Transcriptional regulation of meiosis in budding yeast

Transcriptional regulation of meiosis
in budding yeast
Yona Kassir 1* , Noam Adir 2 , Elisabeth Boger-Nadjar 3 , Noga Guttmann Raviv 1 , Ifat Rubin-
Bejerano 4 , Shira Sagee 1 , and Galit Shenhar 5 .
1. Department of Biology, Technion Haifa Israel; 2. Department of Chemistry, Technion Haifa
Israel; 3. Food Engineering and Biotechnology, Technion Haifa Israel; 4. Whitehead Institute for
Biomedical Research, Cambride USA. 5. Department of Molecular Cell Biology at Weizmann.
Institute, Rehovot, Israel.
*corresponding author. Tel: 972-4-8294214, Fax: 972-4-8225153, e-mail:
ykassir@tx.technion.ac.il
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I. Introduction
II. Transcriptional cascade governs initiation of meiosis.
III. Transcriptional regulation of IME1.
A. The MAT signal
1. Genes whose products transmit the MAT signal to IME1 and meiosis
1.1. RME1.
1.2. IME4.
1.3. RES1.
B. The nitrogen signal
C. The glucose Signal
1. The cAMP signal-transduction pathway.
1.1. The IREu element.
1.1.1. Msn2 and Msn4.
1.1.2. Sok2.
1.2. Rim15
2. The Snf1 signal-transduction pathway
3. Respiration and the transcription of IME1.
D. Additional proteins regulating the transcription of IME1
1. Mck1
2. Tup1/Ssn6
3. Yhp1
4. The Swi/Snf chromatin remodeling complex
E. Positive and negative feedback regulation.
F. Summary.
IV. Transcriptional regulation of early meiosis-specific genes
A. Silencing of early meiosis-specific genes in vegetative growth.
1. The WTM genes.
2. The UME2 gene.
3. The UME3 and UME5 genes.
4. The SIN3, UME6, and RPD3 genes.
5. The ISW2 gene.
B. Expression of early meiosis-specific genes under meiotic conditions.
1. Ume6 is also positive regulator.
2. The function of Ime1.
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3. Proteins required for the association of Ime1 with Ume6.
3.1. The function of Rim11 and its homologs Mck1 and Mrk1.
3.2. The function of Rim15.
4. The function of Gcn5.
5. The transcription of early meiosis-specific genes is also regulated by positive
elements.
6. Additional positive regulators.
7. The role of premeiotic DNA replication and/or recombination in controlling early
meiosis-specific genes expression.
8. The choice between silencing and expression of early meiosis-specific genes.
V. Transcriptional regulation of middle meiosis-specific genes
A. Positive regulators of middle meiosis-specific genes
1. Ndt80
2. Ime2
3. Sin3 and Rpd3
B. Negative regulators of middle meiosis-specific genes
C. The recombination checkpoint
VI. Transcriptional regulation of late meiosis-specific genes
A. Early late genes.
B. Mid late genes
C. Late genes
VII. A feedback loop controlling meiosis.
VIII. Concluding remark. The choice between developmental pathways
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Abstract
Initiation of meiosis in Saccharomyces cerevisiae is regulated by mating type and nutritional
conditions that restrict meiosis to diploid cells grown under starvation conditions. Specifically,
meiosis occurs in MATa/MATα cells shifted to nitrogen depletion media in the absence of glucose
and the presence of a non-fermentable carbon source. These conditions lead to the expression and
activation of Ime1, the master regulator of meiosis. IME1 encodes a transcriptional activator
recruited to promoters of early meiosis-specific genes by association with the DNA binding
protein, Ume6. Under vegetative growth conditions these genes are silent due to recruitment of
the Sin3/Rpd3 histone deacetylase and Isw2 chromatin remodeling complexes by Ume6.
Transcription of these meiotic genes occurs following histone acetylation by Gcn5. Expression of
the early genes promote entry into the meiotic cycle, since they include genes required for
premeiotic DNA synthesis, synapsis of homologous chromosomes, and meiotic recombination.
Two of the early meiosis specific genes, a transcriptional activator, Ndt80, and a CDK2 homolog,
Ime2, are required for the transcription of middle meiosis-specific genes that are involved with
nuclear division and spore formation. Spore maturation depends on late genes whose expression
is indirectly dependent on Ime1, Ime2 and Ndt80. Finally, phosphorylation of Ime1 by Ime2,
leads to its degradation, and consequently to shutting down of the meiotic transcriptional cascade.
This Review is focusing on the regulation of gene expression governing initiation and progression
through meiosis.
Key Word: meiosis, Saccharomyces cerevisiae, transcriptional repression and silencing,
transcriptional activation, glucose, nitrogen, signal transduction pathways.
List of abbreviations: ad – activation domain; bd- DNA binding domain; CDK – cyclin
dependent kinase; EMG – early meiosis-specific genes; id – interaction domain; LMG – late
meiosis-specific genes; MMG – middle meiosis-specific genes; MSE - middle sporulation
element; MAPK – Mitogen activated kinase; NIS – Nuclear import sequence; NLS - Nuclear
localization sequence; PKA – cAMP-dependent protein kinase H; SA – synthetic growth media
with acetate as the sole carbon source; SCB - Swi4/6 cell cycle box; SD – synthetic growth media
with glucose as the sole carbon source; SPM – sporulation media; STRE-stress response element;
UAS - Upstream activation sequence; UCS - Upstream controlling sequence; URS - Upstream
repression sequence.
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I. Introduction
The budding yeast Saccharomyces cerevisiae is a simple unicellular eukaryote exhibiting several
optional developmental pathways. Fig. 1 schematically illustrates the developmental options of
MATa/MATα diploid cells. In the presence of both carbon and nitrogen sources both haploid and
diploid cells adopt the yeast form morphology; upon nitrogen limitation, and in the presence of
high levels of glucose, a dimorphic transition to a filamentous growth takes places [For recent
reviews see (Gancedo, 2001; Lengeler et al., 2000; Pan et al., 2000)]. Haploid as well as diploid
cells manifesting these forms propagate by the mitotic cell cycle. Upon nitrogen depletion the
developmental decision made by the cells depends on the presence or absence of a fermentable
carbon source such as glucose, as well as on the information at the MAT locus. In the presence of
functional MATa1 and MATα2 alleles, regardless of ploidy, i.e. MATa/MATα diploids,
MATa/MATα disomic cells, and haploid cells expressing both MAT alleles, cells enter the meiotic
cycle (Kassir and Simchen, 1976; Kassir and Simchen, 1985; Rine and Herskowitz, 1987; Roman
and Sands, 1953; Roth and Fogel, 1971). In the presence of only a single functional allele, i.e.
MATa and MATα haploids or MATa/MATa and MATα/MATα diploids, depletion of nitrogen
leads to a cell cycle arrest at G1 (Kassir and Simchen, 1976; Roman and Sands, 1953; Strathern et
al., 1981). The last developmental option S. cerevisiae cells possess is that of mating, cells
carrying only one of the two MAT alleles can respond to the pheromone secreted by cells
expressing the other MAT allele by temporal arrest in G1, mate, and then resume cell growth
[reviews on the mating process as well as on how the MAT alleles control cell type are (Fields,
1990; Herskowitz, 1995; Sprague, 1991)].
In this review we focus on how the meiotic signals, namely, the presence of the MATa and
MATα alleles, the presence of a non-fermentable carbon source, the absence of glucose and
nitrogen, control initiation and progression through the meiotic cycle. We focus on transcriptional
regulation rather than the function of the proteins involved with the specific meiotic events [for
reviews on these aspects of meiosis see (Kupiec et al., 1997; Roeder, 1997)].
II. Transcriptional cascade governs initiation of meiosis.
Entry and progression through the meiotic cycle depends on the expression and activity of many
genes that can be roughly divided into 3 groups: meiosis-specific, cell-division cycle (CDC) and
radiation sensitive (RAD) genes. The meiosis-specific genes are expressed only under meiotic
conditions, and their function is required only in meiosis. The cell-division cycle genes that are
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involved with the nuclear cell cycle are required for meiosis (Simchen, 1974), and are expressed
in both the mitotic and meiotic cycles. The RAD genes are involved with DNA repair as well as in
checkpoint surveillance, and in meiosis are required for meiotic recombination and surveillance
mechanisms.
Initiation and progression through the meiotic cycle is regulated by a transcriptional cascade
consisting of a temporal and programmed expression of 6 classes of meiosis-specific genes (early
I, early II, early middle, middle, mid-late, and late) (Chu et al., 1998; Primig et al., 2000). Fig. 2
shows a schematic drawing of this transcriptional cascade, the timing of meiotic events, and the
input of the meiotic signals. IME1 is the master regulator gene absolutely required for entry into
the meiotic cycle and the transcription of all meiosis-specific genes (Kassir et al., 1988; Smith
and Mitchell, 1989). Briefly, in the mitotic cell cycle IME1 is silent, as its transcription is
regulated by the three meiotic signals (Kassir et al., 1988). The translation and activity of Ime1 is
also regulated by nutrients (Rubin-Bejerano et al., 1996; Sherman et al., 1993). IME1 encodes a
transcriptional activator that is directly required for the transcription of early meiosis-specific
genes (EMG) (Mandel et al., 1994; Smith et al., 1993). The transcription of the middle genes
(MMG) depends on the transcription factor, Ndt80, and on the serine/threonine protein kinase,
Ime2, two early meiosis-specific genes whose transcription depends on Ime1 (Chu and
Herskowitz, 1998; Hepworth et al., 1998; Mitchell et al., 1990; Yoshida et al., 1990). In addition,
the function of Ime2 is directly regulated by nutrients (Donzeau and Bandlow, 1999; Mitchell et
al., 1990; Yoshida et al., 1990). Transcriptional regulation of mid-late and late genes (LMG) is
less clear, however, their regulated expression depends on the upstream regulators, Ime1, Ime2,
and Ndt80, as well as on nitrogen depletion (Friesen et al., 1997; Kihara et al., 1991).
There is a good correlation between time of transcription and meiotic function (Chu et al.,
1998; Primig et al., 2000). The transcription of EMG is induced prior to premeiotic DNA
replication, and it includes genes required for pairing of homologous chromosomes (i.e. HOP1),
and meiotic recombination (i.e. DMC1). Furthermore, the transcription of CDC genes required for
premeiotic DNA replication is induced at this time (i.e. POL1) (Johnston et al., 1986). Middle
genes are induced following completion of DNA replication and prior to the first nuclear division.
These genes are involved with nuclear division (i.e. CLB1,3,4, CDC26). The late genes are
expressed following completion of nuclear divisions, and are involved with spore formation and
its maturation (i.e. DIT1, SPS100) (Chu et al., 1998). However, some genes don’t follow this rule.
For example, CLB5,6 are middle genes (Chu et al., 1998) that are required for premeiotic DNA
replication (Dirick et al., 1998; Stuart and Wittenberg, 1998). In the mitotic cell cycle Clb5 is also
required for spindle orientation, a process occurring concomitantly with DNA replication (Segal
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et al., 2000). On the other hand, in the meiotic cycle, separation of the duplicated Spindle-Pole
Bodies and spindle formation occur following completion of DNA replication (Goetsch and
Byers, 1982). It seems, therefore, that the transcription of CLB5/6 correlates with their second
putative meiotic function.
III. Transcriptional regulation of IME1.
The different signal pathways that lead to meiosis converge at IME1, promoting its transcription
(Kassir et al., 1988). In vegetative growth media with glucose as the sole carbon source (SD -
synthetic dextrose) IME1 is not transcribed. A low basal level of IME1 mRNA is present in
growth media when acetate is the sole carbon source (SA - synthetic acetate) (Kassir et al., 1988).
Upon nitrogen depletion and the presence of acetate (SPM - sporulation media), the level of IME1
mRNA is transiently increased in MATa/MATα diploids, but not in cells carrying only a single
active MAT allele (Kassir et al., 1988). In addition, IME1 is subject to both positive and negative
feedback regulation (Shefer-Vaida et al., 1995). An extremely large region, about 2.1 kb long,
consisting of distinct positive and negative elements mediates the regulated transcription of IME1
(Granot et al., 1989; Sagee et al., 1998; Sherman et al., 1993; Smith et al., 1990). By deletion
analysis and insertion of specific elements of IME1 in heterologous reporter genes, the meiotic
signals affecting discrete elements were identified (Fig. 3) (Covitz and Mitchell, 1993; Sagee et
al., 1998; Sherman et al., 1993; Smith et al., 1990). Below is a detailed review on how the MAT,
Glucose, and Nitrogen signals are transmitted to IME1.
A. The MAT signal
The MAT signal is transmitted through two distinct elements, UCS3 and UCS4, which do not
share any sequence homology (Covitz and Mitchell, 1993; Sagee et al., 1998). Deletion of any of
these elements leads to partial derepression, whereas deletion of both elements results in full
expression of IME1 in MATa/MATa cells (Sagee et al., 1998). In addition, UCS3 is apparently
required for complete relief of repression of UCS4. Nested deletion of UCS3 leads to a 2-fold
reduction in the expression of ime1-lacZ in MATa/MATα cells incubated under meiotic
conditions, whereas in concomitant deletion of UCS4 and the entire upstream region, the
expression of ime1-lacZ is complete (Fig. 4, compare line B to lines A and C). The proteins
through which MAT regulation is transmitted to UCS3 are not known. As discussed below, Rme1
transmits the MAT signal to UCS4 (Covitz and Mitchell, 1993) (see section IIIA1.1).
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Full expression of Ime1 in S288C strains deleted for RME1 or UCS3 and UCS4 does not lead
to complete sporulation (Kassir et al., 1988; Sagee et al., 1998), suggesting that the Mata1 Matα2
proteins might be required for a downstream meiotic event. On the other hand, in SK1
background, this is not the case, and the MATa1 and MATα2 alleles are required only for the
transcription of IME1 (Covitz and Mitchell, 1993). There are many reported cases of phenotypic
differences between SK1 and other strains, i.e. S288C or W303. Genomic DNA hybridization
reveals the presence of many deletions and polymorphisms between these strains (Primig et al.,
2000). Thus, the discrepancy between results is attributed to the absence of some functions in the
different strains.
1. Genes whose products transmit the MAT signal to IME1 and meiosis. Three
genes are known to transmit the MAT signal to IME1 and meiosis: RME1, IME4, and RES1.
1.1. RME1. RME1 encodes a Zinc-finger DNA-binding protein (Covitz et al., 1991;
Shimizu et al., 1997a) that is a negative regulator for the transcription of IME1 and meiosis in
cells carrying only one of the MAT alleles (Kassir et al., 1988; Kassir and Simchen, 1976;
Mitchell and Herskowitz, 1986; Rine et al., 1981). Recessive mutations in RME1 and deletion of
RME1 lead to complete expression of IME1 and sporulation in mata1/MATα, MATa/MATa and
MATα/MATα strains (Kassir et al., 1988; Kassir and Simchen, 1976; Mitchell and Herskowitz,
1986). Haploid cells carrying the rme1-1 mutation complete premeiotic DNA replication, but
arrest as mono nucleate cells without loss of viability (Kassir and Simchen, 1976). This is most
probably due to the pachytene checkpoint mechanism that monitors lack of synapsis and/or
recombination [for review on this checkpoint see (Roeder and Bailis, 2000)]. In MATa/MATα
strains relief of repression is due to the substantial reduction (10 to 20-fold) in the level of RME1
mRNA and protein [(Mitchell and Herskowitz, 1986), and L. Johnston, personal communication].
The Mata1/Matα2 complex binds to a specific element in the promoter of RME1 repressing its
transcription (Goutte and Johnson, 1988; Johnson and Herskowitz, 1985; Li et al., 1995; Stark
and Johnson, 1994).
Rme1 binds the IME1 5’ region to two sites localized within UCS4, at -2040 to –2030 and –
1959 to –1949 (Shimizu et al., 1998). The consensus sequence for binding is GWACCTCAARA
(Shimizu et al., 1998). The presence of these two binding sites; defined as the RRE and the
modulation element (Covitz and Mitchell, 1993), is required to repress the transcription of IME1.
Deletion or mutations in a single site cause only partial derepression (Shimizu et al., 1998).
Moreover, deletion of both sites does not lead to complete relief of repression, (Shimizu et al.,
1998), probably due to the repression activity of the UCS3 site (Sagee et al., 1998). Insertion of
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the UCS4 element upstream of the CYC1 UAS results in 17-fold repression, depending on overexpression
of Rme1, as well as on the presence of both the RRE and the modulation sites (Covitz
and Mitchell, 1993). Unlike the intact IME1 gene, repression dependent on over-expression of
Rme1 and dependence on MAT was not reported. It is not surprising, therefore, that in this
heterologous system repression is accomplished by sequestering the binding of a transcriptional
activator (Shimizu et al., 1997b). The mode by which Rme1 represses transcription of IME1 is
not known, however, it repression activity depends on Sin4 and Rgr1 (Covitz et al., 1994), two
components of the RNA polymerase SRB mediator complex (Myer and Young, 1998). These
proteins are not required for the stability of Rme1 (Covitz et al., 1994), or it’s binding to DNA
(Shimizu et al., 1998).
Rme1 also functions as a transcriptional activator; it activates the transcription of CLN2 whose
promoter includes Rme1’s binding site (Toone et al., 1995). Accordingly, Rme1 also activates the
transcription of CYC1 and HIS3 when artificially tethered to their promoters (Covitz and
Mitchell, 1993; Covitz et al., 1994). The transcription of RME1 is mainly induced in G1 (Frenz et
al., 2001), suggesting that although this gene is non-essential, it might contribute to the high level
expression of Cln2 at this stage in the cell cycle. Transcriptional activation is independent of Rgr1
or Sin4 (Blumental-Perry et al., 2002), although the function of Rme1 as an activator or repressor
is mediated through the same domain (Blumental-Perry et al., 2002). These results suggest that
the function of Rme1 is determined by interacting proteins binding to nearby DNA sites.
The effect of MAT on the transcription of IME1 is evident only upon nitrogen depletion; In SA
media the level of IME1 mRNA is low and identical in haploid and MATa/MATα diploid cells
(Kassir et al., 1988), and only SPM and in MATa/MATα that IME1 is fully induced. Moreover,
since nitrogen depletion induces the transcription of RME1 (Frenz et al., 2001), it is possible that
Rme1 represses transcription only in the absence of nitrogen. This hypothesis predicts that in the
absence of Rme1, initiation of meiosis will not depend on nitrogen depletion. However, cells
where either RME1 or UCS3 along with UCS4 (the sites mediating the MAT signal) have been
deleted, induce meiosis only upon nitrogen depletion (Kassir and Simchen, 1976; Sagee et al.,
1998). The effect of Rme1 is mediated through one of two mechanisms: by direct binding and
repression of IME1, and by activation of CLN2 transcription (Frenz et al., 2001; Toone et al.,
1995). The increase in activity of the Cln/Cdc28 complex could result in an increase in
phosphorylation of Ime1, and its sequestering from the nucleus (Colomina et al., 1999).
Consequently, the reduction in positive autoregulation, would lead to a decrease in the level of
IME1 mRNA.
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1.2. IME4. IME4 encodes a positive regulator absolutely required for the transcription of
IME1 (Shah and Clancy, 1992). The transcription of IME4 is regulated by the meiotic signals; it
requires the presence of the MATa1 and MATα2 gene products and nitrogen depletion. Rme1
does not transmit the MAT signal to IME4 (Shah and Clancy, 1992), suggesting that Rme1 and
Ime4 function in two distinct signal pathways. The region within IME1 responding to Ime4, and
the mode by which Ime4 activates the transcription of IME1 is not known. Over expression of
Ime1 partially suppresses ime4∆, suggesting that Ime4 have an additional role in meiosis (Shah
and Clancy, 1992). Ime4 associates with Mum2/SpoT-8 (Uetz et al., 2000) that is required for
premeiotic DNA replication (Engebrecht et al., 1998; Tsuboi, 1983). It is possible that the second
meiotic function of Ime4 is to regulate the function of this protein.
1.3. RES1. The dominant mutation Res1-1 bypasses the requirement for the presence of
both Mata1 and Matα2 for the expression of IME1 and meiosis (Kao et al., 1990). Res1-1 is not
allelic to RME1, IME1 or IME4 (Kao et al., 1990; Shah and Clancy, 1992), neither is Res1 in the
Ime4 or Rme1 signal pathway (Kao et al., 1990; Shah and Clancy, 1992). This gene has not been
cloned, and its normal function is not known.
B. The nitrogen signal
The nitrogen signal is transmitted to IME1 through the UCS1 element (Fig. 3). Nested deletion of
this region leads to a 5 and 15-fold increase in the expression of ime1-lacZ in vegetative growth
media with either glucose (SD) or acetate (SA) as the sole carbon source, respectively [Fig. 4,
compare lines D and E, (Sagee et al., 1998)]. These results suggest that UCS1 functions as a
negative element in the presence of glucose and nitrogen. By itself UCS1 has no UAS activity, it
cannot promote expression of a his4-lacZ reporter gene lacking its own UAS (Nadjar-Boger,
2000). Insertion of UCS1 between the HIS4 UAS and TATA box in the HIS4UAS-HIS4TATA-lacZ
reporter gene leads to a substantial reduction of expression in SD and SA [Fig. 5, (Sagee et al.,
1998)]. Level of repression is 2 to 3-fold higher in SD in comparison to SA, confirming that the
activity of UCS1 is partially regulated by glucose. Upon nitrogen depletion (SPM) a substantial
increase in expression is observed [Fig. 5 and (Sagee et al., 1998)], suggesting that nitrogen is the
major signal regulating the function of UCS1.
The cAMP/PKA pathway transmits a nitrogen signal to IME1 and meiosis (Matsumoto et al.,
1983; Matsuura et al., 1990). Mutations that cause low or no activity of PKA, such as cyr1, ras2,
and cdc25 lead to the expression of IME1 and spore formation in the presence of nitrogen
(Matsumoto et al., 1983; Matsuura et al., 1990; Shilo et al., 1978; Smith and Mitchell, 1989)
[CYR1 encodes adenylate cyclase, RAS2 encodes a small G protein that functions as a positive
9

