Regulated repression, and not activation, governs the cell fate promoter controlling yeast meiosis

: Intrinsic signals and cues from the external environment drive cell fate decisions. In budding yeast, the decision to enter meiosis is controlled by nutrient and mating-type signals that regulate expression of the master transcription factor for meiotic entry, IME1 . How nutrient signals control IME1 expression remains poorly understood. Here we show that IME1 transcription is regulated by multiple sequence-specific transcription factors that mediate association of Tup1-Cyc8 co-repressor to its promoter. We find that at least eight transcription factors bind the IME1 promoter when nutrients are ample. Remarkably, association of these transcription factors is highly regulated by different nutrient cues. Mutant cells lacking three transcription factors (Sok2/Phd1/Yap6) displayed reduced Tup1-Cyc8 association, increased IME1 expression and earlier onset of meiosis. Our data demonstrate that the promoter of a master regulator is primed for rapid activation while repression by multiple transcription factors mediating Tup1-Cyc8 recruitment dictates the fate decision to enter meiosis.


Introduction:
The choice of whether to differentiate into another cell type is directed by multiple cell intrinsic and extrinsic environmental factors.These cues signal to master regulatory genes, which in turn control the initiation of cell differentiation programs.
As a result, multiple signals are transformed into a binary decision: whether or not to undergo cell differentiation.How signalling cues coordinate a cell fate outcome has important implications for the understanding of development and diseases such as cancer.
Diploid budding yeast cells undergo an irreversible differentiation program called gametogenesis or sporulation during which a diploid cell gives rise to four haploid spores.The yeast gametogenesis program is characterized by one round of DNA replication and recombination, two consecutive chromatin segregation events called meiosis followed by spore formation 1 .As a result, an ascus with four haploid spores is produced.In yeast, the decision to enter meiosis is controlled by a master regulatory transcription factor named inducer of meiosis 1, IME1 2,3 .In the absence of IME1, cells cannot enter meiosis and produce gametes.Thus, understanding how IME1 is regulated is key to understanding how the decision to enter meiosis or gametogenesis is made.
Transcriptional control mechanisms regulate IME1 expression.The IME1 gene has an unusually large promoter (over 2.2 kilobases) that integrates multiple signals to control IME1 expression 4 .Nutrient and mating type signals ensure that IME1 is only expressed in the appropriate nutrient environment and in the correct cell type.IME1 is expressed in cells harbouring opposite mating-type loci (MATa and MAT) 5,6 .In cells with a single mating type (MATa or MAT), the transcription factor Rme1 is expressed and induces transcription of the long noncoding RNA (lncRNA) IRT1, which in turn transcribes through the IME1 promoter and thereby represses IME1 expression 7 .In MATa/diploid cells, a second lncRNA upstream of IRT1 named IRT2 interferes with IRT1 transcription forming a positive feedback loop by which Ime1 promotes its own expression 8 .
In addition to mating-type control, environmental cues also play a critical role in regulating IME1 expression.In order to induce IME1 transcription, diploid cells must be starved for glucose and nitrogen, and cells need to be respiring 4,9 .The glucose and nitrogen signals integrate at the IME1 promoter.Several sequence elements in the IME1 promoter are important for control of IME1 transcription.For example, a distinct sequence element mediates IME1 repression by glucose signalling, while other parts of the promoter respond to nitrogen availability 10 .Notably, the transcription factor Sok2 controls IME1 promoter activity via the glucose responding element 11 .In addition, multiple other transcription factors contribute to regulation of IME1 transcription [12][13][14] .Over 50 transcription factors have a conserved consensus site in the IME1 promoter 12 .A genome-wide reporter screen revealed that about 30 transcription factors may directly or indirectly control IME1 transcription 12 .How different transcription factors and functional elements of the IME1 promoter interact to control IME1 expression and thus regulate the decision to enter meiosis is not well understood.
The nutrient control of IME1 expression is mediated by multiple signalling pathways including PKA, TOR complex 1 (TORC1), AMP-activated protein kinase (AMPK) and mitogen-activated protein kinase (MAPK) [15][16][17] .Inhibiting two signalling pathways, PKA and TORC1, is sufficient to induce IME1 expression in cells exposed to a nutrient rich environment where IME1 expression is normally repressed 16 .Thus PKA and TORC1 signalling is essential for controlling IME1 expression and hence the decision to enter meiosis (Figure 1A).Previously, we showed that Tup1 represses the IME1 promoter under nutrient rich conditions.Tup1 is part of the Tup1-Cyc8 corepressor complex, which is involved in repression of more than 300 gene promoters in yeast [18][19][20] .During starvation, however, Tup1 disassociates from the IME1 promoter and IME1 transcription is concomitantly induced.Tup1 binding to the IME1 promoter is controlled by PKA and TORC1 16 .When both signalling pathways are inhibited Tup1 dissociates from the IME1 promoter.Thus understanding how Tup1-Cyc8 binds to the IME1 promoter may reveal how nutrient signalling controls IME1 expression and consequently how cells make the decision to enter meiosis.
