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

在真核细胞中，DNA链与称为组蛋白的结构蛋白结合，形成一种称为染色质的复合体。染色质有助于将遗传物质--染色体--包装成核内的浓缩结构。组蛋白的化学修饰也会影响基因的开启(“转录”)或关闭。例如，我们的实验室以前已经使用小鼠作为一个模型系统，表明向组蛋白H3(H3K9Me)的氨基酸#9(赖氨酸(K))添加甲基会导致凝聚染色质的形成，称为“异染色质”，以关闭转录，并且这种修饰沉积在古代逆转录病毒(内源性逆转录病毒，ERV)的残留物上，以切断这些寄生基因组元件的表达。事实上，新的ERV插入到基因组中的事件会诱导自发的H3K9me从逆转录病毒序列传播到周围的DNA区域，并可能使邻近基因失活。我们假设，组蛋白H3尾部(H3S10ph)上邻近氨基酸丝氨酸10的磷酸化作用可以抑制异染色质的传播，以维持小鼠转录组的完整性，正如以前在果蝇中所显示的那样。保守的H3S10ph以两种矛盾的形式存在--它最为人所知的是细胞分裂中高度浓缩的染色体的标志(有丝分裂)，但也与细胞周期其余部分(间期)活跃转录基因有关。在生物化学方面，H3S10ph阻止了向H3K9添加甲基的酶，但它在间期细胞中的分布和作用在动物细胞中尚未得到广泛的表征。我们使用了细胞周期阶段的荧光标记，并结合“高通量”测序来跟踪H3S10ph随细胞周期的动态变化，并发现，令人惊讶的是，H3S10ph持续处于间期，覆盖了小鼠胚胎干细胞基因组中基因丰富的区域。值得注意的是，我们发现H3S10ph也与首先复制的基因组区域呈正相关，可能导致了一种防止异染色质包装和延迟复制的新机制。我们建议利用全基因组测序方法，鉴定在间期将磷酸基团添加到H3S10中的特定酶，并了解其对H3K9me扩散和异染色质形成的影响，以及对细胞周期中基因组特定区域复制时间的影响。我们的长期目标是利用小鼠作为模型系统，表征界面H3S10ph的功能，并了解其功能作用。为此，我们希望招募一名研究生以及本科生和博士后研究员参与这一项目，并在此过程中获得重要技能。**

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.**