Elucidating the role of SUMO ligase Su(var)2-10 in piRNA-guided transcriptional silencing and repressive chromatin formation

项目摘要：异染色质是指紧凑的和转录抑制的染色质状态 这通常包括染色体臂末端附近的富含重复序列的区域，以及转座元件(TES) 作为某些基因。异染色质起着重要的结构和调节作用，它的错误调节导致 与癌症、衰老和生殖细胞相关的基因表达异常和染色体不稳定， 胚胎致死和不育。从酵母到人类的大部分异染色质都以组蛋白为标志 H3赖氨酸9三甲基化(H3K9me3)。H3K9Me3是由组蛋白标记的“写入者”络合物沉积的，可以 通过DNA结合蛋白或小RNA引导被招募到基因组靶点。异染色质的许多方面 细胞内和发育中的建立和维持仍然知之甚少。异染色质 监管是这项提议的核心焦点。在生殖细胞中，Piwi蛋白及其相关的Piwi相互作用 小RNA(PiRNAs)引导作家复合体安装H3K9me3标记并诱导转录沉默 瞄准TE目标。由piRNAs抑制的TE对于动物的生育是必不可少的，但其机制尚不清楚。 候选人先前的工作表明，保守的相扑E3连接酶SU(Var)2- 10诱导果蝇卵巢生殖细胞异染色质形成。数据导致了一个模型SU(Var)2- 10在基因组靶点与piRNA-Piwi形成复合体，并在尚未建立的因子上沉积相扑(S)， 它又招募了H3K9me3编写器dSetDB1。SU(Var)2-10还控制在piRNA-上的H3K9me3沉积。 独立的基因座，包括几个沉默因子的基因，表明一种新的负反馈机制 异染色质水平和沉默因子之间的关系可以解释生殖细胞如何维持 异染色质水平，以确保适当的基因组功能。这项提议提出了一种战略，以澄清 SU(Var)2-10/SUMO在piRNA引导的沉默中的作用及其自身调节和发育的研究 SU(Var)2-10依赖异染色质的遗传。候选人将描述以下底物的特征 SU(Var)2-10使用最先进的蛋白质组学结合RNAi进行相扑修饰(目标1)，并使用 生化和遗传学方法研究导致SU(Var)2-10定位和 相扑依赖的dSetDB1向基因组靶的重新招募(目标2)。从长远来看，候选人将 研究了负反馈调节异染色质的模型，并研究了该模型的稳定性 在发育过程中抑制Piwi和SU(Var)2-10诱导的染色质状态(目标3)。总而言之，这 该项目将提供对生殖细胞中异染色质形成的深刻机械见解，并解决 与正常细胞功能和疾病状态相关的表观遗传调控的基本原理。目标1和目标2 将在加州理工大学阿列克谢·阿拉文博士的实验室K99阶段启动。这个环境将提供所有 必要的研究设施和培训，以实现拟议的目标，并产生试剂和数据 未来研究，允许平稳过渡到独立研究阶段(AIM 3/R00)。

Project Summary: Heterochromatin refers to the compacted and transcriptionally suppressed chromatin state that typically includes repeat-rich regions near chromosomal arm ends, transposable elements (TEs), as well as some genes. Heterochromatin plays important architectural and regulatory roles, and its misregulation leads to aberrant gene expression and chromosome instability associated with cancers, aging and in germ cells, embryonic lethality and sterility. Large fraction of heterochromatin from yeast to humans is marked by histone H3 lysine 9 trimethylation (H3K9me3). H3K9me3 is deposited by histone mark “writer” complexes which can be recruited to genomic targets by DNA binding proteins or small RNA guides. Many aspects of heterochromatin establishment and maintenance in the cell and in development remain poorly understood. Heterochromatin regulation is the central focus of this proposal. In germ cells, Piwi proteins and associated Piwi-interacting small RNAs (piRNAs) guide a writer complex to install the H3K9me3 mark and induce transcriptional silencing at TE targets. TE repression by piRNAs is essential for animal fertility, yet its mechanism is not known. Candidate's previous work showed that localization to chromatin of the conserved SUMO E3 ligase Su(var)2- 10 induces heterochromatin formation in germ cells of the Drosophila ovary. Data led to a model that Su(var)2- 10 forms a complex with piRNA-Piwi at genomic targets, and deposits SUMO at yet-to-be-established factor(s), which in turn recruits the H3K9me3 writer dSetDB1. Su(var)2-10 also controls H3K9me3 deposition at piRNA- independent loci, including genes of several silencing factors, indicating a novel negative feedback mechanism between heterochromatin levels and silencing factors that can explain how germ cells maintain heterochromatin levels to ensure proper genome function. This proposal presents a strategy to elucidate the role of Su(var)2-10/SUMO in piRNA-guided silencing, and to investigate the auto-regulation and developmental inheritance of Su(var)2-10 dependent heterochromatin. The candidate will characterize the substrates of SUMO modification by Su(var)2-10 using state-of-the-art proteomics coupled with RNAi (Aim 1), and use biochemical and genetic approaches to investigate the mechanisms that lead to Su(var)2-10 localization and SUMO-dependent dSetDB1 recruitment to genomic targets (Aim 2). In the long term, the candidate will investigate the proposed model of heterochromatin regulation by negative feedback, and study the stability of repressed chromatin states induced by Piwi and Su(var)2-10 across development (Aim 3). Together, this project will provide deep mechanistic insight into heterochromatin formation in germ cells, and address fundamental principles of epigenetic regulation relevant to normal cell function and disease states. Aim 1 and 2 will be initiated during the K99 phase in Dr. Alexei Aravin's lab at Caltech. This environment will provide all necessary research facilities and training to achieve the proposed goals, and to generate reagents and data for future studies, allowing a smooth transition to an independent researcher phase (Aim 3/R00).