egulator of Cyr1, CDC25 encodes the Ras GDP/GTP exchange factor, which serves as a positive
regulator of adenylate cyclase (Broach, 1991; Broek et al., 1987; Toda et al., 1985)]. On the other
hand, mutations that cause constitutive PKA activity, such as RAS2-val19 (activated Ras) and
bcy1 [the regulatory subunit of PKA (Broach, 1991)] are sporulation deficient, and are suppressed
by over-expression of IME1 (Matsuura et al., 1990). The repression activity of UCS1 is reduced
in vegetatively grown cdc25-5 cells incubated at the non-permissive temperature (Fig. 5). Upon
nitrogen depletion (SPM), this phenomenon is not observed; the same levels of expression are
observed in cells incubated at the permissive or restrictive temperature (Fig. 5). These results
suggest that Cdc25 transmits the nitrogen signal to UCS1. Cdc25 is a positive regulator of both
the, cAMP/PKA (Broek et al., 1987) and MAPK (Shenhar, 2001) signal transduction pathways.
Therefore, further experiments are required to determine if this effect of Cdc25 is mediated
through PKA, MAPK or a different signal pathway.
MDS3 and its homologue, PMD1, are negative regulators of IME1 transcription in vegetative
growth media with acetate as the sole carbon source. Deletion of these genes results in an
increase in the transcription of IME1 and the EMG IME2 and HOP1 (Benni and Neigeborn,
1997). In addition, when stationary phase mds3∆ pmd1∆ diploid cells are shifted from SD to SA
media, growth is ceased and about 10% of the cells enter and complete the meiotic cycle. These
results suggest that Mds3/Pmd1 function upstream of Ime1, in preventing cell cycle arrest in the
presence of nitrogen (Benni and Neigeborn, 1997). Growth arrest and sporulation are suppressed
in cells carrying the RAS2-val19 mutation, suggesting that these genes might be upstream
activators of the Ras pathway (Benni and Neigeborn, 1997). The region in IME1 that is regulated
by these genes is not known. It will be interesting to determine if their effect is mediated through
the UCS1 element.
As discussed above, the Mata1 and Matα2 gene products are required to induce expression in
the absence of nitrogen, suggesting that UCS1 might also mediate the MAT signal. However, the
repression activity of UCS1 and its relief (in a UASHIS4-UCS1-his4-lacZ reporter) is identical in
isogenic haploid and diploid cells (Boger-Nadjar, 2000), indicating that the activity of UCS1 is
not regulated by MAT. However, it is still possible that within the context of IME1 promoter,
UCS1 might interact with the UCS3 and/or UCS4 elements that mediate MAT regulation.
C. The glucose Signal
The UAS activity of IME1 is confined to UCS2, a region that contains alternate positive and
negative elements [Fig. 3, (Sagee et al., 1998)]. Nested deletions of either one of its three positive
elements, UASru, IREu, and UASrm lead to a reduction in expression of ime1-lacZ in SPM (Fig.
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4, lines F,G,H), confirming their function as UAS elements (Sagee et al., 1998; Shenhar and
Kassir, 2001). In addition, in the presence of glucose UASru and IREu function as repression
elements [Fig. 4, lines F,G and (Shenhar and Kassir, 2001)]. Deletion of UASru increases
expression in SD, but in SA or SPM expression is absent (Fig. 4, line F), suggesting that UASru
functions as a URS element in the presence of glucose and as a UAS element in the absence of
glucose and/or the presence of acetate. Nested deletion of IREu leads to the same levels of
expression of ime1-lacZ in both SD and SA media [Fig. 4, line G, (Sagee et al., 1998)],
suggesting that its URS activity is regulated by either the carbon or nitrogen source. These
UASru, IREu and UASrm element function as a carbon source regulated UAS when inserted
upstream of a his4-lacZ reporter gene (Fig. 6). Decreased levels of expression are observed in SD,
and increased expression is observed in SA, while in SPM there is a slight increase in activity, but
only in the presence of UASru [Fig. 6 and (Sagee et al., 1998)]. These results imply that IREu and
UASrm are regulated only by the glucose signal, and that UASru is mainly regulated by glucose
but nitrogen has also a partial effect on its activity.
1. The cAMP/PKA signal-transduction pathway. Genetic analysis suggests that the
cAMP/PKA signal pathway transmits a nitrogen signal [see section IIIB and (Gancedo, 2001; Pan
et al., 2000)]. However, biochemical and genetic analysis demonstrate that this pathway is one of
the major signal transduction pathways transmitting a glucose signal to yeast cells. This is an
essential pathway that transiently increases the level of cAMP in response to glucose addition [for
reviews see (Broach, 1991; Thevelein and de Winde, 1999)]. Addition of 10mM cAMP to diploid
cells incubated in sporulation conditions leads to a small but significant reduction in the
expression of IME1 and the level of sporulation (Fig. 7). The expression of either UASrm-his4lacZ
or UASru-his4-lacZ is slightly increased by addition of cAMP. However, about 1.6-fold
reduction in the expression of IREu-his4-lacZ is observed, similar to the effect cAMP has on
sporulation and IME1 expression, suggesting that the activity of IREu is negatively regulated by
cAMP. The signal transduction pathways that transmit the glucose signal to UASru and UASrm
are not known.
1.1. The IREu element. The sequence of IREu (-1153 to –1121) reveals that it includes
two known positive elements, a stress response element, STRE, and the Swi4/Swi6 cell cycle
box, SCB (Fig. 8). An almost identical element, IREd, is present at –788 to –756 of IME1 (Fig.
3). IREd differs from IREu in two residues localized to the STRE and SCB elements (Fig. 8). An
ime1-lacZ chimeras whose 5’ end are terminated downstream or upstream of either IREu or IREd
show that IREu functions as a positive element, whereas IREd as a negative element (Sagee et al.,
11

1998). Furthermore, unlike IREu, IREd promotes only low UAS activity when inserted upstream
of his4-lacZ (Fig. 6), suggesting that the STRE and/or the SCB elements are the transcriptional
activation sequences within IREu. The cAMP/PKA pathway negatively regulates IREu,
promoting its URS activity and preventing its UAS activity in the presence of glucose (Sagee et
al., 1998; Shenhar and Kassir, 2001). Deletion of BCY1- the regulatory subunit of PKA (Toda et
al., 1987a) leads to no expression of IREu-his4-lacZ, whereas a temperature sensitive mutations
in CDC25 leads to a substantial increase in the activity of IREu in the presence of either glucose
or acetate as the sole carbon source (Sagee et al., 1998). Transcription factors that regulate the
activity of IREu are the two-homologous DNA-binding proteins, Msn2 and Msn4, Ime1 and
Sok2. Msn2 and Msn4 are absolutely required for the activity of IREu, Ime1 is required for the
complete UAS activity of IREu, and Sok2 is a negative regulator for IREu activity (Sagee et al.,
1998; Shenhar and Kassir, 2001).
1.1.1. Msn2 and Msn4. These C2H2 Zinc-finger proteins bind the STRE site present in
many stress-induced genes (Martinez-Pastor et al., 1996; Schmitt and McEntee, 1996), including
the IREu and IREd elements in IME1 (Sagee et al., 1998). Competition experiments reveal that
IREu is a better competitor than IREd (Sagee et al., 1998), suggesting that Msn2 and Msn4 bind
to the STRE element in IREu. In vitro transcribed and translated Msn2 can bind the STRE
sequence and the IREu element (Martinez-Pastor et al., 1996; Shenhar, 2001), suggesting that
binding is independent of post-translational modifications, and/or the presence of additional
proteins. However, two lines of evidence suggest that Msn2 forms a heterodimer with Sok2 (see
section IIIC1.1.2 below): i. Msn2 physically associates with Sok2, and ii. Deletion of the
postulated Sok2 binding site within IREu (the SCB element) abolishes the activity of IREu
(Shenhar and Kassir, 2001). These results suggest that in vivo, Msn2 forms a heterodimer with
Sok2, and that Sok2 facilitates its binding to the DNA. In the absence of Sok2, an imposter
protein can promote the binding of Msn2/4 to STRE (Fig. 8) (Shenhar and Kassir, 2001). Msn2/4
are apparent targets of the cAMP/PKA pathway. This is concluded from the following
observations: i. Deletion of both MSN2 and MSN4 suppresses the lethality of a strain deleted for
the three TPK genes [TPK1-3 are the three homologous genes encoding the catalytic activity of
PKA (Smith et al., 1998; Toda et al., 1987b)], ii. Msn2 includes sites required for its nuclear
import (NLS) as well as for its export (NIS), whose functions are regulated by glucose through
the cAMP/PKA pathway (Gorner et al., 2002). Glucose starvation and inactivation of the
cAMP/PKA pathway through mutations, leads to nuclear localization, whereas addition of
glucose or cAMP leads to cytoplasmic localization (Gorner et al., 1998; Gorner et al., 2002).
Interestingly, the NIS element is also regulated by the TOR signaling pathway. When this signal
12

pathway is activated, the binding of Msn2/4 to Bmh2, the 14-3-3 adaptor, sequesters it from the
nuclei (Beck and Hall, 1999). 3. The NLS site of Msn2 contains four PKA consensus sites. These
residues are phosphorylated in vivo in response to glucose addition, depending on the presence of
the three TPK genes. Serine to alanine mutations of all these sites lead to constitutive nuclear
localization of Msn2, whereas serine to aspartic acid, mimicking a phosphorylated serine residue,
leads to its cytoplasmic localization (Gorner et al., 2002).
1.1.2. Sok2. SOK2 encodes a DNA-binding protein that is highly homologous to the
DNA-binding domain of Swi4 and Phd1 proteins that are known to bind the SCB consensus
sequence. This homology suggests that Sok2 might also bind the same sequence. Direct binding
of Sok2 to IREu was not reported, however, the following genetic evidence suggests that Sok2
binds IREu under all growth conditions. The IREu-his4-lacZ reporter shows a 10-fold increase in
expression in the triple mutant sok2∆ msn2∆ msn4∆ in comparison to the double mutant msn2∆
msn4∆. On the other hand, IREu-his4-lacZ is not expressed in the sok2T598A msn2∆ msn4∆
triple mutant. The sok2T598A allele, similar to the SOK2 deletion allele is defective in
repression, but unlike the latter, the defective Sok2 protein is present in the cells (Shenhar and
Kassir, 2001). These results imply that Sok2 binds IREu under all growth conditions, and that in
its physical absence an imposter protein can bind and activate transcription. Sok2 functions as a
transcriptional repressor for several PKA target genes such as GAC1, SSA3, SWI5 and IME1 (Pan
and Heitman, 2000; Shenhar and Kassir, 2001; Ward et al., 1995). Furthermore, it functions as a
negative regulator of pseudohyphal growth (Ward et al., 1995) and meiosis (Shenhar and Kassir,
2001), and as a positive regulator in the mitotic cell cycle (Ward et al., 1995). Glucose regulates
both the transcription of SOK2 and the repression activity of Sok2 protein. The latter is concluded
from the observation that a Sok2-Gal4(bd), expressed from the ADH1 promoter, represses the
transcription of UASGAL1-UASHIS4-his4-lacZ, but only in the presence of glucose (SD media)
(Shenhar and Kassir, 2001). The signal pathway regulating the transcription of SOK2 is not
known (Shenhar and Kassir, 2001). However, the signal pathway transmitting the glucose signal
regulating Sok2 repression activity is the cAMP/PKA pathway. This is evident from the
following observations: i. Over-expression of SOK2 suppresses the temperature sensitive growth
defect of a tpk1∆ tpk2-ts tpk3∆ mutant (Ward et al., 1995); ii. Repression of IREu-his4-lacZ and
UASgal1-UAShis4-his4-lacZ by either Sok2 or Gal4(bd)-Sok2, respectively, is relieved in cdc25-
5 cells incubated at the non-permissive temperature, as well as by a threonine to alanine mutation
in a PKA consensus site in Sok2 (Shenhar and Kassir, 2001). Furthermore, Sok2 is a
phosphoprotein whose phosphorylation depends on this same threonine (T598) residue (Shenhar
and Kassir, 2001).
13

