Saccharomyces cerevisiae
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ABSTRACT:
PROVIDER: PRJNA286077 | ENA |
REPOSITORIES: ENA
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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.
2020-11-05 | GSE160821 | GEO
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.
2023-12-06 | GSE249469 | GEO
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.
2023-12-07 | GSE249580 | GEO
Project description:H2A.Z is a highly conserved histone variant involved in several key nuclear processes. It is incorporated into promoters by SWR-C-related chromatin remodeling complexes, but whether it is also actively excluded from non-promoter regions is not clear. Here, we provide genomic and biochemical evidence that RNA polymerase II (RNAPII) elongation-associated histone chaperones FACT and Spt6 both contribute to restricting H2A.Z from intragenic regions. In the absence of FACT or Spt6, the lack of H2A.Z eviction, coupled to its pervasive incorporation by mislocalized SWR-C, alters chromatin composition and contributes to cryptic initiation. Thus, chaperone-mediated H2A.Z removal is crucial for restricting the chromatin signature of gene promoters, which otherwise may license or promote cryptic transcription. We profiled H2A.Z occupancy in S. cerevisiae by ChIP-chip on tiling arrays in different mutants for chromatin remodelers and histone chaperones. In most experiments, H2A.Z ChIP samples (Cy5-labeled) were performed using a polyclonal antibody against H2A.Z and hybridized against H2B ChIP samples (Cy3-labeled), also performed using a polyclonal antibody. Temperature-sensitive mutants for the histone chaperones Spt16 and Spt6 (spt16-197 and spt6-1004 respectively) showed strong H2A.Z localization defects so the role of these factors in H2A.Z localization was further analyzed. H2A.Z ChIP-chip experiments in spt16-197 and spt6-1004 were repeated using different experimental designs [normalized against Input DNA or Mock (IgG) ChIP samples]. The contribution of Spt16 and Spt6 on H2A.Z localization was also confirmed by nuclear depletion of Spt16 and Spt6 using the anchor-away system. H2A.Z ChIP-chip experiments were also performed in sic1Δ and spt16-197/sic1Δ cells in order to rule out any G1-arrest artifact. H2A.Z ChIP-chip experiments were repeated using spike-in controls for normalization, revealing widespread H2A.Z occupancy in Spt16 and Spt6 mutants. In addition, the localization of the chromatin remodeler SWR-C was determined in wild type cells as well as in spt16-197 and spt6-1004 cells. Finally, we also profiled histone H4 occupancy by ChIP-chip in wild type, spt16-197, spt6-1004, spt16-197/htz1Δ and spt6-1004/htz1Δ cells. All ChIP-chip experiments were done in duplicates. Each microarray was normalized using the Lima Loess and replicates were combined using a weighted average method as previously described (Pokholok et al., 2005).
2015-05-12 | E-GEOD-62880 | biostudies-arrayexpress
Project description:The genetic information encoded in DNA is framed by additional layers of information, referred to as the epigenome. Epigenetic marks such as DNA methylation, histone modifications and histone variants are concentrated on specific genomic sites as means to instruct, but also sometimes as a consequence of, gene expression. How this information is maintained, notably in the face of transcription, is not understood. Here we show that the histone chaperones FACT and Spt6 are required for maintaining proper localization of several histone modifications including H3K4me1,2,3, H3K36me3, H3K79me3, H3K14ac, H3K18ac and H2Bub in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates massive nucleosome loss which is partially compensated by increased histone assembly by histone chaperones such as Asf1 and HIR. Because re-incorporation of histones by these histone chaperones in not coupled to transcription, the modified histones are randomly incorporated, leading to scrambling of the epigenetic information. Hence, our work highlights the importance of local nucleosome recycling by FACT and Spt6 during transcription in the maintenance of a proper epigenetic landscape.
2019-07-30 | GSE113213 | GEO
Project description:The genetic information encoded in DNA is framed by additional layers of information, referred to as the epigenome. Epigenetic marks such as DNA methylation, histone modifications and histone variants are concentrated on specific genomic sites as means to instruct, but also sometimes as a consequence of, gene expression. How this information is maintained, notably in the face of transcription, is not understood. Here we show that the histone chaperones FACT and Spt6 are required for maintaining proper localization of several histone modifications including H3K4me1,2,3, H3K36me3, H3K79me3, H3K14ac, H3K18ac and H2Bub in Saccharomyces cerevisiae. In the absence of functional FACT or Spt6, transcription generates massive nucleosome loss which is partially compensated by increased histone assembly by histone chaperones such as Asf1 and HIR. Because re-incorporation of histones by these histone chaperones in not coupled to transcription, the modified histones are randomly incorporated, leading to scrambling of the epigenetic information. Hence, our work highlights the importance of local nucleosome recycling by FACT and Spt6 during transcription in the maintenance of a proper epigenetic landscape.
