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

阐明 SUMO 连接酶 Su(var)2-10 在 piRNA 引导的转录沉默和抑制染色质形成中的作用

• 批准号: 10425661
• 负责人: Maria Antoninova NINOVA
• 金额: $ 24.87万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-01 至 2024-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10425661
• 关键词: Address; Affect; Aging; Animals; Architecture; Biochemical; Biochemical Genetics; Biological Assay; Cell physiology; Cells; Chromatin; Chromatin Remodeling Factor; Chromosomal Instability; Chromosomes; Complex; Coupled; DNA; DNA Damage; DNA Transposable Elements; DNA-Binding Proteins; Data; Defect; Deposition; Development; Disease; Drosophila genus; Embryo; Embryonic Development; Ensure; Environment; Epigenetic Process; Eukaryota; Event; Feedback; Female; Fertility; Future; Gametogenesis; Gene Expression; Gene Silencing; Genes; Genetic; Genetic Recombination; Genetic Transcription; Genome; Genomic Segment; Genomics; Germ Cells; Goals; Guide RNA; Heterochromatin; Histone H3; Histones; Homeostasis; Human; Image; In Vitro; Knowledge; Lead; Ligase; Lysine; Maintenance; Malignant Neoplasms; Methods; Modeling; Modification; Molecular; Monitor; Normal Cell; Ovary; Pathway interactions; Phase; Play; Post-Translational Protein Processing; Process; Proteins; Proteomics; RNA Interference; Reagent; Regulation; Repetitive Sequence; Reporter; Repression; Research Personnel; Research Training; Role; Small RNA; Sterility; Structure; Sumoylation Pathway; System; Testing; Work; Yeasts; arm; discrete time; epigenetic regulation; epigenetic silencing; genetic approach; genetic element; genome-wide; genomic locus; histone methyltransferase; histone modification; in vivo; insight; novel; piRNA; recruit; research facility; sensor; time interval; ubiquitin-protein ligase

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).

项目概述：异染色质是指紧密的和转录抑制的染色质状态 通常包括染色体臂末端附近的重复区，转座因子（TE），以及 就像一些基因一样。异染色质起着重要的结构和调节作用，其失调导致 与癌症、衰老和生殖细胞相关的异常基因表达和染色体不稳定性， 胚胎致死和不育。从酵母到人类的大部分异染色质是由组蛋白标记的 H3赖氨酸9三甲基化（H3 K9 me 3）。H3 K9 me 3由组蛋白标记“写入器”复合物沉积， 通过DNA结合蛋白或小RNA向导募集到基因组靶标。异染色质的许多方面 在细胞和发育中的建立和维持仍然知之甚少。异染色 监管是这项建议的中心重点。在生殖细胞中，Piwi蛋白和相关的Piwi相互作用 小RNA（piRNA）引导写入复合物安装H3 K9 me 3标记并诱导转录沉默 在TE目标。piRNA对TE的抑制对于动物生育力至关重要，但其机制尚不清楚。 候选人先前的工作表明，保守的SUMO E3连接酶Su（var）2- 10诱导果蝇卵巢生殖细胞中异染色质的形成。数据导致了一个模型，苏（var）2- 10与piRNA-Piwi在基因组靶标处形成复合物，并在尚未建立的因子处沉积SUMO， 它又招募H3 K9 me 3写入器dSetDB 1。Su（var）2-10还控制在皮尔纳上的H3 K9 me 3沉积。 独立位点，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联系，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。该提案提出了一项战略， Su（var）2-10/SUMO在piRNA介导的沉默中的作用，并研究其自身调节和发育 Su（var）2-10依赖异染色质遗传候选人将表征以下物质的底物： 使用最先进的蛋白质组学与RNAi偶联的Su（var）2-10的SUMO修饰（Aim 1），以及使用 生物化学和遗传学方法来研究导致Su（var）2-10定位的机制， SUMO依赖性dSetDB 1募集至基因组靶标（目的2）。从长远来看，候选人将 研究所提出的异染色质负反馈调节模型，并研究 Piwi和Su（var）2-10在整个发育过程中诱导的抑制染色质状态（Aim 3）。在一起，这 项目将提供深入的机制洞察异染色质形成在生殖细胞，并解决 与正常细胞功能和疾病状态相关的表观遗传调节的基本原理。目标1和2 将在K99阶段在加州理工学院的阿列克谢·阿拉文博士的实验室启动。这种环境将提供所有 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 未来的研究，允许平稳过渡到独立研究阶段（目标3/R 00）。