Genetic analysis argues for a coactivator function for the Saccharomyces cerevisiae Tup1 corepressor.

The Tup1-Cyc8 corepressor complex of Saccharomyces cerevisiae is recruited to promoters by DNA-binding proteins to repress transcription of genes, including the a-specific mating-type genes. We report here a tup1(S649F) mutant that displays mating irregularities and an α-predominant growth defect. RNA-Seq and ChIP-Seq were used to analyze gene expression and Tup1 occupancy changes in mutant vs wild type in both a and α cells. Increased Tup1(S649F) occupancy tended to occur upstream of upregulated genes, whereas locations with decreased occupancy usually did not show changes in gene expression, suggesting this mutant not only loses corepressor function but also behaves as a coactivator. Based upon studies demonstrating a dual role of Tup1 in both repression and activation, we postulate that the coactivator function of Tup1(S649F) results from diminished interaction with repressor proteins, including α2. We also found that large changes in mating-type-specific gene expression between a and α or between mutant and wild type were not easily explained by the range of Tup1 occupancy levels within their promoters, as predicted by the classic model of a-specific gene repression by Tup1. Most surprisingly, we observed Tup1 occupancy upstream of the a-specific gene MFA2 and the α-specific gene MF(ALPHA)1 in cells in which each gene was expressed rather than repressed. These results, combined with the identification of additional mating-related genes upregulated in the tup1(S649F) α strain, illustrate that the role of Tup1 in distinguishing mating types in yeast appears to be both more comprehensive and more nuanced than previously appreciated.

Ash1 and Tup1 dependent repression of the Saccharomyces cerevisiae HO promoter requires activator-dependent nucleosome eviction.

Transcriptional regulation of the Saccharomyces cerevisiae HO gene is highly complex, requiring a balance of multiple activating and repressing factors to ensure that only a few transcripts are produced in mother cells within a narrow window of the cell cycle. Here, we show that the Ash1 repressor associates with two DNA sequences that are usually concealed within nucleosomes in the HO promoter and recruits the Tup1 corepressor and the Rpd3 histone deacetylase, both of which are required for full repression in daughters. Genome-wide ChIP identified greater than 200 additional sites of co-localization of these factors, primarily within large, intergenic regions from which they could regulate adjacent genes. Most Ash1 binding sites are in nucleosome depleted regions (NDRs), while a small number overlap nucleosomes, similar to HO. We demonstrate that Ash1 binding to the HO promoter does not occur in the absence of the Swi5 transcription factor, which recruits coactivators that evict nucleosomes, including the nucleosomes obscuring the Ash1 binding sites. In the absence of Swi5, artificial nucleosome depletion allowed Ash1 to bind, demonstrating that nucleosomes are inhibitory to Ash1 binding. The location of binding sites within nucleosomes may therefore be a mechanism for limiting repressive activity to periods of nucleosome eviction that are otherwise associated with activation of the promoter. Our results illustrate that activation and repression can be intricately connected, and events set in motion by an activator may also ensure the appropriate level of repression and reset the promoter for the next activation cycle.

FACT and Ash1 promote long-range and bidirectional nucleosome eviction at the HO promoter.

The Saccharomyces cerevisiae HO gene is a model regulatory system with complex transcriptional regulation. Budding yeast divide asymmetrically and HO is expressed only in mother cells where a nucleosome eviction cascade along the promoter during the cell cycle enables activation. HO expression in daughter cells is inhibited by high concentration of Ash1 in daughters. To understand how Ash1 represses transcription, we used a myo4 mutation which boosts Ash1 accumulation in both mothers and daughters and show that Ash1 inhibits promoter recruitment of SWI/SNF and Gcn5. We show Ash1 is also required for the efficient nucleosome repopulation that occurs after eviction, and the strongest effects of Ash1 are seen when Ash1 has been degraded and at promoter locations distant from where Ash1 bound. Additionally, we defined a specific nucleosome/nucleosome-depleted region structure that restricts HO activation to one of two paralogous DNA-binding factors. We also show that nucleosome eviction occurs bidirectionally over a large distance. Significantly, eviction of the more distant nucleosomes is dependent upon the FACT histone chaperone, and FACT is recruited to these regions when eviction is beginning. These last observations, along with ChIP experiments involving the SBF factor, suggest a long-distance loop transiently forms at the HO promoter.

