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In budding yeast, fermentation is the most important pathway for energy production. Under low-glucose conditions, ethanol is used for synthesis of this sugar requiring a shift to respiration. This process is controlled by the transcriptional regulators Cat8, Sip4, Rds2 and Ert1. We characterized Gsm1 (glucose starvation modulator 1), a paralog of Rds2 and Ert1. Genome-wide analysis showed that Gsm1 has a DNA binding profile highly similar to Rds2. Binding of Gsm1 and Rds2 is interdependent at the gluconeogenic gene FBP1. However, Rds2 is required for Gsm1 to bind at other promoters but not the reverse. Gsm1 and Rds2 also bind to DNA independently of each other. Western blot analysis revealed that Rds2 controls expression of Gsm1. In addition, we showed that the DNA binding domains of Gsm1 and Rds2 bind cooperatively in vitro to the FBP1 promoter. In contrast, at the HAP4 gene, Ert1 cooperates with Rds2 for DNA binding. Mutational analysis suggests that Gsm1/Rds2 and Ert1/Rds2 bind to short common DNA stretches, revealing a novel mode of binding for this class of factors. Two-point mutations in a HAP4 site convert it to a Gsm1 binding site. Thus, Rds2 controls binding of Gsm1 at many promoters by two different mechanisms: regulation of Gsm1 levels and increased DNA binding by formation of heterodimers., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Partitioning of active gene loci to the nuclear envelope (NE) is a mechanism by which organisms increase the speed of adaptation and metabolic robustness to fluctuating resources in the environment. In the yeast Saccharomyces cerevisiae, adaptation to nutrient depletion or other stresses, manifests as relocalization of active gene loci from nucleoplasm to the NE, resulting in more efficient transport and translation of mRNA. The mechanism by which this partitioning occurs remains a mystery. Here, we demonstrate that the yeast inositol depletion-responsive gene locus INO1 partitions to the nuclear envelope, driven by local histone acetylation-induced polymer-polymer phase separation from the nucleoplasmic phase. This demixing is consistent with recent evidence for chromatin phase separation by acetylation-mediated dissolution of multivalent histone association and fits a physical model where increased bending stiffness of acetylated chromatin polymer causes its phase separation from de-acetylated chromatin. Increased chromatin spring stiffness could explain nucleation of transcriptional machinery at active gene loci., (© 2023. The Author(s).)

Nuclear export of messenger RNAs (mRNAs) is intimately coupled to their synthesis. pre-mRNAs assemble into dynamic ribonucleoparticles as they are being transcribed, processed, and exported. The role of ubiquitylation in this process is increasingly recognized but, while a few E3 ligases have been shown to regulate nuclear export, evidence for deubiquitylases is currently lacking. Here we identified deubiquitylase Ubp15 as a regulator of nuclear export in Saccharomyces cerevisiae. Ubp15 interacts with both RNA polymerase II and the nuclear pore complex, and its deletion reverts the nuclear export defect of E3 ligase Rsp5 mutants. The deletion of UBP15 leads to hyper-ubiquitylation of the main nuclear export receptor Mex67 and affects its association with THO, a complex coupling transcription to mRNA processing and involved in the recruitment of mRNA export factors to nascent transcripts. Collectively, our data support a role for Ubp15 in coupling transcription to mRNA export., Competing Interests: FE, CJ, JC, FR No competing interests declared, (© 2020, Eyboulet et al.)

Genomic DNA is framed by additional layers of information, referred to as the epigenome. Epigenomic marks such as DNA methylation, histone modifications, and histone variants are concentrated on specific genomic sites, where they can both instruct and reflect gene expression. How this information is maintained, notably in the face of transcription, is not completely understood. Specifically, the extent to which modified histones themselves are retained through RNA polymerase II passage is unclear. Here, we show that several histone modifications are mislocalized when the transcription-coupled histone chaperones FACT or Spt6 are disrupted in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates nucleosome loss, which is partially compensated for by the increased activity of non-transcription-coupled histone chaperones. The random incorporation of transcription-evicted modified histones scrambles epigenomic information. Our work highlights the importance of local recycling of modified histones by FACT and Spt6 during transcription in the maintenance of the epigenomic landscape., (Copyright © 2019 The Author(s). Published by Elsevier Inc. All rights reserved.)

