). Relief <strong>of</strong> repression is regulated by Sum1 availability:<br />
The transcription <strong>of</strong> SUM1 is constitutive, but the level <strong>of</strong> Sum1 prote<strong>in</strong> is reduced prior to<br />
expression <strong>of</strong> MMG, and then it is <strong>in</strong>duced aga<strong>in</strong>. As described above, the transcription <strong>of</strong> NDT80<br />
35
is dependent on Ime2, but <strong>in</strong> cells deleted for both IME2 and SUM1, NDT80 is expressed (Pak<br />
and Segall, 2002a). These results suggest that Ime2 abrogates Sum1 repression (Pak and Segall,<br />
2002a). It is possible, as <strong>in</strong> the case <strong>of</strong> Ime1 (Guttmann-Raviv and Kassir, 2002), that<br />
phosphorylation <strong>of</strong> Sum1 by Ime2 is required for the regulated degradation <strong>of</strong> Sum1. The<br />
Sum1/Hst1 repression activity can be bypassed <strong>in</strong> vegetative growth media by over expression <strong>of</strong><br />
Ndt80 (Xie et al., 1999). Sum1 and Hst1 are not required for <strong>meiosis</strong>, cells deleted for either gene<br />
complete <strong>meiosis</strong> and form viable spores (L<strong>in</strong>dgren et al., 2000).<br />
The S<strong>in</strong>3/Rpd3/Ume6 and Ssn6/Tup1 repression complexes that regulate transcription <strong>of</strong> EMG<br />
are not <strong>in</strong>volved with repression activity <strong>of</strong> MMG (Pierce et al., 1998).<br />
C. The recomb<strong>in</strong>ation checkpo<strong>in</strong>t.<br />
In <strong>meiosis</strong>, a checkpo<strong>in</strong>t mechanism consist<strong>in</strong>g <strong>of</strong> Rad17, Rad24, Mek1, Pch2, Mec3, and Ddc1<br />
prevents the transcription <strong>of</strong> MMG and entry <strong>in</strong>to the first meiotic division <strong>in</strong> cells that have not<br />
properly completed synapsis <strong>of</strong> homologs and meiotic recomb<strong>in</strong>ation (Roeder and Bailis, 2000).<br />
Diploid cells deleted for DMC1 are impaired <strong>in</strong> both meiotic recomb<strong>in</strong>ation (Bishop et al., 1992)<br />
and expression <strong>of</strong> MMG (Chu and Herskowitz, 1998; Hepworth et al., 1998), whereas dmc1 cells<br />
carry<strong>in</strong>g mutations <strong>in</strong> RAD17, or MEK1 express MMG (Chu and Herskowitz, 1998; Hepworth et<br />
al., 1998). Three targets <strong>of</strong> the pachytene checkpo<strong>in</strong>t were identified; these are the prote<strong>in</strong> k<strong>in</strong>ase<br />
Swe1 that phosphorylates and <strong>in</strong>activates Cdc28 (Leu and Roeder, 1999), the transcriptional<br />
activator Ndt80 (Pak and Segall, 2002b; Tung et al., 2000), and the transcriptional repressor<br />
Sum1 (L<strong>in</strong>dgren et al., 2000; Pak and Segall, 2002b). This is concluded from the observations<br />
that <strong>in</strong> the double mutants dmc1 swe1 and dmc1 sum1 MMG are expressed and cells enter the<br />
meiotic nuclear division (Leu and Roeder, 1999; L<strong>in</strong>dgren et al., 2000; Pak and Segall, 2002b;<br />
Tung et al., 2000). Over expression <strong>of</strong> Ndt80 leads to expression <strong>of</strong> MMG and partial entry <strong>in</strong>to<br />
nuclear division (Pak and Segall, 2002b). In this review we focus only on how this surveillance<br />
mechanism affects transcription [for a detailed review on the checkpo<strong>in</strong>t pathway see (Roeder<br />
and Bailis, 2000)].<br />
The pachytene checkpo<strong>in</strong>t leads to a reduction <strong>in</strong> the transcription <strong>of</strong> NDT80 (L<strong>in</strong>dgren et<br />
al., 2000; Pak and Segall, 2002b). However, contradict<strong>in</strong>g results, namely, no effect, were<br />
reported for other different stra<strong>in</strong>s (Chu and Herskowitz, 1998; Hepworth et al., 1998; Tung et al.,<br />
2000). Sum1 mediates the reduction <strong>in</strong> the transcription <strong>of</strong> NDT80. The availability <strong>of</strong> Sum1 is<br />
regulated by the pachytene checkpo<strong>in</strong>t as evident from the observations that <strong>in</strong> dmc1 cells the<br />
steady-state level <strong>of</strong> Sum1 is constitutive throughout the meiotic pathway, whereas <strong>in</strong> the double<br />
mutant dmc1 rad17 the level <strong>of</strong> Sum1 is reduced <strong>in</strong> meiotic prophase, as <strong>in</strong> the wild type stra<strong>in</strong><br />
36
(L<strong>in</strong>dgren et al., 2000). S<strong>in</strong>ce Ime2 mediates the availability <strong>of</strong> Sum1 under normal meiotic<br />
conditions, it is possible that the effect <strong>of</strong> the checkpo<strong>in</strong>t system on Sum1 is mediated through<br />
Ime2. Pak and Segall (Pak and Segall, 2002b) suggest that the effect <strong>of</strong> Sum1 is mediated only<br />
through regulat<strong>in</strong>g the transcription <strong>of</strong> NDT80, s<strong>in</strong>ce the triple mutant dmc1 sum1 ndt80 does not<br />
enter nuclear division (Pak and Segall, 2002b). Morovere, they propose that <strong>in</strong> the dmc1 sum1<br />
cells, expression <strong>of</strong> the B-type cycl<strong>in</strong>s is responsible for entry <strong>in</strong>to meiotic nuclear division (Pak<br />
and Segall, 2002b). This hypothesis is supported by the observation that entry <strong>in</strong>to nuclear<br />
division <strong>in</strong> swe1 hop2 cells is delayed <strong>in</strong> comparison to wild type cells or to swe1 hop2 cells over<br />
express<strong>in</strong>g Clb1 (Leu and Roeder, 1999). However, this model cannot expla<strong>in</strong> why ectopic<br />
expression <strong>of</strong> Ndt80 <strong>in</strong> dmc1 cells leads to <strong>in</strong>efficient entry <strong>in</strong>to nuclear division <strong>in</strong> comparison to<br />
the dmc1 sum1 cells (20% versus 60%) (Pak and Segall, 2002b). These results suggest that an<br />
additional target <strong>of</strong> Sum1 might be required for proper meiotic arrest (L<strong>in</strong>dgren et al., 2000).<br />
S<strong>in</strong>ce the CLB genes are not direct targets <strong>of</strong> Sum1, they are not the genes directly regulated by<br />
Sum1 (L<strong>in</strong>dgren et al., 2000).<br />
The pachytene checkpo<strong>in</strong>t <strong>in</strong>hibits phosphorylation <strong>of</strong> Ntd80 (Tung et al., 2000). In cells<br />
deleted for ZIP1 [a component <strong>of</strong> the synaptonemal complex required for synapsis <strong>of</strong> homologs<br />
(Sym et al., 1993)] Ndt80 is partially phosphorylated (Tung et al., 2000), whereas <strong>in</strong> the double<br />
mutants zip1 pch2 and zip1 mek1 it is highly phosphorylated (Tung et al., 2000). It was suggested<br />
that activation <strong>of</strong> transcription by Ndt80 requires its phosphorylation (Benjam<strong>in</strong> et al., 2002; Chu<br />
and Herskowitz, 1998; Hepworth et al., 1998; Tung et al., 2000). Therefore, lack <strong>of</strong> expression <strong>of</strong><br />
MMG is also due to the reduction <strong>in</strong> the phosphorylation <strong>of</strong> Ndt80. S<strong>in</strong>ce Ime2 is required for<br />
phosphorylation <strong>of</strong> Ndt80, it should be <strong>in</strong>terest<strong>in</strong>g to determ<strong>in</strong>e if Ime2 is the target for the<br />
pachytene check po<strong>in</strong>t system.<br />
In addition, the pachytene checkpo<strong>in</strong>t mediates the transcription <strong>of</strong> MMG through the Cdc28<br />
