Quiescence in Saccharomyces cerevisiae.

Most cells live in environments that are permissive for proliferation only a small fraction of the time. Entering quiescence enables cells to survive long periods of nondivision and reenter the cell cycle when signaled to do so. Here, we describe what is known about the molecular basis for quiescence in Saccharomyces cerevisiae, with emphasis on the progress made in the last decade. Quiescence is triggered by depletion of an essential nutrient. It begins well before nutrient exhaustion, and there is extensive crosstalk between signaling pathways to ensure that all proliferation-specific activities are stopped when any one essential nutrient is limiting. Every aspect of gene expression is modified to redirect and conserve resources. Chromatin structure and composition change on a global scale, from histone modifications to three-dimensional chromatin structure. Thousands of proteins and RNAs aggregate, forming unique structures with unique fates, and the cytoplasm transitions to a glass-like state.

Condensin-Dependent Chromatin Compaction Represses Transcription Globally during Quiescence.

Quiescence is a stress-resistant state in which cells reversibly exit the cell cycle and suspend most processes. Quiescence is essential for stem cell maintenance, and its misregulation is implicated in tumor formation. One of the hallmarks of quiescent cells is highly condensed chromatin. Because condensed chromatin often correlates with transcriptional silencing, it has been hypothesized that chromatin compaction represses transcription during quiescence. However, the technology to test this model by determining chromatin structure within cells at gene resolution has not previously been available. Here, we use Micro-C XL to map chromatin contacts at single-nucleosome resolution genome-wide in quiescent Saccharomyces cerevisiae cells. We describe chromatin domains on the order of 10-60 kilobases that, only in quiescent cells, are formed by condensin-mediated loops. Condensin depletion prevents the compaction of chromatin within domains and leads to widespread transcriptional de-repression. Finally, we demonstrate that condensin-dependent chromatin compaction is conserved in quiescent human fibroblasts.

Post-transcriptional regulation shapes the transcriptome of quiescent budding yeast.

To facilitate long-term survival, cells must exit the cell cycle and enter quiescence, a reversible non-replicative state. Budding yeast cells reprogram their gene expression during quiescence entry to silence transcription, but how the nascent transcriptome changes in quiescence is unknown. By investigating the nascent transcriptome, we identified over a thousand noncoding RNAs in quiescent and G1 yeast cells, and found noncoding transcription represented a larger portion of the quiescent transcriptome than in G1. Additionally, both mRNA and ncRNA are subject to increased post-transcriptional regulation in quiescence compared to G1. We found that, in quiescence, the nuclear exosome-NNS pathway suppresses over one thousand mRNAs, in addition to canonical noncoding RNAs. RNA sequencing through quiescent entry revealed two distinct time points at which the nuclear exosome controls the abundance of mRNAs involved in protein production, cellular organization, and metabolism, thereby facilitating efficient quiescence entry. Our work identified a previously unknown key biological role for the nuclear exosome-NNS pathway in mRNA regulation and uncovered a novel layer of gene-expression control in quiescence.

Opening windows to the genome.

Recent mapping of nucleosome positioning has added a new dimension to the study of transcriptional regulation. Hartley and Madhani (2009) now demonstrate the power of this approach and show that a chromatin regulator alters nucleosome positioning in the promoters of a large number of genes in the budding yeast Saccharomyces cerevisiae.

Novel Classes and Evolutionary Turnover of Histone H2B Variants in the Mammalian Germline.

Histones and their posttranslational modifications facilitate diverse chromatin functions in eukaryotes. Core histones (H2A, H2B, H3, and H4) package genomes after DNA replication. In contrast, variant histones promote specialized chromatin functions, including DNA repair, genome stability, and epigenetic inheritance. Previous studies have identified only a few H2B variants in animals; their roles and evolutionary origins remain largely unknown. Here, using phylogenomic analyses, we reveal the presence of five H2B variants broadly present in mammalian genomes. Three of these variants have been previously described: H2B.1, H2B.L (also called subH2B), and H2B.W. In addition, we identify and describe two new variants: H2B.K and H2B.N. Four of these variants originated in mammals, whereas H2B.K arose prior to the last common ancestor of bony vertebrates. We find that though H2B variants are subject to high gene turnover, most are broadly retained in mammals, including humans. Despite an overall signature of purifying selection, H2B variants evolve more rapidly than core H2B with considerable divergence in sequence and length. All five H2B variants are expressed in the germline. H2B.K and H2B.N are predominantly expressed in oocytes, an atypical expression site for mammalian histone variants. Our findings suggest that H2B variants likely encode potentially redundant but vital functions via unusual chromatin packaging or nonchromatin functions in mammalian germline cells. Our discovery of novel histone variants highlights the advantages of comprehensive phylogenomic analyses and provides unique opportunities to study how innovations in chromatin function evolve.

