FACT and Spt6 Prevent Cryptic Transcription by Impeding H2A.Z Loading in Gene Bodies
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ABSTRACT:
ORGANISM(S): Saccharomyces cerevisiae
PROVIDER: GSE62880 | GEO | 2015/05/12
SECONDARY ACCESSION(S): PRJNA265882
REPOSITORIES: GEO
Similar Datasets
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 variant histone H2A.Z is inserted into nucleosomes immediately downstream of promoters and is important for transcription. The site-specific deposition of H2A.Z is catalyzed by SWR, a conserved chromatin remodeler with affinity for promoter-proximal nucleosome depleted regions (NDRs) and histone acetylation. By comparing the genomic distribution of H2A.Z in wild-type and SWR-deficient cells, we found that SWR is also responsible for depositing H2A.Z at thousands of non-canonical sites not directly linked to NDRs or histone acetylation. To understand the targeting mechanism of H2A.Z, we presented SWR with a library of nucleosomes isolated from yeast and characterized those preferred by SWR. We found that SWR prefers nucleosomes associated with intergenic over coding regions, especially when polyadenine tracks are present. Insertion of polyadenine sequences into recombinant nucleosomes near the H2A-H2B binding site stimulated the H2A.Z insertion activity of SWR. Therefore, the genome is encoded with information contributing to remodeler-mediated targeting of H2A.Z.
2024-01-13 | GSE252824 | GEO
Project description:FACT and Spt6 Prevent Cryptic Transcription by Impeding H2A.Z Loading in Gene Bodies
| PRJNA265882 | ENA
Project description:The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange where either H2A.Z or H2A can be exchanged from nucleosomes. This result is confirmed in vivo, where genome-wide analysis demonstrates widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a M-bM-^@M-^\lockM-bM-^@M-^] that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators. H2A.Z ChIP seq experiments in mutants with constitutive H3K56ac
2013-04-15 | E-GEOD-43935 | biostudies-arrayexpress
Project description:The histone variant H2A.Z plays key roles in gene expression, DNA repair, and centromere function. H2A.Z deposition is controlled by SWR-C chromatin remodeling enzymes that catalyze the nucleosomal exchange of canonical H2A with H2A.Z. Here we report that acetylation of histone H3 lysine 56 (H3-K56Ac) alters the substrate specificity of SWR-C, leading to promiscuous dimer exchange where either H2A.Z or H2A can be exchanged from nucleosomes. This result is confirmed in vivo, where genome-wide analysis demonstrates widespread decreases in H2A.Z levels in yeast mutants with hyperacetylated H3K56. Our work also suggests that a conserved SWR-C subunit may function as a “lock” that prevents removal of H2A.Z from nucleosomes. Our study identifies a histone modification that regulates a chromatin remodeling reaction and provides insights into how histone variants and nucleosome turnover can be controlled by chromatin regulators.
2013-04-15 | GSE43935 | 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: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:Microarray analysis was used to identify all cryptic promoters in the S. cerevisiae genome that are activated in spt6 and spt16 mutants. These experiments showed that cryptic initiation is widespread, occurring in approximately 1,000 genes. We generated 2 microarray profiles from fluor-reversed replicates of wild-type and spt6-1004 or spt16-197 mutants. See Cheung et al., (2008) in revision.
2010-05-16 | E-GEOD-12272 | biostudies-arrayexpress
Project description:Transcription can be quite disruptive for chromatin so cells have evolved mechanisms to preserve chromatin integrity during transcription, hence preventing the emergence of cryptic transcript from spurious promoter sequences. How these transcripts are regulated and processed by cells remains poorly characterized. Notably, very little is known about the termination of cryptic transcription. 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 anti-sense 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 anti-sense cryptic transcripts preferentially terminate at the 3’-end of upstream genes. These findings led us to demonstrate that most terminators in yeast are bidirectional, leading to termination and polyadenylation of transcripts coming from either direction. We propose that S. cerevisiae has evolved this mechanism in order to prevent spurious transcription from invading neighbouring genes, a feature particularly critical for organisms with small compact genomes.
2017-04-03 | GSE89601 | GEO