<strong>in</strong>hibitor, Swe1. In swe1 hop2 diploid cells CLB1 transcription is <strong>in</strong>creased to its normal level,<br />
although with a substantial delay, <strong>in</strong> comparison to wild type or hop2 cells (Leu and Roeder,<br />
1999). It should be <strong>in</strong>terest<strong>in</strong>g to determ<strong>in</strong>e whether this effect <strong>of</strong> Swe1 on Cdc28 is mediated<br />
through Ime2, s<strong>in</strong>ce Cdc28 is required for the activity <strong>of</strong> Ime2 that co<strong>in</strong>cides with nuclear<br />
divisions (Benjam<strong>in</strong> et al., 2002), and Ime2 is absolutely required for the transcription <strong>of</strong> the CLB<br />
genes.<br />
37
VI. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> late <strong>meiosis</strong>-specific genes<br />
A. Early late genes.<br />
SGA1 is an early-late gene encod<strong>in</strong>g a glucoamylase whose transcription <strong>in</strong> growth media is<br />
silenced by a negative element, NRE SGA , which exhibits no UAS activity (Kihara et al., 1991;<br />
Mai and Breeden, 2000). Expression under meiotic conditions depends on a UAS element present<br />
<strong>in</strong> its promoter region (Kihara et al., 1991). In addition, it carries 4 STRE elements (Mai and<br />
Breeden, 2000), but their role <strong>in</strong> the transcription <strong>of</strong> SGA1 is not known. Relief <strong>of</strong> repression by<br />
the NRE SGA element depends on Ime1 and Ime2. However, the activity <strong>of</strong> the UAS element is<br />
<strong>in</strong>dependent <strong>of</strong> these regulators, because it does not require the presence <strong>of</strong> both MATa and<br />
MATα alleles that are required for the expression <strong>of</strong> Ime1 (Kihara et al., 1991). The activity <strong>of</strong> the<br />
positive element requires nitrogen depletion (Kihara et al., 1991), demonstrat<strong>in</strong>g that the effect <strong>of</strong><br />
nitrogen on <strong>meiosis</strong> is mediated not only through Ime1.<br />
Silenc<strong>in</strong>g <strong>of</strong> SGA1 <strong>in</strong> vegetative growth media depends on the chromat<strong>in</strong>-remodel<strong>in</strong>g factor<br />
Isw2 (Goldmark et al., 2000). As described above (section IVA5), Isw2 is also a negative<br />
regulator <strong>of</strong> EMG, and it is recruited by Ume6 to their promoters. S<strong>in</strong>ce the URS1 element to<br />
which Ume6 b<strong>in</strong>ds is not present <strong>in</strong> SGA1, it is not known how Isw2 is recruited to SGA1. The<br />
identities <strong>of</strong> the positive regulators promot<strong>in</strong>g transcriptional activation <strong>of</strong> SGA1 are not known.<br />
However, one positive regulator, SME2, when present on a multi-copy plasmid, <strong>in</strong>creases the<br />
transcription <strong>of</strong> SGA1 <strong>in</strong> the presence <strong>of</strong> nitrogen, thus bypass<strong>in</strong>g also the nitrogen signal<br />
<strong>in</strong>hibit<strong>in</strong>g spore formation (Kawaguchi et al., 1992). Deletion <strong>of</strong> SME2 has no effect on the<br />
regulated expression <strong>of</strong> SGA1, or on the efficiency <strong>of</strong> sporulation. The sequence and mode <strong>of</strong><br />
function <strong>of</strong> SME2 are not known.<br />
B. Mid-late genes. The transcription <strong>of</strong> the mid-late sporulation-specific genes DIT1 and DIT2<br />
is <strong>in</strong>duced upon completion <strong>of</strong> meiotic divisions (Friesen et al., 1997). Time <strong>of</strong> expression is<br />
compatible with function, s<strong>in</strong>ce these divergently transcribed genes (Friesen et al., 1997) encode<br />
enzymes required for biosynthesis <strong>of</strong> the dityros<strong>in</strong>e precursor that is <strong>in</strong>corporated <strong>in</strong>to the<br />
outermost layer <strong>of</strong> the spore wall (Briza et al., 1990). A negative element, designated NRE DIT is<br />
required for repression dur<strong>in</strong>g vegetative growth (Friesen et al., 1997) (Note that NRE DIT is not<br />
identical to NRE SGA1 ). This region carries the middle sporulation-like element MSE, and the DRE<br />
element (Bogengruber et al., 1998; Friesen et al., 1997) that are required for repression <strong>in</strong> growth<br />
conditions and activation under meiotic conditions. Two additional positive elements are required<br />
for high expression under meiotic conditions (Friesen et al., 1997).<br />
38
<strong>Transcriptional</strong> activation <strong>of</strong> the mid late genes depends on three positive regulators, Ndt80<br />
and Ime2 whose effects are most probably mediated through the MSE like sequence (Friesen et<br />
al., 1997), and Rim1 (Rim101) through an unidentified region (Bogengruber et al., 1998). The<br />
effect <strong>of</strong> Rim1 may be <strong>in</strong>direct (see IIIC3). The repression activity <strong>of</strong> NRE DIT depends on Ssn6,<br />
Tup1, Rox3, S<strong>in</strong>4 and Spe3 (Friesen et al., 1997; Friesen et al., 1998). Rox3 and S<strong>in</strong>4 are<br />
subunits <strong>of</strong> the mediator complex <strong>of</strong> RNA polymerase II (Gustafsson et al., 1997; Myer and<br />
Young, 1998) that repress transcription <strong>of</strong> many genes (Hanna-Rose and Hansen, 1996). SPE3<br />
encodes Spermid<strong>in</strong>e synthase (Hamasaki-Katagiri et al., 1997), and accord<strong>in</strong>gly, addition <strong>of</strong><br />
spermid<strong>in</strong>e to the media leads to <strong>in</strong>creased repression (Friesen et al., 1998). The way by which<br />
these complexes are specifically recruited to NRE DIT is not known.<br />
XBP1 is a mid-late gene whose transcription is <strong>in</strong>itiated at the same time as that <strong>of</strong> DIT1,2, but<br />
unlike these genes its transcription is non-transient (Mai and Breeden, 2000). Accord<strong>in</strong>gly, its<br />
regulated transcription is not mediated via a NRE DIT element (Mai and Breeden, 2000). XBP1<br />
encodes a DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> that functions as a repressor when fused to lexA. Deletion <strong>of</strong><br />
XBP1 leads to a delay and reduction <strong>in</strong> the percentage <strong>of</strong> asci, while meiotic divisions are<br />
properly controlled (Mai and Breeden, 2000). It is not known how this effect <strong>of</strong> Xbp1 is<br />
mediated.<br />
C. Late genes<br />
The transcription <strong>of</strong> the 5 late meiotic genes peaks follow<strong>in</strong>g completion <strong>of</strong> meiotic divisions at<br />
8-12 hours <strong>in</strong> sporulation media (Law and Segall, 1988). A representative <strong>of</strong> these genes is<br />
SPS100 that is <strong>in</strong>volved <strong>in</strong> spore wall formation (Law and Segall, 1988). The transcription <strong>of</strong><br />
SPS100 depends on Ime1, Ndt80, and Ama1 (Cooper et al., 2000; Hepworth et al., 1998). AMA1<br />
encodes a <strong>meiosis</strong>-specific subunit <strong>of</strong> APC/C that is required for degradation <strong>of</strong> CLB1 <strong>in</strong> the<br />
meiotic cycle (Cooper et al., 2000). The transcription <strong>of</strong> AMA1 is <strong>in</strong>duced at the same time as<br />