Here we set out to investigate how the Tup1-Cyc8 co-repressor complex regulates IME1 transcription.In short, we found that regulated repression by multiple sequence specific transcription factors mediating the association of Tup1-Cyc8 with the IME1 promoter is the means by which IME1 transcription is controlled.Our data further indicate that nutrient cues highly regulate the association of Tup1-Cyc8 interacting transcription factors with the IME1 promoter, which is key to regulating IME1 expression and thus the cell fate decision to enter meiosis in yeast.Our work provides a framework to understand how nutrient signals integrate at a cell fate promoter and thereby control a developmental decision in yeast.

:
To gain insight into how Tup1 association with the IME1 promoter is controlled, we first examined whether the region of the promoter where Tup1 binds contains key regulatory elements for IME1 transcription.Previously, we found that Tup1 associates between 800 and 1400 base pairs (bp) upstream of the IME1 translation start site 16 .If the region of the IME1 promoter where Tup1 binds is also important for IME1 activation, then deleting that part of the promoter should affect the onset of meiosis.We generated six truncation mutants with a 200 bp interval in the IME1 promoter and examined the ability of these mutants to undergo meiosis (Figure 1B).
The largest truncation mutant that underwent meiosis with comparable kinetics as wild-type cells harboured 1400 bp of the IME1 promoter (pIME1(-1400-2315)) indicating that this region harbours the regulatory elements required for complete activation of the IME1 promoter (Figure 1B).In addition, we found that meiosis in pIME1(-800-2315) was completely impaired, whereas pIME1(-1200-2315) had a much milder effect on meiosis.The result suggests that a region between -800 and -1200 bp harbours regulatory elements essential for IME1 promoter function.We also made smaller truncations in the promoter and found that the region between -800 and -850 bp contains regulatory elements important for IME1 activation because a large fraction of pIME1(-850-2315) cells underwent meiosis (Supplementary Figure 1).In conclusion, the region important for Tup1 binding to the IME1 promoter is also required for transcription of IME1.
Tup1 forms a complex with Cyc8 19,20 .The Tup1-Cyc8 co-repressor complex is conserved and plays various roles in regulating gene transcription 21 .Like Tup1, Cyc8 has also been implicated in regulation of IME1 expression 22 .To investigate how Cyc8 regulates IME1 expression, we first determined Cyc8 binding with the IME1 promoter under nutrient rich conditions.We found that Cyc8 peaked in the same region as Tup1 in the IME1 promoter (Figure 1C and 1D).These data indicate that Tup1 is in a complex with Cyc8 at the IME1 promoter.
Various models for Tup1-Cyc8 mediated repression of target gene promoters have been described 21,23,24 .It has been proposed that Tup1-Cyc8 primarily regulates promoters by masking transcription factors from recruiting co-activators 25 .Thus, Tup1-Cyc8 interacts with transcription factors with activation potential at promoters.We hypothesized that if Tup1-Cyc8 represses the IME1 promoter by shielding coactivators, then transcriptional activators should be at the promoter under repressive conditions.To test this, we measured under nutrient rich repressive conditions the association of a known transcriptional activator of IME1, Pog1 7 .We found that Pog1, like Tup1-Cyc8, is indeed enriched at the IME1 promoter under repressive conditions (Figure 1D).We conclude that Tup1-Cyc8 complex associates with the IME1 promoter, and possibly masks transcriptional activators such as Pog1.
Various transcription factors have been implicated in controlling in IME1 expression 12 .To further examine whether transcriptional activators are readily present at the IME1 promoter, we measured IME1 expression after depletion of Tup1 or Cyc8.We reasoned that if Tup1-Cyc8 represses the IME1 promoter by restraining activating transcription factors, then depletion of Tup1 or Cyc8 should allow activators present to concomitantly induce IME1 transcription.We used the auxin inducible degron (AID) system (TUP1-AID and CYC8-AID) and treated cells with indole-3-acetic acid (IAA) to achieve rapid protein depletion in cells 26 .Rapid and sustained depletion of Tup1 and Cyc8 was achieved within 30 minutes after IAA treatment (Figure 1E).Strikingly, IME1 transcript levels strongly increased concurrently, and were comparable to those in wild-type cells entering meiosis when IME1 expression is typically at its peak (Figure 1F).These data show that Tup1-Cyc8 repressor complex is pivotal for repressing the IME1 promoter under nutrient rich conditions.Our data further suggest that the default state of the IME1 promoter is active due to the presence of transcriptional activators, which are restrained by Tup1-Cyc8 under repressive conditions.