In the absence of glucose Sok2 does not repress transcription. Relief of repression in the
absence of glucose depends on its N-terminal domain, amino acids 1-247 as well as on Ime1 [see
section IIIE and (Shenhar and Kassir, 2001)]. The mode by which Sok2 represses transcription
and how Ime1 relieves repression is not known.
1.2. Rim15. Rim15 encodes a serine/threonine protein kinase whose activity in glucose
growth media is inhibited due to its phosphorylation by PKA (Reinders et al., 1998). In addition,
the transcription of RIM15, and consequently the steady state level of Rim15, is increased in
acetate growth media (Vidan and Mitchell, 1997). Diploid cells deleted for RIM15 or carrying a
kinase-dead point mutation are sporulation deficient (Vidan and Mitchell, 1997). Deletion of
RIM15 causes a drastic reduction in the transcription of IME1. The mode of action of Rim15, and
the IME1 element regulated by it are not known.
2. The Snf1 signal-transduction pathway. SNF1 encodes a protein kinase whose activity as a
kinase is inhibited in the presence of high levels of glucose (Jiang and Carlson, 1996). Snf1
function is required for the expression of glucose-repressed genes (Lesage et al., 1996). Snf1
phosphorylates the DNA-binding protein, Mig1 (Treitel et al., 1998) that recruits the Tup1/Ssn6
repression complex to the glucose-repressed genes to whom it binds (Nehlin et al., 1991). Snf1 is
required for the high level induction in the transcription of IME1 in sporulation conditions, and
diploid cells deleted for SNF1 arrest prior to premeiotic DNA replication, meiotic recombination,
and spore formation (Honigberg and Lee, 1998). Over expression of either Msn2 or Msn4
suppresses a temperature sensitive allele of SNF1 (Estruch and Carlson, 1993), suggesting that
Snf1 effect on IME1 might be mediated through Msn2/4. It is not known, though, whether
Msn2/4 are direct targets of Snf1. Genetic evidence suggests that Snf1 is in a separate pathway
than the cAMP/PKA pathway (Thompson-Jaeger et al., 1991), and both regulate Msn2/4. The
sequence of IME1 shows no homology to the Mig1 binding site, and the region in IME1 regulated
by Snf1 is not known.
In the meiotic cycle Snf1 is also required following the transcription of IME1. When IME1 is
placed on a multi-copy plasmid it is highly expressed in both wild type and snf1∆ diploid cells,
nevertheless, in the SNF1 deleted strain over expressing Ime1, the EMG IME2 is only partially
expressed, 10-15% of the cells complete premeiotic DNA replication and meiotic recombination,
but spores are not formed (Honigberg and Lee, 1998). It was suggested that Snf1 is required in
the meiotic cycle at three points, for the transcription of Ime1, for the transcription of Ime2, and
for spore formation (Honigberg and Lee, 1998).
14

3. Respiration and the transcription of IME1. The presence of glucose prevents the activity
of the mitochondria whose function is required for sporulation (Berger and Yaffe, 2000). The
transcription of IME1 is absent in diploid cells without mitochondria – petites, or in cells treated
with oligomycin - an inhibitor of the mitochondrial ATPase (Treinin and Simchen, 1993). It is not
known whether mitochondrial function is also required at a stage preceding the transcription of
IME1. The site within IME1 that responds to mitochondria function is not known, nor is it known
how the respiration signal is transmitted. The connection between mitochondria function and
IME1 transcription is also evident from the identification of the Rim1 (Rim101) Rim8, Rim9,
Rim13 and Rim20 proteins as positive regulators of IME1 (Li and Mitchell, 1997; Su and
Mitchell, 1993a). RIM1 encodes a C2H2 zinc finger protein that is required for high-level
expression of IME1 as well as for efficient sporulation (Su and Mitchell, 1993b). Activation of
Rim1 requires its cleavage by the products of Rim8, Rim9 and Rim13 (Li and Mitchell, 1997), as
well as the activity of Rim20 that might be required for presenting Rim1 to Rim13 (Xu and
Mitchell, 2001). Rim1 is localized to mitochondria, and is required for mitochondria DNA
replication, and consequently for the maintenance of the mitochondria (Berger and Yaffe, 2000;
Van Dyck et al., 1992). Thus, the effect of these RIM1,8,9,13,20 genes on the transcription of
IME1 may be indirect, through their effect on the maintenance of the mitochondria.
D. Additional proteins regulating the transcription of IME1.
1. Mck1. MCK1 encodes a protein kinase required for efficient transcription of IME1, for
expression of early and middle genes such as IME2 and SPS1,2 respectively, and for spore
maturation (Neigeborn and Mitchell, 1991). Expression of IME1 from the hetrologous GAL1
promoter suppresses the requirement of Mck1 for the transcription of EMG and MMG, but spore
maturation remains defective, suggesting that Mck1 directly regulate IME1 transcription and
spore maturation (Neigeborn and Mitchell, 1991). MCK1 is one of 4 yeast genes showing
homology to mammalian glycogen synthase kinase 3 (GSK3-β), RIM11, YOL128c, and MRK1.
These homologs are not required for the transcription of IME1 (Hajji et al., 1999; Mandel et al.,
1994; Rabitsch et al., 2001), suggesting that the transcription of IME1 is only partially dependent
on Mck1.
Mck1 inhibits the kinase activity of PKA both in vitro and in vivo, through a mechanism that
does not depend on Mck1 kinase activity (Rayner et al., 2002). This result implies that deletion of
MCK1 might increase rather than decrease the transcription of IME1. Therefore, the effect of
Mck1 on the transcription of IME1 is most probably not mediated by PKA. Mck1 is subject to
15

autophosphorylation, including on a conserved tyrosine (Y199) (Rayner et al., 2002).
Phosphorylation of this tyrosine residue in Mck1, similar to other GSK3β homologs, is required
for its kinase activity on exogenous substrates, but not for autophosphorylation (Rayner et al.,
2002). The Ptp2 and Ptp3 tyrosine phosphatases are required for Mck1 dephosphorylation (Zhan
et al., 2000). Deletion of either PTP2 or PTP3 has no effect on meiosis, however, the double
mutant ptp2∆ ptp3∆ is sporulation deficient, it arrests in meiosis prior to premeiotic DNA
replication, with a significant reduction in the expression of IME1 and IME2, and no expression
of middle or late meiosis-specific genes (Zhan et al., 2000). A third tyrosine phosphatase, Yvh1,
might contribute to this regulation (Guan et al., 1992). Deletion of YVH1 leads to minor effects on
the transcription of IME1, a reduced transcription of IME2, and a reduction in the efficiency of
asci formation (Park et al., 1996), but in the double mutant yvh1∆ ptp2∆ sporulation is almost
diminished (Park et al., 1996). Thus, the effect of these tyrosine phosphatases on meiosis may be
partially mediated through Mck1. The transcription of YVH1 is induced upon nitrogen depletion,
suggesting that it might be involved with transmitting the nitrogen signal (Park et al., 1996).
Over expression of Mck1 suppresses the sporulation defect of cells deleted for TPS1 (De Silva-
Udawatta and Cannon, 2001). TPS1 encodes one of the subunits of the trehalose phosphate
synthase complex (Reinders et al., 1997). Thus, Tps1 may either regulate Mck1, or suppression of
mck1∆ by Tps1 is due to unrelated increase in the level of Ime1. The region in IME1 regulated by
Mck1 and its mode of function is not known. However, the additive effects of mutations in
MCK1, MDS3, PMD1, RIM1 and IME4 suggest that these genes affect different regulatory
elements in the IME1 promoter (Su and Mitchell, 1993b).
2. Tup1/Ssn6. The Tup1/Ssn6 complex functions as a general transcriptional repressor upon
recruitment by specific DNA-binding proteins (Keleher et al., 1992). These proteins are also
negative regulators for the transcription of IME1 (Mizuno et al., 1998), but the specific DNAbinding
protein that tethers them to IME1 is not known. Two regions in IME1 promoter respond
to Tup1, the first one is from -914 to – 621 and the second from -1215 to –915 (Mizuno et al.,
1998), functioning as URS in the presence of Tup1, and UAS in the absence of Tup1,
respectively (Mizuno et al., 1998). Since these regions carry the IREd and IREu repeated
elements (Fig. 3) that function as repression and activation sequences, respectively, it should be
interesting to determine if the Tup1/Ssn6 complex mediates its effect through these elements, and
whether Sok2 is the DNA-binding protein that recruits them to these elements.
16

3. Yhp1. YHP1 encodes a homeodomain protein that binds to the UASv element in IME1
promoter, between -702 to –675 (Fig. 3) (Kunoh et al., 2000). This 28 bp element by itself does
not function as a UAS, but when tethered to heterologous reporter genes, it causes a 2-fold
reduction in expression, suggesting that Yhp1 functions as a repressor (Kunoh et al., 2000). The
mode by which Yhp1 represses transcription is not known, but its effect is independent of the
Tup1/Ssn6 repression complex (Kunoh et al., 2000). The transcription of YHP1 is reduced in
vegetative cells grown in the presence of acetate as the sole carbon source in comparison to
glucose (Kunoh et al., 2000), implying that it might transmit a glucose signal. However, deletion
of YHP1 has no effect on either the transcription of IME1, or on the ability of cells to enter and
complete meiosis and sporulation (Kunoh et al., 2000).
4. The Swi/Snf chromatin remodeling complex. Two components of the Swi/Snf
complex, Snf2 and Swi1 are required for high-level expression of IME1 (Yoshimoto et al., 1993).
This complex is involved with transcriptional activation of genes to whom it is tethered through
interaction with specific activation domains [for review see (Carlson and Laurent, 1994)].
E. Positive and Negative feedback regulation.
The transcription of IME1 is subject to positive regulation. Over expression of Ime1 leads to
increase in the level of ime1-lacZ, through its effect on the function of IREu (Shenhar and Kassir,
2001). The effect of Ime1 on additional elements has not yet been determined. This
autoregulation is required to relieve repression by Sok2. This is deduced from the following
results: i. Expression of IREu-his4-lacZ is substantially reduced in diploid cells deleted for IME1
(Shenhar and Kassir, 2001), ii. Gal4(bd)-Sok2 represses the transcription of GAL1UAS-HIS4 UAS -
his4-lacZ in SD but has no repression activity in SA (Shenhar and Kassir, 2001). However, in
diploid cells deleted for IME1 the transcription of the reporter gene is repressed in both SD and
SA media (Shenhar and Kassir, 2001). Since in the presence of glucose Ime1 is not expressed, it
is not surprising that the effect of Ime1 is observed only in SA. As will be detailed in section
IVB2, Ime1 does not bind directly to DNA, and is recruited to the DNA by interacting with a
DNA-binding protein. Since the N-terminal domain of Sok2 is also required to relieve repression
in SA, it is tempting to suggest that Ime1 associates with the N-terminal domain of Sok2. No
information on possible associations between these two proteins has yet been reported.
The transcription of IME1 as well as the steady-state level of Ime1 protein in meiotic
conditions is transient, when induced upon a shift to SPM, the level of IME1 peaks at about 6-8
17

hours in SPM, followed by a decline (Guttmann-Raviv and Kassir, 2002; Kassir et al., 1988;
Shefer-Vaida et al., 1995). This transient transcription depends on both Ime1 and Ime2
(Guttmann-Raviv and Kassir, 2002; Mitchell et al., 1990; Shefer-Vaida et al., 1995; Smith and
Mitchell, 1989; Yoshida et al., 1990). This negative feedback effect is on transcription of IME1
rather than the stability of the mRNA, since insertions of two separate regions, -621 to –924 and –
924 to –1368 (Fig. 3) upstream of cyc1-lacZ exhibit the same feedback regulation (Shefer-Vaida
et al., 1995). Ime1 is a non-stable protein that is degraded by the proteasome and whose half-life
depends on Ime2 (Guttmann-Raviv and Kassir, 2002). IME2 encodes a meiosis-specific protein
kinase that interacts with Ime1 and phosphorylates it in vitro (Guttmann-Raviv and Kassir, 2002).
We suggest that the effect of Ime2 on shutting down the transcription of IME1 is due to its effect
on the stability of Ime1 protein. In the presence of Ime2, degradation of Ime1 would lead to the
establishment of Sok2 repression and silencing. On the other hand, in cells deleted for IME2,
accumulation of Ime1 leads to continues positive autoregulation, and increase in its transcription,
and consequently the availability of Ime1 protein (Guttmann-Raviv and Kassir, 2002).
F. Summary.
IME1 encodes the master regulator required to open a developmental pathway, that of meiosis, in
S. cerevisiae. It is not surprising, therefore, that its transcription is regulated by an unusual large
5’ region that is subject to multiple regulations. Fig. 9 is a summary of the results described
above, on the current knowledge on how the meiotic signals are transmitted to IME1. A
combinatorial effect of at least 10 distinct elements ensures that IME1 will be transcribed only
under the appropriate conditions, namely in MATa/MATα diploids, the absence of glucose, the
presence of acetate, and nitrogen depletion. Two negative elements, UCS3 and UCS4 restrict
elevated transcription of IME1 to cells expressing Mata1 and Matα2 peptides. In vegetative
growth media with glucose as the sole carbon source IME1 is silent. This regulation is
accomplished by using 4 distinct elements, UCS1 IREu UASru, and UASv, whose function as
repression elements is mediated through distinct signal transduction pathways. In acetate growth
media a low level of transcription is accomplished by the positive activity of UASru, IREu,
UASrm and UASv that is opposed by negative regulation from UCS1 as well as from three
constitutive URS elements, URSu, URSd and IREd. Upon nitrogen depletion, relief of UCS1
repression promotes an increase in transcription.
18

IV. Transcriptional regulation of early meiosis-specific genes (EMG)
A. Silencing of early meiosis-specific genes in vegetative growth.
In vegetative growth conditions with either glucose or acetate as the sole carbon source meiosisspecific
genes are silent. Silencing of EMG is not only due to the absence of their main
transcriptional activator, Ime1, but is rather due to active repression. The genes required to
promote this silencing are WTM3, UME2, UME3, SIN3, RPD3, UME5, UME6, and ISW2
(Goldmark et al., 2000; Strich et al., 1989; Strich et al., 1994; Vidal and Gaber, 1991). Except for
Ume6 and Isw2, these genes are not essential for the expression of EMG under meiotic conditions
(Goldmark et al., 2000; Strich et al., 1989).
1. The WTM genes. UME1 (WTM3) and its two homologs WTM1 and WTM2 repress the
transcription of the EMG IME2 synergistically (Pemberton and Blobel, 1997). These proteins also
function as transcriptional repressors of the silent HMR cassette, and when tethered artificially to
heterologous genes. These genes are non-essential; the wtm1 wtm2 wmt3 triple mutant is viable
(Pemberton and Blobel, 1997). The Wtm proteins are expressed constitutively in both mitotic and
meiotic conditions, and are localized to the nucleus (Pemberton and Blobel, 1997). The mode by
which they cause repression is not known.
2. The UME2 gene. UME2 (SRB9, SSN2) is a component of the SRB subcomplex of RNA
polymerase II holoenzyme (Kornberg, 1999). Although it is a negative regulator of EMG under
vegetative growth conditions (Strich et al., 1989), when fused to lexA it activates transcription
(Song and Carlson, 1998).
3. The UME3 and UME5 genes. The UME3 (SRB11, SSN8) and UME5 (SRB10, SSN3)
encode integral components of the SRB subcomplex of RNA polymerase II holoenzyme (Cooper
and Strich, 1998; Kornberg, 1999). UME3 encodes a cyclin C homolog that associates with the
cyclin dependent kinase, Ume5 (Cooper et al., 1997; Cooper and Strich, 1998). Ume5
phosphorylates the C-terminal CTD repeats of RNA polymerase II, but this event is independent
of its repression activity (Cooper and Strich, 1998), thus, it is not know how Ume5 represses the
transcription of EMG. Ume3 is degraded in meiotic conditions, and this degradation, that is
independent of Ume5, is required for complete relief of repression of EMG (Cooper et al., 1997).
19