MMG, but s<strong>in</strong>ce it does not carry the Ndt80 bid<strong>in</strong>g site (Chu et al., 1998) its mode <strong>of</strong> <strong>regulation</strong> is<br />
not known. In addition, <strong>meiosis</strong>-specific splic<strong>in</strong>g by the EMG Mer1 (Engebrecht and Roeder,<br />
1990) determ<strong>in</strong>es the accumulation <strong>of</strong> Ama1 prote<strong>in</strong> (Cooper et al., 2000). Diploid cells deleted<br />
for AMA1 arrest with a short sp<strong>in</strong>dle prior to the first meiotic division (Cooper et al., 2000).<br />
Genetic analysis suggests that Ama1 is dispensable for the second meiotic division, but is<br />
required for spore formation (Cooper et al., 2000). Further work is required to determ<strong>in</strong>e if Ama1<br />
is directly <strong>in</strong>volved with transcriptional <strong>regulation</strong> <strong>of</strong> late genes, for example by degradation <strong>of</strong> a<br />
transcriptional repressor, or if the effect on transcription is mediated by a checkpo<strong>in</strong>t mechanism.<br />
39
The transcriptional activator that b<strong>in</strong>ds to and activates transcription <strong>of</strong> the late genes is not<br />
known.<br />
The late <strong>meiosis</strong>-specific genes SPS100 and DTT1 show basal levels <strong>of</strong> expression <strong>in</strong><br />
vegetative growth conditions (Jung and Lev<strong>in</strong>, 1999). This expression depends on a functional<br />
MAPK cascade that transmits the cell wall <strong>in</strong>tegrity signal (Jung and Lev<strong>in</strong>, 1999). Activation <strong>of</strong><br />
the MAPK Slt2/Mpk1, or deletion <strong>of</strong> Rlm1, the transcription factor whose activity is regulated by<br />
it, reduces the expression the late <strong>meiosis</strong> specific genes (Jung and Lev<strong>in</strong>, 1999). It is not known<br />
whether Rlm1 b<strong>in</strong>ds the promoter region <strong>of</strong> late genes, or if it is required for their expression<br />
under meiotic conditions.<br />
VII. A feedback loop controll<strong>in</strong>g <strong>meiosis</strong>.<br />
The transcription <strong>of</strong> all <strong>meiosis</strong>-specific genes is transient, reflect<strong>in</strong>g the need for the function <strong>of</strong><br />
these genes for a limited period. Nevertheless, it is important to po<strong>in</strong>t that transient transcription<br />
does not necessarily reflect transient availability <strong>of</strong> prote<strong>in</strong>s. For <strong>in</strong>stance, <strong>in</strong> the mitotic cell<br />
cycle, the transcription <strong>of</strong> genes <strong>in</strong>volved <strong>in</strong> DNA replication, such as CLB5, POL1, or POL12 is<br />
periodic, and identically regulated (Lowndes et al., 1991; Schwob and Nasmyth, 1993). But the<br />
steady state level <strong>of</strong> Pol1 and Pol12 is constant (Foiani et al., 1995), whereas the level <strong>of</strong> Clb5 is<br />
periodic (Irniger and Nasmyth, 1997). Periodicity <strong>of</strong> Clb5 is accomplished by its regulated<br />
degradation (Irniger and Nasmyth, 1997). The pattern <strong>of</strong> prote<strong>in</strong> expression <strong>of</strong> many <strong>meiosis</strong>specific<br />
genes is not known, however, the two ma<strong>in</strong> positive regulators <strong>of</strong> <strong>meiosis</strong>, Ime1 and<br />
Ime2 are non-stable prote<strong>in</strong>s whose prote<strong>in</strong> levels mimic the level <strong>of</strong> their RNA. This suggests<br />
that at least <strong>in</strong> the case <strong>of</strong> Ime1 and Ime2, the transient availability might be a sign <strong>of</strong> requirement<br />
for only a short period. In agreement with this view, over expression <strong>of</strong> Ime1 <strong>in</strong> meiotic cultures<br />
leads (<strong>in</strong> some stra<strong>in</strong>s) to <strong>in</strong>efficient sporulation, a substantial <strong>in</strong>crease <strong>in</strong> non-disjunction, the<br />
formation <strong>of</strong> 2-spored rather than 4-spored asci, and a reduction <strong>in</strong> sporulation (Shefer-Vaida et<br />
al., 1995; Sherman, 1992).<br />
Positive feedback loops control the transcription <strong>of</strong> both IME1 and IME2 (Fig. 14). Positive<br />
auto<strong>regulation</strong> by Ime1 is required to relieve repression mediated by Sok2, and thus lead to an<br />
<strong>in</strong>crease <strong>in</strong> Ime1 level (Shenhar and Kassir, 2001). Positive auto<strong>regulation</strong> accelerates the<br />
availability <strong>of</strong> Ime1 and Ime2, and this enhanced expression might be essential for proper entry<br />
and progression through the meiotic cycle.<br />
The transient transcription <strong>of</strong> all <strong>meiosis</strong>-specific genes is expla<strong>in</strong>ed by the transient<br />
availability <strong>of</strong> Ime1, Ime2, and Ndt80. Phosphorylation <strong>of</strong> Ime1 by Ime2 leads to its degradation<br />
40
y the 26S proteasome (Guttmann-Raviv and Kassir, 2002). In the absence <strong>of</strong> Ime1, Sok2<br />
represses the transcription <strong>of</strong> IME1, lead<strong>in</strong>g to a decrease <strong>in</strong> IME1 mRNA. It is not known<br />
whether the negative feedback <strong>regulation</strong> <strong>of</strong> Ime2 on the transcription <strong>of</strong> IME1 is mediated only<br />
through its effect on Ime1. S<strong>in</strong>ce the transcription <strong>of</strong> IME2 depends on Ime1 (Mitchell et al.,<br />
1990; Yoshida et al., 1990) and the half-life <strong>of</strong> Ime2 is very short (Bolte et al., 2002; Guttmann-<br />
Raviv and Kassir, 2002), there are sufficient levels <strong>of</strong> Ime1 to relieve repression by S<strong>in</strong>3<br />
(Washburn and Esposito, 2001) and to activate transcription <strong>of</strong> EMG. In the absence <strong>of</strong> Ime1 lack<br />
<strong>of</strong> these two functions leads to shutt<strong>in</strong>g down the transcription <strong>of</strong> EMG. The transcription <strong>of</strong><br />
MMG and LMG depends on Ime2 (Chu et al., 1998; Chu and Herskowitz, 1998; Friesen et al.,<br />
1997; Hepworth et al., 1998; Kihara et al., 1991; Ozsarac et al., 1997; Smith and Mitchell, 1989).<br />
In cells depleted for Ime1, Ime2 is absent, and the transcription <strong>of</strong> the early late and late genes is<br />
closed.<br />
VIII. Conclud<strong>in</strong>g remark. The choice between developmental pathways<br />
S. cerevisiae is a simple eukaryote with limited developmental options. Diploid cells may chose<br />
between growth <strong>in</strong> <strong>yeast</strong> or pseudohyphal forms, and <strong>meiosis</strong>. Two crucial mechanisms regard<strong>in</strong>g<br />
developmental decisions are required for faithful growth, entry <strong>in</strong>to a specific developmental<br />
pathway should take place only <strong>in</strong> response to specific signals, and should be a signal to block<br />
entry <strong>in</strong>to an alternative pathway. Concomitant entry <strong>in</strong>to two developmental pathways could lead<br />
to chaos. In S. cerevisiae, the MATa and MATα gene products as well as the pachytene<br />
surveillance mechanisms ensure that only diploid cells can <strong>in</strong>itiate <strong>meiosis</strong>. This is a crucial<br />
decision, because <strong>meiosis</strong> <strong>in</strong> haploid cells would lead to the formation <strong>of</strong> uneuploid gametes.<br />
