Project description:A compendium of nucleosome and transcript profiles reveals determinants of chromatin architecture and transcription [transcript profiling]
Project description:Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors including the CAF-1 complex, Spt10 and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning and these data provide a valuable resource for future studies. We examined 50 single-gene loss-of-function strains, comprised of gene deletions (del) and temperature-sensitive (ts) or tetracycline promoter-shutoff (tet) alleles. These genes were selected based on their known or potential role in nucleosome biology and include remodeler ATPases and chaperones, histones and histone modifiers, transcription and elongation factors, and components of RNA polymerase I and II. The compendium also includes 4 compounds targeting transcription and histone deacetylases, as well as a histone depletion time course performed with a strain in which H4 gene expression is exclusively under the control of a GAL1 promoter. Genome-wide nucleosome occupancy profiles were generated using Affymetrix Tiling arrays with probes spaced every 4 bp [PMID:16569694], or next-generation sequencing. Identically prepared samples for each strain and treatment were analyzed on the same tiling arrays for strand-specific expression differences. Each compendium condition was compared to a matched wild-type (WT) reference grown in parallel.
Project description:Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors including the CAF-1 complex, Spt10 and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning and these data provide a valuable resource for future studies. We examined 50 single-gene loss-of-function strains, comprised of gene deletions (del) and temperature-sensitive (ts) or tetracycline promoter-shutoff (tet) alleles. These genes were selected based on their known or potential role in nucleosome biology and include remodeler ATPases and chaperones, histones and histone modifiers, transcription and elongation factors, and components of RNA polymerase I and II. The compendium also includes 4 compounds targeting transcription and histone deacetylases, as well as a histone depletion time course performed with a strain in which H4 gene expression is exclusively under the control of a GAL1 promoter. Genome-wide nucleosome occupancy profiles were generated using Affymetrix Tiling arrays with probes spaced every 4 bp [PMID:16569694], or next-generation sequencing. Identically prepared samples for each strain and treatment were analyzed on the same tiling arrays for strand-specific expression differences. Each compendium condition was compared to a matched wild-type (WT) reference grown in parallel.
Project description:Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors including the CAF-1 complex, Spt10 and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning and these data provide a valuable resource for future studies. We examined 50 single-gene loss-of-function strains, comprised of gene deletions (del) and temperature-sensitive (ts) or tetracycline promoter-shutoff (tet) alleles. These genes were selected based on their known or potential role in nucleosome biology and include remodeler ATPases and chaperones, histones and histone modifiers, transcription and elongation factors, and components of RNA polymerase I and II. The compendium also includes 4 compounds targeting transcription and histone deacetylases, as well as a histone depletion time course performed with a strain in which H4 gene expression is exclusively under the control of a GAL1 promoter. Genome-wide nucleosome occupancy profiles were generated using Affymetrix Tiling arrays with probes spaced every 4 bp [PMID:16569694], or next-generation sequencing. Identically prepared samples for each strain and treatment were analyzed on the same tiling arrays for strand-specific expression differences. Each compendium condition was compared to a matched wild-type (WT) reference grown in parallel.
Project description:FACT is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone:DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome structure, chromatin integrity, and transcription output. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT in vitro and in vivo as expected, but H3-K56 and FACT mutations caused surprisingly similar changes in transcription of individual genes. Notably, neither the changes in transcript levels nor the effects on nucleosome occupancy resulting from mutations conformed to the model that FACT is needed to overcome nucleosomal barriers during transcription initiation or elongation. Instead, the results suggest that both FACT and H3-K56ac are involved in establishing chromatin architecture prior to transcription and restoring it afterwards. They contribute to a process that optimizes transcription frequency, especially at conditionally expressed genes, and restores chromatin integrity after transcription, especially at the +1 nucleosome to block antisense transcription, but FACT appears to be less involved than expected in directly promoting transcription.
Project description:FACT is a histone chaperone that can destabilize or assemble nucleosomes. Acetylation of histone H3-K56 weakens a histone:DNA contact that is central to FACT activity, suggesting that this modification could affect FACT functions. We tested this by asking how mutations of H3-K56 and FACT affect nucleosome structure, chromatin integrity, and transcription output. Mimics of unacetylated or permanently acetylated H3-K56 had different effects on FACT in vitro and in vivo as expected, but H3-K56 and FACT mutations caused surprisingly similar changes in transcription of individual genes. Notably, neither the changes in transcript levels nor the effects on nucleosome occupancy resulting from mutations conformed to the model that FACT is needed to overcome nucleosomal barriers during transcription initiation or elongation. Instead, the results suggest that both FACT and H3-K56ac are involved in establishing chromatin architecture prior to transcription and restoring it afterwards. They contribute to a process that optimizes transcription frequency, especially at conditionally expressed genes, and restores chromatin integrity after transcription, especially at the +1 nucleosome to block antisense transcription, but FACT appears to be less involved than expected in directly promoting transcription.
Project description:Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors including the CAF-1 complex, Spt10 and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning and these data provide a valuable resource for future studies.
Project description:Nucleosomes in all eukaryotes examined to date adopt a characteristic architecture within genes and play fundamental roles in regulating transcription, yet the identity and precise roles of many of the trans-acting factors responsible for the establishment and maintenance of this organization remain to be identified. We profiled a compendium of 50 yeast strains carrying conditional alleles or complete deletions of genes involved in transcriptional regulation, histone biology and chromatin remodeling, as well as compounds that target transcription and histone deacetylases, to assess their respective roles in nucleosome positioning and transcription. We find that nucleosome patterning in genes is affected by many factors including the CAF-1 complex, Spt10 and Spt21, in addition to previously reported remodeler ATPases and histone chaperones. Disruption of these factors or reductions in histone levels led genic nucleosomes to assume positions more consistent with their intrinsic sequence preferences, with pronounced and specific shifts of the +1 nucleosome relative to the transcription start site. These shifts of +1 nucleosomes appear to have functional consequences, as several affected genes in Ino80 mutants exhibited altered expression responses. Our parallel expression profiling compendium revealed extensive transcription changes in intergenic and antisense regions, most of which occur in regions with altered nucleosome occupancy and positioning. We show that the nucleosome-excluding transcription factors Reb1, Abf1, Tbf1, and Rsc3 suppress cryptic transcripts at their target promoters, while a combined analysis of nucleosome and expression profiles identified 36 novel transcripts that are normally repressed by Tup1/Cyc8. Our data confirm and extend the roles of chromatin remodelers and chaperones as major determinants of genic nucleosome positioning and these data provide a valuable resource for future studies.