Although the transcriptional activator Pog1 is already bound in repressive conditions the at IME1 promoter, it is possible that other transcriptional activators factors associate with the IME1 promoter after Tup1-Cyc8 dissociates.This may results in a delay between Tup1-Cyc8 depletion and activation of IME1 transcription.We therefore decided to monitor IME1 expression by single molecule RNA fluorescence in situ hybridization (smFISH).This technique can detect individual transcripts in single cells 27 .We found that as soon as Tup1 was depleted, IME1 transcripts were detected in single cells (Figure 1G, 1H, 1I, and Supplementary Figure 2A).An increase in IME1 transcripts was detected 10 minutes after IAA treatment when Tup1 was partially depleted (Figure 1G).After 15 min of inducing Tup1 depletion, 5.3 IME1 transcripts were detected per cell on average, and 12% of the cells (Tup1-AID+IAA) had more than 10 IME1 transcripts compared to 2% in control cells (Figure 1H and   1I).At 30 minutes after IAA treatment, more than 55% of cells expressed more than 10 IME1 transcripts.It is worth noting that the AID-tag fused to Tup1 had some effect on IME1 expression in the absence of IAA as IME1 transcript levels were increased by five-fold in TUP1-AID compared to wild-type cells (Figure 1H, 1I, and Supplementary Figure 2B and 2C).We conclude that there is little to no temporal delay between Tup1-Cyc8 depletion and IME1 expression suggesting that transcriptional activators are bound or readily available for recruitment to the IME1 promoter.
The Tup1-Cyc8 complex also controls gene promoters by altering chromatin states [28][29][30] .Specifically, Tup1-Cyc8 directly interacts with class I and II histone deacetylases, which in turn confer repression through deacetylation of nucleosomes [31][32][33] .For example, repression of the FLO1 promoter is achieved by Tup1-Cyc8 mediated recruitment of Hda1 and Rpd3 34 .In hda1 rpd3 cells significant derepression of the FLO1 gene can be observed.To examine whether histone deacetylases mediate Tup1-Cyc8 repression at the IME1 promoter, we generated single and double mutants of known Tup1-Cyc8 interacting histone deacetylases (Rpd3, Hda1, Hos1, and Hos2) and measured IME1 expression levels by smFISH (Figure 1J and Supplementary Figure 2C).Deletion of individual HDACs (rpd3, hda1, hos1, and hos2) did not increase expression of IME1.About 10% of rpd3hda1 cells expressed four or more IME1 transcripts, a marginal increase when compared to Tup1 depleted cells (Supplementary Figure 2C).Two other double mutants (rpd3hos1 and rpd3hos2) displayed no detectable increase in IME1 expression.We conclude that HDACs that are known to interact with Tup1-Cyc8 play only a marginal role in repressing the IME1 promoter.
The Tup1-Cyc8 interacts with DNA sequence specific transcription factors to form co-repressor complexes at promoters [35][36][37][38][39][40][41] .These transcription factors facilitate Tup1-Cyc8 association with promoters and thereby mediate repression of target genes.Our observation that Tup1-Cyc8 is the key repressor of the IME1 promoter prompted us to examine which transcription factors recruit Tup1-Cyc8 to the IME1 promoter and how they control IME1 transcription.First, we assembled a list of transcription factors previously reported to interact with either Tup1 or Cyc8.In addition, we examined the region of the IME1 promoter (-600 to -1200 bp) where Tup1-Cyc8 binds, and scanned for sequence motifs among transcription factors known to interact with Tup1-Cyc8 (Figure 2A, Supplementary Figure 3A and 3B).We identified 13 candidate transcription factors that were known or implicated to interact with Tup1-Cyc8 and have binding sites in the IME1 promoter (Figure 2A and Supplementary Figure 3A).We also included the transcription factor Sok2 in our analyses because it has been proposed to interact with Tup1-Cyc8 and Sok2 is known to directly repress IME1 transcription 11,35 .After the curation of the list of transcription factors, we subsequently measured the binding of under nutrient rich conditions by epitope tagging each transcription factor and performing ChIP.
Remarkably, eight transcription factors displayed enrichment at the IME1 promoter (Figure 2B).As expected, a known regulator of the IME1 promoter, Sok2, was strongly enriched 11 .In addition, Phd1 (a paralog of Sok2) and Yap6 also displayed strong enrichment (Figure 2B).The transcription factor Sut1, which is known to interact with Cyc8, was also enriched 38 .The transcription factors Mot3, Sko1, Nrg1, and Nrg2 displayed a milder enrichment, but their binding was above background levels.For all transcription factors that displayed enrichment, we also assessed their binding to other parts of the IME1 promoter (Figure 2C).Transcription factors exclusively co-localised with Tup1-Cyc8 in the same region of the IME1 promoter around 1000 bp upstream of the IME1 start codon.Thus, at least eight transcription factors that are known or have been implicated to interact with Tup1-Cyc8 associate with the IME1 promoter.
Next, we examined whether the candidate transcription factors are responsible for recruiting Tup1 to the IME1 promoter.We reasoned that candidate transcription factors should associate independently of Tup1-Cyc8 to the IME1 promoter, while the binding of transcription factors and Tup1-Cyc8 should depend on the presence of specific sequence motifs (Figure 3A).First, we depleted Tup1 (TUP1-AID+IAA), and measured binding for a subset of the bound transcription factors (Supplementary Figure 4A).Except for Sko1, the binding of the transcription factors to the IME1 promoter was not affected by Tup1 depletion (Figure 3B).Thus, multiple transcription factors known to interact with Tup1-Cyc8 associate with the IME1 promoter independently of Tup1-Cyc8.Second, we examined whether the candidate transcription factors contribute to Tup1-Cyc8 recruitment.We mutated putative binding sites of the transcription factors that showed binding to the IME1 promoter.