In S. cerevisiae, the meiotic cycle ends with the formation <strong>of</strong> dormant spores resistant to<br />
hazardous conditions. The decision to exit the mitotic cell cycle depends on the availability <strong>of</strong><br />
nutrients. Entry <strong>in</strong>to the meiotic cycle <strong>in</strong> the presence <strong>of</strong> nutrients can be a “waste”. Several<br />
redundant mechanisms ensure that <strong>in</strong> the presence <strong>of</strong> glucose the meiotic pathway will be<br />
repressed: i. The transcription <strong>of</strong> IME1 is repressed <strong>in</strong> the presence <strong>of</strong> glucose due to the presence<br />
<strong>of</strong> dist<strong>in</strong>ct negative elements (i.e. UASru, IREu, UCS1), each respond<strong>in</strong>g to a different signal<br />
pathway. Thus, if one signal pathway is malfunction<strong>in</strong>g, the activity <strong>of</strong> the additional signal<br />
pathways will ensure repression. Moreover, several UAS elements (i.e. UASru, IREu, UASrm),<br />
each regulated <strong>in</strong> a dist<strong>in</strong>ct manner, activates transcription <strong>in</strong> the absence <strong>of</strong> glucose. ii. The<br />
activity <strong>of</strong> Ime1 is repressed <strong>in</strong> the presence <strong>of</strong> glucose by two dist<strong>in</strong>ct mechanisms. a.<br />
Phosphorylation <strong>of</strong> Ime1 by the Cln/Cdc28 k<strong>in</strong>ase sequesters the prote<strong>in</strong> from the nucleus, and b.<br />
41
Functional <strong>in</strong>teraction between Ime1 and Ume6 that promote transcriptional activation is<br />
<strong>in</strong>hibited <strong>in</strong> the presence <strong>of</strong> glucose and nitrogen. The presence <strong>of</strong> nitrogen also prevents<br />
translation <strong>of</strong> IME1 mRNA and entry <strong>of</strong> Ime1 <strong>in</strong>to the nucleus.<br />
The presence <strong>of</strong> glucose also <strong>in</strong>hibits the function <strong>of</strong> Ime2. Normally Ime2 is available only<br />
under meiotic conditions, but cells developed mechanisms to ensure that <strong>in</strong> the presence <strong>of</strong><br />
nutrients accidental expression <strong>of</strong> Ime2 will not lead to entry and completion <strong>of</strong> <strong>meiosis</strong>. In the<br />
presence <strong>of</strong> glucose activated Gpa2 b<strong>in</strong>ds to and <strong>in</strong>hibits the function <strong>of</strong> Ime2. Furthermore, Ime2<br />
is a non-stabile prote<strong>in</strong>, thus under growth conditions the low level <strong>of</strong> Ime2 does not suffice for<br />
<strong>in</strong>itiation <strong>of</strong> <strong>meiosis</strong> <strong>in</strong> the absence <strong>of</strong> Ime1.<br />
Yeast cells use the same regulators, but <strong>in</strong> reverse directions, to control alternative<br />
developmental pathways. Ime2 is a positive regulator <strong>of</strong> <strong>meiosis</strong> that prevents pseudohyphae<br />
development <strong>in</strong> growth media with acetate as the sole carbon source (Donzeau and Bandlow,<br />
1999). The activity <strong>of</strong> the cAMP/PKA signal pathway is a major player <strong>in</strong> determ<strong>in</strong><strong>in</strong>g the<br />
developmental choice <strong>yeast</strong> cells make. Entry <strong>in</strong>to mitosis with either the <strong>yeast</strong> form or<br />
filamentous morphology requires high activity <strong>of</strong> this pathway [for reviews see (Gancedo, 2001)],<br />
whereas entry <strong>in</strong>to <strong>meiosis</strong> requires low PKA activity (Matsumoto et al., 1983). Sok2 is a positive<br />
regulator <strong>of</strong> mitosis (Ward et al., 1995) and a negative regulator for the transcription <strong>of</strong> IME1,<br />
<strong>meiosis</strong>, and filamentous growth (Shenhar and Kassir, 2001; Ward et al., 1995). The negative role<br />
<strong>of</strong> Sok2 <strong>in</strong> pseudohyphae formation is not clear s<strong>in</strong>ce its C. albicans homolog, Efg1, that is a<br />
negative regulator <strong>of</strong> <strong>meiosis</strong> when expressed <strong>in</strong> S. cerevisiae [complement<strong>in</strong>g sok2∆ (Shenhar<br />
and Kassir, 2001)], is a positive regulator <strong>of</strong> filamentous growth <strong>in</strong> both S. cerevisiae and C.<br />
albicans (Stoldt et al., 1997). Msn2/4 exhibit reverse tasks, it functions as a negative regulator <strong>of</strong><br />
the mitotic cell cycle (Smith et al., 1998) and filamentous growth (Stanhill et al., 1999) and as a<br />
positive regulator for the transcription <strong>of</strong> IME1 and <strong>meiosis</strong> (Shenhar and Kassir, 2001). The<br />
activity <strong>of</strong> Sok2 and Msn2/4 <strong>in</strong> grow<strong>in</strong>g cells requires their phosphorylation by PKA (Gorner et<br />
al., 1998; Shenhar and Kassir, 2001; Smith et al., 1998; Ward et al., 1995). The use <strong>of</strong> the same<br />
regulators, but <strong>in</strong> reverse directions for different developmental pathways, ensures that one<br />
pathway will be an alternative to the other one. When cells enter mitosis, <strong>meiosis</strong> will be<br />
repressed, whereas when cells enter <strong>meiosis</strong>, mitosis will be blocked. Thus, a s<strong>in</strong>gle signal<br />
transduction pathway, the cAMP/PKA, is sufficient to control two alternative developmental<br />
pathways.<br />
Acknowledgment. We thank M. Foiani for his hospitality while writ<strong>in</strong>g this review, for fruitful<br />
discussions and critical read<strong>in</strong>g <strong>of</strong> the review.<br />
42
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Figure legends<br />
Fig. 1. Developmental choices <strong>of</strong> MATa/MATα diploid cells <strong>of</strong> Saccharomyces cerevisiae.<br />
In the presence <strong>of</strong> carbon and nitrogen sources cells adopt the <strong>yeast</strong> form morphology; upon<br />
nitrogen limitation and <strong>in</strong> the presence <strong>of</strong> high levels <strong>of</strong> glucose, a dimorphic transition to a<br />
filamentous growth rem<strong>in</strong>iscent <strong>of</strong> hyphae takes places. In the absence <strong>of</strong> nitrogen and glucose<br />
and the presence <strong>of</strong> acetate as the sole carbon source cell enter the meiotic cycle, form<strong>in</strong>g four<br />
haploid spores engulfed <strong>in</strong> a sac, the ascus.<br />
Fig. 2. A transcriptional cascade governs <strong>in</strong>itiation <strong>of</strong> <strong>meiosis</strong>.<br />
The meiotic signals, i.e. the presence <strong>of</strong> Mata1 and Matα2, the absence <strong>of</strong> glucose and nitrogen<br />
and the presence <strong>of</strong> acetate leads to G1 arrest as well as to the expression and activity <strong>of</strong> Ime1.<br />
IME1 encodes a transcriptional activator required for the transcription <strong>of</strong> early <strong>meiosis</strong>-specific<br />
genes (EMG). EMG are <strong>in</strong>volved with premeiotic DNA replication and meiotic recomb<strong>in</strong>ation.<br />
Ndt80 and Ime2, a transcriptional activator and a prote<strong>in</strong> k<strong>in</strong>ase, whose transcription depends on<br />
Ime1, are required for the transcription <strong>of</strong> middle <strong>meiosis</strong>-specific genes (MMG). MMG are<br />