2019-07-30 | GSE113025 | GEO
Project description:RSC (remodels the structure of chromatin) is an essential ATP-dependent chromatin remodeling complex in Saccharomyces cerevisiae. The catalytic subunit of RSC, Sth1 uses its ATPase activity to slide or remove nucleosomes. RSC has been shown to regulate the width of the nucleosome-depleted regions (NDRs) by sliding the flanking nucleosomes away from NDRs. As such the nucleosomes encroach NDRs when RSC is depleted and leads to transcription initiation defects. In this study, we examined the effects of the catalytic-dead Sth1 on transcription and compared them to the effects observed during acute and rapid Sth1 depletion by auxin-induced degron strategy. We found that rapid depletion of Sth1 reduces recruitment of TBP and Pol II in highly transcribed genes, as would be expected considering its role in regulating chromatin structure at promoters. In contrast, cells harboring the catalytic-dead Sth1 exhibited a severe reduction in TBP binding, but surprisingly, also displayed a substantial accumulation in Pol II occupancies within coding regions. After depleting endogenous Sth1 in the catalytic dead mutant, we observed a further increase in Pol II occupancies, suggesting that the inactive Sth1 contributed to the observed accumulation of Pol II in coding regions. Notwithstanding the Pol II increase, the ORF occupancies of histone chaperones FACT and Spt6 were significantly reduced in the mutant. These results suggest a potential role for RSC in recruiting/retaining these chaperones in coding regions. Pol II accumulation despite substantial reductions in TBP, FACT, and Spt6 occupancies in the catalytic-dead mutant could be indicative of severe transcription elongation and termination defects. Such defects would be consistent with studies showing that RSC is recruited to coding regions in a transcription-dependent manner. Thus, these findings imply a role for RSC in transcription elongation and termination processes, in addition to its established role in transcription initiation.
2023-11-07 | GSE245521 | GEO
Project description:The FACT complex and Spt6 are conserved histone chaperones that are recruited to the open reading frames of transcribed genes. In this study, we provide evidence that FACT interaction with acetylated H3 tail is important for its localization to the coding sequences. Pol II CTD kinase Kin28 additionally stimulates FACT recruitment to a subset of genes. Pol II occupancies in the 5’ ends of transcribed genes are greatly reduced on depleting FACT, whereas reduced occupancies at the 3’ ends were observed upon Spt6 depletion indicating that these factors modulate Pol II progression through distinct regions of transcribed coding sequences. While FACT is largely responsible for reassembling histones, we uncover a role for Spt6 in promoting histone eviction in addition to widely-accepted role for Spt6 in histone reassembly. Consistent with their localization in the coding regions, simultaneously impairing FACT and Spt6 function severely dampens histone eviction and impairs transcription genome-wide. ChIP-chip experiments to measure Spt16 occupancies in WT and kin28as mutant, as well Rpb3 and histone H3 occupancies in undepleted or depleted cells for Spt16 and Spt6, and also in the strain lacking Spt6 tandem SH2 domain
2018-05-21 | GSE69642 | GEO
Project description:FACT and Spt6 Prevent Cryptic Transcription by Impeding H2A.Z Loading in Gene Bodies
| PRJNA265882 | ENA
Project description:The Paf1 complex (Paf1C) is a conserved transcription elongation factor that regulates transcription elongation efficiency, facilitates co-transcriptional histone modifications, and impacts molecular processes linked to RNA synthesis, such as polyA site selection. Coupling of the activities of Paf1C to transcription elongation requires its association with RNA polymerase II (Pol II). Mutational studies in yeast identified Paf1C subunits Cdc73 and Rtf1 as important mediators of Paf1C association with Pol II on active genes. While the interaction between Rtf1 and the general elongation factor Spt5 is relatively well-understood, the interactions involving Cdc73 have not been fully elucidated. Using a site-specific protein cross-linking strategy in yeast cells, we identified direct interactions between Cdc73 and two components of the Pol II elongation complex, the elongation factor Spt6 and the largest subunit of Pol II. Both of these interactions require the tandem SH2 domain of Spt6. We also show that Cdc73 and Spt6 can interact in vitro and that rapid depletion of Spt6 dissociates Paf1 from chromatin, altering patterns of Paf1C-dependent histone modifications genome-wide. These results reveal interactions between Cdc73 and the Pol II elongation complex and identify Spt6 as a key factor contributing to the occupancy of Paf1C at active genes in Saccharomyces cerevisiae.
2023-02-24 | GSE201436 | GEO