Proteogenomics analysis reveals specific genomic orientations at distal regulatory regions composed by non-canonical histone variants
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE64652 | GEO | 2015/12/22
SECONDARY ACCESSION(S): PRJNA271569
REPOSITORIES: GEO
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Project description:Histone variants (H3.1, H3.3, H2A, and macroH2A) were studied about their proteomic and genomic distribution in Hela cells HeLa S3 cells stably expressing either FLAG-tagged H3.1, and H3.3 were grown in suspension in Joklik media containing 10% newborn calf serum (Hyclone), 1% GlutaMAX (Invitrogen), and 1% penicillin-streptomycin, and cells were harvested at log phase. Nuclei were isolated, mononucleosomes were subsequently obtained from these cell lines. Cells were lysed in hypotonic TMSD buffer to isolate nuclei, which were then digested with micrococcal nuclease. The resulting mononucleosomes were immunoprecipitated with anti-FLAG M2-agarose beads (Sigma) and eluted with FLAG peptide (Sigma)
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Project description:Histone variants play further important roles in DNA packaging and controlling gene expression. However, our understanding about their composition and their functions is limited.Integrating proteomic and genomic approaches, we performed a comprehensive analysis of the epigenetic landscapes containing the four histone variants H3.1, H3.3, H2A.Z, and macroH2A. These histones were FLAG-tagged in HeLa cells and purified using chromatin immunoprecipitation (ChIP). By adopting ChIP followed by mass spectrometry (ChIP-MS), we quantified histone post-translational modifications (PTMs) and histone variant nucleosomal ratios in highly purified mononucleosomes. Subsequent ChIP followed by next-generation sequencing (ChIP-seq) was used to map the genome-wide localization of the analyzed histone variants and define their chromatin domains. Finally, we included in our study large datasets contained in the ENCODE database. We newly identified a group of regulatory regions enriched in H3.1 and the histone variant associated with repressive marks macroH2A. Systematic analysis identified both symmetric and asymmetric patterns of histone variant occupancies at intergenic regulatory regions. Strikingly, these directional patterns were associated with RNA polymerase II (PolII). These asymmetric patterns correlated with the enhancer activities measured using global run-on sequencing (GRO-seq) data.Our studies show that H2A.Z and H3.3 delineate the orientation of transcription at enhancers as observed at promoters. We also showed that enhancers with skewed histone variant patterns well facilitate enhancer activity. Collectively, our study indicates that histone variants are deposited at regulatory regions to assist gene regulation.
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Project description:Epigenetic inheritance refers to the faithful replication of DNA and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferase during S phase, unless they are remodeled by Decrease in DNA methylation 1 (DDM1Lsh/HELLS), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1. Inddm1mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a di-sulphide bond in the helicase domain is essential for activity in vivo and in vitro. Differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1Dnmt1. DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome and contributes to epigenetic inheritance.
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Project description:Epigenetic inheritance refers to the faithful replication of DNA and histone modification independent of DNA sequence. Nucleosomes block access to DNA methyltransferase during S phase, unless they are remodeled by Decrease in DNA methylation 1 (DDM1[Lsh/HELLS]), a Snf2-like master regulator of epigenetic inheritance. We show that DDM1 activity results in replacement of the transcriptional histone variant H3.3 for the replicative variant H3.1. Inddm1mutants, DNA methylation can be restored by loss of the H3.3 chaperone HIRA, while the H3.1 chaperone CAF-1 becomes essential. The single-particle cryo-EM structure at 3.2 Å of DDM1 with a variant nucleosome reveals direct engagement at SHL2 with histone H3.3 at or near variant residues required for assembly, as well as with the deacetylated H4 tail. An N-terminal autoinhibitory domain binds H2A variants to allow remodeling, while a di-sulphide bond in the helicase domain is essential for activity in vivo and in vitro. Differential remodeling of H3 and H2A variants in vitro reflects preferential deposition in vivo. DDM1 co-localizes with H3.1 and H3.3 during the cell cycle, and with the DNA methyltransferase MET1[Dnmt1]. DDM1 localization to the chromosome is blocked by H4K16 acetylation, which accumulates at DDM1 targets in ddm1 mutants, as does the sperm cell specific H3.3 variant MGH3 in pollen, which acts as a placeholder nucleosome and contributes to epigenetic inheritance.
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