A Role for Mediator Core in Limiting Coactivator Recruitment in Saccharomyces cerevisiae.

Mediator is an essential, multisubunit complex that functions as a transcriptional coactivator in yeast and other eukaryotic organisms. Mediator has four conserved modules, Head, Middle, Tail, and Kinase, and has been implicated in nearly all aspects of gene regulation. The Tail module has been shown to recruit the Mediator complex to the enhancer or upstream activating sequence (UAS) regions of genes via interactions with transcription factors, and the Kinase module facilitates the transition of Mediator from the UAS/enhancer to the preinitiation complex via protein phosphorylation. Here, we analyze expression of the Saccharomyces cerevisiaeHO gene using a sin4 Mediator Tail mutation that separates the Tail module from the rest of the complex; the sin4 mutation permits independent recruitment of the Tail module to promoters without the rest of Mediator. Significant increases in recruitment of the SWI/SNF and SAGA coactivators to the HO promoter UAS were observed in a sin4 mutant, along with increased gene activation. These results are consistent with recent studies that have suggested that the Kinase module functions negatively to inhibit activation by the Tail. However, we found that Kinase module mutations did not mimic the effect of a sin4 mutation on HO expression. This suggests that at HO the core Mediator complex (Middle and Head modules) must play a role in limiting Tail binding to the promoter UAS and gene activation. We propose that the core Mediator complex helps modulate Mediator binding to the UAS regions of genes to limit coactivator recruitment and ensure proper regulation of gene transcription.

Multiple Negative Regulators Restrict Recruitment of the SWI/SNF Chromatin Remodeler to the HO Promoter in Saccharomyces cerevisiae.

Activation of the Saccharomyces cerevisiae HO promoter is highly regulated, requiring the ordered recruitment of activators and coactivators and allowing production of only a few transcripts in mother cells within a short cell cycle window. We conducted genetic screens to identify the negative regulators of HO expression necessary to limit HO transcription. Known repressors of HO (Ash1 and Rpd3) were identified, as well as several additional chromatin-associated factors including the Hda1 histone deacetylase, the Isw2 chromatin remodeler, and the corepressor Tup1 We also identified clusters of HO promoter mutations that suggested roles for the Dot6/Tod6 (PAC site) and Ume6 repression pathways. We used ChIP assays with synchronized cells to validate the involvement of these factors and map the association of Ash1, Dot6, and Ume6 with the HO promoter to a brief window in the cell cycle between binding of the initial activating transcription factor and initiation of transcription. We found that Ash1 and Ume6 each recruit the Rpd3 histone deacetylase to HO, and their effects are additive. In contrast, Rpd3 was not recruited significantly to the PAC site, suggesting this site has a distinct mechanism for repression. Increases in HO expression and SWI/SNF recruitment were all additive upon loss of Ash1, Ume6, and PAC site factors, indicating the convergence of independent pathways for repression. Our results demonstrate that multiple protein complexes are important for limiting the spread of SWI/SNF-mediated nucleosome eviction across the HO promoter, suggesting that regulation requires a delicate balance of activities that promote and repress transcription.

Establishment and Maintenance of Chromatin Architecture Are Promoted Independently of Transcription by the Histone Chaperone FACT and H3-K56 Acetylation in Saccharomyces cerevisiae.

