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).

项目摘要：异染色质是指压实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域，可转座元素（TES）， 作为一些基因。异染色质扮演着重要的建筑和调节作用，其不正当引线是 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， 胚胎致死性和无菌性。从酵母到人类的异染色质的大部分是组蛋白 H3赖氨酸9三甲基化（H3K9ME3）。 H3K9me3由Hisstone Mark“作家”复合体存放 通过DNA结合蛋白或小的RNA指南募集到基因组靶标。异染色质的许多方面 在细胞和开发中的建立和维护仍然知之甚少。异染色质 法规是该提议的核心重点。在生殖细胞中，PIWI蛋白和相关的PIWI相互作用 小型RNA（PIRNA）指南的作者建筑群以安装H3K9me3标记并引起转录沉默 在TE目标。 PIRNA的TE表达对于动物的生育至关重要，但其机制尚不清楚。 候选人以前的工作表明，在配置的Sumo E3连接酶SU（var）2-的染色质上定位 10在果蝇卵巢的生殖细胞中诱导异染色质形成。数据导致了SU（var）2-的模型 10在基因组靶标处与pirna-piwi形成一个复合物，并在尚未建立的因子（s）中沉积Sumo（Sumo） 这反过来招募了H3K9ME3作者DSETDB1。 SU（var）2-10还控制pirna-的H3K9me3沉积 独立的地方，包括几个沉默因素的基因，表明一种新型的负反馈机制 在异染色质水平和沉默因素之间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。该提案提出了阐明的策略 SU（VAR）2-10/SUMO在PIRNA引导的沉默中的作用，并研究自动调节和发育 SU（VAR）2-10依赖异染色质的继承。候选人将表征 SU（VAR）2-10使用最先进的蛋白质组学与RNAi（AIM 1）进行的SU（VAR）2-10修改，并使用 生化和遗传方法研究导致SU（VAR）2-10定位的机制和 SUMO依赖性DSETDB1募集到基因组靶标（AIM 2）。从长远来看，候选人将 通过负反馈调查异染色质调节的拟议模型，并研究 在发育过程中，PIWI和SU（VAR）2-10诱导的压抑染色质状态（AIM 3）。一起，这个 项目将提供有关生殖细胞中异染色质形成的深度机械洞察力，并解决 与正常细胞功能和疾病状态有关的表观遗传调节的基本原理。目标1和2 将在Caltech的Alexei Aravin博士实验室的K99阶段开始。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，并生成试剂和数据 未来的研究，可以平稳过渡到独立的研究人员阶段（AIM 3/R00）。