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Expression profiles of the NuB4/HAT-B complex and histone knockouts

ABSTRACT: Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics.

ORGANISM(S): Saccharomyces cerevisiae

PROVIDER: GSE30168 | GEO | 2012/05/15

SECONDARY ACCESSION(S): PRJNA143909

REPOSITORIES: GEO

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Expression profiles of the NuB4/HAT-B complex and histone knockouts

Project description:Dynamic modification of histone proteins plays a key role in regulating gene expression. However, histones themselves can also be dynamic, which potentially affects the stability of histone modifications. To determine the molecular mechanisms of histone turnover we developed a parallel screening method for epigenetic regulators by analyzing chromatin states on DNA barcodes. Histone turnover was quantified by employing a genetic pulse-chase technique called RITE, which was combined with chromatin immunoprecipitation and high-throughput sequencing. In this screen, the NuB4/HAT-B complex, containing the conserved type B histone acetyltransferase Hat1, was found to promote histone turnover. Unexpectedly, the three members of this complex could be functionally separated from each other as well as from the known interacting factor and histone chaperone Asf1. Thus, systematic and direct interrogation of chromatin structure on DNA barcodes can lead to the discovery of genes and pathways involved in chromatin modification and dynamics. RNA isolated from a large amount of wt yeast from a single culture was used as a common reference. This common reference was used for each separate hybridization and used in the statistical analysis to obtain an average expression-profile for each deletion mutant relative to the wt. Two independent cultures were hybridized on two separate microarrays. For the first hybridization the Cy5 (red) labeled cRNA from the deletion mutant is hybridized together with the Cy3 (green) labeled cRNA from the common reference. For the replicate hybridization, the labels are swapped. Each gene is represented twice on the microarray, resulting in four measurements per mutant. Up to five deletion strains were grown on a single day. Wt cultures were grown parallel to the deletion mutants to assess day-to-day variance.

2012-05-14 | E-GEOD-30168 | biostudies-arrayexpress

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Hat mutants

Project description:Transcriptomes from the hat1, hat2 and hat1 hat2 mutants in S288C yeast background.

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A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin [ChIP-exo]

Project description:Nucleosome composition actively contribute to the chromatin structure and accessibility. To preserve chromatin state during replication, transcription and DNA repair, cells have evolved mechanisms to evict or recycle histones, generating a landscape of differentially aged nucleosomes. To map the stability of nucleosomes, we have adapted the recombination induced tag exchange (RITE) method to Schizosaccharomyces pombe histone H3. The RITE method allows us to study replication-independent protein turnover both through the occurrence of, in our case, new histone H3 and the disappearance or preservation of old histone H3. We contrast the RITE system to nucleosome turnover measured by chromatin incorporation of an epitope-tagged H3 under an inducible promoter. We confirm previous findings that stable nucleosomes are found at heterochromatin, but also at coding regions of actively transcribed genes. Genome-wide comparisons with several chromatin marks showed that high turnover nucleosomes correlate with H2A.Z, acetylated H4 and H3K4me2. The histones with high turnover are primarily found at the nucleosomes on the 5´and/or 3´ edges of the transcribed unit. In addition, in this study we have determined genome-wide maps of all three methylation marks at H4K20. All methylation of H4K20 appeared in low turnover nucleosomes and particularly in euchromatic regions. H4K20me1 marks stable nucleosomes at loci proximal to nucleosome depleted regions (NDR) and H4K20me2/3 were found further inside of transcribed units, especially at coding regions of long genes expressed at low levels. Further, this transcription-dependent accumulation of histone methylations was dependent on the histone chaperone complex FACT (Facilitates Chromatin Transcription), as predicited from its role in recycling nucleosomes during transcription.

2015-03-13 | GSE66865 | GEO

A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depend on histone recycling in transcribed chromatin [ChIP]

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Expression profiles of the NuB4/HAT-B complex and histone knockouts

Project description:Single/double/triple mutants of hat1, hat2, hif2 and histone mutants have been analyzed by expression profiling in high glucose and under starvation