FACT (FAcilitates Chromatin Transcription/Transactions) is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone-DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome reorganization activity in vitro, and chromatin integrity and transcript output in vivo Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT activity as expected, but the same mutations had surprisingly similar effects on global transcript levels. The results are consistent with emerging models that emphasize FACT's importance in establishing global chromatin architecture prior to transcription, promoting transitions among different states as transcription profiles change, and restoring chromatin integrity after it is disturbed. Optimal FACT activity required the availability of both modified and unmodified states of H3-K56. Perturbing this balance was especially detrimental for maintaining repression of genes with high nucleosome occupancy over their promoters and for blocking antisense transcription at the +1 nucleosome. The results reveal a complex collaboration between H3-K56 modification status and multiple FACT functions, and support roles for nucleosome reorganization by FACT before, during, and after transcription.

A member of the gut mycobiota modulates host purine metabolism exacerbating colitis in mice.

The commensal microbiota has an important impact on host health, which is only beginning to be elucidated. Despite the presence of fungal, archaeal, and viral members, most studies have focused solely on the bacterial microbiota. Antibodies against the yeast Saccharomyces cerevisiae are found in some patients with Crohn's disease (CD), suggesting that the mycobiota may contribute to disease severity. We report that S. cerevisiae exacerbated intestinal disease in a mouse model of colitis and increased gut barrier permeability. Transcriptome analysis of colon tissue from germ-free mice inoculated with S. cerevisiae or another fungus, Rhodotorula aurantiaca, revealed that S. cerevisiae colonization affected the intestinal barrier and host metabolism. A fecal metabolomics screen of germ-free animals demonstrated that S. cerevisiae colonization enhanced host purine metabolism, leading to an increase in uric acid production. Treatment with uric acid alone worsened disease and increased gut permeability. Allopurinol, a clinical drug used to reduce uric acid, ameliorated colitis induced by S. cerevisiae in mice. In addition, we found a positive correlation between elevated uric acid and anti-yeast antibodies in human sera. Thus, yeast in the gut may be able to potentiate metabolite production that negatively affects the course of inflammatory bowel disease.

Disruption of promoter memory by synthesis of a long noncoding RNA.

The yeast HO endonuclease is expressed in late G1 in haploid mother cells to initiate mating-type interconversion. Cells can be arrested in G1 by nutrient deprivation or by pheromone exposure, but cells that resume cycling after nutrient deprivation or cyclin-dependent kinase (CDK) inactivation express HO in the first cell cycle, whereas HO is not expressed until the second cycle after release from pheromone arrest. Here, we show that transcription of a long noncoding RNA (lncRNA) mediates this differential response. The SBF and Mediator factors remain bound to the inactive promoter during arrest due to CDK inactivation, and these bound factors allow the cell to remember a transcriptional decision made before arrest. If the presence of mating pheromone indicates that this decision is no longer appropriate, a lncRNA originating at -2700 upstream of the HO gene is induced, and the transcription machinery displaces promoter-bound SBF, preventing HO transcription in the subsequent cell cycle. Further, we find that the displaced SBF is blocked from rebinding due to incorporation of its recognition sites within nucleosomes. Expressing the pHO-lncRNA in trans is ineffective, indicating that transcription in cis is required. Factor displacement during lncRNA transcription could be a general mechanism for regulating memory of previous events at promoters.

Nucleosomes Are Essential for Proper Regulation of a Multigated Promoter in Saccharomyces cerevisiae.

Nucleosome-depleted regions (NDRs) are present immediately adjacent to the transcription start site in most eukaryotic promoters. Here we show that NDRs in the upstream promoter region can profoundly affect gene regulation. Chromatin at the yeast HO promoter is highly repressive and numerous coactivators are required for expression. We modified the HO promoter with segments from the well-studied CLN2 NDR, creating chimeric promoters differing in nucleosome occupancy but with binding sites for the same activator, SBF. Nucleosome depletion resulted in substantial increases in both factor binding and gene expression and allowed activation from a much longer distance, probably by allowing recruited coactivators to act further downstream. Nucleosome depletion also affected sequential activation of the HO promoter; HO activation typically requires the ordered recruitment of activators first to URS1, second to the left-half of URS2 (URS2-L), and finally to the right-half of URS2 (URS2-R), with each region representing distinct gates that must be unlocked to achieve activation. The absence of nucleosomes at URS2-L resulted in promoters no longer requiring both the URS1 and URS2-L gates, as either gate alone is now sufficient to promote binding of the SBF factor to URS2-R. Furthermore, nucleosome depletion at URS2 altered the timing of HO expression and bypassed the regulation that restricts expression to mother cells. Our results reveal insight into how nucleosomes can create a requirement for ordered recruitment of factors to facilitate complex transcriptional regulation.