In Saccharomyces cerevisiae, transcription termination at protein-coding genes is coupled to the cleavage of the nascent transcript, whereas most non-coding RNA transcription relies on a cleavage-independent termination pathway involving Nrd1, Nab3, and Sen1 (NNS). Termination involves RNA polymerase II CTD phosphorylation, but a systematic analysis of the contribution of individual residues would improve our understanding of the role of the CTD in this process. Here we investigated the effect of mutating phosphorylation sites in the CTD on termination. We observed widespread termination defects at protein-coding genes in mutants for Ser2 or Thr4 but rare defects in Tyr1 mutants for this genes class. Instead, mutating Tyr1 led to widespread termination defects at non-coding genes terminating via NNS. Finally, we showed that Tyr1 is important for pausing in the 5' end of genes and that slowing down transcription suppresses termination defects. Our work highlights the importance of Tyr1-mediated pausing in NNS-dependent termination., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Recent experimental and computational work revealed that transcriptional terminators in Saccharomyces cerevisiae can terminate transcription coming from both directions. This mechanism helps budding yeast cope with the pervasive nature of transcription by limiting aberrant transcription from invading neighboring genes.

Transcription can be quite disruptive for chromatin so cells have evolved mechanisms to preserve chromatin integrity during transcription, thereby preventing the emergence of cryptic transcripts from spurious promoter sequences. How these transcripts are regulated and processed remains poorly characterized. Notably, very little is known about the termination of cryptic transcripts. Here, we used RNA-Seq to identify and characterize cryptic transcripts in Spt6 mutant cells (spt6-1004) in Saccharomyces cerevisiae. We found polyadenylated cryptic transcripts running both sense and antisense relative to genes in this mutant. Cryptic promoters were enriched for TATA boxes, suggesting that the underlying DNA sequence defines the location of cryptic promoters. While intragenic sense cryptic transcripts terminate at the terminator of the genes that host them, we found that antisense cryptic transcripts preferentially terminate near the 3΄-end of the upstream gene. This finding led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from both directions. We propose that S. cerevisiae has evolved this mechanism in order to prevent/attenuate spurious transcription from invading neighbouring genes, a feature that is particularly critical for organisms with small compact genomes., (© The Author(s) 2017. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Chromatin immunoprecipitation coupled to DNA microarrays (ChIP-chip) is widely used in the chromatin field, notably to map the position of histone variants or histone modifications along the genome. Often, the position and the occupancy of these epigenetic marks are to be compared between different experiments. It is now increasingly recognized that such cross-sample comparison is better done using externally added exogenous controls for normalization but no such method has been described for ChIP-chip. Here we describe a spiking normalization strategy that makes use of phiX174 phage DNA as a spiked control for normalization of ChIP-chip signals across different experiments.

