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Interphase histone H3 serine 10 phosphorylation in mouse embryonic stem cells Chen, Carol Chia-Lu

Abstract

Phosphorylation of histone H3 at serine 10 (H3S10ph) has been observed in two paradoxical contexts, depending on the stage of the cell cycle – it is a hallmark of condensed mitotic chromatin, but is also found at specific mitogen-inducible genic promoters and distal enhancers. In the work presented in this thesis, I derived a mouse embryonic stem cell (ESC) line harbouring an endogenous fluorescent cell cycle reporter (FUCCI) in combination with next-generation sequencing to comprehensively map H3S10ph at distinct stages of the mammalian cell cycle and examined the crosstalk between this mark and the repressive mark, H3K9me. I found that H3S10ph broadly demarcates gene-rich, early-replicating euchromatic regions in G1, marking up to 30% of the ESC genome. Reminiscent of H3S10ph deposited by JIL-1 kinase in Drosophila, interphase H3S10ph pervasively marks gene-dense regions to prevent the accumulation of H3K9me2 at actively transcribed genes in ESCs. In contrast to H3S10ph at euchromatin, mitotic phosphorylation mediated by Aurora kinase is also detectable in ESCs in G1 at young endogenous retroviruses (ERVs), specifically in combination with H3K9me3. Finally, to identify the kinases responsible for interphase H3S10ph, I generated knockout (KO) ESC lines of serine kinases homologous to JIL-1, including Msk1/2 and Rsk1/2/3/4. Msk2-/- ESCs showed the greatest loss of interphase H3S10ph at active promoter/enhancers. Furthermore, known H3K9me3 repressed targets, including imprinted genes, young ERVs and germline genes, were downregulated in MSK2-deficient cells. Taken together, this work revealed that H3S10ph plays a previously unappreciated role in interphase chromatin architecture and facilitates appropriate genic transcription by countering the repressive effects exerted by H3K9 methyltransferases.

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