Spatiotemporal cascade of transcription factor binding required for promoter activation.

Promoters often contain multiple binding sites for a single factor. The yeast HO gene contains nine highly conserved binding sites for the SCB (Swi4/6-dependent cell cycle box) binding factor (SBF) complex (composed of Swi4 and Swi6) in the 700-bp upstream regulatory sequence 2 (URS2) promoter region. Here, we show that the distal and proximal SBF sites in URS2 function differently. Chromatin immunoprecipitation (ChIP) experiments show that SBF binds preferentially to the left side of URS2 (URS2-L), despite equivalent binding to the left-half and right-half SBF sites in vitro. SBF binding at URS2-L sites depends on prior chromatin remodeling events at the upstream URS1 region. These signals from URS1 influence chromatin changes at URS2 but only at sites within a defined distance. SBF bound at URS2-L, however, is unable to activate transcription but instead facilitates SBF binding to sites in the right half (URS2-R), which are required for transcriptional activation. Factor binding at HO, therefore, follows a temporal cascade, with SBF bound at URS2-L serving to relay a signal from URS1 to the SBF sites in URS2-R that ultimately activate gene expression. Taken together, we describe a novel property of a transcription factor that can have two distinct roles in gene activation, depending on its location within a promoter.

The Rts1 regulatory subunit of PP2A phosphatase controls expression of the HO endonuclease via localization of the Ace2 transcription factor.

The RTS1 gene encodes a subunit of the PP2A phosphatase that regulates cell cycle progression. Ace2 and Swi5 are cell cycle-regulated transcription factors, and we recently showed that phosphorylation of Ace2 and Swi5 is altered in an rts1 mutant. Here we examine expression of Ace2 and Swi5 target genes and find that an rts1 mutation markedly reduces expression of the HO gene. The decreased HO expression in an rts1 mutant is significantly restored by an additional ace2 mutation, a surprising result because HO is normally activated by Swi5 but not by Ace2. Ace2 normally accumulates only in daughter cells, and only activates transcription in daughters. However, in an rts1 mutant, Ace2 is present in both mother and daughter cells. One of the genes activated by Ace2 is ASH1, a protein that normally accumulates mostly in daughter cells; Ash1 is a transcriptional repressor, and it blocks HO expression in daughters. We show that in the rts1 mutant, Ace2 accumulation in mother cells results in Ash1 expression in mothers, and the Ash1 can now repress HO expression in mothers.

PP2ARts1 is a master regulator of pathways that control cell size.

Cell size checkpoints ensure that passage through G1 and mitosis occurs only when sufficient growth has occurred. The mechanisms by which these checkpoints work are largely unknown. PP2A associated with the Rts1 regulatory subunit (PP2A(Rts1)) is required for cell size control in budding yeast, but the relevant targets are unknown. In this paper, we used quantitative proteome-wide mass spectrometry to identify proteins controlled by PP2A(Rts1). This revealed that PP2A(Rts1) controls the two key checkpoint pathways thought to regulate the cell cycle in response to cell growth. To investigate the role of PP2A(Rts1) in these pathways, we focused on the Ace2 transcription factor, which is thought to delay cell cycle entry by repressing transcription of the G1 cyclin CLN3. Diverse experiments suggest that PP2A(Rts1) promotes cell cycle entry by inhibiting the repressor functions of Ace2. We hypothesize that control of Ace2 by PP2A(Rts1) plays a role in mechanisms that link G1 cyclin accumulation to cell growth.