4. The SIN3, UME6, and RPD3 genes. UME6 encodes a C6Zn2 protein that binds the
URS1 sequence present in many genes including EMG (Anderson et al., 1995; Strich et al.,
1994). The C6Zn2 domain is sufficient for binding, and is required for its repression activity
(Anderson et al., 1995; Strich et al., 1994). Deletion of UME6 causes high expression of EMG, as
well as additional non-meiotic genes carrying the URS1 element, in vegetative growth conditions
(Bowdish and Mitchell, 1993; Lopes et al., 1993; Park et al., 1992; Strich et al., 1994). In
addition, Ume6 functions as a transcriptional repressor when tethered to heterologous genes
following its fusion to lexA (Kadosh and Struhl, 1997). This repression activity of Ume6 depends
on the availability of Sin3 (Ume4, Rpd1) and Rpd3 (Kadosh and Struhl, 1997).
Coimmunoprecipitation and two-hybrid assays demonstrate physical association between
Ume6(515-530) and Sin3(426-472) (the PAH2 domain), as well as between Sin3 and Rpd3
(Kadosh and Struhl, 1997; Kasten et al., 1997; Washburn and Esposito, 2001).
Sin3 functions as a transcriptional repressor when tethered to heterologous genes following its
fusion to lexA (Wang and Stillman, 1993). The repression activity of Sin3 depends on Rpd3
(Kadosh and Struhl, 1997; Kasten et al., 1997). In accord, the double mutant sin3 rpd3 have the
same phenotype as either single mutant (Vidal and Gaber, 1991).
RPD3 encodes histone deacetylase whose deletion, similar to SIN3 deletion, causes an increase
in acetylation of lysines 5 and 12 in histone H4 in various genes carrying the URS1 element
(Burgess et al., 1999; Rundlett et al., 1998). Recruitment of the Sin3/Rpd3 complex by Ume6 to
DNA results in decreased acetylation of histone H3 and H4 in a restricted region, up to two
nucleosomes from the binding site of Ume6 (Kadosh and Struhl, 1998).
5. The ISW2 gene. Isw2 in a complex with Itc1 functions as an ATP dependent chromatinremodeling
factor that is required for the repression of EMG under vegetative growth conditions
(Goldmark et al., 2000). Isw2 functions in parallel to the Sin3/Rpd3 histone deacetylase complex,
as deduced from the observation that the level of expression of EMG are dramatically increased
in the sin3 isw2 double mutant in comparison to the single mutants (Goldmark et al., 2000). Isw2
physically associates with Ume6, and since it’s binding to the URS1 element depends on Ume6,
it was concluded that Ume6 recruits the Isw2 complex to URS1 (Goldmark et al., 2000). DNaseI
and micrococcal nuclease digestion of the chromatin near the EMG REC104 show dependence on
Isw2, suggesting that the Isw2 complex forms an inaccessible chromatin structure near the URS1
element (Goldmark et al., 2000). These results suggest that Isw2 would be localized to the
nucleus in vegetative growth media. However, this is not the case, Isw2 is localized to the
cytoplasm in vegetative growth cultures, and to the nucleus, the cytoplasmic and spindle
microtubuli, in meiotic cultures (Trachtulcova et al., 2000). There is no good explanation to
20

clarify these contradicting results. Isw2 is required for meiosis, cells deleted for ISW2 arrest under
meiotic conditions prior to premeiotic DNA replication, and asci are not formed (Trachtulcova et
al., 2000). It is not known whether Isw2 is required for the transcription of EMG under meiotic
conditions, nor why cells deleted for ISW2 are sporulation deficient.
B. Expression of early MSG under meiotic conditions.
1. Ume6 is also a positive regulator. Ume6 is required for the transcription of EMG under
meiotic conditions (Bowdish and Mitchell, 1993; Bowdish et al., 1995; Steber and Esposito,
1995; Strich et al., 1994; Szent-Gyorgyi, 1995). In addition, Sin3 and Rpd3 are required for their
high level of expression (Lamb and Mitchell, 2001). Ume6, Sin3 and Rpd3 are present and
physically associate in both vegetative growth and meiotic conditions (Lamb and Mitchell, 2001).
When tethered to heterologous genes, Ume6 can serve as either a transcriptional repressor or
activator, depending on Sin3/Rpd3 and Ime1, respectively (Bowdish et al., 1995; Kadosh and
Struhl, 1997). Different domains in Ume6 are required for transcriptional repression and
activation (Bowdish et al., 1995; Washburn and Esposito, 2001). The Ume6T99N mutant protein
is competent in repression of EMG in growth conditions, but is defective in their expression
under meiotic conditions (Bowdish et al., 1995). On the other hand, the M530T mutation in
UME6 has a defect only in repression (Washburn and Esposito, 2001). In agreement, the
transcriptional activator, Ime1 associates with Ume6(1-232) (Rubin-Bejerano et al., 1996), while
the transcriptional repressor, Sin3, with Ume6(515-530) (Kadosh and Struhl, 1997; Kasten et al.,
1997; Washburn and Esposito, 2001). The positive and negative roles of Ume6 are mediated
through its binding to the URS1 element whose presence is required for complete expression of
EMG in starved cells (Bowdish et al., 1995).
2. The function of Ime1. IME1 encodes as positive regulator of meiosis. Over-expression
of Ime1 bypasses the requirement for MATa1 and MATα2 gene products for sporulation (Kassir
et al., 1988). Diploid cells deleted for IME1 arrest under meiotic conditions in G1, as unbudded
cells (Foiani et al., 1996; Kassir et al., 1988), prior to execution of any meiotic event, namely,
expression of meiosis-specific genes, premeiotic DNA replication, commitment to meiotic
recombination, meiotic divisions, and spore formation (Foiani et al., 1996; Kassir et al., 1988;
Mitchell et al., 1990; Smith and Mitchell, 1989). The requirement of IME1 for transcription
suggests that it might encode a transcription factor. However, its predicted amino acid sequence
does not show any homology to known DNA-binding motifs (Sherman et al., 1993; Smith et al.,
21

1993). Nevertheless, when Ime1 is tethered to heterologous genes it functions as a potent
transcriptional activator (Mandel et al., 1994; Smith et al., 1993).
As described above, the transcription of IME1 is subject to extensive regulation by the MAT
alleles and nutrients. On top of it, translation of IME1 mRNA is regulated by nutrients (Sherman
et al., 1993). In vegetative growth media with acetate as the sole carbon source low but
substantial levels of IME1 or ime1-lacZ mRNAs are observed, but Ime1-lacZ protein is not
detected. On the other hand, upon nitrogen depletion, the level of IME1 mRNA is not induced in
MATa/MATa cells, but Ime1-lacZ protein is readily observed (Sherman et al., 1993). Furthermore,
α-factor and heat-shock treatment increases the transcription of IME1, but translation occurs only
in cells arrested in G1 by either α-factor, or the CDC mutations cdc28-4 and cdc4-3 (Sherman et
al., 1993). These results suggest that nitrogen and/or cell cycle progression inhibits translation of
IME1 mRNA. Since nitrogen depletion leads to a G1 arrest, it is possible that the effect of
nitrogen is indirect, and that a G1 arrest is a prerequisite for efficient translation. IME1 has an
atypical 5 UTR (untranslated region), 229 bp long (Sherman et al., 1993), which might mediate
this regulation. Sequence analysis reveals that this RNA region can form stem-and-loop
secondary structure that might inhibit translation. However, deletion of this region has no effect
on the translation of IME1 mRNA (Ben- Dov, 1994). Thus, it is not known how nitrogen and/or
G1 phase controls the efficiency of translation of IME1 mRNA.
Over expression of Ime1 in acetate growth media does not induce meiosis and sporulation in
logarithmic cultures of wild-type diploids (Colomina et al., 1999; Sherman et al., 1993). On the
other hand, in diploid cells arrested in G1 by recessive temperature sensitive mutations in
CDC28, CDC4, CDC25, CDC35 (CYR1), or concomitant deletion of the three CLN genes, high
percentages of asci are formed (Colomina et al., 1999; Sherman et al., 1993; Shilo et al., 1978),
suggesting that post-translational modification of Ime1 or another factor required for meiosis
depends on lack of function of these proteins. Indeed, in vegetative cultures, depending on the
Cdc28/Cln function, Ime1 is phosphorylated and sequestered from the nucleus (Colomina et al.,
1999). The localization of Ime1 in the cytoplasm prevents its transcriptional activation function,
and entry into meiosis. Cdc4 is an F-box protein [for review see (Patton et al., 1998)] required for
degradation of specific targets in G1. Interestingly, one of its substrates is Far1, an inhibitor of
Cln/Cdc28 function (Henchoz et al., 1997). Thus, the effect of Cdc4 on sporulation may be
mediated through Cdc28/Cln function. The role of Cdc25 and Cdc35, the two positive regulators
of PKA on the function of Ime1 is most probably through their effect on the association of Ime1
with Ume6 (see below).
22

It is not clear if the activity of Ime1 as a transcriptional activator is subject to regulation. Using
the SK1 strain and a LexA-Ime1 fusion, it was shown that transcriptional activation of the
reporter gene lexAop-lacZ depends on nitrogen depletion and the presence of a protein kinase,
Rim11 (Smith et al., 1993). Genetic analysis showed that in this system Rim11 was required to
relieve a repression activity of Ime1 that was modulated by the C-terminal domain of Ime1. This
conclusion was based on the following results: i. A lexA-Ime1 fusion truncated for the C-terminal
66 amino acids activated transcription of lexAop-lacZ in rim11∆ cells (Smith et al., 1993). ii.
LexA-Ime1L321F mutant protein is impaired in both association with Rim11 and transcriptional
activation (Malathi et al., 1997). On the other hand, using the S288C strain and a Gal4(bd)-Ime1
fusion, it was shown that transcriptional activation of the reporter gene gal1-lacZ is independent
of growth conditions or Rim11 (Mandel et al., 1994; Rubin-Bejerano et al., 1996). In both strains
Rim11 is required for the transcription of EMG and sporulation (Mandel et al., 1994; Mitchell
and Bowdish, 1992). The reasons for the disparity between these reports are not known, but
several possibilities can be suggested. i. The use of different strain backgrounds, S288C and SK1.
ii. The binding of the Gal4(bd) and lexA to the DNA requires its dimerization. The gal4(bd)-
IME1 gene includes the Gal4 dimerization signal, whereas the lexA-IME1 gene might lack the
lexA dimerization sequence (Golemis and Brent, 1992). Under meiotic conditions Ime1 can
oligomerize, and this activity depends on Rim11 (Rubin-Bejerano et al., 1996). Therefore, it is
possible that the ability of the lexA-Ime1 protein to activate transcription only under meiotic
conditions and only in the RIM11 strain is due to the ability of the protein to dimerize and bind
the DNA only under these conditions.
Deletion and mutation analysis reveals that Ime1 is composed of at least two domains essential
for meiosis: a transcriptional activation domain (ad) (amino acids 165-228), and an interaction
domain (id) (amino acids 270-360) (Mandel et al., 1994; Smith et al., 1993). The N-terminal 160
amino acids are not essential for meiosis (Mandel et al., 1994). However, an Ime1 protein
truncated for this domain gives rise to lower levels of asci in comparison to cells expressing this
truncated protein fused to the Gal4(bd), suggesting that the later might either provide a nuclear
localization signal or increase the stability of the protein (Mandel et al., 1994).
Two hybrid assays reveal that Ime1 interacts with Ume6, and that this interaction is through
amino acids 270-360 of Ime1 [Ime1(id)] and amino acids 1-232 of Ume6 [Ume6(id)] (Colomina
et al., 1999; Rubin-Bejerano et al., 1996; Xiao and Mitchell, 2000). The validity of this
interaction is evident from the use of different strain backgrounds and different two-hybrid
systems, namely, the Gal4(bd)-Ime1(id) with Ume6(id)-Gal4(ad) (Rubin-Bejerano et al., 1996;
Xiao and Mitchell, 2000), and the tetR-Ime1(id) with Ume6(id)-VP16 (Colomina et al., 1999).
23

However, direct demonstration of physical association between Ime1 and Ume6 is still missing.
The use of the two-hybrid assay enables the demonstration that this interaction is negatively
regulated by both glucose and nitrogen. The interaction is absent in vegetative growth conditions
with glucose as the sole carbon source, and excessive interaction takes place under meiotic
conditions (SPM media) (Colomina et al., 1999; Rubin-Bejerano et al., 1996). Shifting glucose
grown stationary cells to acetate growth media leads to partial interaction (Rubin-Bejerano et al.,
1996), suggesting that the interaction of Ime1 with Ume6 depends on the absence of both glucose
and nitrogen. Furthermore, this interaction is absolutely dependent on Rim11 (Colomina et al.,
1999; Rubin-Bejerano et al., 1996), and partially dependent on Rim15 (Vidan and Mitchell,
1997).
Two models were suggested for the role of Ime1 Ume6 and Rim11 in the transcription of
EMG. I. Bowdish et al proposed that Ume6 is converted from a repressor to an activator
following phosphorylation by Rim11, and that Ime1, which associates with Rim11 (Bowdish et
al., 1994; Rubin-Bejerano et al., 1996), is required to recruit Rim11 to Ume6 (Bowdish et al.,
1995). ii. Rubin-Bejerano et al proposed that Ume6 recruits Ime1 to the URS1 element present in
EMG, and that this recruitment promotes the Ime1 transcriptional activation domain to induce the
transcription of EMG. According to the later, Rim11 is required for the association between Ime1
and Ume6 (Rubin-Bejerano et al., 1996). The following results support the second model: i. Ime1
functions as a transcriptional activator (Mandel et al., 1994; Smith et al., 1993). ii. Fusion of an
heterologous transcriptional activation domain, that of Gal4, to Ime1(id), leads to the expression
of the EMG, IME2, and sporulation (Mandel et al., 1994). Moreover, this Ime1(id)-Gal4(ad)
fusion protein promotes meiosis of ime1∆ diploid cells only when galactose is added to the
sporulation media (Mandel et al., 1994). Since galactose is required to relieve repression of Gal80
on the transcriptional activation of Gal4(ad) (Johnston and Carlson, 1992), it was suggested that
the normal function of Ime1 is to activate transcription (Mandel et al., 1994). iii. A Gal4(ad)-
Ume6(159-836) fusion protein that is expressed from the IME1 promoter bypasses the
requirement for IME1 for the transcription of EMG and sporulation: it leads to expression of the
EMG, HOP1, and to 40% sporulation (Rubin-Bejerano et al., 1996). iv. A Gal4(ad)-Ume6 fusion
protein can activate the transcription of the EMG SPO13 in SD media if it carries mutations that
prevent its association with Sin3 (Washburn and Esposito, 2001). In the absence of the Gal4(ad),
these ume6 mutants do not promote expression of EMG (Washburn and Esposito, 2001). These
results suggest that by itself Ume6 does not function as a transcriptional activator, and its
conversion into a positive regulator depends on the recruitment of the transcriptional activation
domain of Ime1 (Rubin-Bejerano et al., 1996; Washburn and Esposito, 2001). Currently, this is
24

the established model, however, the ultimate proof would be the demonstration that Ime1 is
present on the complex formed on URS1, and/or that it associates with the transcription
machinery. Both models assume that since Ime1 functions through Ume6, deletions of either one
of these genes would have a similar phenotype in meiotic cultures. However, ime1∆ diploids
arrest in G1, whereas ume6∆ cells mainly arrest at G2. We assume that the expression of EMG in
vegetative cultures promotes the entry and progression through some meiotic events.
Ime1 has at least two functions, it supplies a transcriptional activation domain, but it is also
required to relieve repression of Sin3/Rpd3 (Washburn and Esposito, 2001). Diploid cells deleted
for IME1 and expressing Gal4(ad)-Ume6 from the ADH1 promoter are sporulation deficient
(Washburn and Esposito, 2001), but the Gal4(ad)-Ume6(M530T) mutant protein promotes low
levels of sporulation (10%). Since the latter protein is defective in association with Sin3, it was
concluded that Ime1 is required to relieve repression mediated by the Sin3/Rpd3 complex
(Washburn and Esposito, 2001).
The interaction between Ime1 and Ume6 is regulated by nutrients, Rim11 and Rim15. Since
two-hybrid assays were used to determine this interaction, the regulated expression of the reporter
genes might result from regulation at any of the following levels: i. Regulated physical
association between Ime1 and Ume6, or ii. Regulated transcriptional activation by the
Ume6/Ime1 complex. The latter model assumes that Ime1 and Ume6 physically associate under
all growth conditions, but in glucose growth media the resulting complex does not function as a
transcriptional activator. This hypothesis is supported by the following results. i. In vegetative
growth media association of Ume6 with the Sin3/Rpd3 complex represses transcription (Kadosh
and Struhl, 1997). ii. A point mutation in Ume6 that prevents its association with Sin3 promote
transcriptional activation when the mutant protein is fused to Gal4(ad) (Washburn and Esposito,
2001). Direct demonstration of regulated (or not) physical association between Ime1 and Ume6 is
required to distinguish between these models.
In accord with the role of Ime1 as a transcriptional activator, the protein is localized to the
nucleus (Colomina et al., 1999; Rubin-Bejerano et al., 1996; Smith et al., 1993). Localization of
Ime1 is independent of Rim11 (Rubin-Bejerano et al., 1996), but as described above, it is
inhibited by the Cln/Cdc28 function (Colomina et al., 1999). Fig. 10 schematically illustrates how
nutrients control the availability and activity of Ime1
25