To do so, we generated a construct containing the full-length promoter, followed by sfGFP and the IME1 gene (pIME1-WT).Subsequently, we mutated 103 nucleotides distributed across a region of 400 bp in the IME1 promoter (pIME1-bs∆) (Figure 3C and Supplementary Figure 4B).We integrated the constructs in the TRP1 locus in cells harbouring a deletion of the endogenous IME1 gene and promoter sequence.
Tup1 association with the IME1 promoter was nearly completely lost in pIME1-bs∆ cells (Figure 3C).As expected, control cells (pIME1-WT) did not show a decrease in Tup1 binding.Finally, we assessed how IME1 expression and the onset of meiosis is affected in pIME1-bs∆ cells.Surprisingly, IME1 expression in pIME1-bs∆ cells was reduced, suggesting the regulatory elements essential for Tup1-Cyc8 recruitment are also important for IME1 activation (Figure 3D).In conclusion, DNA sequence motifs of transcription factors bound to the IME1 promoter are required for Tup1-Cyc8 association with the IME1 promoter.So far, our analyses of the IME1 promoter showed that multiple transcription factors and multiple binding sites are essential for Tup1-Cyc8 recruitment.Next, we examined how the different transcription factors control IME1 expression and mediate Tup1-Cyc8 recruitment under nutrient rich conditions.First, we assessed how the paralogs Sok2 and Phd1 control IME1 expression.sok2 cells displayed a negligible increase in IME1 expression (average transcripts per cell: 0.6 for sok2 versus 0.3 for control) (Figure 4A and 4B).In the sok2phd1 double mutant, IME1 expression was marginally increased (average transcripts per cell: 2.2) and about 5% of cells displayed more than 10 transcripts per cell suggesting that Sok2 and Phd1 play redundant roles in tightly repressing the IME1 promoter (Figure 4A and   4B).We also measured IME1 expression in yap6 cells or in combination with the sok2phd1 mutation.IME1 repression was not affected in cells containing yap6but in the sok2phd1yap6 triple deletion mutant IME1 expression was increased (average transcripts per cell: 2.8 for sok2phd1yap6 versus 2.2 for sok2phd1) (Figure 4A and B).About 8% of sok2phd1yap6 cells expressed more than 10 IME1 transcripts per cell.It is worth noting that IME1 transcript levels in sok2phd1yap6 cells were much lower than in cells depleted for Tup1 (Figure 1F and 1H) -suggesting that additional transcription factors contribute to IME1 repression.
Our data demonstrate that Sok2, Phd1 and Yap6 associate with the IME1 promoter and contribute to IME1 repression in nutrient rich conditions.Yet, IME1 expression was reduced in cells with DNA sequence motifs mutated (pIME1-bs).One explanation is that the mutated binding sites in pIME1-bs important for Tup1-Cyc8 recruitment also facilitate binding for transcriptional activators.Another possibility is that transcription factors important for Tup1-Cyc8 recruitment are also required for IME1 activation.To discriminate between both possibilities, we generated a construct that contained binding sites for Sok2, Phd1, and Yap6 (pIME1-spy), while the other transcription factor binding sites remained mutated (Figure 4C and Supplementary Figure 5).By combining pIME1-spy with sok2phd1yap6, we could then determine whether Sok2, Phd1, and Yap6 are important for IME1 activation or repression.Tup1 binding was restored in cells harbouring pIME1-spy (Figure 4C).Furthermore, Yap6, Sok2, and Phd1 were enriched at the IME1 promoter in pIME1-spy cells but their binding was reduced compared to the wild-type promoter -suggesting that there are additional binding sites (Figure 4C).Next, we measured IME1 expression in wild-type and sok2phd1yap6 mutant cells harbouring pIME1-spy (Figure 4D).We found that IME1 expression was further derepressed in cells harbouring both pIME1-spy and sok2phd1yap6 compared to cells expressing the three transcription factors (Figure 4D and 4E).About 17% of cells harbouring pIME1-spy and sok2phd1yap6 expressed more than 10 IME1 transcripts per cell.As expected, the sok2phd1yap6 had only a mild effect on IME1 levels in cells expressing pIME1-WT or pIME1-bs.We conclude that Sok2, Phd1 and Yap6 are important for repression of the IME1 promoter, and play little role in IME1 activation.
