Genome-wide profiling and functional analysis of the covalent histone mark H3S10P in interphase mouse cells

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In eukaryotic cells, DNA strands are bound to structural proteins called histones, forming a complex called chromatin. Chromatin facilitates the packaging of the genetic material- the chromsomes- into condensed structures in the nucleus. Chemical modification of histones also influences whether genes are turned on (“transcribing”) or off. For example, our lab has previously shown using the mouse as a model system that addition of methyl groups to amino acid #9, a lysine (K), of histone H3 (H3K9me) causes formation of condensed chromatin called “heterochromatin” to shut off transcription, and this modification is deposited over remnants of ancient retroviruses (Endogenous Retroviruses, ERVs) that now reside in the genome to shut off the expression of these parasitic genomic elements. In fact, new ERV insertion events into the genome induce spontaneous H3K9me spreading beyond the retroviral sequences into the surrounding DNA regions, and could potentially inactivate neighbouring genes. We hypothesize that the phosphorylation of the adjacent amino acid, serine 10, on the histone H3 tail (H3S10ph) acts to counter the propagation of heterochromatin to maintain the integrity of the mouse transcriptome, as has been shown previously in fruit flies. The conserved H3S10ph exist in two paradoxical forms - it is best known as a hallmark of highly condensed chromosomes in dividing cells (mitosis), but is also associated with actively transcribing genes in the rest of the cell cycle (interphase). Biochemically, H3S10ph blocks the enzymes that add a methyl group to H3K9, but its distribution and role in interphase cells have not been extensively characterized in animal cells. We used a fluorescent marker of cell cycle stages coupled with “high-throughput” sequencing to follow the dynamics of H3S10ph with the cell cycle, and found that, surprisingly, H3S10ph persists in interphase to blanket gene-rich regions of the genome in mouse embryonic stem cells. Strikingly, we found H3S10ph also positively correlates with genomic regions that are replicated first, possibly resulting in a novel mechanism to prevent heterochromatin packaging and delayed replication. We propose to identify the specific enzyme that adds the phosphate group to H3S10 in interphase and to understand its influence on H3K9me spreading and in turn formation of heterochromatin as well as on the timing of replication of specific regions of the genome during the cell cycle, using genome-wide sequencing methodologies. Our long-term goal is to characterize the function of interphase H3S10ph and understand its functional role, using the mouse as a model system. Towards this end, we hope to recruit a graduate student as well as undergraduate students and a postdoctoral fellow to work on this project and gain important skills in the process.