3. Proteins required for the association of Ime1 with Ume6.
3.1. The function of Rim11 and its homologs Mck1 and Mrk1. Rim11 is absolutely
required for the interaction between Ime1 and Ume6 (Colomina et al., 1999; Rubin-Bejerano et
al., 1996), the expression of EMG, and spore formation (Bowdish et al., 1994; Rubin-Bejerano et
al., 1996). Rim11 associates with Ime1(id) under all growth conditions (Bowdish et al., 1994;
Rubin-Bejerano et al., 1996), and in vitro it phosphorylates both Ime1 (Bowdish et al., 1994) and
Ume6 (Malathi et al., 1997). The Rim11 homologs Mck1 and Mrk1 have only minor effects on
these processes (Neigeborn and Mitchell, 1991; Rabitsch et al., 2001).
The transcription of RIM11 is constitutive in vegetative and meiotic conditions; however, in
ime1∆ cells its expression in meiotic cultures is reduced, probably reflecting the use of the URS1
like site present in its promoter (Bowdish et al., 1994). The level of Rim11 protein is slightly
reduced in glucose growth media in comparison to acetate (Bowdish et al., 1994). Rim11
functions as a kinase that shows autophosphorylation (Bowdish et al., 1994). Rim11 is
phosphorylated on tyrosine 199 (Zhan et al., 2000). Tyrosine to phenylalanine mutation in this
residue results in impaired expression of ime2-lacZ in vivo, and in a defect in phosphorylation
Ume6 in vitro (Zhan et al., 2000). This mutation does not impair autophosphorylation (Zhan et
al., 2000), suggesting different requirement for phosphorylation of exogenous substrates (see
section IIID1).
Rim11 associates with the C-terminal domain of Ime1, amino acids 270-360 (Malathi et al.,
1999; Rubin-Bejerano et al., 1996). In vitro, Rim11 phosphorylates the C-terminal 20 amino
acids residues in this region of Ime1 (Malathi et al., 1999). Simultaneous mutations of 8 serine
threonine and tyrosine of these residues have the following effects: i. In vitro phosphorylation of
Ime1 by Rim11 is absent, ii. Ime1 does not interact with Ume6, iii. IME2 is not expressed, and iv.
Asci are not formed (Malathi et al., 1999). Ime1 is detected by antibodies directed against
phosphotyrosines, and tyrosine to phenylalanine mutation in this domain results in impaired
expression of ime2-lacZ in vivo, suggesting that Rim11 phosphorylates these tyrosine resides in
Ime1.
Rim11 controls meiosis by regulating not only Ime1, but also Ume6. Using the two-hybrid
method, coIP, and in vitro kinase assay, Malathi et al show that Rim11 physically associates with
Ume6, it phosphorylates Ume6(1-232), and the association between Rim11 and Ume6 is
independent of Ime1 (Malathi et al., 1997). In addition, the two-hybrid assay reveals interaction
between Ume6 and Mck1 (Xiao and Mitchell, 2000). In vivo, Ume6 is a phosphoprotein, whose
level of phosphorylation is increased in vegetative growth media with acetate as the sole carbon
source in comparison to glucose (Xiao and Mitchell, 2000). Moreover, hyper phosphorylation is
26

observed upon nitrogen starvation (Xiao and Mitchell, 2000). Single deletion of RIM11, MCK1 or
MRK1 has no effect on the in vivo pattern of phosphorylation of Ume6 (Xiao and Mitchell, 2000),
but a substantial reduction in the pattern of phosphorylation is observed for the rim11∆ mck1∆
mrk1∆ triple mutant (Xiao and Mitchell, 2000). The redundant function of these GSK3-β
homologs is also evident in cells carrying the partially active rim11K68R allele. Efficient
sporulation is observed for cells carrying the MCK1 MRK1 wild type alleles, and no sporulation
in either mck1∆ MRK1 or MCK1 mrk1∆ strains (Xiao and Mitchell, 2000). Ume6 sequence
reveals several consensus sites for phosphorylation by the mammalian GSK3-β homolog.
Simultaneous substitution of serine and threonine residues to alanine in one such site (T99A
T103A T107) leads to a reduction in phosphorylation, and concomitantly a dramatic reduction in
the ability of Ume6(1-232) to interact with Ime1, suggesting that phosphorylation is a prerequisite
for interaction (Xiao and Mitchell, 2000).
The two-hybrid method demonstrates that the interaction between Rim11 and Ume6 is
regulated by the carbon source; the interaction is low in glucose growth media and it is increased
in acetate growth media (Malathi et al., 1997). However, coIP shows physical association
between Ume6 and Rim11 that is independent on nutrients (Malathi et al., 1997). The authors
suggest that this is due to lower levels of Galbd-Ume6 in glucose versus acetate growth media
(Malathi et al., 1997). However, it is also possible that this is due to the inability of the Gal4(bd)-
Rim11/Gal4(ad)-Ume6 complex to activate transcription, due to the activity of Sin3/Rpd3.
Nevertheless, the ability of Rim11 to in vitro phosphorylate Ume6 is increased when both
proteins are isolated from acetate grown cells, suggesting that the carbon source regulates either
the activity of Rim11, or the ability of its substrate, Ume6, to be phosphorylated (Malathi et al.,
1997).
The association of Ime1 with Ume6, and the association of Rim11 with both Ime1 and Ume6
suggest that one association may serve as a scaffold for the second association. There are no
direct evidence for association of Rim11 with Ime1 and Ume6 in cells deleted for UME6 or
IME1, respectively. However, a Gal4ad-Ume6T99N mutant protein shows no interaction with
Rim11 in a two-hybrid assay, and reduced interaction with Ime1 (Malathi et al., 1997). These
results suggest that either the association between Ume6 and Rim11 is required for the interaction
between Ime1 and Ume6 (Malathi et al., 1997), or that the Ume6 T99 residue is required for the
association of Ume6 with both Rim11 and Ime1.
3.2. The function of Rim15. Rim15 is absolutely required for the transcription of EMG
such as IME2, HOP1 and SPO13 (Vidan and Mitchell, 1997). As described above (section
IIIC1.2), Rim15 is required for the transcription of IME1, however, expression of IME1 from an
27

heterologous promoter does not suppress this phenotype, suggesting that Rim15 is required for an
additional event. Indeed, Rim15 is required for efficient interaction between Ime1 and Ume6, in
cells deleted for RIM15 5-fold reduction in their interaction is observed (Vidan and Mitchell,
1997). This effect is probably due to the effect of Rim15 on Ume6. It is required for complete in
vivo phosphorylation of Ume6 in SA and SPM media (Xiao and Mitchell, 2000). Rim15 is not
required for the in vitro kinase activity of Rim11 on Ime1 (Vidan and Mitchell, 1997).
4. The function of Gcn5. The conversion of URS1 from a repression to activation element
requires the function of the histone acetylase, Gcn5 (Burgess et al., 1999). Diploid cells carrying
a recessive mutation in GCN5 arrest under meiotic conditions prior to premeiotic DNA
replication and meiotic recombination (Burgess et al., 1999). These cells express IME1, but are
defective in the transcription of IME2 (Burgess et al., 1999). Using antibodies directed against
acetylated histones H3 and H4, Burges et al., show that under meiotic conditions, depending on
Gcn5, specific hyper acetylation on histone H3 is observed in the IME2 promoter (Burgess et al.,
1999). Deletion of RPD3 that is required for deacetylation of Histone H4 in the IME2 promote,
does not suppress gcn5, suggesting that acetylation of histones is a prerequisite for the
transcription of EMG (Burgess et al., 1999). It is not known how Gcn5 is recruited to the
promoter of IME2 or additional EMG.
5. The transcription of EMG is also regulated by positive elements. In addition to the
negative element, URS1, the promoters of EMG carry positive elements required for their
transcription under meiotic conditions: UASH element in HOP1, and the T4C sequence in IME2
(Bowdish and Mitchell, 1993; Vershon et al., 1992). Homologous sequences are found in
additional EMG (Chu et al., 1998). Deletion or mutations of these elements prevent high-level
expression of EMG (Bowdish and Mitchell, 1993; Vershon et al., 1992). Abf1 binds to the UASH
element (Gailus-Durner et al., 1996). ABF1 encodes an essential DNA-binding transcriptional
activator required for the transcription of various genes as well as for DNA replication; it binds to
origins of DNA replication (Svetlov and Cooper, 1995). The protein that binds the T4C element in
IME2 is not known.
6. Additional positive regulators. Ime2, Rim4, and Ndt80 are early and early late genes
required for high-level transcription of EMG. Detailed description on their functions is given in
section VA.
28

7. The role of premeiotic DNA replication and/or recombination in controlling EMG
expression. Hydroxyurea (HU) is routinely used to inhibit DNA synthesis as it inhibits
ribonucleotide reductase (Elford, 1968; Slater, 1973). Shifting cells to meiotic conditions in the
presence of 40 mM and 200 mM hydroxyurea leads to a reduction or no expression, respectively,
of EMG (Davis et al., 2001; Lamb and Mitchell, 2001). It was suggested that perturbation of
premeiotic DNA replication inhibits the transcription of EMG. However, cells deleted for CLB5
along with CLB6 or MUM2/SPOT8 arrest prior to premeiotic DNA replication, with complete
expression of EMG (Davis et al., 2001; Dirick et al., 1998; Stuart and Wittenberg, 1998). These
results imply that HU blocks DNA replication at different point than clb5 clb6 and mum2, and
that only the HU block transmits a checkpoint signal to repress transcription. However, it is also
possible, that the effect of HU is not mediated through DNA replication. The latter hypothesis is
strengthened by the observation that treatment of yeast cells with HU leads to a substantial
decrease in RNA and protein synthesis (Slater, 1973).
The effect of HU is mediated through Rpd3 and Sin3 whose presence is required for the
reduction in transcription (Lamb and Mitchell, 2001). In addition, in the presence of HU there is a
reduction in phosphorylation of Ume6, a phenomenon that is associated with reduction in the
activity of Ume6 (Lamb and Mitchell, 2001).
8. The choice between silencing and expression of EMG. Silencing and expression of
EMG are dependent on the URS1 element present in their promoter. URS1 serves as a silencing
element in vegetative growth media with glucose as the sole carbon source, and is converted into
an activation element under meiotic conditions, i.e. nitrogen depletion in the presence of acetate.
Fig. 11 summarizes the current knowledge and model for how this regulation is accomplished.
Under all growth conditions Ume6 binds to URS1. However, nutrients control the protein
complexes that are recruited by Ume6 to the URS1 element. In glucose growth media Ume6
recruits two repression complexes, the HDAC Sin3/Rpd3 that leads to deacetylation of lysines in
histone H4, and the Isw2 complex that leads to chromatin remodeling. These two activities are
required for complete silencing of EMG. The Sin3/Rpd3 complex is conserved, and functions as a
transcriptional silencer in all eukaryotes (Ng and Bird, 2000; Pazin and Kadonaga, 1997).
However, Ume6 is not conserved, and in mammals, it is the Mad/Max and nuclear receptors that
recruit Sin3/Rpd3 to the DNA (Ng and Bird, 2000; Pazin and Kadonaga, 1997). Similarly, the
Isw2 complex is also conserved and functional in all eukaryotes [for review see (Cairns, 1998)].
Under meiotic conditions (acetate media and nitrogen depletion) the Sin3/Rpd3 and Isw2
repression complexes are not functional. Relief of repression of Sin3 depends on Ime1 that is
29

expressed only in the absence of glucose. The mode by which Ime1 relieves repression of
Sin3/Rpd3 and how Isw2 does not repress transcription under these conditions are not known.
Relief of repression does not suffice for transcriptional activation, and requires Gcn5, the histone
acetylase, as well as a transcriptional activation protein, Ime1. Ume6 recruits Ime1 to URS1, and
the interaction between these two proteins depends on the absence of glucose and nitrogen. These
nutrient signals are transmitted to both Ime1 and Ume6, by two protein kinases, Rim15 and
Rim11. Since Rim15 is required for complete phosphorylation of Ume6 in SA and SPM, and
since its kinase activity is inhibited in the presence of glucose by PKA, it is assumed that the
glucose signal that inhibits the association of Ime1 with Ume6 is transmitted, at least partially,
through Rim15. Since Rim15 is only partially required for the interaction of Ime1 with Ume6, the
glucose signal must be transmitted through an additional protein. Rim11 phosphorylates both
Ime1 and Ume6, and this phosphorylation is essential for their interaction. It is not known how
nutrients regulate the function of Rim11.
V. Transcriptional regulation of middle meiosis-specific genes (MMG).
The regulated transcription of middle meiosis-specific genes (MMG) depends on the presence of
two positive elements, MSE (Middle Sporulation Element) (gNCRCAAAA/T) and an Abf1
binding site (Chu et al., 1998; Chu and Herskowitz, 1998; Hepworth et al., 1995; Hepworth et al.,
1998; Ozsarac et al., 1995; Ozsarac et al., 1997; Pierce et al., 1998). The MSE elements present in
different MMG are not identical, some, for example that in CLB1, function only as positive
elements, whereas others, for example that of SMK1 or NDT80 (designated inhere as MSE*),
function also as repression elements in vegetative growth conditions and early meiotic times
(Pierce et al., 1998; Xie et al., 1999). Schematic illustration on the regulation of MMG is
illustrated in Fig. 13, and discussed in the text below.
A. Positive regulators of MMG.
Ndt80 and Ime2 are two meiosis-specific positive regulators absolutely required for the
transcription of MMG (Chu et al., 1998; Chu and Herskowitz, 1998; Hepworth et al., 1998;
Ozsarac et al., 1997). The sequential transcription of MMG following transcription of the early
genes is due to the regulated transcription of both NDT80 and IME2 (Chu and Herskowitz, 1998;
Hepworth et al., 1998).
30

1. Ndt80. NDT80 encodes a transcriptional activator that binds to the MSE and MSE* elements
and activates transcription (Chu and Herskowitz, 1998). Cells deleted for NDT80 arrest following
the completion of premeiotic DNA replication and meiotic recombination, at pachytene, as mono
nucleate cells (Chu and Herskowitz, 1998; Hepworth et al., 1998; Xu et al., 1995). Ectopic
expression of Ndt80 in vegetative growth conditions is sufficient to induce the transcription of
most MMG (probably the ones carrying the MSE rather than the MSE* element) (Chu et al.,
1998; Chu and Herskowitz, 1998), suggesting that under growth conditions, the second positive
regulator, Ime2, is non-essential. Ime2 is also non-essential under meiotic conditions. When
Ndt80 is expressed from a heterologous, IME2 independent promoter, the MMG, SPS4, is
expressed in both IME2 and ime2∆ cells (Pak and Segall, 2002a). Nevertheless, in the latter,
expression of SPS4 is both delayed and lower than in the IME2 diploid cells, suggesting that Ime2
may be required for high and efficient activity of Ndt80 (Pak and Segall, 2002a). This hypothesis
is supported by the observation that Ime2 phosphorylates Ndt80 (Benjamin et al., 2002). Ndt80 is
a phosphoprotein (Benjamin et al., 2002; Tung et al., 2000), whose in vivo extent of
phosphorylation depends on IME2 (Benjamin et al., 2002). Furthermore, in vitro kinase assay
demonstrates that Ime2 directly phosphorylates Ndt80 (Benjamin et al., 2002).
Ndt80 is also required for high levels of transcription of IME1 as well as for its transient
expression (Hepworth et al., 1998). This effect might be mediated through Ime2, since Ndt80
regulates the transcription of IME2, through an MSE element present in its promoter (Chu and
Herskowitz, 1998).
NDT80 is a meiosis-specific gene whose transcription is dependent on Ime1 and Ime2 (Fig. 13)
(Chu and Herskowitz, 1998; Hepworth et al., 1998; Pak and Segall, 2002a). This regulated
transcription is mediated by two URS1 and two MSE elements (Chu and Herskowitz, 1998;
Hepworth et al., 1998; Pak and Segall, 2002a). One MSE element functions either as a repression
or activation element under growth and meiotic condition, respectively, whereas the second one
functions only as an activation element (Pak and Segall, 2002a; Xie et al., 1999). Furthermore,
both URS1 elements contribute to repression of NDT80 in vegetative cultures and for its high
level expression under meiotic conditions [(Pak and Segall, 2002a) and Fig. 13]. The presence of
the MSE elements delays NDT80 transcription by the Ume6/Ime1 complex in comparison to
other EMG that carry only the URS1 element (Chu and Herskowitz, 1998; Hepworth et al., 1998;
Mitchell et al., 1990; Pak and Segall, 2002a; Yoshida et al., 1990).
2. Ime2. IME2 encodes a meiosis-specific protein kinase that shows 58.9% similarity and
37% identity to the human cyclin-dependent kinase, CDK2 (Kominami et al., 1993). The crystal
31