The Tup1-Cyc8 complex dissociates from the IME1 promoter in cells exposed to nutrient starvation 16 .We hypothesized that transcription factors interacting with Tup1-Cyc8 at the IME1 promoter control Tup1-Cyc8 dissociation during IME1 activation.For example, one possibility is that the physical interaction between Tup1-Cyc8 and transcription factors is altered during activation IME1.Alternatively, during activation of the IME1 promoter transcription factors dissociate together with Tup1-Cyc8.To examine this, we measured the binding of the eight transcription factors during activation of the IME1 promoter.In order to induce IME1 expression and meiotic entry in a synchronous manner in all cells, we typically grow cells in rich medium conditions containing glucose until saturation, then shift and grow cells in pre-sporulation medium containing acetate.Cellular respiration is required for meiosis, and growth in medium with acetate but lacking glucose ensures that cells are respiring and not subject to repressive glucose signalling to the IME1 promoter 4,9 .Subsequently, cells are starved by shifting them to sporulation medium (0.3% acetate), which induces IME1 transcription and drives meiotic entry.First, we measured the binding of Tup1 and Pog1 at the IME1 promoter prior to induction of IME1 (0 hours in SPO), and during meiotic entry when IME1 is induced (4 hours in SPO) (Figure 5A).Both Pog1 and Tup1 were enriched at 0 hours in SPO prior to IME1 induction.As expected, during meiotic entry (4 hours in SPO) Tup1 dissociated from the IME1 promoter completely while Pog1 binding was maintained albeit to a reduced level (Figure 5A).Next, we determined how binding of the different transcription factors with the IME1 promoter was regulated.We found that all eight transcription factors, were enriched at the IME1 promoter prior to induction of IME1 (Figure 5A).Strikingly, five transcription factors showed no binding to the IME1 promoter upon entry into meiosis, while three transcription factors (Yap6, Phd1, and Nrg1) displayed marginal enrichment (4 hours in SPO).These data suggest that Tup1-Cyc8 dissociates from the IME1 promoter due to the loss of binding of multiple transcription factors.In conclusion, activation of IME1 transcription correlates with the dissociation of transcription factors important for Tup1-Cyc8 recruitment, while a transcriptional activator remains present at the IME1 promoter.
We showed that Sok2, Phd1 and Yap6 were strongly enriched at the IME1 promoter under nutrient rich conditions and prior to meiotic entry (Figure 2B, Figure 5A).In addition, in sok2phd1yap6cells, IME1 expression was partially de-repressed when nutrients were ample (Figure 4A and 4B).These observations indicate that Sok2, Phd1 and Yap6 are important transcription factors for IME1 repression.Next, we investigated how the three transcription factors control Tup1-Cyc8 recruitment in different nutrient conditions.Specifically, we examined Tup1 binding to the IME1 promoter in the absence of the three transcription factors in cells grown in rich medium containing glucose (YPD) and in acetate-containing medium prior to meiotic entry.We found that Tup1 binding to the IME1 promoter was not affected in rich medium containing glucose in sok2, phd1, and yap6 single/double/triple deletion mutants, which is in line with IME1 expression data described in (Figure 5B, 4A, and   4B).This suggests that other transcription factors contribute to IME1 repression via Tup1-Cyc8 when glucose is used by cells as the carbon source.In contrast, prior to meiotic entry (0h in SPO) Tup1 binding was significantly reduced in sok2 and sok2phd1 cells, but not in yap6 and phd1 cells (Figure 5B).Strikingly, Tup1 association with the IME1 promoter was reduced to nearly background levels in sok2yap6 and sok2phd1yap6 cells at 0 hours in SPO.IME1 expression inversely correlated with Tup1-Cyc8 recruitment to the IME1 promoter (Figure 5C).
For example, sok2yap6 cells displayed significant de-repression of IME1 expression prior to entry into meiosis compared to WT control cells.Likewise, cells harbouring sok2phd1 or sok2phd1yap6 also displayed de-repression of IME1 expression at 0 hours in SPO (Figure 5C).Finally, we examined how Sok2, Phd1, and Yap6 mediated Tup1-Cyc8 recruitment to IME1 promoter is important for meiosis.In general, the onset of meiosis in the different mutants correlated well with IME1 expression levels (Figure 5D).Cells harbouring the sok2or sok2phd1 underwent meiosis much faster than wild-type cells.For example after 3 hours in SPO, 50% of sok2cells completed at least one meiotic division while this took more than 6 hours in wild-type cells.There was little effect on the onset of meiosis in the yap6 or yap6phd1 mutants.In sok2yap6 and sok2phd1yap6 cells the kinetics of meiosis was slightly faster than sok2phd1 cells.Approximately 50% of cells underwent meiotic divisions within 2 hours in SPO for sok2phd1yap6 cells compared to 3 hours for sok2phd1 cells.
During meiotic entry (4 hours in SPO), Yap6, Phd1 and Nrg1 showed some enrichment at the IME1 promoter suggesting that they could contribute to activation of IME1 transcription.Therefore, we also analysed how the onset of meiosis is affected in sok2phd1yap6nrg1 cells (Supplementary Figure 6).We found no difference in meiosis between sok2phd1yap6nrg1 and sok2phd1yap6 cells suggesting that these transcription factors do not contribute to activation of IME1 transcription in cells induced to enter meiosis synchronously.Taken together, we conclude that Sok2, Phd1, and Yap6 direct Tup1-Cyc8 association to the IME1 promoter to ensure timely expression of IME1 in cells grown in acetate containing medium.Our data suggest that the IME1 promoter is regulated by multiple Tup1-Cyc8 co-repressor complexes.
