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

间期小鼠细胞中共价组蛋白标记 H3S10P 的全基因组分析和功能分析

• 批准号: RGPIN-2015-05228
• 负责人: Lorincz, Matthew
• 金额: $ 3.79万
• 依托单位: University of British Columbia
• 依托单位国家: 加拿大
• 项目类别: Discovery Grants Program - Individual
• 财政年份: 2017
• 资助国家: 加拿大
• 起止时间: 2017-01-01 至 2018-12-31
• 项目状态: 已结题
• 来源: https://www.nserc-crsng.gc.ca/ase-oro/Details-Detailles_eng.asp?id=629076
• 关键词: Genome; wide; profiling; functional; analysis

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.

在真核细胞中，DNA链与称为组蛋白的结构蛋白结合，形成称为染色质的复合体。染色质有助于将遗传物质-染色体-包装成细胞核中的浓缩结构。组蛋白的化学修饰也影响基因是开启（“转录”）还是关闭。例如，我们的实验室以前使用小鼠作为模型系统，在组蛋白H3 （H3K9me）的氨基酸#9（赖氨酸(K)）上添加甲基，会导致称为“异染色质”的浓缩染色质的形成，从而关闭转录，这种修饰沉积在现在驻留在基因组中的古代逆转录病毒（内源性逆转录病毒，erv）的残余上，以关闭这些寄生基因组元件的表达。事实上，新的ERV插入事件会诱导自发的H3K9me从逆转录病毒序列扩散到周围的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的磷酸化可以对抗异染色质的繁殖，以维持小鼠转录组的完整性，正如之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——它最著名的是作为分裂细胞（有丝分裂）中高度浓缩的染色体的标志，但也与细胞周期（间期）其余部分的活跃转录基因有关。生物化学上，H3S10ph可以阻断向H3K9添加甲基的酶，但其在间期细胞中的分布和作用尚未在动物细胞中得到广泛的表征。我们使用细胞周期阶段荧光标记结合“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持续存在于小鼠胚胎干细胞的间期，以覆盖基因组中富含基因的区域。引人注目的是，我们发现H3S10ph也与首先复制的基因组区域呈正相关，这可能导致一种防止异染色质包装和延迟复制的新机制。我们建议使用全基因组测序方法，确定在间期将磷酸基团添加到H3S10上的特定酶，并了解其对H3K9me扩散的影响，进而影响异染色质的形成，以及细胞周期中基因组特定区域复制的时间。我们的长期目标是利用小鼠作为模型系统，表征间期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、一名本科生和一名博士后参与这个项目，并在这个过程中获得重要的技能。