structure of hCDK2 reveals that cyclin A binds to two regions: PSTAIRE and the T-loop (Jeffrey
et al., 1995). This binding is required to induce a conformational change that promotes activation
of the kinase. Ime2 does not carry the PSTAIRE sequence, but it shows 67% similarity and
52.3% identity to the T-loop region of hCDK2 (Fig. 12). This homology raises the possibility that
Ime2 might be activated by binding to a cyclin like molecule, and that in the meiotic cycle it
might replace Cdc28, the yeast CDK that regulates initiation and progression in the cell cycle
(Lew et al., 1997). The following results support the hypothesis that in the meiotic cycle Ime2
might replace Cdc28: i. Similarly to Cdc28, phosphorylation by Ime2 affects stability of Ime1
Cdh1 and Sic1, the latter two proteins are known substrates of Cdc28 in the mitotic cell cycle
(Bolte et al., 2002; Dirick et al., 1998; Guttmann-Raviv and Kassir, 2002). ii. Ime2 is absolutely
required for premeiotic DNA replication and meiotic recombination in cdc28-4 cells incubated at
the non-permissive temperature (Guttmann-Raviv et al., 2001). However, there are no reports on
any yeast proteins that bind to the T-loop like sequence in Ime2 and are required for its kinase
activity. Moreover, a recombinant Ime2 protein isolated from E. coli is active in phosphorylating
histone H1 (Donzeau and Bandlow, 1999), suggesting that unlike Cdc28, Ime2 is active in the
absence of additional yeast proteins. However, phosphorylation of its native substrate, Gpa2 (see
below) requires the isolation of Ime2 from yeast cells (Donzeau and Bandlow, 1999), suggesting
that specificity, for at least for some substrates, might require post-translation modification or the
presence of an activator.
Ime2 is a positive regulator of meiosis required for multiple functions: the correct timing and
high level transcription of early meiosis-specific genes, the transcription of middle and late genes
(Mitchell et al., 1990; Yoshida et al., 1990), the correct timing of entry into premeiotic DNA
replication meiotic recombination and nuclear division (Benjamin et al., 2002; Foiani et al.,
1996), as well as for ascus formation (Benjamin et al., 2002; Foiani et al., 1996; Mitchell et al.,
1990; Yoshida et al., 1990). When Ime2 is over expressed, but only in the SK1 background, it
bypasses the requirement for Ime1 for the transcription of EMG and sporulation (Mitchell et al.,
1990), suggesting an Ime1 independent pathway for activating the transcription of these genes
(Mitchell et al., 1990). It is not known how Ime2, that encodes a kinase, replaces a transcriptional
activator, nor if under normal conditions this pathway has any contribution to the transcription of
these genes.
Ime2 functions also as a negative regulator for the following functions: i. Phosphorylation of
Ime1 by Ime2 leads to its degradation by the 26S proteasome (Guttmann-Raviv and Kassir,
2002). It is possible that the absence of Ime1 reestablishes repression by Sok2 and Sin3 [see
sections IIIC1.1.2, IVB1.2, and (Shenhar and Kassir, 2001; Washburn and Esposito, 2001)],
32

esulting in the transient transcription of IME1 and EMG. According to this model, in cells
deleted for IME2, the continuous presence of Ime1 leads to the non-transient expression of IME1
and EMG (Mitchell et al., 1990; Yoshida et al., 1990). ii. In the meiotic cycle, upon completion of
premeiotic DNA replication and the presence of Ime2, two subunits of the DNA polymerase αprimase
complex, Pol1 and Pol12, are degraded (Foiani et al., 1996). iii. The level of the
Clb/Cdc28 inhibitor, Sic1, is reduced under meiotic conditions, a process regulated by Ime2
(Dirick et al., 1998). iv. When Ime2 is ectopically expressed in the cell cycle it phosphorylates
Cdh1, an event causing dissociation of Cdh1 from APC (anaphase promoting complex – the
ubiquitin ligase required for proteolysis of specific substrates), and consequently stabilization of
its substrates, such as Clb2 and Cdc5 (Bolte et al., 2002). It is not known if Ime2 phosphorylate
Cdh1 in the meiotic cycle, and what are the consequences of this event for entry into the meiotic
divisions. v. In the meiotic cycle Ime2 is required to limit premeiotic DNA replication and
nuclear division to one and two rounds, respectively (Foiani et al., 1996). It is assumed that this
phenomenon results from stabilization of specific proteins or regulators of DNA replication and
nuclear divisions.
The availability and function of Ime2 is regulated in several levels:
Transcriptional regulation: The transcription of IME2 is silent in vegetative growth conditions by
the binding and activity of the Ume6/Sin3/Rpd3 and Ume6/Isw2 complexes to the URS1 element
present in its promoter. Under meiotic condition, recruitment of Ime1 by Ume6 promotes the
transcription of Ime2. An additional histone deacetylase activity, that of the Set3/Hos2 complex,
regulates the transcription of IME2 as well as NDT80 early in meiosis (Pijnappel et al., 2001).
Deletion of SET3 or HOS2 has no effect during vegetative growth (although this might be due to
repression by Sin3/Rpd3), but it leads to increased and advanced transcription of IME2 and
NDT80, as well as moderate increase in the transcription of IME1 (Pijnappel et al., 2001).
Consequently, these cells progress faster through MI, MII and asci formation (Pijnappel et al.,
2001). Ime2 is subject to positive autoregulation that is most probably mediated by the MSE
element present in its promoter and Ndt80 (see above, VA1).
Protein stability: Ime2 is an extremely non-stable protein (Bolte et al., 2002; Guttmann-Raviv and
Kassir, 2002) with a half-life of about 5 minutes (Guttmann-Raviv and Kassir, 2002). It is not
known if the stability of Ime2 is subject to any regulation.
Activity regulation: Ime2 is subject to phosphorylation (Benjamin et al., 2002; Guttmann-Raviv
and Kassir, 2002). In vitro kinase assays reveals autophosphorylation (Benjamin et al., 2002;
Guttmann-Raviv and Kassir, 2002; Kominami et al., 1993), but its effect on the activity and/or
stability of Ime2 is not known. The kinase activity of Ime2 is negatively regulated by its C-
33

terminal non-essential domain (Kominami et al., 1993). This is deduced from the following
observations: i. Over expression of an Ime2 protein truncated for this domain promotes meiosis in
the presence of either nitrogen or glucose (Kominami et al., 1993), and ii. The in vitro-kinase
activity of Ime2 is higher for a truncated Ime2 protein in comparison to the wt protein (Kominami
et al., 1993). Thus, in the presence of nutrients Ime2 is less, or non-active. The nutrient signal is
transmitted to the C-terminal domain through the Gα protein, Gpa2 (Donzeau and Bandlow,
1999). Ime2 specifically associates with the GTP bound Gpa2 (Donzeau and Bandlow, 1999), a
form whose level is increased in the presence of glucose [for review see (Versele et al., 2001)]. In
addition, the association between Gpa2 and Ime2 requires the presence of nitrogen (Donzeau and
Bandlow, 1999). Cells deleted for GPA2 show similar phenotypes to cells over expressing the
truncated Ime2 protein, namely, sporulation in the presence of glucose and nitrogen (Donzeau
and Bandlow, 1999). Gpa2 associates with the glucose receptor, Gpr1, and transmits the glucose
signal to adenylate cyclase [for review see (Pan et al., 2000)], however, the effect of Gpa2 on
Ime2 is independent of PKA (Donzeau and Bandlow, 1999). This is deduced from the
observation that the bcy1-tpk1 w1 mutation that leads to low activity of PKA does not suppress the
reduction in sporulation exhibited by cells expressing the constitutive active Gpa2G132V protein
(Donzeau and Bandlow, 1999). The in vitro kinase activity of Ime2 on histone H1 is decreased by
the addition of recombinant Gpa2γS (Donzeau and Bandlow, 1999), suggesting that Gpa2 inhibits
the kinase activity of Ime2 (Donzeau and Bandlow, 1999). In vitro kinase assays also demonstrate
that Ime2 phosphorylates Gpa2 (Donzeau and Bandlow, 1999), but the role of this
phosphorylation is not known.
Ime2 kinase activity fluctuates in the meiotic cycle, it first peaks concomitantly with
premeiotic DNA replication and then, concomitantly with nuclear division (Benjamin et al.,
2002). The second increase in activity is regulated by Cdc28 (Benjamin et al., 2002).
The activity of Ime2 is also regulated by Ids2 (Sia and Mitchell, 1995). Over expression of
Ime2 in vegetative growth media is toxic (Bolte et al., 2002; Guttmann-Raviv and Kassir, 2002;
Sia and Mitchell, 1995), and this toxicity is relived in cells deleted for IDS2 (Sia and Mitchell,
1995), suggesting that Ids2 is a positive regulator of Ime2. Ids2 is not required for meiosis (Sia
and Mitchell, 1995), however, in its absence over expression of Ime2 (in the SK1 strain) leads to
the transcription of only the early meiosis-specific genes, while middle and late genes remain
silent and spores are not formed (Sia and Mitchell, 1995). This result suggests that the activity of
Ime2 is distinctly regulated at early and middle meiotic times (Sia and Mitchell, 1995), in
agreement with the finding of Benjamin et al., (Benjamin et al., 2002) reported above. The effect
of Ids2 is mediated through the C-terminal domain of Ime2: Over expression of Ime2 truncated
34

for this domain suppresses ime1∆ in both wt and ids2∆ cells (Sia and Mitchell, 1995). The steadystate
level of Ids2 is decreased and then disappears in cells shifted to meiotic conditions (Sia and
Mitchell, 1995).
RIM4 is an EMG required for high-level expression of EMG, premeiotic DNA replication,
timely and efficient commitment to meiotic recombination, nuclear division, and spore formation
(Deng and Saunders, 2001; Soushko and Mitchell, 2000). Rim4 function is mediated through
both the Ime1 and Ime2 transcriptional activation pathways (Soushko and Mitchell, 2000). rim4∆
diploids over-expressing Ime2 are sporulation proficient, suggesting that Rim4 functions
upstream of Ime2 (Soushko and Mitchell, 2000). Rim4 carries two RNA recognition motifs that
are important for its role in meiosis (Soushko and Mitchell, 2000). The mode by which Rim4
affects transcription is not known, though it was proposed that it might stabilize IME2 mRNA
(Deng and Saunders, 2001; Soushko and Mitchell, 2000).
3. Sin3 and Rpd3. Random screening for mutations preventing expression of MMG
identified NDT80 and SIN3 (Hepworth et al., 1998). Sin3 as well as Rpd3 are required for the
transcription of MMG, suggesting that they function also as positive regulators, or that their
function as positive regulators might be due to repression of a negative regulator (Hepworth et al.,
1998). Diploid cells deleted for SIN3 or RPD3 arrest at the same point as ndt80 cells, namely,
following the completion of premeiotic DNA replication, as mono nucleate cells (Hepworth et al.,
1998). Point of arrest is not due to defects in recombination, since concomitant deletion of SPO13
and SPO11 does not allow a bypass of the arrest, and the triple mutant cells remain sporulation
deficient (Hepworth et al., 1998; Xu et al., 1995).
B. Negative regulators of MMG.
SMK1 is a middle gene subject to silencing by binding of Sum1 to the MSE* element in its
promoter (Xie et al., 1999). Sum1 also represses the transcription of NDT80, in cells deleted for
SUM1 premature expression of NDT80 is observed (Pak and Segall, 2002a). Sum1 associates
with Hst1, and the resulting complex, containing additional proteins, exhibits NAD + dependent
histone deacetylase activity (Pierce et al., 1998; Pijnappel et al., 2001; Rusche and Rine, 2001).
Deletion of either SUM1 or HST1 leads to expression of SMK1 in vegetative growth conditions
(Xie et al., 1999). This expression is probably independent of Ndt80, since NDT80 remains silent
in sum1∆ or hst1∆ cells (Xie et al., 1999). Relief of repression is regulated by Sum1 availability:
The transcription of SUM1 is constitutive, but the level of Sum1 protein is reduced prior to
expression of MMG, and then it is induced again. As described above, the transcription of NDT80
35

is dependent on Ime2, but in cells deleted for both IME2 and SUM1, NDT80 is expressed (Pak
and Segall, 2002a). These results suggest that Ime2 abrogates Sum1 repression (Pak and Segall,
2002a). It is possible, as in the case of Ime1 (Guttmann-Raviv and Kassir, 2002), that
phosphorylation of Sum1 by Ime2 is required for the regulated degradation of Sum1. The
Sum1/Hst1 repression activity can be bypassed in vegetative growth media by over expression of
Ndt80 (Xie et al., 1999). Sum1 and Hst1 are not required for meiosis, cells deleted for either gene
complete meiosis and form viable spores (Lindgren et al., 2000).
The Sin3/Rpd3/Ume6 and Ssn6/Tup1 repression complexes that regulate transcription of EMG
are not involved with repression activity of MMG (Pierce et al., 1998).
C. The recombination checkpoint.
In meiosis, a checkpoint mechanism consisting of Rad17, Rad24, Mek1, Pch2, Mec3, and Ddc1
prevents the transcription of MMG and entry into the first meiotic division in cells that have not
properly completed synapsis of homologs and meiotic recombination (Roeder and Bailis, 2000).
Diploid cells deleted for DMC1 are impaired in both meiotic recombination (Bishop et al., 1992)
and expression of MMG (Chu and Herskowitz, 1998; Hepworth et al., 1998), whereas dmc1 cells
carrying mutations in RAD17, or MEK1 express MMG (Chu and Herskowitz, 1998; Hepworth et
al., 1998). Three targets of the pachytene checkpoint were identified; these are the protein kinase
Swe1 that phosphorylates and inactivates Cdc28 (Leu and Roeder, 1999), the transcriptional
activator Ndt80 (Pak and Segall, 2002b; Tung et al., 2000), and the transcriptional repressor
Sum1 (Lindgren et al., 2000; Pak and Segall, 2002b). This is concluded from the observations
that in the double mutants dmc1 swe1 and dmc1 sum1 MMG are expressed and cells enter the
meiotic nuclear division (Leu and Roeder, 1999; Lindgren et al., 2000; Pak and Segall, 2002b;
Tung et al., 2000). Over expression of Ndt80 leads to expression of MMG and partial entry into
nuclear division (Pak and Segall, 2002b). In this review we focus only on how this surveillance
mechanism affects transcription [for a detailed review on the checkpoint pathway see (Roeder
and Bailis, 2000)].
The pachytene checkpoint leads to a reduction in the transcription of NDT80 (Lindgren et
al., 2000; Pak and Segall, 2002b). However, contradicting results, namely, no effect, were
reported for other different strains (Chu and Herskowitz, 1998; Hepworth et al., 1998; Tung et al.,
2000). Sum1 mediates the reduction in the transcription of NDT80. The availability of Sum1 is
regulated by the pachytene checkpoint as evident from the observations that in dmc1 cells the
steady-state level of Sum1 is constitutive throughout the meiotic pathway, whereas in the double
mutant dmc1 rad17 the level of Sum1 is reduced in meiotic prophase, as in the wild type strain
36

(Lindgren et al., 2000). Since Ime2 mediates the availability of Sum1 under normal meiotic
conditions, it is possible that the effect of the checkpoint system on Sum1 is mediated through
Ime2. Pak and Segall (Pak and Segall, 2002b) suggest that the effect of Sum1 is mediated only
through regulating the transcription of NDT80, since the triple mutant dmc1 sum1 ndt80 does not
enter nuclear division (Pak and Segall, 2002b). Morovere, they propose that in the dmc1 sum1
cells, expression of the B-type cyclins is responsible for entry into meiotic nuclear division (Pak
and Segall, 2002b). This hypothesis is supported by the observation that entry into nuclear
division in swe1 hop2 cells is delayed in comparison to wild type cells or to swe1 hop2 cells over
expressing Clb1 (Leu and Roeder, 1999). However, this model cannot explain why ectopic
expression of Ndt80 in dmc1 cells leads to inefficient entry into nuclear division in comparison to
the dmc1 sum1 cells (20% versus 60%) (Pak and Segall, 2002b). These results suggest that an
additional target of Sum1 might be required for proper meiotic arrest (Lindgren et al., 2000).
Since the CLB genes are not direct targets of Sum1, they are not the genes directly regulated by
Sum1 (Lindgren et al., 2000).
The pachytene checkpoint inhibits phosphorylation of Ntd80 (Tung et al., 2000). In cells
deleted for ZIP1 [a component of the synaptonemal complex required for synapsis of homologs
(Sym et al., 1993)] Ndt80 is partially phosphorylated (Tung et al., 2000), whereas in the double
mutants zip1 pch2 and zip1 mek1 it is highly phosphorylated (Tung et al., 2000). It was suggested
that activation of transcription by Ndt80 requires its phosphorylation (Benjamin et al., 2002; Chu
and Herskowitz, 1998; Hepworth et al., 1998; Tung et al., 2000). Therefore, lack of expression of
MMG is also due to the reduction in the phosphorylation of Ndt80. Since Ime2 is required for
phosphorylation of Ndt80, it should be interesting to determine if Ime2 is the target for the
pachytene check point system.
In addition, the pachytene checkpoint mediates the transcription of MMG through the Cdc28
inhibitor, Swe1. In swe1 hop2 diploid cells CLB1 transcription is increased to its normal level,
although with a substantial delay, in comparison to wild type or hop2 cells (Leu and Roeder,
1999). It should be interesting to determine whether this effect of Swe1 on Cdc28 is mediated
through Ime2, since Cdc28 is required for the activity of Ime2 that coincides with nuclear
divisions (Benjamin et al., 2002), and Ime2 is absolutely required for the transcription of the CLB
genes.
37