Mediator is a highly conserved transcriptional coactivator organized into four modules, namely Tail, Middle, Head, and Kinase (CKM). Previous work suggests regulatory roles for Tail and CKM, but an integrated model for these activities is lacking. Here, we analyzed the genome-wide distribution of Mediator subunits in wild-type and mutant yeast cells in which RNA polymerase II promoter escape is blocked, allowing detection of transient Mediator forms. We found that although all modules are recruited to upstream activated regions (UAS), assembly of Mediator within the pre-initiation complex is accompanied by the release of CKM. Interestingly, our data show that CKM regulates Mediator-UAS interaction rather than Mediator-promoter association. In addition, although Tail is required for Mediator recruitment to UAS, Tailless Mediator nevertheless interacts with core promoters. Collectively, our data suggest that the essential function of Mediator is mediated by Head and Middle at core promoters, while Tail and CKM play regulatory roles., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Recognition of histone marks by reader modules is thought to be at the heart of epigenetic mechanisms. These protein domains are considered to function by targeting regulators to chromosomal loci carrying specific histone modifications. This is important for proper gene regulation as well as propagation of epigenetic information. The NuA4 acetyltransferase complex contains two of these reader modules, an H3K4me3-specific plant homeodomain (PHD) within the Yng2 subunit and an H3K36me2/3-specific chromodomain in the Eaf3 subunit. While each domain showed a close functional interaction with the respective histone mark that it recognizes, at the biochemical level, genetic level (as assessed with epistatic miniarray profile screens), and phenotypic level, cells with the combined loss of both readers showed greatly enhanced phenotypes. Chromatin immunoprecipitation coupled with next-generation sequencing experiments demonstrated that the Yng2 PHD specifically directs H4 acetylation near the transcription start site of highly expressed genes, while Eaf3 is important downstream on the body of the genes. Strikingly, the recruitment of the NuA4 complex to these loci was not significantly affected. Furthermore, RNA polymerase II occupancy was decreased only under conditions where both PHD and chromodomains were lost, generally in the second half of the gene coding regions. Altogether, these results argue that methylated histone reader modules in NuA4 are not responsible for its recruitment to the promoter or coding regions but, rather, are required to orient its acetyltransferase catalytic site to the methylated histone 3-bearing nucleosomes in the surrounding chromatin, cooperating to allow proper transition from transcription initiation to elongation., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

Histone variants are specialized histones which replace their canonical counterparts in specific nucleosomes. Together with histone post-translational modifications and DNA methylation, they contribute to the epigenome. Histone variants are incorporated at specific locations by the concerted action of histone chaperones and ATP-dependent chromatin remodelers. Recent studies have shown that the histone chaperone FACT plays key roles in preventing pervasive incorporation of two histone variants: H2A.Z and CenH3/CENP-A. In addition, Spt6, another histone chaperone, was also shown to be important for appropriate H2A.Z localization. FACT and Spt6 are both associated with elongating RNA polymerase II. Based on these two examples, we propose that the establishment and maintenance of histone variant genomic distributions depend on a transcription-coupled epigenome editing (or surveillance) function of histone chaperones., (© 2016 WILEY Periodicals, Inc.)

