Project description:Senescence is a state of stable cell cycle exit that has important implications for development, physiology and disease. It is distinct from quiescence in which cells can be induced to re-enter the cell cycle. Although it is well known that there are massive changes in the heterochromatin of senescent cells, the molecular mechanisms underpinning the transition from reversible quiescence into irreversible senescence have remained elusive. Here, we demonstrate that the chromatin-remodeling enzyme ATRX is required for senescence. ATRX accumulates in nuclear foci during both replicative and cellular senescence. Using ChIP-seq and RNA-seq we identified HRAS as part of an ATRX regulated gene expression program associated with senescence. Repression of HRAS is sufficient to promote the transition of quiescent cells into senescence. Thus we conclude that the repression of HRAS is likely a direct consequence of ATRX binding and critical to how it mediates its role in senescence.
Project description:Senescence is a state of stable cell cycle exit that has important implications for development, physiology and disease. It is distinct from quiescence in which cells can be induced to re-enter the cell cycle. Although it is well known that there are massive changes in the heterochromatin of senescent cells, the molecular mechanisms underpinning the transition from reversible quiescence into irreversible senescence have remained elusive. Here, we demonstrate that the chromatin-remodeling enzyme ATRX is required for senescence. ATRX accumulates in nuclear foci during both replicative and cellular senescence. Using ChIP-seq and RNA-seq we identified HRAS as part of an ATRX regulated gene expression program associated with senescence. Repression of HRAS is sufficient to promote the transition of quiescent cells into senescence. Thus we conclude that the repression of HRAS is likely a direct consequence of ATRX binding and critical to how it mediates its role in senescence.
Project description:We applied in parallel RNA-Seq and Ribosome-profiling analyses to immortalized human primary BJ fibroblast cells under the following conditions: normal proliferation, quiescence (induced by serum depletion), senescence (induced by activation of the oncogenic RASG12V gene, and examined at early (5 days; pre-senescent state) and late (14 days; fully senescent state) time points), and neoplastic transformation (induced by RASG12V in the background of stable p53 and p16INK4A knockdowns and SV40 small-T expression. RNA-seq, using Illumina HiSeq 2000, was applied to BJ cells under 5 conditions: proliferation, quiescence, pre-senescence, full-senescence, and transfomed. Ribosome profiling, using Illumina HiSeq 2000, was applied to BJ cells under 5 conditions: proliferation, quiescence, pre-senescence, full-senescence, and transfomed.
Project description:ATRX is a member of the SWI2/SNF2 family of chromatin remodeling proteins and primarily functions at heterochromatic loci via its recognition of M-bM-^@M-^XrepressiveM-bM-^@M-^Y histone modifications (e.g., H3K9me3). Despite significant roles for ATRX during normal neural development, as well as its relationship to human disease, ATRX function in the central nervous system is not well understood. Here, we describe ATRXM-bM-^@M-^Ys ability to recognize an activity-dependent combinatorial histone modification, H3K9me3S10ph, in post-mitotic neurons. In neurons, this M-bM-^@M-^\methyl/phosM-bM-^@M-^] switch occurs exclusively following periods of stimulation and is highly enriched at heterochromatic repeats associated with centromeres. Using a multifaceted approach, we reveal that H3K9me3S10ph bound Atrx represses non-coding transcription of centromeric minor satellite sequences during instances of heightened activity. Our results indicate an essential interaction between ATRX and a previously uncharacterized histone modification in the central nervous system and suggest a potential role for abnormal repetitive element transcription in pathological states manifested by ATRX dysfunction. For Atrx ChIP-seq, IPs were performed on three control vs. three KCl stimulated (all representing biological, and not technical replicates) primary cultured mouse cortical neurons at DIV 8. All samples were normalized to background input levels. For H3K9me3S10phos ChIP-seq, biological singlecates (control vs. forskolin) were analyzed against respective inputs.