VI. Transcriptional regulation of late meiosis-specific genes
A. Early late genes.
SGA1 is an early-late gene encoding a glucoamylase whose transcription in growth media is
silenced by a negative element, NRE SGA , which exhibits no UAS activity (Kihara et al., 1991;
Mai and Breeden, 2000). Expression under meiotic conditions depends on a UAS element present
in its promoter region (Kihara et al., 1991). In addition, it carries 4 STRE elements (Mai and
Breeden, 2000), but their role in the transcription of SGA1 is not known. Relief of repression by
the NRE SGA element depends on Ime1 and Ime2. However, the activity of the UAS element is
independent of these regulators, because it does not require the presence of both MATa and
MATα alleles that are required for the expression of Ime1 (Kihara et al., 1991). The activity of the
positive element requires nitrogen depletion (Kihara et al., 1991), demonstrating that the effect of
nitrogen on meiosis is mediated not only through Ime1.
Silencing of SGA1 in vegetative growth media depends on the chromatin-remodeling factor
Isw2 (Goldmark et al., 2000). As described above (section IVA5), Isw2 is also a negative
regulator of EMG, and it is recruited by Ume6 to their promoters. Since the URS1 element to
which Ume6 binds is not present in SGA1, it is not known how Isw2 is recruited to SGA1. The
identities of the positive regulators promoting transcriptional activation of SGA1 are not known.
However, one positive regulator, SME2, when present on a multi-copy plasmid, increases the
transcription of SGA1 in the presence of nitrogen, thus bypassing also the nitrogen signal
inhibiting spore formation (Kawaguchi et al., 1992). Deletion of SME2 has no effect on the
regulated expression of SGA1, or on the efficiency of sporulation. The sequence and mode of
function of SME2 are not known.
B. Mid-late genes. The transcription of the mid-late sporulation-specific genes DIT1 and DIT2
is induced upon completion of meiotic divisions (Friesen et al., 1997). Time of expression is
compatible with function, since these divergently transcribed genes (Friesen et al., 1997) encode
enzymes required for biosynthesis of the dityrosine precursor that is incorporated into the
outermost layer of the spore wall (Briza et al., 1990). A negative element, designated NRE DIT is
required for repression during vegetative growth (Friesen et al., 1997) (Note that NRE DIT is not
identical to NRE SGA1 ). This region carries the middle sporulation-like element MSE, and the DRE
element (Bogengruber et al., 1998; Friesen et al., 1997) that are required for repression in growth
conditions and activation under meiotic conditions. Two additional positive elements are required
for high expression under meiotic conditions (Friesen et al., 1997).
38

Transcriptional activation of the mid late genes depends on three positive regulators, Ndt80
and Ime2 whose effects are most probably mediated through the MSE like sequence (Friesen et
al., 1997), and Rim1 (Rim101) through an unidentified region (Bogengruber et al., 1998). The
effect of Rim1 may be indirect (see IIIC3). The repression activity of NRE DIT depends on Ssn6,
Tup1, Rox3, Sin4 and Spe3 (Friesen et al., 1997; Friesen et al., 1998). Rox3 and Sin4 are
subunits of the mediator complex of RNA polymerase II (Gustafsson et al., 1997; Myer and
Young, 1998) that repress transcription of many genes (Hanna-Rose and Hansen, 1996). SPE3
encodes Spermidine synthase (Hamasaki-Katagiri et al., 1997), and accordingly, addition of
spermidine to the media leads to increased repression (Friesen et al., 1998). The way by which
these complexes are specifically recruited to NRE DIT is not known.
XBP1 is a mid-late gene whose transcription is initiated at the same time as that of DIT1,2, but
unlike these genes its transcription is non-transient (Mai and Breeden, 2000). Accordingly, its
regulated transcription is not mediated via a NRE DIT element (Mai and Breeden, 2000). XBP1
encodes a DNA-binding protein that functions as a repressor when fused to lexA. Deletion of
XBP1 leads to a delay and reduction in the percentage of asci, while meiotic divisions are
properly controlled (Mai and Breeden, 2000). It is not known how this effect of Xbp1 is
mediated.
C. Late genes
The transcription of the 5 late meiotic genes peaks following completion of meiotic divisions at
8-12 hours in sporulation media (Law and Segall, 1988). A representative of these genes is
SPS100 that is involved in spore wall formation (Law and Segall, 1988). The transcription of
SPS100 depends on Ime1, Ndt80, and Ama1 (Cooper et al., 2000; Hepworth et al., 1998). AMA1
encodes a meiosis-specific subunit of APC/C that is required for degradation of CLB1 in the
meiotic cycle (Cooper et al., 2000). The transcription of AMA1 is induced at the same time as
MMG, but since it does not carry the Ndt80 biding site (Chu et al., 1998) its mode of regulation is
not known. In addition, meiosis-specific splicing by the EMG Mer1 (Engebrecht and Roeder,
1990) determines the accumulation of Ama1 protein (Cooper et al., 2000). Diploid cells deleted
for AMA1 arrest with a short spindle prior to the first meiotic division (Cooper et al., 2000).
Genetic analysis suggests that Ama1 is dispensable for the second meiotic division, but is
required for spore formation (Cooper et al., 2000). Further work is required to determine if Ama1
is directly involved with transcriptional regulation of late genes, for example by degradation of a
transcriptional repressor, or if the effect on transcription is mediated by a checkpoint mechanism.
39

The transcriptional activator that binds to and activates transcription of the late genes is not
known.
The late meiosis-specific genes SPS100 and DTT1 show basal levels of expression in
vegetative growth conditions (Jung and Levin, 1999). This expression depends on a functional
MAPK cascade that transmits the cell wall integrity signal (Jung and Levin, 1999). Activation of
the MAPK Slt2/Mpk1, or deletion of Rlm1, the transcription factor whose activity is regulated by
it, reduces the expression the late meiosis specific genes (Jung and Levin, 1999). It is not known
whether Rlm1 binds the promoter region of late genes, or if it is required for their expression
under meiotic conditions.
VII. A feedback loop controlling meiosis.
The transcription of all meiosis-specific genes is transient, reflecting the need for the function of
these genes for a limited period. Nevertheless, it is important to point that transient transcription
does not necessarily reflect transient availability of proteins. For instance, in the mitotic cell
cycle, the transcription of genes involved in DNA replication, such as CLB5, POL1, or POL12 is
periodic, and identically regulated (Lowndes et al., 1991; Schwob and Nasmyth, 1993). But the
steady state level of Pol1 and Pol12 is constant (Foiani et al., 1995), whereas the level of Clb5 is
periodic (Irniger and Nasmyth, 1997). Periodicity of Clb5 is accomplished by its regulated
degradation (Irniger and Nasmyth, 1997). The pattern of protein expression of many meiosisspecific
genes is not known, however, the two main positive regulators of meiosis, Ime1 and
Ime2 are non-stable proteins whose protein levels mimic the level of their RNA. This suggests
that at least in the case of Ime1 and Ime2, the transient availability might be a sign of requirement
for only a short period. In agreement with this view, over expression of Ime1 in meiotic cultures
leads (in some strains) to inefficient sporulation, a substantial increase in non-disjunction, the
formation of 2-spored rather than 4-spored asci, and a reduction in sporulation (Shefer-Vaida et
al., 1995; Sherman, 1992).
Positive feedback loops control the transcription of both IME1 and IME2 (Fig. 14). Positive
autoregulation by Ime1 is required to relieve repression mediated by Sok2, and thus lead to an
increase in Ime1 level (Shenhar and Kassir, 2001). Positive autoregulation accelerates the
availability of Ime1 and Ime2, and this enhanced expression might be essential for proper entry
and progression through the meiotic cycle.
The transient transcription of all meiosis-specific genes is explained by the transient
availability of Ime1, Ime2, and Ndt80. Phosphorylation of Ime1 by Ime2 leads to its degradation
40

y the 26S proteasome (Guttmann-Raviv and Kassir, 2002). In the absence of Ime1, Sok2
represses the transcription of IME1, leading to a decrease in IME1 mRNA. It is not known
whether the negative feedback regulation of Ime2 on the transcription of IME1 is mediated only
through its effect on Ime1. Since the transcription of IME2 depends on Ime1 (Mitchell et al.,
1990; Yoshida et al., 1990) and the half-life of Ime2 is very short (Bolte et al., 2002; Guttmann-
Raviv and Kassir, 2002), there are sufficient levels of Ime1 to relieve repression by Sin3
(Washburn and Esposito, 2001) and to activate transcription of EMG. In the absence of Ime1 lack
of these two functions leads to shutting down the transcription of EMG. The transcription of
MMG and LMG depends on Ime2 (Chu et al., 1998; Chu and Herskowitz, 1998; Friesen et al.,
1997; Hepworth et al., 1998; Kihara et al., 1991; Ozsarac et al., 1997; Smith and Mitchell, 1989).
In cells depleted for Ime1, Ime2 is absent, and the transcription of the early late and late genes is
closed.
VIII. Concluding remark. The choice between developmental pathways
S. cerevisiae is a simple eukaryote with limited developmental options. Diploid cells may chose
between growth in yeast or pseudohyphal forms, and meiosis. Two crucial mechanisms regarding
developmental decisions are required for faithful growth, entry into a specific developmental
pathway should take place only in response to specific signals, and should be a signal to block
entry into an alternative pathway. Concomitant entry into two developmental pathways could lead
to chaos. In S. cerevisiae, the MATa and MATα gene products as well as the pachytene
surveillance mechanisms ensure that only diploid cells can initiate meiosis. This is a crucial
decision, because meiosis in haploid cells would lead to the formation of uneuploid gametes.
In S. cerevisiae, the meiotic cycle ends with the formation of dormant spores resistant to
hazardous conditions. The decision to exit the mitotic cell cycle depends on the availability of
nutrients. Entry into the meiotic cycle in the presence of nutrients can be a “waste”. Several
redundant mechanisms ensure that in the presence of glucose the meiotic pathway will be
repressed: i. The transcription of IME1 is repressed in the presence of glucose due to the presence
of distinct negative elements (i.e. UASru, IREu, UCS1), each responding to a different signal
pathway. Thus, if one signal pathway is malfunctioning, the activity of the additional signal
pathways will ensure repression. Moreover, several UAS elements (i.e. UASru, IREu, UASrm),
each regulated in a distinct manner, activates transcription in the absence of glucose. ii. The
activity of Ime1 is repressed in the presence of glucose by two distinct mechanisms. a.
Phosphorylation of Ime1 by the Cln/Cdc28 kinase sequesters the protein from the nucleus, and b.
41

Functional interaction between Ime1 and Ume6 that promote transcriptional activation is
inhibited in the presence of glucose and nitrogen. The presence of nitrogen also prevents
translation of IME1 mRNA and entry of Ime1 into the nucleus.
The presence of glucose also inhibits the function of Ime2. Normally Ime2 is available only
under meiotic conditions, but cells developed mechanisms to ensure that in the presence of
nutrients accidental expression of Ime2 will not lead to entry and completion of meiosis. In the
presence of glucose activated Gpa2 binds to and inhibits the function of Ime2. Furthermore, Ime2
is a non-stabile protein, thus under growth conditions the low level of Ime2 does not suffice for
initiation of meiosis in the absence of Ime1.
Yeast cells use the same regulators, but in reverse directions, to control alternative
developmental pathways. Ime2 is a positive regulator of meiosis that prevents pseudohyphae
development in growth media with acetate as the sole carbon source (Donzeau and Bandlow,
1999). The activity of the cAMP/PKA signal pathway is a major player in determining the
developmental choice yeast cells make. Entry into mitosis with either the yeast form or
filamentous morphology requires high activity of this pathway [for reviews see (Gancedo, 2001)],
whereas entry into meiosis requires low PKA activity (Matsumoto et al., 1983). Sok2 is a positive
regulator of mitosis (Ward et al., 1995) and a negative regulator for the transcription of IME1,
meiosis, and filamentous growth (Shenhar and Kassir, 2001; Ward et al., 1995). The negative role
of Sok2 in pseudohyphae formation is not clear since its C. albicans homolog, Efg1, that is a
negative regulator of meiosis when expressed in S. cerevisiae [complementing sok2∆ (Shenhar
and Kassir, 2001)], is a positive regulator of filamentous growth in both S. cerevisiae and C.
albicans (Stoldt et al., 1997). Msn2/4 exhibit reverse tasks, it functions as a negative regulator of
the mitotic cell cycle (Smith et al., 1998) and filamentous growth (Stanhill et al., 1999) and as a
positive regulator for the transcription of IME1 and meiosis (Shenhar and Kassir, 2001). The
activity of Sok2 and Msn2/4 in growing cells requires their phosphorylation by PKA (Gorner et
al., 1998; Shenhar and Kassir, 2001; Smith et al., 1998; Ward et al., 1995). The use of the same
regulators, but in reverse directions for different developmental pathways, ensures that one
pathway will be an alternative to the other one. When cells enter mitosis, meiosis will be
repressed, whereas when cells enter meiosis, mitosis will be blocked. Thus, a single signal
transduction pathway, the cAMP/PKA, is sufficient to control two alternative developmental
pathways.
Acknowledgment. We thank M. Foiani for his hospitality while writing this review, for fruitful
discussions and critical reading of the review.
42

