Capturing the dynamic epigenome using single molecule and single cell approaches

摘要 我们的身体由数十亿个基因相同的细胞组成，这些细胞可以表现出不同的表型或表观遗传 states.组蛋白的共价和可逆修饰使细胞能够建立遗传基因 而不改变它们的基因蓝图。控制基因的表观遗传机制 表达对于维持细胞身份和程序多细胞分化是必需的。组蛋白H3赖氨酸 9甲基化（H3K9me）与转录沉默和异染色质形成相关。裂殖酵母 （S.粟酒裂殖酵母）具有最低限度的异染色质结构，其适合于高通量遗传学， 生物化学三种保守蛋白调节异染色质，包括：1）H3K9me特异性 2）H3K9me特异性“阅读器”，结合H3K9me的Swi6HP 1 3）H3K9me特异性的“擦除器”Epe1JmjC，它与H3K9me的转录相反， 异染色质装配和表观遗传。Clr4与四环素诱导的TetR DNA的融合 结合结构域促进异染色质装配的快速和可逆控制。我的实验室很创新 遗传策略使我们能够识别具有独特作用的染色质相关因子，这些因子仅限于 异染色质维持作为促进健康相关领域多样性的研究补充材料的一部分， 研究计划，补充资金申请将调查影响表观遗传的机制 在H3K9甲基化建立的下游沉默。具体来说，受此支持的研究生 应用将使用体外重建和单分子方法来研究动态染色质 导致表观遗传沉默的基因重排。补充奖将促进研究，职业生涯和 属于NIH指定的代表性不足类别的研究生的专业培训。的 本申请中概述的研究计划与 候选人未来的职业抱负。此外，这里描述的所有科学目标都是完全一致的。 家长奖的研究方向。

ABSTRACT Our bodies consist of billions of genetically identical cells that can exhibit distinct phenotypic or epigenetic states. The covalent and reversible modification of histones enables cells to establish heritable gene expression patterns without altering their genetic blueprint. Epigenetic mechanisms that control gene expression are essential to maintain cellular identity and program multicellular differentiation. Histone H3 lysine 9 methylation (H3K9me) is associated with transcription silencing and heterochromatin formation. Fission yeast (S. pombe) has a minimalist heterochromatin architecture that is amenable to high-throughput genetics and biochemistry. A trio of conserved proteins regulates heterochromatin, which includes, 1) an H3K9me specific ''writer,'' Clr4Suv39h that catalyzes H3K9me 2) an H3K9me specific ''reader,'' Swi6HP1 that binds to H3K9me chromatin and silences transcription and, 3) an H3K9me specific ''eraser,'' Epe1JmjC, that opposes heterochromatin assembly and epigenetic inheritance. Fusing Clr4 to the tetracycline-inducible TetR DNA binding domain facilitates rapid and reversible control of heterochromatin assembly. My lab’s innovative genetic strategy has enabled us to identify chromatin-associated factors with unique roles that are restricted to heterochromatin maintenance. As part of the Research Supplements to Promote Diversity in Health-Related Research program, the supplemental funding request will investigate the mechanisms that influence epigenetic silencing downstream of H3K9 methylation establishment. Specifically, the graduate student supported by this application will use in vitro reconstitution and single-molecule approaches to investigate the dynamic chromatin rearrangements that lead to epigenetic silencing. The supplement award will promote the research, career and professional training of a graduate student belonging to an NIH designated underrepresented category. The program of research outlined in this application is well-aligned and collaboratively developed with the candidate’s future career aspirations. Furthermore, all of the scientific goals described here are fully consistent with the proposed research directions of the parent award.