Inhibition of transcription leads to rewiring of locus-specific chromatin proteomes.

Transcription of a chromatin template involves the concerted interaction of many different proteins and protein complexes. Analyses of specific factors showed that these interactions change during stress and upon developmental switches. However, how the binding of multiple factors at any given locus is coordinated has been technically challenging to investigate. Here we used Epi-Decoder in yeast to systematically decode, at one transcribed locus, the chromatin binding changes of hundreds of proteins in parallel upon perturbation of transcription. By taking advantage of improved Epi-Decoder libraries, we observed broad rewiring of local chromatin proteomes following chemical inhibition of RNA polymerase. Rapid reduction of RNA polymerase II binding was accompanied by reduced binding of many other core transcription proteins and gain of chromatin remodelers. In quiescent cells, where strong transcriptional repression is induced by physiological signals, eviction of the core transcriptional machinery was accompanied by the appearance of quiescent cell-specific repressors and rewiring of the interactions of protein-folding factors and metabolic enzymes. These results show that Epi-Decoder provides a powerful strategy for capturing the temporal binding dynamics of multiple chromatin proteins under varying conditions and cell states. The systematic and comprehensive delineation of dynamic local chromatin proteomes will greatly aid in uncovering protein-protein relationships and protein functions at the chromatin template.

Global Promoter Targeting of a Conserved Lysine Deacetylase for Transcriptional Shutoff during Quiescence Entry.

Quiescence is a conserved cell-cycle state characterized by cell-cycle arrest, increased stress resistance, enhanced longevity, and decreased transcriptional, translational, and metabolic output. Although quiescence plays essential roles in cell survival and normal differentiation, the molecular mechanisms leading to this state are not well understood. Here, we determined changes in the transcriptome and chromatin structure of S. cerevisiae upon quiescence entry. Our analyses revealed transcriptional shutoff that is far more robust than previously believed and an unprecedented global chromatin transition, which are tightly correlated. These changes require Rpd3 lysine deacetylase targeting to at least half of gene promoters via quiescence-specific transcription factors including Xbp1 and Stb3. Deletion of RPD3 prevents cells from establishing transcriptional quiescence, leading to defects in quiescence entry and shortening of chronological lifespan. Our results define a molecular mechanism for global reprogramming of transcriptome and chromatin structure for quiescence driven by a highly conserved chromatin regulator.

ATP-dependent chromatin remodeling shapes the long noncoding RNA landscape.

Long noncoding RNAs (lncRNAs) are pervasively transcribed across eukaryotic genomes, but functions of only a very small subset of them have been demonstrated. This has led to active debate about whether many of them have any biological functions. In addition, very few regulators of lncRNAs have been identified. We developed a novel genetic screen using reconstituted RNAi in Saccharomyces cerevisiae and systematically identified a large number of putative lncRNA repressors. Among them, we found that four highly conserved chromatin remodeling factors are global lncRNA repressors that play major roles in shaping the eukaryotic lncRNA transcriptome. Importantly, we identified >250 antisense lncRNAs (CRRATs [chromatin remodeling-repressed antisense transcripts]) whose repression by these chromatin remodeling factors is required for the maintenance of normal levels of overlapping mRNA transcripts. Our results strongly suggest that regulation of mRNA through repression of antisense lncRNAs is far more broadly used than previously appreciated.

DNA looping facilitates targeting of a chromatin remodeling enzyme.

ATP-dependent chromatin remodeling enzymes are highly abundant and play pivotal roles regulating DNA-dependent processes. The mechanisms by which they are targeted to specific loci have not been well understood on a genome-wide scale. Here, we present evidence that a major targeting mechanism for the Isw2 chromatin remodeling enzyme to specific genomic loci is through sequence-specific transcription factor (TF)-dependent recruitment. Unexpectedly, Isw2 is recruited in a TF-dependent fashion to a large number of loci without TF binding sites. Using the 3C assay, we show that Isw2 can be targeted by Ume6- and TFIIB-dependent DNA looping. These results identify DNA looping as a mechanism for the recruitment of a chromatin remodeling enzyme and define a function for DNA looping. We also present evidence suggesting that Ume6-dependent DNA looping is involved in chromatin remodeling and transcriptional repression, revealing a mechanism by which the three-dimensional folding of chromatin affects DNA-dependent processes.

ATR-like kinase Mec1 facilitates both chromatin accessibility at DNA replication forks and replication fork progression during replication stress.

