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A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae [MNase-seq]

ABSTRACT: The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which requires a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in S. cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE249469 | GEO | 2023/12/06

REPOSITORIES: GEO

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A fluorescent assay for cryptic transcription in Saccharomyces cerevisiae [RNA-seq]

Project description:The disruption of chromatin structure can result in transcription initiation from cryptic promoters within gene bodies. While the passage of RNA polymerase II is a well-characterized chromatin-disrupting force, numerous factors, including histone chaperones, normally stabilize chromatin on transcribed genes, thereby repressing cryptic transcription. DNA replication, which requires a partially overlapping set of histone chaperones, is also inherently disruptive to chromatin, but a role for DNA replication in cryptic transcription has never been examined. In this study, we tested the hypothesis that, in the absence of chromatin-stabilizing factors, DNA replication can promote cryptic transcription in S. cerevisiae. Using a novel fluorescent reporter assay, we show that multiple factors, including Asf1, CAF-1, Rtt106, Spt6, and FACT, block transcription from a cryptic promoter, but are entirely or partially dispensable in G1-arrested cells, suggesting a requirement for DNA replication in chromatin disruption. Collectively, these results demonstrate that transcription fidelity is dependent on numerous factors that function to assemble chromatin on nascent DNA.

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Transcriptome profiling reveals a novel role for Trichostatin A in antagonizing histone chaperone Chz1 mediated telomere anti-silencing.

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Identification of Elg1 interaction partners and effects on post-replication chromatin re-formation

Project description:Elg1, the major subunit of a Replication Factor C-like complex, is critical to ensure genomic stability during DNA replication, and is implicated in controlling chromatin structure. We investigated the consequences of Elg1 loss for the dynamics of chromatin re-formation following DNA replication. Measurement of Okazaki fragment length and the micrococcal nuclease sensitivity of newly replicated DNA revealed a defect in nucleosome re-assembly in the absence of Elg1. Using a proteomic approach to identify Elg1 binding partners, we discovered that Elg1 interacts with Rtt106, a histone chaperone implicated in replication-coupled nucleosome assembly that also regulates transcription. We find that Rtt106 recruitment to a number of promoters depends on Elg1. A central role for Elg1 is the unloading of PCNA from chromatin following DNA replication, so we examined the relative importance of Rtt106 and PCNA unloading for chromatin reassembly following DNA replication. We find that the major cause of the chromatin assembly defects of an elg1 mutant is PCNA retention on DNA following replication, with Rtt106-Elg1 interaction potentially playing a contributory role.

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Transcriptome profiling reveals a novel role for Trichostatin A in antagonizing histone chaperone Chz1 mediated telomere anti-silencing.

Project description:The histone chaperones play an important role in chromatin assembly and disassembly during replication and transcription. We assessed the global roles of histone chaperones in Saccharomyces cerevisiae. Microarray transcriptional analyses indicate that histone chaperones have their own specific target genes, and various histone chaperones have partially overlapping functions during transcriptional regulation. Histone deacetylase inhibitor TSA and histone chaperones Asf1, Vps75 and Rtt106 can function in parallel pathways to regulate transcription. Moreover, TSA can specifically antagonize histone chaperone Chz1-mediated telomere anti-silencing. This study demonstrates that a mutual cross-talk mechanism exists between histone chaperones and histone deacetylation in transcriptional regulation. All yeast strains used in this study are listed in the paper (Table S1). CHZ1, NAP1, ASF1, VPS75 and RTT106 genes were deleted individually by homologous recombination in BY4742 (WT) by using a previously described in (Wan, Y. et al. MCB, 2009) PCR-based procedure. The strains were cultured at 30°C in YPD (1% yeast extract, 2% peptone, 2% glucose) media. For TSA treatments, WT and deletion mutants were grown to mid-log phase (OD600=0.5), TSA (Sigma-Aldrich) was then added to the yeast cultures at a final concentration of 10 ?M and cells were cultured for additional hour.Total RNA was isolated by hot acid phenol extraction protocol as previously described in Wan, Y. et al. MCB, 2009. Microarray labeling and hybridization reactions were performed as previously described in Wan, Y. et al. MCB, 2009 and Wan, Y. et al. NAR, 2010. Two color microarrays, comparing RNA from the experimental conditions (deletion mutations grown in YPD, WT and deletion mutations grown in YPD with TSA treatment) to RNA from the control WT cells grown in glucose-containing medium (YPD), were performed using Agilent whole-genome S. cerevisiae arrays.

2011-06-28 | E-GEOD-30228 | biostudies-arrayexpress

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Project description:We have previously shown that Okazaki fragments in Saccharomyces cerevisiae are sized according to the chromatin repeat. Here we demonstrate that nucleosome positioning is rapidly established on newly synthesized DNA. Using deep sequencing, we demonstrate that ATP-dependent chromatin remodeling enzymes, Isw1 and Chd1, collaborate with histone chaperones, such as CAF-1 and Rtt106, to remodel nucleosomes, as they are loaded. Importantly, we find that nucleosome positioning is ultimately specified by select sequence-specific DNA-binding factors, which serve as physical cues for chromatin remodeling. Our results provide a mechanistic understanding of how chromatin structures are replicated in vivo, and show that chromatin structure at gene promoters is rapidly established after DNA replication. Altogether, our data provide evidence for coordinated “loading and remodeling” of nucleosomes behind the replication fork, in collaboration with packing of nucleosomes against a barrier.

2016-04-14 | GSE63583 | GEO

Transcription and FACT facilitate the restoration of replication-coupled chromatin assembly defects

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Project description:Histone chaperones are critical for controlling chromatin integrity during transcription, DNA replication, and DNA repair. We have discovered that the physical interaction between two essential histone chaperones, Spt6 and Spn1/Iws1, is required for transcriptional accuracy and nucleosome organization. To understand this requirement, we have isolated suppressors of an spt6 mutation that disrupts the Spt6-Spn1 interaction. Several suppressors are in a third essential histone chaperone, FACT, while another suppressor is in the transcription elongation factor Spt5/DSIF. The FACT suppressors weaken FACT-nucleosome interactions and bypass the requirement for Spn1, possibly by restoring a necessary balance between Spt6 and FACT on chromatin. In contrast, the Spt5 suppressor modulates Spt6 function in a Spn1-dependent manner. Despite these distinct mechanisms, both suppressors alleviate the nucleosome organization defects caused by disruption of the Spt6-Spn1 interaction. Taken together, we have uncovered a network in which histone chaperones and other elongation factors coordinate transcriptional integrity and chromatin structure.

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Project description:A two-colour cell array screen reveals interdependent roles for histone chaperones and a chromatin boundary regulator in histone gene repression. We describe a fluorescent reporter system that exploits the functional genomic tools available in budding yeast to systematically assess consequences of genetic perturbations on gene expression. We used our Reporter-Synthetic Genetic Array (R-SGA) method to screen for regulators of core histone gene expression. We discovered that the histone chaperone Rtt106 functions in a pathway with two other chaperones, Asf1 and the HIR complex, to create a repressive chromatin structure at core histone promoters. We found that activation of histone (HTA1) gene expression involves both relief of Rtt106-mediated repression by the activity of the histone acetyltransferase Rtt109 and restriction of Rtt106 to the promoter region by the bromodomain-containing protein Yta7. We propose that the maintenance of Asf1/HIR/Rtt106-mediated repressive chromatin domains is the primary mechanism of cell cycle regulation of histone promoters. Our data suggest that this pathway may represent a chromatin regulatory mechanism that is broadly used across the genome.

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