How do nutrient signals regulate the association and dissociation of Tup1-Cyc8 recruiting transcription factors with the IME1 promoter?While it is known that nutrients mediate PKA and TORC1 signalling pathways and thereby control IME1 expression, little is known about the mechanisms that mediate PKA and TORC1 signalling at the IME1 promoter 4,16 .Regulated localization or abundance of transcription factors are prevalent mechanisms for controlling gene expression via PKA and TORC1 42,43 .Among the transcription factors that associate with the IME1 promoter, Sok2 protein abundance is regulated by glucose signalling via the Ras/PKA pathway 11,44 .To gain insight into how Tup1-Cyc8 recruiting transcription factors (Sok2, Phd1, and Yap6) dissociate from the IME1 promoter, we sought to investigate whether their abundance or localization is altered during entry into meiosis.First, we examined protein expression levels in exponentially grown cells (E), cells grown to saturation (S), and in cells just prior to and during meiotic entry (0h and 4h SPO).The levels of Sok2, Phd1 and Yap6 as well as Cyc8 were reduced in cells grown to saturation compared to exponentially growing cells (Figure 6A).However, in cells grown until saturation, Sok2, Phd1 and Yap6 were bound to the IME1 promoter (Supplementary Figure 7).Interestingly, reduced Cyc8 levels mirrored the reduced binding of Cyc8 at the IME1 promoter (Figure 6A and Supplementary Figure 7).Importantly, Sok2, Phd1 and Yap6 abundance was not altered in cells between the 0 and 4 hour time points (in SPO) when IME1 expression is induced.These data indicate that cellular changes in Sok2, Phd1 and Yap6 abundance are unlikely to explain the dissociation of these transcription factors from the IME1 promoter during entry into meiosis.To determine whether these transcription factors are evicted from the IME1 promoter due to nuclear export, we determined the cellular localization for each transcription factor.We fused mNeongreen to Sok2, Phd1, Yap6, Tup1 and Cyc8 (Supplementary Figure 8A).As expected, Sok2, Phd1, and Yap6 were concentrated in the nucleus.Neither the nuclear-to-cytoplasmic ratios nor total protein abundance in the nucleus were significantly altered in cells prior to (0h in SPO) and during entry into meiosis (4h in SPO) (Figure 6B and Supplementary Figure 8B).Furthermore, the overall localization of Tup1 and Cyc8 was also not altered during meiotic entry (Figure 6B and Supplementary Figure 8B).We conclude that cellular changes in protein abundance or localization cannot solely account for the dissociation of Tup1-Cyc8 and transcription factors from the IME1 promoter during entry in meiosis.
There is evidence that signalling kinases can act locally at gene promoters.For example, Tpk1 and Tpk2 kinases of PKA can associate with specific gene promoters and regulate transcription locally 45 .We reasoned that our measurements of transcription factor abundance and localization may be masked if PKA and TORC1 signalling is heterogeneous within cells.Therefore, perhaps inhibiting PKA and TORC1 altogether could reveal how both signalling pathways regulate transcription factors important for repressing the IME1 promoter.To inhibit PKA we used the tpk1as allele previously described, while we used rapamycin to inhibit TORC1 16 .
Strikingly, Sok2, Phd1 and Yap6 protein levels were reduced between five to ten-fold when PKA was inhibited (tpk1-as + NMPP1) (Figure 6C).Inhibiting TORC1 activity with rapamycin also lowered Yap6 and Phd1, but not Sok2 levels.We conclude that inhibiting PKA and TORC1 affects the abundance of transcription factors important for repressing the IME1 promoter, and coincides with Tup1 disassociation and activation of IME1 transcription as described previously 16 .Our data suggest that the association of transcription factors with the IME1 promoter may be regulated locally by PKA and TORC1 signalling.
Our data indicate that at least eight transcription factors that are known or implicated to interact with Tup1 or Cyc8, associate with the IME1 promoter (Figure 2B).Motif analyses revealed that there are 52 corresponding binding sites present in the region of the IME1 promoter where Tup1-Cyc8 associates (Supplementary Figure 3).In addition, there are multiple binding motifs for almost every transcription factor present suggesting that multiple copies of each transcription factors bind to the IME1 promoter (Supplementary Figure 3A).Why is there need for so many binding sites in the IME1 promoter?One possibility is that the transcription factors are important for facilitating Tup1-Cyc8 recruitment under different nutrient conditions.With this logic, the repression of the IME1 promoter can be maintained under various nutrient conditions, and will only be fully activated when all the nutrient signalling requirements are met.In agreement with this model, IME1 expression was only marginally increased in sok2 phd1yap6 cells grown in the presence of ample nutrients with glucose as the carbon source (YPD), while the IME1 promoter was nearly completely de-repressed in sok2phd1yap6 cells grown in acetatecontaining medium (Figure 4A, 4B, 5B and 5C).To examine how the different transcription factors respond to nutrient signalling at the IME1 promoter more systematically, we measured their association under different nutrient conditions (Figure 7A).We grew cells until the pre-sporulation stage, and subsequently shifted cells to sporulation medium (SPO) (1), SPO plus 2% glucose (2), YP (3), and YP plus 2% glucose (YPD) (4).First, we measured Tup1 association with the IME1 promoter.We found that in SPO plus glucose, Tup1 binding to the IME1 promoter was partially restored (Figure 7B).The association of Tup1 with the IME1 promoter was further increased in YP and was the highest in YPD growth medium.The transcriptional activator of IME1, Pog1, was enriched in all four nutrient conditions, but at higher levels in YP and YPD (Figure 7B).Interestingly, transcription factors important for Tup1-Cyc8 recruitment to IME1 promoter responded to nutrient signals in distinct ways (Figure 7C).For example, Yap6, Sok2, Sko1 and Nrg1 did not associate with the IME1 promoter in response to glucose, but their binding was restored due to nutrient cues present in YP.Phd1 binding partially recovered in the presence of glucose, and showed the strongest enrichment in cells exposed to YP and YPD.The carbon source, glucose, but not YP, induced association of Mot3 and Nrg2 with the IME1 promoter, while Sut1 association with the IME1 promoter was restored in YPD only.