A role for FACT in repopulation of nucleosomes at inducible genes.

Xenobiotic drugs induce Pleiotropic Drug Resistance (PDR) genes via the orthologous Pdr1/Pdr3 transcription activators. We previously identified the Mediator transcription co-activator complex as a key target of Pdr1 orthologs and demonstrated that Pdr1 interacts directly with the Gal11/Med15 subunit of the Mediator complex. Based on an interaction between Pdr1 and the FACT complex, we show that strains with spt16 or pob3 mutations are sensitive to xenobiotic drugs and display diminished PDR gene induction. Although FACT acts during the activation of some genes by assisting in the nucleosomes eviction at promoters, PDR promoters already contain nucleosome-depleted regions (NDRs) before induction. To determine the function of FACT at PDR genes, we examined the kinetics of RNA accumulation and changes in nucleosome occupancy following exposure to a xenobiotic drug in wild type and FACT mutant yeast strains. In the presence of normal FACT, PDR genes are transcribed within 5 minutes of xenobiotic stimulation and transcription returns to basal levels by 30-40 min. Nucleosomes are constitutively depleted in the promoter regions, are lost from the open reading frames during transcription, and the ORFs are wholly repopulated with nucleosomes as transcription ceases. While FACT mutations cause minor delays in activation of PDR genes, much more pronounced and significant defects in nucleosome repopulation in the ORFs are observed in FACT mutants upon transcription termination. FACT therefore has a major role in nucleosome redeposition following cessation of transcription at the PDR genes, the opposite of its better-known function in nucleosome disassembly.

Stochastic expression and epigenetic memory at the yeast HO promoter.

Eukaryotic gene regulation usually involves sequence-specific transcription factors and sequence-nonspecific cofactors. A large effort has been made to understand how these factors affect the average gene expression level among a population. However, little is known about how they regulate gene expression in individual cells. In this work, we address this question by mutating multiple factors in the regulatory pathway of the yeast HO promoter (HOpr) and probing the corresponding promoter activity in single cells using time-lapse fluorescence microscopy. We show that the HOpr fires in an "on/off" fashion in WT cells as well as in different genetic backgrounds. Many chromatin-related cofactors that affect the average level of HO expression do not actually affect the firing amplitude of the HOpr; instead, they affect the firing frequency among individual cell cycles. With certain mutations, the bimodal expression exhibits short-term epigenetic memory across the mitotic boundary. This memory is propagated in "cis" and reflects enhanced activator binding after a previous "on" cycle. We present evidence that the memory results from slow turnover of the histone acetylation marks.

Dancing the cell cycle two-step: regulation of yeast G1-cell-cycle genes by chromatin structure.

The chromatin structure at a promoter can define how a gene is regulated. Studies of two yeast genes expressed in the G1 phase of the cell cycle, HO and CLN2, have provided important paradigms for transcriptional regulation. Although the SBF (Swi4/Swi6 box factor) transcription factor activates both genes, the chromatin landscapes that regulate SBF binding are different. Specifically, the CLN2 promoter is constitutively available for SBF binding, whereas HO has a complex two-step promoter in which chromatin changes in one region allow SBF to bind at a downstream location. These studies reveal the role of chromatin in defining the regulatory properties of promoters.

Shields up: the Tup1-Cyc8 repressor complex blocks coactivator recruitment.

The Tup1-Cyc8 complex is responsible for repression of a large and diverse collection of genes in Saccharomyces cerevisiae. The predominant view has been that Tup1-Cyc8 functions as a corepressor, actively associating with regulatory proteins and organizing chromatin to block transcription. A new study by Wong and Struhl in this issue of Genes & Development (pp. 2525-2539) challenges nearly 20 years of models by demonstrating that Tup1-Cyc8 functions primarily as a shield to block DNA-binding proteins from recruiting transcriptional coactivators.

Repressive chromatin affects factor binding at yeast HO (homothallic switching) promoter.