<strong>in</strong>volved with nuclear division and spore formation. The transcription <strong>of</strong> the late <strong>meiosis</strong>-specific<br />
genes (LMG) depends on Ime1 and Ime2, and these genes are required for spore maturation. The<br />
nutrient signal has a direct affect also on the activity <strong>of</strong> Ime2 and the transcription <strong>of</strong> LMG.<br />
An arrow represents a positive role, a l<strong>in</strong>e a negative role. A scissors symbolizes the negative<br />
feedback role <strong>of</strong> Ime2 <strong>in</strong> regulat<strong>in</strong>g degradation <strong>of</strong> Ime1.<br />
Fig. 3. Schematic structure <strong>of</strong> IME1 5’ untranslated region.<br />
The regulated transcription <strong>of</strong> IME1 is mediated by the comb<strong>in</strong>atorial effect <strong>of</strong> dist<strong>in</strong>ct elements.<br />
The MAT signal mediates repression activity <strong>of</strong> 2 elements UCS3 and UCS4. The carbon source<br />
signal is transmitted to four elements. UCS1, UASru and IREu function as repression elements <strong>in</strong><br />
the presence <strong>of</strong> glucose. In addition, UASru IREu, as well as UASrm function as activation<br />
elements <strong>in</strong> the absence <strong>of</strong> glucose and the presence <strong>of</strong> acetate as the sole carbon source. UCS1<br />
functions as a negative element <strong>in</strong> the presence <strong>of</strong> nitrogen.<br />
Filled boxs - elements required for transcriptional activation. Open boxs – elements whose<br />
function is only to repress transcription. A positive role is marked with an arrow, a negative role<br />
by a l<strong>in</strong>e. Larger effects are denoted by heavier l<strong>in</strong>es while lesser effects are denoted by slender<br />
l<strong>in</strong>es.<br />
59
Fig. 4. The function <strong>of</strong> positive and negative elements regulat<strong>in</strong>g the transcription <strong>of</strong> IME1.<br />
IME1 with various portions <strong>of</strong> its 5’ untranslated region were fused at the sixth am<strong>in</strong>o acid to the<br />
E.coli lacZ gene. The chimeric genes were <strong>in</strong>tegrated at the LEU2 loci <strong>of</strong> a MATa/MATα diploid<br />
(stra<strong>in</strong> Y4122, S288C background). Samples were taken from 1x10 7 cells grown <strong>in</strong> either SD<br />
(synthetic glucose) or PSP2 (SA – synthetic acetate (Kassir and Simchen, 1991). In addition, cells<br />
grown <strong>in</strong> PSP2 to 1x10 7 were washed once <strong>in</strong> water and resuspended <strong>in</strong> SPM (sporulation media<br />
(Kassir and Simchen, 1991). Samples were taken to extract prote<strong>in</strong>s and measure lacZ levels after<br />
3 and 6 h <strong>in</strong> SPM. The level <strong>of</strong> β-Galactosidase is given <strong>in</strong> Miller units. The results are the<br />
averages <strong>of</strong> three to five <strong>in</strong>dependent transformants. Standard deviations were less than 10%. The<br />
studied elements are marked as filled boxes. A nested deletion is marked by a l<strong>in</strong>e. NT – not<br />
tested.<br />
Fig. 5. Cdc25 transmits the nitrogen signal that modulates the repression activity <strong>of</strong> UCS1, a<br />
URS element <strong>in</strong> IME1 5’ region.<br />
UCS1 was <strong>in</strong>serted between HIS4 UAS and the TATA box <strong>in</strong> a his4-lacZ chimera. The level <strong>of</strong><br />
β-Galactosidase was determ<strong>in</strong>ed <strong>in</strong> three to five <strong>in</strong>dependent transformants. Standard deviations<br />
were less than 10%. The results are given as relative levels <strong>in</strong> comparison (<strong>in</strong> each case) to the<br />
level <strong>of</strong> expression <strong>of</strong> UASHIS4-his4-lacZ (control).<br />
Insertion <strong>of</strong> UCS1 <strong>in</strong> the heterologous UASHIS4-his4-lacZ chimera leads to a substantial reduction<br />
<strong>in</strong> its expression <strong>in</strong> vegetative growth conditions. Partial relief <strong>of</strong> repression is observed upon<br />
nitrogen depletion, suggest<strong>in</strong>g that the activity <strong>of</strong> UCS1 as a URS element is mediated by<br />
nitrogen. Depletion <strong>of</strong> Cdc25 by shift<strong>in</strong>g cdc25-5 cells to the non-permissive temperature leads to<br />
relief <strong>of</strong> repression <strong>in</strong> vegetative growth media, and has no effect upon nitrogen depletion (SPM),<br />
suggest<strong>in</strong>g that Cdc25 transmits the nitrogen signal to UCS1.<br />
Fig. 6: The UAS activity <strong>of</strong> the IME1 elements UASru, IREu and UASrm is modulated by<br />
the carbon source.<br />
His4-lacZ fusions carry<strong>in</strong>g various elements from IME1 5’ untranslated region were <strong>in</strong>tegrated at<br />
the LEU2 loci <strong>of</strong> a MATa/MATα diploid (stra<strong>in</strong> Y4122, S288C background). Cells were grown <strong>in</strong><br />
either SD (synthetic glucose) or PSP2 (SA – synthetic acetate (Kassir and Simchen, 1991). In<br />
addition, cells grown <strong>in</strong> PSP2 to 1x10 7 were washed once <strong>in</strong> water and resuspended <strong>in</strong> SPM<br />
[sporulation media (Kassir and Simchen, 1991)] and <strong>in</strong>cubated for 6 hours. Samples were taken<br />
from 1x10 7 cells/ml to extract prote<strong>in</strong>s and measure lacZ levels. The level <strong>of</strong> β-Galactosidase is<br />
60
given <strong>in</strong> Miller units. The results are the averages <strong>of</strong> three to five <strong>in</strong>dependent transformants.<br />
Standard deviations were less than 10%.<br />
UASru, IREu and UASrm support the expression <strong>of</strong> his4-lacZ, suggest<strong>in</strong>g that all three elements<br />
possess a UAS activity. The UAS activity is low <strong>in</strong> SD, and <strong>in</strong>creased activity is observed <strong>in</strong> SA<br />
and SPM media, suggest<strong>in</strong>g that their activity is repressed by glucose and/or depends on acetate.<br />
The IREd element that is homologus to IREu (see Fig. 8) shows very low UAS activity.<br />
Fig. 7. cAMP reduces sporulation and the expression <strong>of</strong> IME1 through the IREu element.<br />
MATa/MATα diploid cells (stra<strong>in</strong> Y4122, S288C background) carry<strong>in</strong>g ime1-lacZ or his4-lacZ<br />
fusions with various elements from IME1 5’ untranslated region <strong>in</strong>tegrated at the LEU2 loci.<br />
Cells were grown <strong>in</strong> PSP2 [synthetic acetate (Kassir and Simchen, 1991)] with or without 10mM<br />
cAMP to 1x10 7 cells/ml, washed once <strong>in</strong> water and resuspended <strong>in</strong> SPM [sporulation media<br />
(Kassir and Simchen, 1991)] with or without 10mM cAMP, respectively. Samples were taken at 6<br />
hours <strong>in</strong> SPM. The level <strong>of</strong> β-Galactosidase is given <strong>in</strong> Miller units. The results are the averages<br />
<strong>of</strong> three to five <strong>in</strong>dependent transformants. Standard deviations were less than 10%. At 24 hours<br />
<strong>in</strong> SPM samples were taken to count the percentage <strong>of</strong> asci. Results are given as relative levels.<br />
Addition <strong>of</strong> cAMP leads to a reduction <strong>in</strong> sporulation (72.6% and 49.8% <strong>in</strong> the absence and<br />