Given that Sok2, Phd1, and Yap6 were strongly enriched in cells exposed to YP medium (Figure 7C), we hypothesized that Tup1-Cyc8 association with the IME1 promoter is affected in sok2phd1yap6 cells in YP, but not in SPO containing glucose.We therefore examined how Tup1-Cyc8 association with IME1 promoter was affected in sok2phd1yap6 cells under different nutrient conditions.Indeed, in sok2phd1yap6 cells, Tup1 binding was detected in SPO plus glucose, but not in YP medium (Figure 7D).These data indicate that Sok2, Phd1, and Yap6 are important for mediating Tup1-Cyc8 association in YP, while other transcription factors are required for glucose signalling to the IME1 promoter (Figure 7E).In

Model for Tup1-Cyc8 mediated regulation of the IME1 promoter
Repression of IME1 transcription, and not activation, is highly regulated.Depletion of either Tup1 or Cyc8 completely de-repressed IME1 expression (Figure 1).
Remarkably, there was little or no delay between depletion of Tup1 and IME1 transcription in the presence of ample nutrients.From these two observations, we can infer two important features of the IME1 promoter.First, the transcriptional activators are bound to the IME1 promoter or readily available prior to activation of IME1 transcription.Second, transcriptional activators do not require nutrient or environmental signalling to activate the IME1 promoter.Indeed, we found that the activator Pog1 is bound to the IME1 promoter prior to activation.Pog1 does not have clear DNA binding domain, and thus Pog1 likely interacts with other proteins to associate with the IME1 promoter.A pog1 mutant has only a mild effect on IME1 expression indicating that there must be other transcriptional activators controlling IME1 transcription 7 .Several transcriptional activators have been implicated in regulating IME1 transcription that have not been linked with Tup1-Cyc8 12 .
How does Tup1-Cyc8 control the IME1 promoter?The Tup1-Cyc8 regulates transcription of a subset of promoters in yeast 18,46 .Several mechanisms have been described for Tup1-Cyc8 mediated gene repression 21,23,24 .For example, Tup1-Cyc8 interacts with HDACs, which in turn facilitate repression of promoters by deacetylating nucleosomes.However, we find that deletion mutants of HDACs have little effect on IME1 expression and do not mimic IME1 expression levels detected in Tup1 or Cyc8 depleted cells.Our data are largely consistent with a model in which Tup1-Cyc8 masks or shields activating transcription factors from recruiting coactivators at promoters 25 .In line with Tup1-Cyc8 depletion experiments described in this work, IME1 was among the genes that showed increased transcription upon rapid depletion of Tup1-Cyc8 from nucleus 25 .Furthermore, we show that the transcription activator Pog1 is bound to the IME1 promoter prior to activation, and remains bound during activation of IME1 transcription.
Transcription factors that interact with Tup1-Cyc8 can converted into transcriptional activators in the absence of Tup1-Cyc8 25,39 .We showed that multiple (at least eight) transcription factors that are known to interact with Tup1-Cyc8 associate with the IME1 promoter.However, our data suggest these transcription factors are important for facilitating Tup1-Cyc8 binding but play little role in IME1 transcriptional activation.
Multiple lines of evidence indicate that Tup1-Cyc8 acts predominantly as a corepressor at the IME1 promoter.First, almost all transcription factors involved in Tup1-Cyc8 recruitment dissociate from the IME1 promoter upon activation of IME1 transcription (Figure 5A, SPO 4h).Second, deleting multiple transcription factors led to activation, not repression of IME1 transcription.For example, cells lacking four of the transcription factors that showed the strongest enrichment with the IME1 promoter (sok2phd1yap6nrg1) displayed an earlier onset of meiosis (Supplementary Figure 6).We cannot exclude that the Tup1-Cyc8 recruiting transcription factors can function as transcriptional activators in some conditions.Indeed, Yap6 and Sok2 have both been implicated as activators of transcription at some promoters 47,48  We propose that multiple transcription factors ensure IME1 repression under various environmental conditions.First, we found that distinct sets of transcription factors associate with the IME1 promoter in different nutrient environments (Figure 7).
Second, deleting three transcription factors (Sok2, Phd1, and Yap6) led to very mild IME1 expression in rich medium containing glucose, but IME1 was almost fully expressed in cells grown in an acetate containing medium (Figure 4A, 4B and 5C).