Proper modulation of promoter chromatin architecture is crucial for gene regulation in order to precisely and efficiently orchestrate various cellular activities. Previous studies have identified the stimulatory effect of the histone-modifying complex NuA4 on the incorporation of the histone variant H2A.Z (Htz1) at the PHO5 promoter (A. Auger, L. Galarneau, M. Altaf, A. Nourani, Y. Doyon, R. T. Utley, D. Cronier, S. Allard, and J. Côté, Mol Cell Biol 28:2257-2270, 2008, http://dx.doi.org/10.1128/MCB.01755-07). In vitro studies with a reconstituted system also indicated an intriguing cross talk between NuA4 and the H2A.Z-loading complex, SWR-C (M. Altaf, A. Auger, J. Monnet-Saksouk, J. Brodeur, S. Piquet, M. Cramet, N. Bouchard, N. Lacoste, R. T. Utley, L. Gaudreau, J. Côté, J Biol Chem 285:15966-15977, 2010, http://dx.doi.org/10.1074/jbc.M110.117069). In this work, we investigated the role of the NuA4 scaffold subunit Eaf1 in global gene expression and genome-wide incorporation of Htz1. We found that loss of Eaf1 affects Htz1 levels mostly at the promoters that are normally highly enriched in the histone variant. Analysis of eaf1 mutant cells by expression array unveiled a relationship between NuA4 and the gene network implicated in the purine biosynthesis pathway, as EAF1 deletion cripples induction of several ADE genes. NuA4 directly interacts with Bas1 activation domain, a key transcription factor of adenine genes. Chromatin immunoprecipitation (ChIP) experiments demonstrate that nucleosomes on the inactive ADE17 promoter are acetylated already by NuA4 and enriched in Htz1. Upon derepression, these poised nucleosomes respond rapidly to activate ADE gene expression in a mechanism likely reminiscent of the PHO5 promoter, leading to nucleosome disassembly. These detailed molecular events depict a specific case of cross talk between NuA4-dependent acetylation and incorporation of histone variant Htz1, presetting the chromatin structure over ADE promoters for subsequent chromatin remodeling and activated transcription., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Spt6 is a multifunctional histone chaperone involved in the maintenance of chromatin structure during elongation by RNA polymerase II (Pol II). Spt6 has a tandem SH2 (tSH2) domain within its C terminus that recognizes Pol II C-terminal domain (CTD) peptides phosphorylated on Ser2, Ser5, or Try1 in vitro. Deleting the tSH2 domain, however, only has a partial effect on Spt6 occupancy in vivo, suggesting that more complex mechanisms are involved in the Spt6 recruitment. Our results show that the Ser2 kinases Bur1 and Ctk1, but not the Ser5 kinase Kin28, cooperate in recruiting Spt6, genome-wide. Interestingly, the Ser2 kinases promote the association of Spt6 in early transcribed regions and not toward the 3' ends of genes, where phosphorylated Ser2 reaches its maximum level. In addition, our results uncover an unexpected role for histone deacetylases (Rpd3 and Hos2) in promoting Spt6 interaction with elongating Pol II. Finally, our data suggest that phosphorylation of the Pol II CTD on Tyr1 promotes the association of Spt6 with the 3' ends of transcribed genes, independently of Ser2 phosphorylation. Collectively, our results show that a complex network of interactions, involving the Spt6 tSH2 domain, CTD phosphorylation, and histone deacetylases, coordinate the recruitment of Spt6 to transcribed genes in vivo., (Copyright © 2014, American Society for Microbiology. All Rights Reserved.)

In the yeast Saccharomyces cerevisiae, fermentation is the major pathway for energy production, even under aerobic conditions. However, when glucose becomes scarce, ethanol produced during fermentation is used as a carbon source, requiring a shift to respiration. This adaptation results in massive reprogramming of gene expression. Increased expression of genes for gluconeogenesis and the glyoxylate cycle is observed upon a shift to ethanol and, conversely, expression of some fermentation genes is reduced. The zinc cluster proteins Cat8, Sip4, and Rds2, as well as Adr1, have been shown to mediate this reprogramming of gene expression. In this study, we have characterized the gene YBR239C encoding a putative zinc cluster protein and it was named ERT1 (ethanol regulated transcription factor 1). ChIP-chip analysis showed that Ert1 binds to a limited number of targets in the presence of glucose. The strongest enrichment was observed at the promoter of PCK1 encoding an important gluconeogenic enzyme. With ethanol as the carbon source, enrichment was observed with many additional genes involved in gluconeogenesis and mitochondrial function. Use of lacZ reporters and quantitative RT-PCR analyses demonstrated that Ert1 regulates expression of its target genes in a manner that is highly redundant with other regulators of gluconeogenesis. Interestingly, in the presence of ethanol, Ert1 is a repressor of PDC1 encoding an important enzyme for fermentation. We also show that Ert1 binds directly to the PCK1 and PDC1 promoters. In summary, Ert1 is a novel factor involved in the regulation of gluconeogenesis as well as a key fermentation gene., (Copyright © 2014 by the Genetics Society of America.)