presence <strong>of</strong> cAMP, respectively) as well as a reduction <strong>in</strong> the expression <strong>of</strong> ime1-lacZ. We<br />
assume that the m<strong>in</strong>or effect is due to the function <strong>of</strong> the cAMP specific phosphodiesterases.<br />
Addition <strong>of</strong> cAMP reduces the UAS activity <strong>of</strong> IREu while it has no effect on the activity <strong>of</strong><br />
either UASru or UASrm.<br />
Fig. 8. IREu and IREd are almost identical repeats <strong>in</strong> IME1 5’ region carry<strong>in</strong>g the known<br />
UAS sequences STRE and SCB.<br />
Sequence alignments <strong>of</strong> IREu (upstream) and IREd (downstream) to the stress response element<br />
(STRE) and the Swi4/6 Cycle box (SCB). We suggest that Msn2 b<strong>in</strong>ds the STRE element <strong>in</strong><br />
IREu s<strong>in</strong>ce Msn2 b<strong>in</strong>ds IREu as well as STRE elements <strong>in</strong> stress response genes. We further<br />
suggest that Sok2 b<strong>in</strong>ds the SCB element s<strong>in</strong>ce it is highly homologous to the DNA-b<strong>in</strong>d<strong>in</strong>g<br />
doma<strong>in</strong> <strong>of</strong> Swi4 that b<strong>in</strong>ds such elements, and s<strong>in</strong>ce genetic analysis reveals that it b<strong>in</strong>ds IREu<br />
(see text). The physical association between Sok2 and Msn2 suggests that they b<strong>in</strong>d the DNA as a<br />
heterodimer.<br />
61
Fig. 9. A schematic structure <strong>of</strong> IME1 5’ untranslated region show<strong>in</strong>g the positive and<br />
negative transcription factors which regulate its expression.<br />
The regulated transcription <strong>of</strong> IME1 is mediated by the comb<strong>in</strong>atorial effect <strong>of</strong> dist<strong>in</strong>ct elements.<br />
The MAT signal is transmitted to UCS4 by Rme1. The activity <strong>of</strong> Rme1 as a repressor requires<br />
Rgr1 and S<strong>in</strong>4. The cAMP/PKA signal transduction pathway transmits the glucose signal to IREu<br />
through two transcription factors, Sok2 and Msn2. In glucose growth media phosphorylation <strong>of</strong><br />
Sok2 by PKA promotes its repression activity, whereas phosphorylation <strong>of</strong> Msn2 sequesters the<br />
prote<strong>in</strong> <strong>in</strong> the cytoplasm. In acetate growth media Sok2 repression is relieved by a decrease <strong>in</strong> the<br />
activity <strong>of</strong> PKA, as well as by the function <strong>of</strong> Ime1. The <strong>in</strong>creased b<strong>in</strong>d<strong>in</strong>g <strong>of</strong> Msn2 to IREu leads<br />
to transcriptional activation. Yhp1 b<strong>in</strong>ds to UASv, however, its activity is not required for the<br />
transcription <strong>of</strong> IME1. Nitrogen, via Cdc25, regulates the URS activity <strong>of</strong> UCS1. It is not known<br />
whether the effect <strong>of</strong> Cdc25 is mediated through the cAMP/PKA and/or MAPK pathways.<br />
Filled box - elements required for transcriptional activation. Open box – elements that whose<br />
function is only to repress transcription. A positive role is marked with an arrow, a negative role<br />
<strong>in</strong> l<strong>in</strong>e. Dashed l<strong>in</strong>es and a question mark – prelim<strong>in</strong>ary data.<br />
Fig. 10. The expression and activity <strong>of</strong> Ime1 are regulated by nutrients.<br />
Schematic representation on how glucose and nitrogen regulates the availability and activity <strong>of</strong><br />
Ime1. The nucleus is depicted between the two circular l<strong>in</strong>es, and cell membrane by a heavy l<strong>in</strong>e.<br />
A positive role is marked with an arrow, a negative role by a straight l<strong>in</strong>e. The transcription <strong>of</strong><br />
IME1 is <strong>in</strong>hibited <strong>in</strong> the presence <strong>of</strong> glucose and nitrogen. Translation <strong>of</strong> IME1 mRNA is<br />
<strong>in</strong>hibited <strong>in</strong> the presence <strong>of</strong> a nitrogen source. In the presence <strong>of</strong> nitrogen, Ime1is sequestered<br />
from the nuclei due to phosphorylation by Cdc28/Cln. Interaction <strong>of</strong> Ime1 with Ume6, and<br />
consequently transcriptional activation <strong>of</strong> early <strong>meiosis</strong>-specific genes (EMG) is <strong>in</strong>hibited by<br />
glucose and nitrogen.<br />
Fig. 11. Conditions lead<strong>in</strong>g to the choice between silenc<strong>in</strong>g and expression <strong>of</strong> early <strong>meiosis</strong>specific<br />
genes.<br />
A. Vegetative growth conditions B. Meiotic conditions.<br />
Ume6 b<strong>in</strong>ds to the URS1 element <strong>in</strong> early-<strong>meiosis</strong> specific genes (EMG) under all growth<br />
conditions. In vegetative growth conditions Ume6 recruits to repression complexes, S<strong>in</strong>3/Rpd3<br />
and Isw2, lead<strong>in</strong>g to histone H4 deacetylase and chromat<strong>in</strong> remodel<strong>in</strong>g, respectively, and<br />
silenc<strong>in</strong>g. Under these conditions Rim15 and most probably Rim11 are non-active. The activity<br />
<strong>of</strong> Rim15 is repressed by PKA (Tpk1,2,3) phosphorylation. Under meiotic conditions (acetate<br />
62
media and nitrogen depletion) phosphorylation <strong>of</strong> Ime1and Ume6 by Rim11 and Rim15 promotes<br />
the <strong>in</strong>teraction <strong>of</strong> Ime1 with Ume6. Ime1 function is required to relieve repression by S<strong>in</strong>3 and to<br />
activate transcription. In addition, transcriptional activation depends on histone acetylation by<br />
Gcn5.<br />
Fig. 12. Ime2 is highly homologous to hCDK2.<br />
A. Sequence alignment <strong>of</strong> Ime2 to hCDK2. B, Ribbon depiction <strong>of</strong> the crystal structure <strong>of</strong><br />
hCDK2 (Prote<strong>in</strong> Data Bank structure code 1HCL). C. Ribbon depiction <strong>of</strong> the homology based<br />
model <strong>of</strong> Ime2 based on the CDK structure. The follow<strong>in</strong>g stretches <strong>of</strong> residues are colored for<br />
clarity: the ATP b<strong>in</strong>d<strong>in</strong>g site (yellow), The PSTAIRE site (orange), the Catalytic site (red) and the<br />
T-loop (blue). The Ime2 model was prepared us<strong>in</strong>g the 3D-PSSM model<strong>in</strong>g algorithm (Kelley et<br />
al., 2000). The figures were prepared us<strong>in</strong>g InsightII (Accelrys).<br />
Fig. 13. <strong>Transcriptional</strong> <strong>regulation</strong> <strong>of</strong> middle <strong>meiosis</strong>-specific genes.<br />
Ndt80 is the transcriptional activator b<strong>in</strong>d<strong>in</strong>g to the MSE element present <strong>in</strong> the 5’ region <strong>of</strong> all<br />
middle <strong>meiosis</strong>-specific genes (MMG). A subset <strong>of</strong> MMG carries an MSE* site (SMK1 <strong>in</strong><br />
comparison to CLB1) that can be bound by Sum1. Sum1 associates with Hst1 that functions as<br />
histone deacetylase, thus lead<strong>in</strong>g to silenc<strong>in</strong>g under vegetative growth conditions and at early<br />
meiotic times. Under meiotic conditions relief <strong>of</strong> repression is due to degradation <strong>of</strong> Sum1 by<br />
Ime2. Sum1 also represses the transcription <strong>of</strong> NDT80, s<strong>in</strong>ce it carries both an MSE and MSE*<br />