Thus, additional transcription factors facilitate Tup1-Cyc8 association with the IME1 promoter in rich medium containing glucose.

How nutrient signalling controls the association of transcription factors directing
Tup1-Cyc8 to the IME1 promoter is key to dissecting how meiotic entry is regulated.
We previously showed that inhibiting PKA and TORC1 is sufficient to drive entry into meiosis despite that cells were exposed to a nutrient rich medium 16 .Here we showed that inhibiting PKA and TORC1 lowers the abundances of transcription factors important for Tup1-Cyc8 recruitment to the IME1 promoter (Sok2, Phd1 and Yap6) (Figure 6C), however the mechanisms remains unclear.One possibility is that PKA or TORC1 phosphorylation controls the stability of Sok2, Phd1 and Yap6.Indeed, Sok2 is a direct substrate of PKA 11,49 .It is also possible that nutrient or stress induced phosphorylation regulates the interaction between transcription factors and Tup1-Cyc8 39,50 .In addition, nutrient signalling may regulate Tup1-Cyc8 itself.Several studies have shown that sumoylation modulates the function of Tup1-Cyc8 51,52 .Our data further suggest that PKA and TORC1 locally regulates the IME1 promoter.We observed no change in cellular localization and only a minor change in abundance during meiotic entry, while chemical inhibition of PKA and TORC1 resulted in much lower levels of Sok2, Phd1 and Yap6 (Figure 6).Perhaps, PKA and TORC1 associate with IME1 promoter directly.Future work will pinpoint the mechanism by which nutrient signalling pathways control the transcription factors regulating Tup1-Cyc8 binding with the IME1 promoter.

Concluding remarks
Our observation that regulated repression by multiple transcription factors controls a cell fate decision in yeast shows similarities with how multicellular organisms undertake developmental decisions.In Drosophila, plants, and mammals, transcriptional repressors of the Groucho family (structurally related to Tup1) are important for regulating of various developmental processes such as body patterning and determination of organ identity [53][54][55] .Like Tup1-Cyc8, the association of Groucho repressor with promoters relies on sequence specific transcription factors, and Groucho repressor integrates multiple signals to control gene expression and cell fate outcomes.Regulation of the IME1 promoter also demonstrates features of enhancer-directed transcriptional control of cell-fate master regulators in mammalian cells [56][57][58] .Like the IME1 promoter, an array of transcription factors associate and control the activity of enhancers.In addition, developmentally controlled enhancers are typically regulated by multiple upstream signalling pathways and are often primed for activation 59 .Our findings in yeast may provide insights to better understand how signal integration controls master regulatory genes and developmental decisions in all eukaryotic cells.
summary, our analyses revealed that the association of one set of transcription factors (i.e.Mot3 and Nrg2) with the IME1 promoter is induced by glucose signalling, while another set of transcription factors (i.e.Yap6, Sok2, Phd1, Sko1, and Nrg1) was recruited to the IME1 promoter in cells exposed to YP.Thus, only when all the required nutrient signalling pathways are repressed, all transcription factors interacting with Tup1-Cyc8 dissociate from the IME1 promoter allowing activation of IME1 transcription.Taken together, we propose that transcription factors important for Tup1-Cyc8 recruitment to the IME1 promoter respond to different environmental cues to ensure tight control of IME1 expression and thereby regulate the fate decision to enter meiosis in yeast.Discussion: Our study of the IME1 promoter sheds new light on how the fate decision to enter meiosis is regulated.We report that the Tup1-Cyc8 complex together with multiple sequence-specific transcription factors constitute the essential components that control repression of the IME1 promoter.The decision to enter meiosis and produce gametes is remarkably simple in yeast: environmental signals regulate the association and disassociation of transcription factors that recruit Tup1-Cyc8 to the IME1 promoter.We propose that multiple transcription factors ensures repression of IME1 expression under various environmental conditions and thus establishes tight control of entry into meiosis in yeast.
. In the context of the IME1 promoter, however, transcription factors mediating Tup1-Cyc8 recruitment and transcriptional activators are likely not the same.Each transcription factor likely has a designated function in either repression or activation of IME1 transcription.We propose that multiple transcription factors ensure that Tup1-Cyc8 co-repressor is bound to the IME1 promoter.The Tup1-Cyc8 co-repressor complexes, in turn, mask transcriptional activators (which are different from Tup1-Cyc8 recruiting transcription factors) and prevent them from recruiting co-activators to induce IME1 transcription.The regulatory logic of employing multiple sequence specific transcription factors to repress the IME1 promoterWhy are multiple transcription factors required for recruiting Tup1-Cyc8 to the IME1 promoter?Expression of IME1 only occurs when the cells are starved for nitrogen and glucose4 .Under other environmental conditions, the IME1 promoter must be repressed to prevent cells from inappropriately entering meiosis and form gametes.

Figure 3 .
Figure 3. Tup1-Cyc8 is recruited by transcription factors associated with the

Figure 7 .
Figure 7. Nutrient signalling triggers distinct responses of transcription factors

Figure 1 A
Figure 1