2012-06-19 | E-TABM-1175 | biostudies-arrayexpress

Cell cycle- and chaperone-mediated regulation of H3K56ac incorporation in yeast

Project description:Acetylation of histone H3 lysine 56 is a covalent modification best-known as a mark of newly-replicated chromatin, but has also been linked to replication-independent histone replacement. Here, we measured H3K56ac levels at single-nucleosome resolution in asynchronously growing yeast cultures, as well as in yeast proceeding synchronously through the cell cycle. We developed a quantitative model of H3K56ac kinetics, which shows that H3K56ac is largely explained by the genomic replication timing and the turnover rate of each nucleosome, suggesting that cell cycle profiles of H3K56ac should reveal most first-time nucleosome incorporation events. However, since the deacetylases Hst3/4 prevent use of H3K56ac as a marker for histone deposition during M phase, we also directly measured M phase histone replacement rates. We report a global decrease in turnover rates during M phase, and a further specific decrease in turnover among early origins of replication, which switch from rapidly-replaced in G1 phase to stable-bound during M phase. Finally, by measuring H3 replacement in yeast deleted for the H3K56 acetyltransferase Rtt109 and its two co-chaperones Asf1 and Vps75, we find evidence that Rtt109 and Asf1 preferentially enhance histone replacement at rapidly-replaced nucleosomes, whereas Vps75 appears to inhibit histone turnover at those loci. These results provide a broad perspective on histone replacement/incorporation throughout the cell cycle, and suggest that H3K56 acetylation provides a positive feedback loop by which replacement of a nucleosome enhances subsequent replacement at the same location.

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Histone chaperone ASF1 mediates H3.3-H4 deposition in Arabidopsis

Project description:Histone chaperones and chromatin remodelers control nucleosome dynamics, essential for transcription, replication, and DNA repair. The histone chaperone Anti-Silencing Factor 1 (ASF1) plays a central role in facilitating CAF-1-mediated replication-dependent H3.1 deposition and HIRA-mediated replication-independent H3.3 deposition in yeast and metazoans. Whether ASF1 function is evolutionarily conserved in plants is unknown. Here, we show that Arabidopsis ASF1 proteins display an exclusive preference for the H3.3-depositing HIRA complex. Simultaneous mutation of both Arabidopsis ASF1 genes caused a decrease in chromatin density and ectopic H3.1 occupancy at loci typically enriched with H3.3. Genetic, transcriptomic, and proteomic data indicate that ASF1 proteins strongly prefer the HIRA complex over CAF-1. asf1 mutants also displayed an increase in spurious Pol II transcriptional initiation, and showed defects in the maintenance of gene body CG DNA methylation and in the distribution of histone modifications. Furthermore, ectopic targeting of ASF1 caused excessive histone deposition, less accessible chromatin, and gene silencing. These findings reveal the importance of ASF1-mediated H3.3-H4 deposition via the HIRA pathway for proper epigenetic regulation of the genome.

2022-10-31 | PXD035578 | Pride

RIF1-ASF1-mediated high-order chromatin structure safeguards genome integrity

Project description:The 53BP1-RIF1 pathway antagonizes resection of DNA broken ends and confers PARP inhibitor sensitivity on BRCA1-mutated tumors. However, it is unclear how this pathway suppresses initiation of resection. Here, we identify ASF1 as a partner of RIF1 via an interacting manner similar to its interactions with histone chaperones CAF-1 and HIRA. ASF1 is recruited to distal chromatin flanking DNA breaks by 53BP1-RIF1 and promotes non-homologous end joining (NHEJ) using its histone chaperone activity. Epistasis analysis shows that ASF1 acts in the same NHEJ pathway as RIF1, but via a parallel pathway with the shieldin complex, which suppresses resection after initiation. Moreover, defects in end resection and homologous recombination (HR) in BRCA1-deficient cells are largely suppressed by ASF1 deficiency. Mechanistically, ASF1 compacts adjacent chromatin by heterochromatinization to protect broken DNA ends from BRCA1-mediated resection. Taken together, our findings identified a RIF1-ASF1 histone chaperone complex that promotes changes in high-order chromatin structure to stimulate the NHEJ pathway for DSB repair.

2022-02-04 | PXD031311 | Pride

HAT1 coordinates histone production and acetylation via H4 promoter binding

Project description:The energetic costs of duplicating chromatin are large and therefore likely depend on nutrient sensing checkpoints and metabolic inputs. By studying chromatin modifiers regulated by epithelial growth factor, we identified histone acetyltransferase 1 (HAT1) as an induced gene that enhances proliferation through coordinating histone production, acetylation and glucose metabolism. In addition to its canonical role as a cytoplasmic histone H4 acetyltransferase, we isolated a HAT1-containing complex bound specifically at promoters of H4 genes. HAT1-dependent transcription of H4 genes required an acetate-sensitive promoter element. HAT1 expression was critical for S-phase progression and maintenance of H3 lysine 9 acetylation at proliferation-associated genes, including histone genes. Therefore, these data describe a feed-forward circuit whereby HAT1 captures acetyl-groups on nascent histones and drives H4 production by chromatin binding to support chromatin replication and acetylation. These findings have important implications for human disease, since high HAT1 levels associate with poor outcomes across multiple cancer types.

2019-07-02 | GSE117472 | GEO

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