Faithful DNA replication is essential for normal cell division and differentiation. In eukaryotic cells, DNA replication takes place on chromatin. This poses the critical question as to how DNA replication can progress through chromatin, which is inhibitory to all DNA-dependent processes. Here, we developed a novel genome-wide method to measure chromatin accessibility to micrococcal nuclease (MNase) that is normalized for nucleosome density, the NCAM (normalized chromatin accessibility to MNase) assay. This method enabled us to discover that chromatin accessibility increases specifically at and ahead of DNA replication forks in normal S phase and during replication stress. We further found that Mec1, a key regulatory ATR-like kinase in the S-phase checkpoint, is required for both normal chromatin accessibility around replication forks and replication fork rate during replication stress, revealing novel functions for the kinase in replication stress response. These results suggest a possibility that Mec1 may facilitate DNA replication fork progression during replication stress by increasing chromatin accessibility around replication forks.

Nucleosome occupancy as a novel chromatin parameter for replication origin functions.

Eukaryotic DNA replication initiates from multiple discrete sites in the genome, termed origins of replication (origins). Prior to S phase, multiple origins are poised to initiate replication by recruitment of the pre-replicative complex (pre-RC). For proper replication to occur, origin activation must be tightly regulated. At the population level, each origin has a distinct firing time and frequency of activation within S phase. Many studies have shown that chromatin can strongly influence initiation of DNA replication. However, the chromatin parameters that affect properties of origins have not been thoroughly established. We found that nucleosome occupancy in G1 varies greatly around origins across the S. cerevisiae genome, and nucleosome occupancy around origins significantly correlates with the activation time and efficiency of origins, as well as pre-RC formation. We further demonstrate that nucleosome occupancy around origins in G1 is established during transition from G2/M to G1 in a pre-RC-dependent manner. Importantly, the diminished cell-cycle changes in nucleosome occupancy around origins in the orc1-161 mutant are associated with an abnormal global origin usage profile, suggesting that proper establishment of nucleosome occupancy around origins is a critical step for regulation of global origin activities. Our work thus establishes nucleosome occupancy as a novel and key chromatin parameter for proper origin regulation.

Unraveling quiescence-specific repressive chromatin domains.

Quiescence is a highly conserved inactive life stage in which the cell reversibly exits the cell cycle in response to external cues. Quiescence is essential for diverse processes such as the maintenance of adult stem cell stores, stress resistance, and longevity, and its misregulation has been implicated in cancer. Although the non-cycling nature of quiescent cells has made obtaining sufficient quantities of quiescent cells for study difficult, the development of a Saccharomyces cerevisiae model of quiescence has recently enabled detailed investigation into mechanisms underlying the quiescent state. Like their metazoan counterparts, quiescent budding yeast exhibit widespread transcriptional silencing and dramatic chromatin condensation. We have recently found that the structural maintenance of chromosomes (SMC) complex condensin binds throughout the quiescent budding yeast genome and induces the formation of large chromatin loop domains. In the absence of condensin, quiescent cell chromatin is decondensed and transcription is de-repressed. Here, we briefly discuss our findings in the larger context of the genome organization field.

Sequence-targeted nucleosome sliding in vivo by a hybrid Chd1 chromatin remodeler.

ATP-dependent chromatin remodelers regulate chromatin dynamics by modifying nucleosome positions and occupancy. DNA-dependent processes such as replication and transcription rely on chromatin to faithfully regulate DNA accessibility, yet how chromatin remodelers achieve well-defined nucleosome positioning in vivo is poorly understood. Here, we report a simple method for site-specifically altering nucleosome positions in live cells. By fusing the Chd1 remodeler to the DNA binding domain of the Saccharomyces cerevisiae Ume6 repressor, we have engineered a fusion remodeler that selectively positions nucleosomes on top of adjacent Ume6 binding motifs in a highly predictable and reproducible manner. Positioning of nucleosomes by the fusion remodeler recapitulates closed chromatin structure at Ume6-sensitive genes analogous to the endogenous Isw2 remodeler. Strikingly, highly precise positioning of single founder nucleosomes by either chimeric Chd1-Ume6 or endogenous Isw2 shifts phased chromatin arrays in cooperation with endogenous chromatin remodelers. Our results demonstrate feasibility of engineering precise nucleosome rearrangements through sequence-targeted chromatin remodeling and provide insight into targeted action and cooperation of endogenous chromatin remodelers in vivo.

ISWI and CHD chromatin remodelers bind promoters but act in gene bodies.