elements. In addition, NDT80 carries two URS1 elements that are bound by Ume6. In vegetative<br />
growth media Ume6 recruits the S<strong>in</strong>3/Rpd3 histone deacetylase complex that represses<br />
transcription. Under meiotic conditions Ume6 recruits Ime1 that can activate transcription, but<br />
only <strong>in</strong> the absence <strong>of</strong> Sum1. Ime2 phosphorylates Ndt80, and this phosphorylation may<br />
contribute to the complete transcriptional activity <strong>of</strong> Ndt80. In the absence <strong>of</strong> meiotic<br />
recomb<strong>in</strong>ation the pachytene checkpo<strong>in</strong>t <strong>in</strong>hibits degradation <strong>of</strong> Sum1 and phosphorylation <strong>of</strong><br />
Ndt80. Consequently, the level <strong>of</strong> Ndt80 is reduced, and the unphosphorylated Ndt80 is impaired<br />
<strong>in</strong> activat<strong>in</strong>g the transcription <strong>of</strong> MMG.<br />
Fig. 14. Positive and negative feedback loops control <strong>meiosis</strong>.<br />
Ime1 shows positive auto<strong>regulation</strong> that is required to relieve repression mediated by Sok2 and<br />
thus for its high-level transcription. Expression <strong>of</strong> early <strong>meiosis</strong>-specific genes (EMG) requires<br />
relief <strong>of</strong> repression <strong>of</strong> S<strong>in</strong>3 and transcriptional activation, two functions that are promoted by<br />
Ime1. The transcription <strong>of</strong> middle genes (MMG) depends on Ime2 and Ndt80. <strong>Transcriptional</strong><br />
63
activation by Ime2 is due to phosphorylation <strong>of</strong> the transcriptional activator, Ndt80, and<br />
degradation <strong>of</strong> the transcriptional repressor, Sum1. Ime2 exhibits a negative feedback role, it is<br />
required to shut down the transcription <strong>of</strong> IME1. S<strong>in</strong>ce Ime2 directly phosphorylates Ime1, and<br />
s<strong>in</strong>ce this phosphorylation is required for degradation <strong>of</strong> Ime1 by the 26S proteasome, it was<br />
suggested that the reduction <strong>in</strong> the transcription <strong>of</strong> IME1 as well as EMG is due to the absence <strong>of</strong><br />
Ime1 and reestablishment <strong>of</strong> repression by Sok2 and S<strong>in</strong>3, respectively. The transcription <strong>of</strong><br />
MMG is repressed by the pachytene checkpo<strong>in</strong>t that <strong>in</strong>hibits phosphorylation <strong>of</strong> Ndt80 and<br />
degradation <strong>of</strong> Sum1.<br />
64
Table 1. List <strong>of</strong> positive and negative regulators <strong>of</strong> transcription <strong>of</strong> <strong>meiosis</strong>-specific genes.<br />
Name Brief description <strong>of</strong> function<br />
ABF1 An essential DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> required for DNA replication and the transcription <strong>of</strong> several<br />
<strong>meiosis</strong>-specific genes carry<strong>in</strong>g its b<strong>in</strong>d<strong>in</strong>g site (UASH).<br />
AMA1 A <strong>meiosis</strong>-specific subunit <strong>of</strong> APC/C that is required for degradation <strong>of</strong> CLB1 <strong>in</strong> the meiotic cycle.<br />
Required for the transcription <strong>of</strong> LMG.<br />
BCY1 The regulatory subunit <strong>of</strong> PKA. Positive regulator for IME1 transcription and sporulation.<br />
CDC25 Ras GDP/GTP exchange factor. Positive activator <strong>of</strong> PKA, negative regulator <strong>of</strong> IME1<br />
transcription. Transmits the nitrogen signal to UCS1 and the glucose signal to IREu elements <strong>in</strong><br />
IME1 promoter.<br />
CDC28 Cycl<strong>in</strong> dependent k<strong>in</strong>ase that regulates <strong>in</strong>itiation and progression <strong>in</strong> the mitotic cell cycle. In a<br />
complex with Cln phosphorylates Ime1 and sequesters it out <strong>of</strong> the nuclei. Required to activate<br />
Ime2 at the time cells enter the meiotic division.<br />
CDC35 Adenylate cyclase. Positive activator <strong>of</strong> PKA, negative regulator <strong>of</strong> IME1 transcription and<br />
sporulation<br />
GCN5 Histone acetylase required for the transcription <strong>of</strong> EMG.<br />
GPA2 A Gα prote<strong>in</strong> that associates, when GTP bound, with Ime2. An <strong>in</strong>hibitor <strong>of</strong> Ime2 k<strong>in</strong>ase activity.<br />
HST1 Histone deacetylase. Represses the transcription <strong>of</strong> MMG follow<strong>in</strong>g recruitment by Sum1.<br />
IDS2 A positive regulator <strong>of</strong> Ime2 activity.<br />
IME1 The master regulator gene <strong>of</strong> <strong>meiosis</strong>. A transcriptional activator; recruited by Ume6 to the URS1<br />
element <strong>in</strong> EMG, and is required for their transcription. The transcription <strong>of</strong> IME1 depends on the<br />
presence <strong>of</strong> Mata1 and Matα2 as well as on glucose and nitrogen depletion. Translation and<br />
activity <strong>of</strong> Ime1 are regulated by nutrients.<br />
IME2 An early <strong>meiosis</strong>-specific gene encod<strong>in</strong>g ser<strong>in</strong>e/threon<strong>in</strong>e k<strong>in</strong>ase, homologous to the human CDK2.<br />
Required for timely and high level transcription <strong>of</strong> EMG, and for the transcription <strong>of</strong> MMG and<br />
LMG. Negative regulator <strong>of</strong> Ime1 stability.<br />
IME4 A positive transcriptional regulator <strong>of</strong> IME1 whose transcription depends on the presence <strong>of</strong> Mata1<br />
and Matα2 and nitrogen depletion.<br />
ISW2 In a complex with Itc1 functions as an ATP dependent chromat<strong>in</strong>-remodel<strong>in</strong>g factor. Required for<br />
the repression <strong>of</strong> EMG under vegetative growth conditions. Physically associate with Ume6 that<br />
recruits it to URS1 elements.<br />
ITC1 In a complex with Isw2 functions as an ATP dependent chromat<strong>in</strong>-remodel<strong>in</strong>g factor. Required for<br />
the repression <strong>of</strong> EMG under vegetative growth conditions.<br />
65
MCK1 Prote<strong>in</strong> k<strong>in</strong>ase required for efficient transcription <strong>of</strong> IME1, expression <strong>of</strong> EMG, MMG and spore<br />
maturation. Homologous to mammalian glycogen synthase k<strong>in</strong>ase 3 and S. cerevisiae, RIM11,<br />
YOL128c, and MRK1. Required for phosphorylation <strong>of</strong> Ume6 and sporulation <strong>in</strong> cells deleted for<br />
RIM11 and MRK1.<br />
MDS3 Negative regulator <strong>of</strong> IME1 transcription. Redundant function with its homolog, PMD1<br />
MSN2/4 Z<strong>in</strong>c f<strong>in</strong>ger transcriptional activators, which share redundant function and b<strong>in</strong>d STRE elements.<br />
Negative regulators <strong>of</strong> mitosis. Positive regulators <strong>of</strong> <strong>meiosis</strong> and IME1 transcription through the<br />
IREu element <strong>in</strong> IME1 promoter. Targets <strong>of</strong> the cAMP/PKA pathway.<br />
MRK1 Prote<strong>in</strong> k<strong>in</strong>ase homologous to mammalian glycogen synthase k<strong>in</strong>ase 3 and S. cerevisiae, RIM11,<br />
YOL128c, and MCK1.Required for phosphorylation <strong>of</strong> Ume6 and sporulation <strong>in</strong> cells deleted for<br />
RIM11 and MCK1.<br />