Mediator is an essential, broadly used eukaryotic transcriptional coactivator. How and what Mediator communicates from activators to RNA polymerase II (RNAPII) remains an open question. Here we performed genome-wide location profiling of Saccharomyces cerevisiae Mediator subunits. Mediator is not found at core promoters but rather occupies the upstream activating sequence, upstream of the pre-initiation complex. In the absence of Kin28 (CDK7) kinase activity or in cells in which the RNAPII C-terminal domain is mutated to replace Ser5 with alanine, however, Mediator accumulates at core promoters together with RNAPII. We propose that Mediator is released quickly from promoters after phosphorylation of Ser5 by Kin28 (CDK7), which also allows for RNAPII to escape from the promoter.

Upon glucose depletion, a massive reprogramming of gene expression occurs in the yeast Saccharomyces cerevisiae for the use of alternate carbon sources such as the nonfermentable compounds ethanol and glycerol. This process is mediated by the master kinase Snf1 that controls the activity of various targets including the transcriptional regulators Cat8, Sip4 and Adr1. We have recently identified Rds2 as an additional player in this pathway. Here, we have performed genome-wide location analysis of Rds2 in cells grown in the presence of glycerol. We show that Rds2 binds to promoters of genes involved in gluconeogenesis, the glyoxylate shunt, and the TCA cycle as well as some genes encoding mitochondrial components or some involved in the stress response. Interestingly, we also detected Rds2 at the promoters of SIP4, ADR1 and HAP4 which encodes the limiting subunit of the Hap2/3/4/5 complex, a regulator of respiration. Strikingly, we observed an important overlap between the targets of Rds2 and Adr1. Finally, we provide a model to account for the complex interplay among these transcriptional regulators., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Spt6 is a highly conserved factor required for normal transcription and chromatin structure. To gain new insights into the roles of Spt6, we measured nucleosome occupancy along Saccharomyces cerevisiae chromosome III in an spt6 mutant. We found that the level of nucleosomes is greatly reduced across some, but not all, coding regions in an spt6 mutant, with nucleosome loss preferentially occurring over highly transcribed genes. This result provides strong support for recent studies that have suggested that transcription at low levels does not displace nucleosomes, while transcription at high levels does, and adds the idea that Spt6 is required for restoration of nucleosomes at the highly transcribed genes. Unexpectedly, our studies have also suggested that the spt6 effects on nucleosome levels across coding regions do not cause the spt6 effects on mRNA levels, suggesting that the role of Spt6 across coding regions is separate from its role in transcriptional regulation. In the case of the CHA1 gene, regulation by Spt6 likely occurs by controlling the position of the +1 nucleosome. These results, along with previous studies, suggest that Spt6 regulates transcription by controlling chromatin structure over regulatory regions, and its effects on nucleosome levels over coding regions likely serve an independent function.

Rapamycin is an anticancer agent and immunosuppressant that acts by inhibiting the TOR signaling pathway. In yeast, rapamycin mediates a profound transcriptional response for which the RRD1 gene is required. To further investigate this connection, we performed genome-wide location analysis of RNA polymerase II (RNAPII) and Rrd1 in response to rapamycin and found that Rrd1 colocalizes with RNAPII on actively transcribed genes and that both are recruited to rapamycin responsive genes. Strikingly, when Rrd1 is lacking, RNAPII remains inappropriately associated to ribosomal genes and fails to be recruited to rapamycin responsive genes. This occurs independently of TATA box binding protein recruitment but involves the modulation of the phosphorylation status of RNAPII CTD by Rrd1. Further, we demonstrate that Rrd1 is also involved in various other transcriptional stress responses besides rapamycin. We propose that Rrd1 is a novel transcription elongation factor that fine-tunes the transcriptional stress response of RNAPII.

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication-fork stalling results in phosphorylation of histone H2A yielding gamma-H2A (yeast gamma-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Here, we describe the genome-wide location analysis of gamma-H2A as a strategy to identify loci prone to engaging the Mec1 and Tel1 pathways. Notably, gamma-H2A enrichment overlaps with loci prone to replication-fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for gamma-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of gamma-H2A at these loci. Finally, our work indicates that high-resolution mapping of gamma-H2AX is a fruitful route to map fragile sites in eukaryotic genomes.