ATP-dependent nucleosome remodelers influence genetic processes by altering nucleosome occupancy, positioning, and composition. In vitro, Saccharomyces cerevisiae ISWI and CHD remodelers require ∼30-85 bp of extranucleosomal DNA to reposition nucleosomes, but linker DNA in S. cerevisiae averages <20 bp. To address this discrepancy between in vitro and in vivo observations, we have mapped the genomic distributions of the yeast Isw1, Isw2, and Chd1 remodelers at base-pair resolution on native chromatin. Although these remodelers act in gene bodies, we find that they are also highly enriched at nucleosome-depleted regions (NDRs), where they bind to extended regions of DNA adjacent to particular transcription factors. Surprisingly, catalytically inactive remodelers show similar binding patterns. We find that remodeler occupancy at NDRs and gene bodies is associated with nucleosome turnover and transcriptional elongation rate, suggesting that remodelers act on regions of transient nucleosome unwrapping or depletion within gene bodies subsequent to transcriptional elongation.

Initial acidic media promotes quiescence entry in Saccharomyces cerevisiae.

Quiescence is a conserved cellular state wherein cells cease proliferation and remain poised to re-enter the cell cycle when conditions are appropriate. Budding yeast is a powerful model for studying cellular quiescence. In this work, we demonstrate that the pH of the YPD media strongly affects quiescence entry efficiency in Saccharomyces cerevisiae. Adjusting the initial media pH to 5.5 significantly improves quiescence entry efficiency compared to unadjusted YPD media. Thermotolerance of the produced quiescence yeast are similar, suggesting the media pH influences the quantity of quiescent cells more than quality of quiescence reached.

Chromatin remodelling at promoters suppresses antisense transcription.

Chromatin allows the eukaryotic cell to package its DNA efficiently. To understand how chromatin structure is controlled across the Saccharomyces cerevisiae genome, we have investigated the role of the ATP-dependent chromatin remodelling complex Isw2 in positioning nucleosomes. We find that Isw2 functions adjacent to promoter regions where it repositions nucleosomes at the interface between genic and intergenic sequences. Nucleosome repositioning by Isw2 is directional and results in increased nucleosome occupancy of the intergenic region. Loss of Isw2 activity leads to inappropriate transcription, resulting in the generation of both coding and noncoding transcripts. Here we show that Isw2 repositions nucleosomes to enforce directionality on transcription by preventing transcription initiation from cryptic sites. Our analyses reveal how chromatin is organized on a global scale and advance our understanding of how transcription is regulated.

Acidic media promotes quiescence entry in Saccharomyces cerevisiae.

Quiescence is a conserved cellular state wherein cells cease proliferation and remain poised to re-enter the cell cycle when conditions are appropriate. Budding yeast is a powerful model for studying cellular quiescence. In this work, we demonstrate that the pH of the YPD media strongly affects quiescence entry efficiency in Saccharomyces cerevisiae. Adjusting media pH to 5.5 significantly improves quiescence entry efficiency compared to unadjusted YPD media. Thermotolerance of the produced quiescence yeast are similar, suggesting the media pH influences the quantity of quiescent cells more than quality of quiescence reached.

Antagonistic forces that position nucleosomes in vivo.

ATP-dependent chromatin remodeling complexes are implicated in many areas of chromosome biology. However, the physiological role of many of these enzymes is still unclear. In budding yeast, the Isw2 complex slides nucleosomes along DNA. By analyzing the native chromatin structure of Isw2 targets, we have found that nucleosomes adopt default, DNA-directed positions when ISW2 is deleted. We provide evidence that Isw2 targets contain DNA sequences that are inhibitory to nucleosome formation and that these sequences facilitate the formation of nuclease-accessible open chromatin in the absence of Isw2. Our data show that the biological function of Isw2 is to position nucleosomes onto unfavorable DNA. These results reveal that antagonistic forces of Isw2 and the DNA sequence can control nucleosome positioning and genomic access in vivo.

RSC primes the quiescent genome for hypertranscription upon cell-cycle re-entry.

Quiescence is a reversible G0 state essential for differentiation, regeneration, stem-cell renewal, and immune cell activation. Necessary for long-term survival, quiescent chromatin is compact, hypoacetylated, and transcriptionally inactive. How transcription activates upon cell-cycle re-entry is undefined. Here we report robust, widespread transcription within the first minutes of quiescence exit. During quiescence, the chromatin-remodeling enzyme RSC was already bound to the genes induced upon quiescence exit. RSC depletion caused severe quiescence exit defects: a global decrease in RNA polymerase II (Pol II) loading, Pol II accumulation at transcription start sites, initiation from ectopic upstream loci, and aberrant antisense transcription. These phenomena were due to a combination of highly robust Pol II transcription and severe chromatin defects in the promoter regions and gene bodies. Together, these results uncovered multiple mechanisms by which RSC facilitates initiation and maintenance of large-scale, rapid gene expression despite a globally repressive chromatin state.