The yeast HO gene is tightly regulated, with multiple activators and coactivators needed to overcome repressive chromatin structures that form over this promoter. Coactivator binding is strongly interdependent, as loss of one factor sharply reduces recruitment of other factors. The Rpd3(L) histone deacetylase is recruited to HO at two distinct times during the cell cycle, first by Ash1 to the URS1 region of the promoter and then by SBF/Whi5/Stb1 to URS2. SBF itself is localized to only a subset of its potential binding sites in URS2, and this localization takes longer and is less robust than at other SBF target genes, suggesting that binding to the HO promoter is limited by chromatin structures that dynamically change as the cell cycle progresses. Ash1 only binds at the URS1 region of the promoter, but an ash1 mutation results in markedly increased binding of SBF and Rpd3(L) at URS2, some 450 bp distant from the site of Ash1 binding, suggesting these two regions of the promoter interact. An ash1 mutation also results in increased coactivator recruitment, Swi/Snf and Mediator localization in the absence of the normally required Gcn5 histone acetyltransferase, and HO expression even in the presence of a taf1 mutation affecting TFIID activity that otherwise blocks HO transcription. Ash1 therefore appears to play a central role in generating the strongly repressive environment at the HO promoter, which limits the binding of several coactivators at URS2 and TATA region.

Insight into the mechanism of nucleosome reorganization from histone mutants that suppress defects in the FACT histone chaperone.

FACT (FAcilitates Chromatin Transcription/Transactions) plays a central role in transcription and replication in eukaryotes by both establishing and overcoming the repressive properties of chromatin. FACT promotes these opposing goals by interconverting nucleosomes between the canonical form and a more open reorganized form. In the forward direction, reorganization destabilizes nucleosomes, while the reverse reaction promotes nucleosome assembly. Nucleosome destabilization involves disrupting contacts among histone H2A-H2B dimers, (H3-H4)(2) tetramers, and DNA. Here we show that mutations that weaken the dimer:tetramer interface in nucleosomes suppress defects caused by FACT deficiency in vivo in the yeast Saccharomyces cerevisiae. Mutating the gene that encodes the Spt16 subunit of FACT causes phenotypes associated with defects in transcription and replication, and we identify histone mutants that selectively suppress those associated with replication. Analysis of purified components suggests that the defective version of FACT is unable to maintain the reorganized nucleosome state efficiently, whereas nucleosomes with mutant histones are reorganized more easily than normal. The genetic suppression observed when the FACT defect is combined with the histone defect therefore reveals the importance of the dynamic reorganization of contacts within nucleosomes to the function of FACT in vivo, especially to FACT's apparent role in promoting progression of DNA replication complexes. We also show that an H2B mutation causes different phenotypes, depending on which of the two similar genes that encode this protein are altered, revealing unexpected functional differences between these duplicated genes and calling into question the practice of examining the effects of histone mutants by expressing them from a single plasmid-borne allele.

First time, every time: nucleosomes at a promoter can determine the probability of gene activation.

Transcription factor binding sites are found in either nucleosome-free or nucleosome-embedded locations, thus in vivo relationships between nucleosome position and gene activation are not fully understood. In this issue of Developmental Cell, Bai et al. show that binding sites located in nucleosome depleted regions guarantee high reliability, not amplitude, of promoter firing.

Nhp6: a small but powerful effector of chromatin structure in Saccharomyces cerevisiae.

The small Nhp6 protein from budding yeast is an abundant protein that binds DNA non-specifically and bends DNA sharply. It contains only a single HMGB domain that binds DNA in the minor groove and a basic N-terminal extension that wraps around DNA to contact the major groove. This review describes the genetic and biochemical experiments that indicate Nhp6 functions in promoting RNA pol III transcription, in formation of preinitiation complexes at promoters transcribed by RNA pol II, and in facilitating the activity of chromatin modifying complexes. The FACT complex may provide a paradigm for how Nhp6 functions with chromatin factors, as Nhp6 allows Spt16-Pob3 to bind to and reorganize nucleosomes in vitro.