Saccharomyces cerevisiae preferentially uses glucose as a carbon source, but following its depletion, it can utilize a wide variety of other carbons including nonfermentable compounds such as ethanol. A shift to a nonfermentable carbon source results in massive reprogramming of gene expression including genes involved in gluconeogenesis, the glyoxylate cycle, and the tricarboxylic acid cycle. This review is aimed at describing the recent progress made toward understanding the mechanism of transcriptional regulation of genes responsible for utilization of nonfermentable carbon sources. A central player for the use of nonfermentable carbons is the Snf1 kinase, which becomes activated under low glucose levels. Snf1 phosphorylates various targets including the transcriptional repressor Mig1, resulting in its inactivation allowing derepression of gene expression. For example, the expression of CAT8, encoding a member of the zinc cluster family of transcriptional regulators, is then no longer repressed by Mig1. Cat8 becomes activated through phosphorylation by Snf1, allowing upregulation of the zinc cluster gene SIP4. These regulators control the expression of various genes including those involved in gluconeogenesis. Recent data show that another zinc cluster protein, Rds2, plays a key role in regulating genes involved in gluconeogenesis and the glyoxylate pathway. Finally, the role of additional regulators such as Adr1, Ert1, Oaf1, and Pip2 is also discussed.

Transcription termination of messenger RNA (mRNA) is normally achieved by polyadenylation followed by Rat1p-dependent 5'-3' exoribonuleolytic degradation of the downstream transcript. Here we show that the yeast ortholog of the dsRNA-specific ribonuclease III (Rnt1p) may trigger Rat1p-dependent termination of RNA transcripts that fail to terminate near polyadenylation signals. Rnt1p cleavage sites were found downstream of several genes, and the deletion of RNT1 resulted in transcription readthrough. Inactivation of Rat1p impaired Rnt1p-dependent termination and resulted in the accumulation of 3' end cleavage products. These results support a model for transcription termination in which cotranscriptional cleavage by Rnt1p provides access for exoribonucleases in the absence of polyadenylation signals.

Chromatin immunoprecipitation coupled to DNA microarray has become a widely used method to study transcription factors and chromatin structure. Here, we provide a detailed protocol for the localization of the variant histone Htz1 in the S. cerevisiae genome. This protocol can easily be adapted to fit other purposes such as profiling histone modifications.

In Saccharomyces cerevisiae, RDS2 encodes a zinc cluster transcription factor with unknown function. Here, we unravel a key function of Rds2 in gluconeogenesis using chromatin immunoprecipitation-chip technology. While we observed that Rds2 binds to only a few promoters in glucose-containing medium, it binds many additional genes when the medium is shifted to ethanol, a nonfermentable carbon source. Interestingly, many of these genes are involved in gluconeogenesis, the tricarboxylic acid cycle, and the glyoxylate cycle. Importantly, we show that Rds2 has a dual function: it directly activates the expression of gluconeogenic structural genes while it represses the expression of negative regulators of this pathway. We also show that the purified DNA binding domain of Rds2 binds in vitro to carbon source response elements found in the promoters of target genes. Finally, we show that upon a shift to ethanol, Rds2 activation is correlated with its hyperphosphorylation by the Snf1 kinase. In summary, we have characterized Rds2 as a novel major regulator of gluconeogenesis.

In yeast, histone H3/H4 exchange independent of replication is poorly understood. Here, we analyzed the deposition of histone H3 molecules, synthesized during G1, using a high-density microarray histone exchange assay. While we found that H3 exchange in coding regions requires high levels of transcription, promoters exchange H3 molecules in the absence of transcription. In inactive promoters, H3 is deposited predominantly in well-positioned nucleosomes surrounding nucleosome-free regions, indicating that some nucleosomes in promoters are dynamic. This could facilitate induction of repressed genes. Importantly, we show that histone H3 K56 acetylation, a replication-associated mark, is also present in replication-independent newly assembled nucleosomes and correlates perfectly with the deposition of new H3. Finally, we found that transcription-dependent incorporation of H3 at promoters is highly dependent on Asf1. Taken together, our data underline the dynamic nature of replication-independent nucleosome assembly/disassembly, specify a link to transcription, and implicate Asf1 and H3 K56 acetylation.

