Histone H3.3 and its proteolytically processed form drive a cellular senescence program
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE55949 | GEO | 2014/10/01
SECONDARY ACCESSION(S): PRJNA241413
REPOSITORIES: GEO
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Project description:In this study, we have uncovered novel proteolytic processing of the histone H3 tail in senescence models in primary fibroblasts and melanocytes. Cleavage of H3 tail occurs at two distinct residues and is mediated by Cathepsin L. We show that variant H3.3 is preferentially cleaved, and that cleaved histones are associated with chromatin and incorporated into nucleosomes. We also found that the histone chaperone ASF1a is required for chromatin incorporation of the cleaved histone species. Further, we show that overexpression of cleaved H3.3 induces a senescence program in fibroblasts in the absensence of oncogenic signaling. For the RNA-seq studies, growing IMR90 fibroblasts were compared to cells induced to senesce via oncogene activation or cleaved H3.3 overexpression. Growing controls consist of IMR90 cells infected with empty retroviral construct pBabe and grown under normal conditions for 13 days prior to RNA isolation. For oncogene-induced senescence samples, IMR90s carrying a tamoxifen-inducible H-RasV12 retroviral construct were induced to senesce by addition of 10nM tamoxifen to the media for 8 days. Finally, IMR90s were infected with a retroviral construct expressing the cleaved form of H3.3 with a C-terminal Flag tag. RNA samples form this group were isolated at days 3 (early) and 13 (late) post-infection. In all cases, total RNA samples were isolated using RNeasy kit (Qiagen) and prepared at the Icahn School of Medicine at Mount Sinai Genomics Core Facility for poly A library construction and sequencing on IlluminaHiSeq 2500.
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Project description:Hypoxia increases histone methylation by inhibiting O2- and a-ketoglutarate-dependent histone lysine demethylases (KDMs). This study is the first to demonstrate how the hypoxic increment of methylated histones cross-talks with other epigenetic changes, such as histone clipping, and heterochromatin redistribution (senescence-associated heterochromatin foci, SAHF) found during oncogene-induced senescence (OIS). Raf activation in primary human fibroblasts IMR90 increased cathepsin L (CTSL)-mediated clipping of histone 3 (H3), H2B and H4 at H3 A21/T22, H2B T19/K20, and H4 G11/K12, respectively. Hypoxia protected H3 from CTSL by increasing histone methylation, especially at H3K23me3 without reducing the activity of CTSL. The maintenance of methylated histones is sufficient for protecting histones from CTSL, not sufficient but necessary for inhibiting SAHFs. Expression of cleaved H3 induces senescence even under hypoxia, suggesting that hypoxia disrupts this positive feedback loop of OIS by increasing histone methylation. Thus, hypoxia protects histones and chromatin from dramatic epigenetic changes by increasing histone methylation. These observations suggest that the OIS process increases the accessibility of chromatin, which hypoxic conditions inhibited.
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