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Figure legends
Fig. 1. Developmental choices of MATa/MATα diploid cells of Saccharomyces cerevisiae.
In the presence of carbon and nitrogen sources cells adopt the yeast form morphology; upon
nitrogen limitation and in the presence of high levels of glucose, a dimorphic transition to a
filamentous growth reminiscent of hyphae takes places. In the absence of nitrogen and glucose
and the presence of acetate as the sole carbon source cell enter the meiotic cycle, forming four
haploid spores engulfed in a sac, the ascus.
Fig. 2. A transcriptional cascade governs initiation of meiosis.
The meiotic signals, i.e. the presence of Mata1 and Matα2, the absence of glucose and nitrogen
and the presence of acetate leads to G1 arrest as well as to the expression and activity of Ime1.
IME1 encodes a transcriptional activator required for the transcription of early meiosis-specific
genes (EMG). EMG are involved with premeiotic DNA replication and meiotic recombination.
Ndt80 and Ime2, a transcriptional activator and a protein kinase, whose transcription depends on
Ime1, are required for the transcription of middle meiosis-specific genes (MMG). MMG are
involved with nuclear division and spore formation. The transcription of the late meiosis-specific
genes (LMG) depends on Ime1 and Ime2, and these genes are required for spore maturation. The
nutrient signal has a direct affect also on the activity of Ime2 and the transcription of LMG.
An arrow represents a positive role, a line a negative role. A scissors symbolizes the negative
feedback role of Ime2 in regulating degradation of Ime1.
Fig. 3. Schematic structure of IME1 5’ untranslated region.
The regulated transcription of IME1 is mediated by the combinatorial effect of distinct elements.
The MAT signal mediates repression activity of 2 elements UCS3 and UCS4. The carbon source
signal is transmitted to four elements. UCS1, UASru and IREu function as repression elements in
the presence of glucose. In addition, UASru IREu, as well as UASrm function as activation
elements in the absence of glucose and the presence of acetate as the sole carbon source. UCS1
functions as a negative element in the presence of nitrogen.
Filled boxs - elements required for transcriptional activation. Open boxs – elements whose
function is only to repress transcription. A positive role is marked with an arrow, a negative role
by a line. Larger effects are denoted by heavier lines while lesser effects are denoted by slender
lines.
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Fig. 4. The function of positive and negative elements regulating the transcription of IME1.
IME1 with various portions of its 5’ untranslated region were fused at the sixth amino acid to the
E.coli lacZ gene. The chimeric genes were integrated at the LEU2 loci of a MATa/MATα diploid
(strain Y4122, S288C background). Samples were taken from 1x10 7 cells grown in either SD
(synthetic glucose) or PSP2 (SA – synthetic acetate (Kassir and Simchen, 1991). In addition, cells
grown in PSP2 to 1x10 7 were washed once in water and resuspended in SPM (sporulation media
(Kassir and Simchen, 1991). Samples were taken to extract proteins and measure lacZ levels after
3 and 6 h in SPM. The level of β-Galactosidase is given in Miller units. The results are the
averages of three to five independent transformants. Standard deviations were less than 10%. The
studied elements are marked as filled boxes. A nested deletion is marked by a line. NT – not
tested.
Fig. 5. Cdc25 transmits the nitrogen signal that modulates the repression activity of UCS1, a
URS element in IME1 5’ region.
UCS1 was inserted between HIS4 UAS and the TATA box in a his4-lacZ chimera. The level of
β-Galactosidase was determined in three to five independent transformants. Standard deviations
were less than 10%. The results are given as relative levels in comparison (in each case) to the
level of expression of UASHIS4-his4-lacZ (control).
Insertion of UCS1 in the heterologous UASHIS4-his4-lacZ chimera leads to a substantial reduction
in its expression in vegetative growth conditions. Partial relief of repression is observed upon
nitrogen depletion, suggesting that the activity of UCS1 as a URS element is mediated by
nitrogen. Depletion of Cdc25 by shifting cdc25-5 cells to the non-permissive temperature leads to
relief of repression in vegetative growth media, and has no effect upon nitrogen depletion (SPM),
suggesting that Cdc25 transmits the nitrogen signal to UCS1.
Fig. 6: The UAS activity of the IME1 elements UASru, IREu and UASrm is modulated by
the carbon source.
His4-lacZ fusions carrying various elements from IME1 5’ untranslated region were integrated at
the LEU2 loci of a MATa/MATα diploid (strain Y4122, S288C background). Cells were grown in
either SD (synthetic glucose) or PSP2 (SA – synthetic acetate (Kassir and Simchen, 1991). In
addition, cells grown in PSP2 to 1x10 7 were washed once in water and resuspended in SPM
[sporulation media (Kassir and Simchen, 1991)] and incubated for 6 hours. Samples were taken
from 1x10 7 cells/ml to extract proteins and measure lacZ levels. The level of β-Galactosidase is
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given in Miller units. The results are the averages of three to five independent transformants.
Standard deviations were less than 10%.
UASru, IREu and UASrm support the expression of his4-lacZ, suggesting that all three elements
possess a UAS activity. The UAS activity is low in SD, and increased activity is observed in SA
and SPM media, suggesting that their activity is repressed by glucose and/or depends on acetate.
The IREd element that is homologus to IREu (see Fig. 8) shows very low UAS activity.
Fig. 7. cAMP reduces sporulation and the expression of IME1 through the IREu element.
MATa/MATα diploid cells (strain Y4122, S288C background) carrying ime1-lacZ or his4-lacZ
fusions with various elements from IME1 5’ untranslated region integrated at the LEU2 loci.
Cells were grown in PSP2 [synthetic acetate (Kassir and Simchen, 1991)] with or without 10mM
cAMP to 1x10 7 cells/ml, washed once in water and resuspended in SPM [sporulation media
(Kassir and Simchen, 1991)] with or without 10mM cAMP, respectively. Samples were taken at 6
hours in SPM. The level of β-Galactosidase is given in Miller units. The results are the averages
of three to five independent transformants. Standard deviations were less than 10%. At 24 hours
in SPM samples were taken to count the percentage of asci. Results are given as relative levels.
Addition of cAMP leads to a reduction in sporulation (72.6% and 49.8% in the absence and
presence of cAMP, respectively) as well as a reduction in the expression of ime1-lacZ. We
assume that the minor effect is due to the function of the cAMP specific phosphodiesterases.
Addition of cAMP reduces the UAS activity of IREu while it has no effect on the activity of
either UASru or UASrm.
Fig. 8. IREu and IREd are almost identical repeats in IME1 5’ region carrying the known
UAS sequences STRE and SCB.
Sequence alignments of IREu (upstream) and IREd (downstream) to the stress response element
(STRE) and the Swi4/6 Cycle box (SCB). We suggest that Msn2 binds the STRE element in
IREu since Msn2 binds IREu as well as STRE elements in stress response genes. We further
suggest that Sok2 binds the SCB element since it is highly homologous to the DNA-binding
domain of Swi4 that binds such elements, and since genetic analysis reveals that it binds IREu
(see text). The physical association between Sok2 and Msn2 suggests that they bind the DNA as a
heterodimer.
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Fig. 9. A schematic structure of IME1 5’ untranslated region showing the positive and
negative transcription factors which regulate its expression.
The regulated transcription of IME1 is mediated by the combinatorial effect of distinct elements.
The MAT signal is transmitted to UCS4 by Rme1. The activity of Rme1 as a repressor requires
Rgr1 and Sin4. The cAMP/PKA signal transduction pathway transmits the glucose signal to IREu
through two transcription factors, Sok2 and Msn2. In glucose growth media phosphorylation of
Sok2 by PKA promotes its repression activity, whereas phosphorylation of Msn2 sequesters the
protein in the cytoplasm. In acetate growth media Sok2 repression is relieved by a decrease in the
activity of PKA, as well as by the function of Ime1. The increased binding of Msn2 to IREu leads
to transcriptional activation. Yhp1 binds to UASv, however, its activity is not required for the
transcription of IME1. Nitrogen, via Cdc25, regulates the URS activity of UCS1. It is not known
whether the effect of Cdc25 is mediated through the cAMP/PKA and/or MAPK pathways.
Filled box - elements required for transcriptional activation. Open box – elements that whose
function is only to repress transcription. A positive role is marked with an arrow, a negative role
in line. Dashed lines and a question mark – preliminary data.
Fig. 10. The expression and activity of Ime1 are regulated by nutrients.
Schematic representation on how glucose and nitrogen regulates the availability and activity of
Ime1. The nucleus is depicted between the two circular lines, and cell membrane by a heavy line.
A positive role is marked with an arrow, a negative role by a straight line. The transcription of
IME1 is inhibited in the presence of glucose and nitrogen. Translation of IME1 mRNA is
inhibited in the presence of a nitrogen source. In the presence of nitrogen, Ime1is sequestered
from the nuclei due to phosphorylation by Cdc28/Cln. Interaction of Ime1 with Ume6, and
consequently transcriptional activation of early meiosis-specific genes (EMG) is inhibited by
glucose and nitrogen.
Fig. 11. Conditions leading to the choice between silencing and expression of early meiosisspecific
genes.
A. Vegetative growth conditions B. Meiotic conditions.
Ume6 binds to the URS1 element in early-meiosis specific genes (EMG) under all growth
conditions. In vegetative growth conditions Ume6 recruits to repression complexes, Sin3/Rpd3
and Isw2, leading to histone H4 deacetylase and chromatin remodeling, respectively, and
silencing. Under these conditions Rim15 and most probably Rim11 are non-active. The activity
of Rim15 is repressed by PKA (Tpk1,2,3) phosphorylation. Under meiotic conditions (acetate
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media and nitrogen depletion) phosphorylation of Ime1and Ume6 by Rim11 and Rim15 promotes
the interaction of Ime1 with Ume6. Ime1 function is required to relieve repression by Sin3 and to
activate transcription. In addition, transcriptional activation depends on histone acetylation by
Gcn5.
Fig. 12. Ime2 is highly homologous to hCDK2.
A. Sequence alignment of Ime2 to hCDK2. B, Ribbon depiction of the crystal structure of
hCDK2 (Protein Data Bank structure code 1HCL). C. Ribbon depiction of the homology based
model of Ime2 based on the CDK structure. The following stretches of residues are colored for
clarity: the ATP binding site (yellow), The PSTAIRE site (orange), the Catalytic site (red) and the
T-loop (blue). The Ime2 model was prepared using the 3D-PSSM modeling algorithm (Kelley et
al., 2000). The figures were prepared using InsightII (Accelrys).
Fig. 13. Transcriptional regulation of middle meiosis-specific genes.
Ndt80 is the transcriptional activator binding to the MSE element present in the 5’ region of all
middle meiosis-specific genes (MMG). A subset of MMG carries an MSE* site (SMK1 in
comparison to CLB1) that can be bound by Sum1. Sum1 associates with Hst1 that functions as
histone deacetylase, thus leading to silencing under vegetative growth conditions and at early
meiotic times. Under meiotic conditions relief of repression is due to degradation of Sum1 by
Ime2. Sum1 also represses the transcription of NDT80, since it carries both an MSE and MSE*
elements. In addition, NDT80 carries two URS1 elements that are bound by Ume6. In vegetative
growth media Ume6 recruits the Sin3/Rpd3 histone deacetylase complex that represses
transcription. Under meiotic conditions Ume6 recruits Ime1 that can activate transcription, but
only in the absence of Sum1. Ime2 phosphorylates Ndt80, and this phosphorylation may
contribute to the complete transcriptional activity of Ndt80. In the absence of meiotic
recombination the pachytene checkpoint inhibits degradation of Sum1 and phosphorylation of
Ndt80. Consequently, the level of Ndt80 is reduced, and the unphosphorylated Ndt80 is impaired
in activating the transcription of MMG.
Fig. 14. Positive and negative feedback loops control meiosis.
Ime1 shows positive autoregulation that is required to relieve repression mediated by Sok2 and
thus for its high-level transcription. Expression of early meiosis-specific genes (EMG) requires
relief of repression of Sin3 and transcriptional activation, two functions that are promoted by
Ime1. The transcription of middle genes (MMG) depends on Ime2 and Ndt80. Transcriptional
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activation by Ime2 is due to phosphorylation of the transcriptional activator, Ndt80, and
degradation of the transcriptional repressor, Sum1. Ime2 exhibits a negative feedback role, it is
required to shut down the transcription of IME1. Since Ime2 directly phosphorylates Ime1, and
since this phosphorylation is required for degradation of Ime1 by the 26S proteasome, it was
suggested that the reduction in the transcription of IME1 as well as EMG is due to the absence of
Ime1 and reestablishment of repression by Sok2 and Sin3, respectively. The transcription of
MMG is repressed by the pachytene checkpoint that inhibits phosphorylation of Ndt80 and
degradation of Sum1.
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Table 1. List of positive and negative regulators of transcription of meiosis-specific genes.
Name Brief description of function
ABF1 An essential DNA-binding protein required for DNA replication and the transcription of several
meiosis-specific genes carrying its binding site (UASH).
AMA1 A meiosis-specific subunit of APC/C that is required for degradation of CLB1 in the meiotic cycle.
Required for the transcription of LMG.
BCY1 The regulatory subunit of PKA. Positive regulator for IME1 transcription and sporulation.
CDC25 Ras GDP/GTP exchange factor. Positive activator of PKA, negative regulator of IME1
transcription. Transmits the nitrogen signal to UCS1 and the glucose signal to IREu elements in
IME1 promoter.
CDC28 Cyclin dependent kinase that regulates initiation and progression in the mitotic cell cycle. In a
complex with Cln phosphorylates Ime1 and sequesters it out of the nuclei. Required to activate
Ime2 at the time cells enter the meiotic division.
CDC35 Adenylate cyclase. Positive activator of PKA, negative regulator of IME1 transcription and
sporulation
GCN5 Histone acetylase required for the transcription of EMG.
GPA2 A Gα protein that associates, when GTP bound, with Ime2. An inhibitor of Ime2 kinase activity.
HST1 Histone deacetylase. Represses the transcription of MMG following recruitment by Sum1.
IDS2 A positive regulator of Ime2 activity.
IME1 The master regulator gene of meiosis. A transcriptional activator; recruited by Ume6 to the URS1
element in EMG, and is required for their transcription. The transcription of IME1 depends on the
presence of Mata1 and Matα2 as well as on glucose and nitrogen depletion. Translation and
activity of Ime1 are regulated by nutrients.
IME2 An early meiosis-specific gene encoding serine/threonine kinase, homologous to the human CDK2.
Required for timely and high level transcription of EMG, and for the transcription of MMG and
LMG. Negative regulator of Ime1 stability.
IME4 A positive transcriptional regulator of IME1 whose transcription depends on the presence of Mata1
and Matα2 and nitrogen depletion.
ISW2 In a complex with Itc1 functions as an ATP dependent chromatin-remodeling factor. Required for
the repression of EMG under vegetative growth conditions. Physically associate with Ume6 that
recruits it to URS1 elements.
ITC1 In a complex with Isw2 functions as an ATP dependent chromatin-remodeling factor. Required for
the repression of EMG under vegetative growth conditions.
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MCK1 Protein kinase required for efficient transcription of IME1, expression of EMG, MMG and spore
maturation. Homologous to mammalian glycogen synthase kinase 3 and S. cerevisiae, RIM11,
YOL128c, and MRK1. Required for phosphorylation of Ume6 and sporulation in cells deleted for
RIM11 and MRK1.
MDS3 Negative regulator of IME1 transcription. Redundant function with its homolog, PMD1
MSN2/4 Zinc finger transcriptional activators, which share redundant function and bind STRE elements.
Negative regulators of mitosis. Positive regulators of meiosis and IME1 transcription through the
IREu element in IME1 promoter. Targets of the cAMP/PKA pathway.
MRK1 Protein kinase homologous to mammalian glycogen synthase kinase 3 and S. cerevisiae, RIM11,
YOL128c, and MCK1.Required for phosphorylation of Ume6 and sporulation in cells deleted for
RIM11 and MCK1.
NDT80 A meiosis-specific transcriptional activator that binds the MSE element in MMG. An early MMG.
PMD1 Negative regulator of IME1 transcription. Redundant function with its homolog, MDS3
PTP2,3 Tyrosine phosphatases, required for Mck1 dephosphorylation as well as for high-level transcription
of IME1 and IME2, and for expression of MMG and LMG.
RAS2 Small G protein that activates the cAMP/PKA and the MAPK signal pathways. Negative regulator
of IME1 transcription.
RES1 A dominant mutation in this gene bypasses MAT regulation for the transcription of IME1.
RGR1 A component of the RNA polymerase SRB mediator complex. Required for the repression activity
of Rme1.
RIM1 A C2H2 zinc finger protein required for high-level expression of IME1, and efficient sporulation.
Required for mitochondria DNA replication and maintenance. Activity is regulated by cleavage
that depends on Rim8, Rim9, Rim13 and Rim20.
RIM4 An early meiosis-specific gene required for high-level expression of EMG. Sequence identifies two
RNA recognition motifs that are essential for function.
RIM8 Positive regulator of IME1 transcription. Required for activating Rim1.
RIM9 Positive regulator of IME1 transcription. Required for activating Rim1.
RIM11 Tyrosine/serine/threonine kinase that is homologous to mammalian glycogen synthase kinase 3 and
S. cerevisiae, MCK1, YOL128c, and MRK1.Required for the interaction between Ime1 and Ume6,
and consequently for the transcription of EMG. Associates and phosphorylates both Ime1 and
Ume6.
RIM13 Positive regulator of IME1 transcription. Required for activating Rim1.
RIM20 Positive regulator of IME1 transcription. Required for activating Rim1.
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RIM15 A serine/threonine kinase whose activity is inhibited by PKA phosphorylation. Positive regulator
for the transcription of IME1 and for the interaction between Ime1 and Ume6. Phosphorylates
Ume6.
RME1 A Zn finger DNA-binding protein that represses the transcription of IME1 through the UCS4
element in IME1 promoter. The transcription of RME1 is negatively regulated by the Mata1/Matα2
complex.
ROX3 A component of the RNA polymerase SRB mediator complex. Required for repressing the
transcription of mid-late meiosis-specific genes.
RPD3 Histone deacetylase. Required for repression of EMG in media supporting vegetative growth.
Positive regulator of MMG.
SIN3 Negative regulator that is required for repression of EMG in media supporting vegetative growth.
Recruits Rpd3 to EMG due to its association with both Ume6 and Rpd3. Positive regulator of
MMG.
SIN4 A component of the RNA polymerase SRB mediator complex. Required for the repression activity
of Rme1, and for repressing the transcription of mid-late meiosis-specific genes.
SNF1 Serine/threonine protein kinase whose activity is inhibited by high levels of glucose. Required for
the transcription of both IME1 and IME2, and for spore formation.
SNF2 Component of the Swi/Snf transcriptional activation complex. Required for high-level expression
of IME1.
SOK2 Transcriptional repressor that binds SCB like sequences. Positive regulator in the mitotic cell cycle
Negative regulator of pseudohyphal growth, meiosis and IME1 transcription. Sok2 function as a
repressor depends on phosphorylation by PKA.
SSN6 A general transcriptional repressor that regulates the transcription of IME1. Forms a repression
complex with Tup1.
SUM1 A transcriptional repressor of MMG that binds the MSE* element. Recruits histone deacetylase
activity by association with Hst1.
SWI1 Component of the Swi/Snf transcriptional activation complex. Required for high-level expression
of IME1.
TPK1,2,3 Three genes encoding the catalytic subunit of PKA.
TUP1 A general transcriptional repressor that regulates the transcription of IME1. Forms a repression
complex with Ssn6.
UME2 A component of the SRB subcomplex of RNA polymerase II holoenzyme. Negative regulator of
EMG transcription.
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UME3 A cyclin C homolog that associates with the cyclin dependent kinase, Ume5. An integral
component of the SRB subcomplex of RNA polymerase II holoenzyme. Required to repress
transcription of EMG in vegetative growth media. Degraded in meiotic conditions.
UME5 A cyclin dependent kinase that associates with the cyclin C homolog, Ume3. An integral
component of the SRB subcomplex of RNA polymerase II holoenzyme. Required to repress
transcription of EMG in vegetative growth media.
UME6 A C6Zn2 DNA-binding protein. Binds to URS1 element. Functions as a negative regulator by
recruiting the Sin3/Rpd3 and Isw2 complexes, and as a positive regulator by recruiting Ime1.
YHP1 Homeodomain protein that binds to UASv, a specific region in IME1 5’ region. It is not required
for the transcription of IME1.
WTM1,2,3 Transcriptional repressors that repress the transcription of IME2 synergistically.
YVH1 Tyrosine phosphatase whose transcription is induced upon nitrogen depletions. Has redundant
function with Ptp2 in regulating sporulation.
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