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.

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.

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.

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.

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.

Local chromatin fiber folding represses transcription and loop extrusion in quiescent cells.

A longstanding hypothesis is that chromatin fiber folding mediated by interactions between nearby nucleosomes represses transcription. However, it has been difficult to determine the relationship between local chromatin fiber compaction and transcription in cells. Further, global changes in fiber diameters have not been observed, even between interphase and mitotic chromosomes. We show that an increase in the range of local inter-nucleosomal contacts in quiescent yeast drives the compaction of chromatin fibers genome-wide. Unlike actively dividing cells, inter-nucleosomal interactions in quiescent cells require a basic patch in the histone H4 tail. This quiescence-specific fiber folding globally represses transcription and inhibits chromatin loop extrusion by condensin. These results reveal that global changes in chromatin fiber compaction can occur during cell state transitions, and establish physiological roles for local chromatin fiber folding in regulating transcription and chromatin domain formation.

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.

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.

H3K4 methylation at active genes mitigates transcription-replication conflicts during replication stress.

Transcription-replication conflicts (TRCs) occur when intensive transcriptional activity compromises replication fork stability, potentially leading to gene mutations. Transcription-deposited H3K4 methylation (H3K4me) is associated with regions that are susceptible to TRCs; however, the interplay between H3K4me and TRCs is unknown. Here we show that H3K4me aggravates TRC-induced replication failure in checkpoint-defective cells, and the presence of methylated H3K4 slows down ongoing replication. Both S-phase checkpoint activity and H3K4me are crucial for faithful DNA synthesis under replication stress, especially in highly transcribed regions where the presence of H3K4me is highest and TRCs most often occur. H3K4me mitigates TRCs by decelerating ongoing replication, analogous to how speed bumps slow down cars. These findings establish the concept that H3K4me defines the transcriptional status of a genomic region and defends the genome from TRC-mediated replication stress and instability.

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.

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.

Chromatin Remodeling Factors Isw2 and Ino80 Regulate Chromatin, Replication, and Copy Number of the Saccharomyces cerevisiae Ribosomal DNA Locus.

In the budding yeast Saccharomyces cerevisiae, ribosomal RNA genes are encoded in a highly repetitive tandem array referred to as the ribosomal DNA (rDNA) locus. The yeast rDNA is the site of a diverse set of DNA-dependent processes, including transcription of ribosomal RNAs by RNA polymerases I and III, transcription of noncoding RNAs by RNA polymerase II, DNA replication initiation, replication fork blocking, and recombination-mediated regulation of rDNA repeat copy number. All of this takes place in the context of chromatin, but little is known about the roles played by ATP-dependent chromatin remodeling factors at the yeast rDNA. In this work, we report that the Isw2 and Ino80 chromatin remodeling factors are targeted to this highly repetitive locus. We characterize for the first time their function in modifying local chromatin structure, finding that loss of these factors decreases the fraction of actively transcribed 35S ribosomal RNA genes and the positioning of nucleosomes flanking the ribosomal origin of replication. In addition, we report that Isw2 and Ino80 promote efficient firing of the ribosomal origin of replication and facilitate the regulated increase of rDNA repeat copy number. This work significantly expands our understanding of the importance of ATP-dependent chromatin remodeling for rDNA biology.

Preparation and Analysis of Saccharomyces cerevisiae Quiescent Cells.

Saccharomyces cerevisiae enter quiescence during extended growth in culture (greater than 7 days). Here, we describe a method to separate quiescent from non-quiescent cells by density gradient. We also describe approaches for DAPI staining the chromatin of quiescent cells, measuring quiescent cell viability, and extracting RNA from quiescent cells for use in genomics experiments.

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.

Expansion of antisense lncRNA transcriptomes in budding yeast species since the loss of RNAi.

Antisense long noncoding RNAs (ASlncRNAs) have been implicated in regulating gene expression in response to physiological cues. However, little is known about the evolutionary dynamics of ASlncRNA and what underlies the evolution of their expression. Here, using budding yeast Saccharomyces spp. and Naumovozyma castellii as models, we show that ASlncRNA repertoires have expanded since the loss of RNA interference (RNAi), in terms of their expression levels, their lengths and their degree of overlap with coding genes. Furthermore, we show that RNAi is inhibitory to ASlncRNA transcriptomes and that increased expression of ASlncRNAs in the presence of RNAi is deleterious to N. castellii, which has retained RNAi. Together, our results suggest that the loss of RNAi had substantial effects on the genome-wide increase in expression of ASlncRNAs during the evolution of budding yeasts.

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.

Systematic approaches to identify functional lncRNAs.

Long noncoding RNAs (lncRNAs) were discovered in eukaryotes more than 30 years ago [1]. Recent advances in genomics have led to the discovery that lncRNAs are transcribed pervasively across the genome [2(•),3,4,5(•)]. There are an increasing number of reports that identify lncRNAs whose expression is modulated during cell differentiation or in disease states. However, biological functions for the vast majority of them are yet to be determined. Here, we propose two ways to identify lncRNAs that have biological functions: to identify lncRNAs with dedicated preinitiation complexes (PICs), and to focus on those whose transcription is highly regulated.

The conserved HDAC Rpd3 drives transcriptional quiescence in S. cerevisiae.

Quiescence is a ubiquitous cell cycle stage conserved from microbes through humans and is essential to normal cellular function and response to changing environmental conditions. We recently reported a massive repressive event associated with quiescence in Saccharomyces cerevisiae, where Rpd3 establishes repressive chromatin structure that drives transcriptional shutoff [6]. Here, we describe in detail the experimental procedures, data collection, and data analysis related to our characterization of transcriptional quiescence in budding yeast (GEO: GSE67151). Our results provide a bona fide molecular event driven by widespread changes in chromatin structure through action of Rpd3 that distinguishes quiescence as a unique cell cycle stage in S. cerevisiae.

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.