In Saccharomyces cerevisiae, zinc cluster protein Pdr1 can form homodimers as well as heterodimers with Pdr3 and Stb5, suggesting that different combinations of these proteins may regulate the expression of different genes. To gain insight into the interplay among these regulators, we performed genome-wide location analysis (chromatin immunoprecipitation with hybridization to DNA microarrays) and gene expression profiling. Unexpectedly, we observed that Stb5 shares only a few target genes with Pdr1 or Pdr3 in rich medium. Interestingly, upon oxidative stress, Stb5 binds and regulates the expression of most genes of the pentose phosphate pathway as well as of genes involved in the production of NADPH, a metabolite required for oxidative stress resistance. Importantly, deletion of STB5 results in sensitivity to diamide and hydrogen peroxide. Our data suggest that Stb5 acts both as an activator and as a repressor in the presence of oxidative stress. Furthermore, we show that Stb5 activation is not mediated by known regulators of the oxidative stress response. Integrity of the pentose phosphate pathway is required for the activation of Stb5 target genes but is not necessary for the increased DNA binding of Stb5 in the presence of diamide. These data suggest that Stb5 is a key player in the control of NADPH production for resistance to oxidative stress.

H2A.Z is an evolutionary conserved histone variant involved in transcriptional regulation, antisilencing, silencing, and genome stability. The mechanism(s) by which H2A.Z regulates these various biological functions remains poorly defined, in part due to the lack of knowledge regarding its physical location along chromosomes and the bearing it has in regulating chromatin structure. Here we mapped H2A.Z across the yeast genome at an approximately 300-bp resolution, using chromatin immunoprecipitation combined with tiling microarrays. We have identified 4,862 small regions--typically one or two nucleosomes wide--decorated with H2A.Z. Those "Z loci" are predominantly found within specific nucleosomes in the promoter of inactive genes all across the genome. Furthermore, we have shown that H2A.Z can regulate nucleosome positioning at the GAL1 promoter. Within HZAD domains, the regions where H2A.Z shows an antisilencing function, H2A.Z is localized in a wider pattern, suggesting that the variant histone regulates a silencing and transcriptional activation via different mechanisms. Our data suggest that the incorporation of H2A.Z into specific promoter-bound nucleosomes configures chromatin structure to poise genes for transcriptional activation. The relevance of these findings to higher eukaryotes is discussed.

Chromatin regulators play fundamental roles in the regulation of gene expression and chromosome maintenance, but the regions of the genome where most of these regulators function has not been established. We explored the genome-wide occupancy of four different chromatin regulators encoded in Saccharomyces cerevisiae. The results reveal that the histone acetyltransferases Gcn5 and Esa1 are both generally recruited to the promoters of active protein-coding genes. In contrast, the histone deacetylases Hst1 and Rpd3 are recruited to specific sets of genes associated with distinct cellular functions. Our results provide new insights into the association of histone acetyltransferases and histone deacetylases with the yeast genome, and together with previous studies, suggest how these chromatin regulators are recruited to specific regions of the genome.

We have determined how most of the transcriptional regulators encoded in the eukaryote Saccharomyces cerevisiae associate with genes across the genome in living cells. Just as maps of metabolic networks describe the potential pathways that may be used by a cell to accomplish metabolic processes, this network of regulator-gene interactions describes potential pathways yeast cells can use to regulate global gene expression programs. We use this information to identify network motifs, the simplest units of network architecture, and demonstrate that an automated process can use motifs to assemble a transcriptional regulatory network structure. Our results reveal that eukaryotic cellular functions are highly connected through networks of transcriptional regulators that regulate other transcriptional regulators.

