Abstract
Cellular senescence involves epigenetic alteration, e.g. loss of H3K27me3 in Ink4a-Arf locus. Using mouse embryonic fibroblast (MEF), we here analyzed transcription and epigenetic alteration during Ras-induced senescence on genome-wide scale by chromatin immunoprecipitation (ChIP)-sequencing and microarray. Bmp2 was the most activated secreted factor with H3K4me3 gain and H3K27me3 loss, whereas H3K4me3 loss and de novo formation of H3K27me3 occurred inversely in repression of nine genes, including two BMP-SMAD inhibitors Smad6 and Noggin. DNA methylation alteration unlikely occurred. Ras-activated cells senesced with nuclear accumulation of phosphorylated SMAD1/5/8. Senescence was bypassed in Ras-activated cells when Bmp2/Smad1 signal was blocked by Bmp2 knockdown, Smad6 induction, or Noggin induction. Senescence was induced when recombinant BMP2 protein was added to Bmp2-knocked-down Ras-activated cells. Downstream Bmp2-Smad1 target genes were then analyzed genome-wide by ChIP-sequencing using anti-Smad1 antibody in MEF that was exposed to BMP2. Smad1 target sites were enriched nearby transcription start sites of genes, which significantly correlated to upregulation by BMP2 stimulation. While Smad6 was one of Smad1 target genes to be upregulated by BMP2 exposure, Smad6 repression in Ras-activated cells with increased enrichment of Ezh2 and gain of H3K27me3 suggested epigenetic disruption of negative feedback by Polycomb. Among Smad1 target genes that were upregulated in Ras-activated cells without increased repressive mark, Parvb was found to contribute to growth inhibition as Parvb knockdown lead to escape from senescence. It was revealed through genome-wide analyses in this study that Bmp2-Smad1 signal and its regulation by harmonized epigenomic alteration play an important role in Ras-induced senescence.
Highlights
Cellular senescence was first described as the limited replicative capacity of primary cells in culture [1]
We identify that Bmp2-Smad1 signal is critical
We show dynamic and coordinated H3K27me3 alteration, e.g. activation of Bmp2 by loss of H3K27me3, repression of the signal inhibitors and the negative feedback loop by gain of H3K27me3, and selective activation of downstream target genes that may contribute to growth arrest
Summary
Cellular senescence was first described as the limited replicative capacity of primary cells in culture [1]. Activated oncogenes can induce premature form of cellular senescence, and cells fall into irreversible arrest to block cellular proliferation [2,3]. In addition to cell death programs such as apoptosis and autophagy, oncogene-induced senescence is recognized as a potent barrier against oncogenic transformation, suppressing unscheduled proliferation of early neoplastic cells [4,5,6,7]. Replicative senescence and oncogene-induced senescence are known to comprise activation of tumor suppressor pathways including p16Ink4a-Rb and p19Arf (p14ARF in human)-p53 signaling cascades. The roles of RB and p53 signaling pathways in senescence are undisputed, it has become clear that other factors are involved. The induction of senescence required several secreted factors including members of Wnt, insulin, transforming growth factor-b, plasmin and interleukin signaling cascades [11]
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