NDT80 A <strong>meiosis</strong>-specific transcriptional activator that b<strong>in</strong>ds the MSE element <strong>in</strong> MMG. An early MMG.<br />
PMD1 Negative regulator <strong>of</strong> IME1 transcription. Redundant function with its homolog, MDS3<br />
PTP2,3 Tyros<strong>in</strong>e phosphatases, required for Mck1 dephosphorylation as well as for high-level transcription<br />
<strong>of</strong> IME1 and IME2, and for expression <strong>of</strong> MMG and LMG.<br />
RAS2 Small G prote<strong>in</strong> that activates the cAMP/PKA and the MAPK signal pathways. Negative regulator<br />
<strong>of</strong> IME1 transcription.<br />
RES1 A dom<strong>in</strong>ant mutation <strong>in</strong> this gene bypasses MAT <strong>regulation</strong> for the transcription <strong>of</strong> IME1.<br />
RGR1 A component <strong>of</strong> the RNA polymerase SRB mediator complex. Required for the repression activity<br />
<strong>of</strong> Rme1.<br />
RIM1 A C2H2 z<strong>in</strong>c f<strong>in</strong>ger prote<strong>in</strong> required for high-level expression <strong>of</strong> IME1, and efficient sporulation.<br />
Required for mitochondria DNA replication and ma<strong>in</strong>tenance. Activity is regulated by cleavage<br />
that depends on Rim8, Rim9, Rim13 and Rim20.<br />
RIM4 An early <strong>meiosis</strong>-specific gene required for high-level expression <strong>of</strong> EMG. Sequence identifies two<br />
RNA recognition motifs that are essential for function.<br />
RIM8 Positive regulator <strong>of</strong> IME1 transcription. Required for activat<strong>in</strong>g Rim1.<br />
RIM9 Positive regulator <strong>of</strong> IME1 transcription. Required for activat<strong>in</strong>g Rim1.<br />
RIM11 Tyros<strong>in</strong>e/ser<strong>in</strong>e/threon<strong>in</strong>e k<strong>in</strong>ase that is homologous to mammalian glycogen synthase k<strong>in</strong>ase 3 and<br />
S. cerevisiae, MCK1, YOL128c, and MRK1.Required for the <strong>in</strong>teraction between Ime1 and Ume6,<br />
and consequently for the transcription <strong>of</strong> EMG. Associates and phosphorylates both Ime1 and<br />
Ume6.<br />
RIM13 Positive regulator <strong>of</strong> IME1 transcription. Required for activat<strong>in</strong>g Rim1.<br />
RIM20 Positive regulator <strong>of</strong> IME1 transcription. Required for activat<strong>in</strong>g Rim1.<br />
66
RIM15 A ser<strong>in</strong>e/threon<strong>in</strong>e k<strong>in</strong>ase whose activity is <strong>in</strong>hibited by PKA phosphorylation. Positive regulator<br />
for the transcription <strong>of</strong> IME1 and for the <strong>in</strong>teraction between Ime1 and Ume6. Phosphorylates<br />
Ume6.<br />
RME1 A Zn f<strong>in</strong>ger DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong> that represses the transcription <strong>of</strong> IME1 through the UCS4<br />
element <strong>in</strong> IME1 promoter. The transcription <strong>of</strong> RME1 is negatively regulated by the Mata1/Matα2<br />
complex.<br />
ROX3 A component <strong>of</strong> the RNA polymerase SRB mediator complex. Required for repress<strong>in</strong>g the<br />
transcription <strong>of</strong> mid-late <strong>meiosis</strong>-specific genes.<br />
RPD3 Histone deacetylase. Required for repression <strong>of</strong> EMG <strong>in</strong> media support<strong>in</strong>g vegetative growth.<br />
Positive regulator <strong>of</strong> MMG.<br />
SIN3 Negative regulator that is required for repression <strong>of</strong> EMG <strong>in</strong> media support<strong>in</strong>g vegetative growth.<br />
Recruits Rpd3 to EMG due to its association with both Ume6 and Rpd3. Positive regulator <strong>of</strong><br />
MMG.<br />
SIN4 A component <strong>of</strong> the RNA polymerase SRB mediator complex. Required for the repression activity<br />
<strong>of</strong> Rme1, and for repress<strong>in</strong>g the transcription <strong>of</strong> mid-late <strong>meiosis</strong>-specific genes.<br />
SNF1 Ser<strong>in</strong>e/threon<strong>in</strong>e prote<strong>in</strong> k<strong>in</strong>ase whose activity is <strong>in</strong>hibited by high levels <strong>of</strong> glucose. Required for<br />
the transcription <strong>of</strong> both IME1 and IME2, and for spore formation.<br />
SNF2 Component <strong>of</strong> the Swi/Snf transcriptional activation complex. Required for high-level expression<br />
<strong>of</strong> IME1.<br />
SOK2 <strong>Transcriptional</strong> repressor that b<strong>in</strong>ds SCB like sequences. Positive regulator <strong>in</strong> the mitotic cell cycle<br />
Negative regulator <strong>of</strong> pseudohyphal growth, <strong>meiosis</strong> and IME1 transcription. Sok2 function as a<br />
repressor depends on phosphorylation by PKA.<br />
SSN6 A general transcriptional repressor that regulates the transcription <strong>of</strong> IME1. Forms a repression<br />
complex with Tup1.<br />
SUM1 A transcriptional repressor <strong>of</strong> MMG that b<strong>in</strong>ds the MSE* element. Recruits histone deacetylase<br />
activity by association with Hst1.<br />
SWI1 Component <strong>of</strong> the Swi/Snf transcriptional activation complex. Required for high-level expression<br />
<strong>of</strong> IME1.<br />
TPK1,2,3 Three genes encod<strong>in</strong>g the catalytic subunit <strong>of</strong> PKA.<br />
TUP1 A general transcriptional repressor that regulates the transcription <strong>of</strong> IME1. Forms a repression<br />
complex with Ssn6.<br />
UME2 A component <strong>of</strong> the SRB subcomplex <strong>of</strong> RNA polymerase II holoenzyme. Negative regulator <strong>of</strong><br />
EMG transcription.<br />
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UME3 A cycl<strong>in</strong> C homolog that associates with the cycl<strong>in</strong> dependent k<strong>in</strong>ase, Ume5. An <strong>in</strong>tegral<br />
component <strong>of</strong> the SRB subcomplex <strong>of</strong> RNA polymerase II holoenzyme. Required to repress<br />
transcription <strong>of</strong> EMG <strong>in</strong> vegetative growth media. Degraded <strong>in</strong> meiotic conditions.<br />
UME5 A cycl<strong>in</strong> dependent k<strong>in</strong>ase that associates with the cycl<strong>in</strong> C homolog, Ume3. An <strong>in</strong>tegral<br />
component <strong>of</strong> the SRB subcomplex <strong>of</strong> RNA polymerase II holoenzyme. Required to repress<br />
transcription <strong>of</strong> EMG <strong>in</strong> vegetative growth media.<br />
UME6 A C6Zn2 DNA-b<strong>in</strong>d<strong>in</strong>g prote<strong>in</strong>. B<strong>in</strong>ds to URS1 element. Functions as a negative regulator by<br />
recruit<strong>in</strong>g the S<strong>in</strong>3/Rpd3 and Isw2 complexes, and as a positive regulator by recruit<strong>in</strong>g Ime1.<br />
YHP1 Homeodoma<strong>in</strong> prote<strong>in</strong> that b<strong>in</strong>ds to UASv, a specific region <strong>in</strong> IME1 5’ region. It is not required<br />
for the transcription <strong>of</strong> IME1.<br />
WTM1,2,3 <strong>Transcriptional</strong> repressors that repress the transcription <strong>of</strong> IME2 synergistically.<br />
YVH1 Tyros<strong>in</strong>e phosphatase whose transcription is <strong>in</strong>duced upon nitrogen depletions. Has redundant<br />
function with Ptp2 <strong>in</strong> regulat<strong>in</strong>g sporulation.<br />
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