The Peer Review file and Supplementary Information file have been corrected. Correction to: I Nature Communications i https://doi.org/10.1038/s41467-023-36391-6, published online 28 February 2023 The original version of this article contained formatting errors in the Peer Review file and the Supplementary Information. [Extracted from the article]

Phosphorylation of histone H2AX is an early response to DNA damage in eukaryotes. In Saccharomyces cerevisiae, DNA damage or replication-fork stalling results in phosphorylation of histone H2A yielding γ-H2A (yeast γ-H2AX) in a Mec1 (ATR)- and Tel1 (ATM)-dependent manner. Here, we describe the genome-wide location analysis of γ-H2A as a strategy to identify loci prone to engaging the Mec1 and Tel1 pathways. Notably, γ-H2A enrichment overlaps with loci prone to replication-fork stalling and is caused by the action of Mec1 and Tel1, indicating that these loci are prone to breakage. Moreover, about half the sites enriched for γ-H2A map to repressed protein-coding genes, and histone deacetylases are necessary for formation of γ-H2A at these loci. Finally, our work indicates that high-resolution mapping of γ-H2AX is a fruitful route to map fragile sites in eukaryotic genomes. [ABSTRACT FROM AUTHOR]

Summary: MNase-Seq and ChIP-Seq have evolved as popular techniques to study chromatin and histone modification. Although many tools have been developed to identify enriched regions, software tools for nucleosome positioning are still limited. We introduce a flexible and powerful open-source R package, PING 2.0, for nucleosome positioning using MNase-Seq data or MNase– or sonicated– ChIP-Seq data combined with either single-end or paired-end sequencing. PING uses a model-based approach, which enables nucleosome predictions even in the presence of low read counts. We illustrate PING using two paired-end datasets from Saccharomyces cerevisiae and compare its performance with nucleR and ChIPseqR.Availability: PING 2.0 is available from the Bioconductor website at http://bioconductor.org. It can run on Linux, Mac and Windows.Contact: rgottard@fhcrc.orgSupplementary Information: Supplementary material is available at Bioinformatics online. [ABSTRACT FROM AUTHOR]

The histone chaperone FACT occupies transcribed regions where it plays prominent roles in maintaining chromatin integrity and preserving epigenetic information. How it is targeted to transcribed regions, however, remains unclear. Proposed models include docking on the RNA polymerase II (RNAPII) C-terminal domain (CTD), recruitment by elongation factors, recognition of modified histone tails, and binding partially disassembled nucleosomes. Here, we systematically test these and other scenarios in Saccharomyces cerevisiae and find that FACT binds transcribed chromatin, not RNAPII. Through a combination of high-resolution genome-wide mapping, single-molecule tracking, and mathematical modeling, we propose that FACT recognizes the +1 nucleosome, as it is partially unwrapped by the engaging RNAPII, and spreads to downstream nucleosomes aided by the chromatin remodeler Chd1. Our work clarifies how FACT interacts with genes, suggests a processive mechanism for FACT function, and provides a framework to further dissect the molecular mechanisms of transcription-coupled histone chaperoning. [Display omitted] • High-resolution mapping and single-molecule tracking of FACT in yeast • FACT binds partially unwrapped nucleosomes in transcribed genes, not RNAPII • FACT distribution along genes requires Chd1 • Processive mechanism for FACT function Preserving nucleosomes during transcription elongation requires histone chaperone FACT and chromatin remodeler Chd1, but their interplay is unclear. Here, Jeronimo et al. show that nucleosomal DNA unraveling by RNAPII drives the recruitment of FACT, while the remodeling activity of Chd1 promotes FACT movement along genes, enabling a processive mechanism. [ABSTRACT FROM AUTHOR]