Investigating the role of Sumo in piRNA-mediated germline heterochromatin maintenance in C.elegans

研究 Sumo 在 piRNA 介导的线虫种系异染色质维持中的作用

• 批准号: 10750099
• 负责人: Johan Girgenrath
• 金额: $ 3.25万
• 依托单位: UNIV OF MASSACHUSETTS MED SCH WORCESTER
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-07 至 2026-07-06
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10750099
• 关键词: Affect; Animals; Binding; Biochemistry; Bioinformatics; Biological Assay; Biological Process; Caenorhabditis elegans; Chromatin; Clustered Regularly Interspaced Short Palindromic Repeats; Co-Immunoprecipitations; Collaborations; Complex; Data; Deacetylase; Defect; Development; Developmental Biology; Embryo; Ensure; Fertility; Future; Gene Expression; Gene Silencing; Generations; Genetic; Genome Stability; Germ Cells; Goals; Gonadal structure; Heterochromatin; Histone Deacetylase; Human; Hybrids; Immunofluorescence Immunologic; Knock-out; Laboratory Finding; Maintenance; Mass Spectrum Analysis; Mediating; Messenger RNA; Microscopy; Mus; Mutate; Nuclear; Nucleosomes; Pathway interactions; Plants; Proteins; Publishing; Regulation; Research Personnel; Role; Site; Small RNA; Sumoylation Pathway; Testing; Tissues; Training; Transcript; Ubiquitin; Work; career; embryo tissue; genetic information; induced pluripotent stem cell; insight; mRNA sequencing; mutant; piRNA; prevent; recruit; self-renewal; transmission process

PROJECT SUMMARY Germline immortality is essential for species survival. Evidence from plants to animals suggests germline immortality depends on small RNA pathways that promote genome stability. In Caenorhabditis elegans germline, small RNAs that interact with the PIWI Argonaute protein PRG-1—called piRNAs—initiate silencing of foreign sequences that is then maintained by Worm Argonautes (WAGOs). Nuclear WAGO-9 binds the nucleosome remodeling and deacetylase (NuRD) complex via histone deacetylase (HDA-1), which initiates heterochromatin formation on piRNA targets. piRNA pathway defects activate expression of foreign sequences and disrupt germ cell development and fertility. How collaboration between the piRNA pathway and heterochromatin machinery is regulated to promote germline immortality is unclear. In the C. elegans germline, assembly of HDA-1 into the NuRD complex depends on the small ubiquitin-like modifier (SUMO). In worms expressing mutant HDA-1 that cannot be SUMOylated, piRNA-mediated silencing, germline heterochromatin formation, and fertility are lost. Interestingly, HDA-1 SUMOylation is not needed for its assembly into the NuRD complex in somatic embryo. Together, these findings suggest that the piRNA pathway relies on SUMO to co-opt NuRD complex assembly in the germline for silencing of piRNA targets. This proposal seeks to explore how germline NuRD complex assembly and its recruitment by the piRNA pathway are regulated. Aim 1 will explore the role of SUMOylated and germline-specific factors in preventing default (SUMO independent) assembly of the NuRD complex in germline. The highly-conserved MRG-1 chromodomain protein is essential for piRNA-mediated silencing and is SUMOylated. Preliminary data suggest SUMOylated MRG-1 in germline renders HDA-1 function to be SUMO-dependent. Co-immunoprecipitation assays will be used to determine whether SUMOylated MRG-1 prevents unmodified HDA-1 from assembling into the germline NuRD complex. Because SUMOylated MRG-1 is abundant in embryonic tissue, this aim will also use mass spectrometry and immunofluorescence assays to identify potential germline-specific factors interacting with SUMOylated MRG-1 to render germline HDA-1 function SUMO dependent. Aim 2 will determine whether HDA- 1 SUMOylation promotes interaction with WAGO-9. Putative SUMO-interacting motifs of WAGO-9 will be mutated and the effect on piRNA-mediated silencing and HDA-1interaction will be determined. This aim will also computationally analyze published data characterizing strength of piRNA-target interactions to explore how the piRNA pathway may utilize its targeting of transcripts to enact downstream silencing via heterochromatin. This work will reveal how highly conserved small RNA and chromatin factors coordinate to promote germline immortality, and provide the fellow with training in genetics, microscopy, biochemistry, and bioinformatics to prepare them for a future career as an independent investigator.

项目概要 种系永生对于物种生存至关重要。从植物到动物的证据表明种系 永生取决于促进基因组稳定性的小RNA途径。在秀丽隐杆线虫种系中， 与 PIWI Argonaute 蛋白 PRG-1 相互作用的小 RNA（称为 piRNA）会启动外源基因的沉默 然后由 Worm Argonautes (WAGO) 维护的序列。核 WAGO-9 结合核小体 通过组蛋白脱乙酰酶 (HDA-1) 形成的重塑和脱乙酰酶 (NuRD) 复合物，启动异染色质 piRNA 靶标上的形成。 piRNA 通路缺陷激活外源序列的表达并破坏细菌 细胞发育和生育能力。 piRNA 途径和异染色质机制之间的协作如何 促进种系永生的调节尚不清楚。 在秀丽隐杆线虫种系中，HDA-1 组装到 NuRD 复合体中取决于小的泛素样 修饰符（相扑）。在表达突变 HDA-1 的蠕虫中，HDA-1 不能被 SUMO 化，piRNA 介导的沉默， 种系异染色质形成，生育能力丧失。有趣的是，HDA-1 SUMOylation 不需要其 在体细胞胚胎中组装成 NuRD 复合体。总之，这些发现表明 piRNA 途径 依靠 SUMO 在种系中选择 NuRD 复合物组装来沉默 piRNA 靶点。 该提案旨在探索种系 NuRD 复合物如何组装及其通过 piRNA 途径的招募 受到监管。目标 1 将探讨 SUMO 化和种系特异性因子在预防违约方面的作用 （独立于 SUMO）NuRD 复合物在种系中的组装。高度保守的 MRG-1 染色结构域 蛋白质对于 piRNA 介导的沉默至关重要，并且被 SUMO 化。初步数据表明 SUMO 化 种系中的 MRG-1 使 HDA-1 功能具有 SUMO 依赖性。免疫共沉淀分析将 用于确定 SUMOylated MRG-1 是否阻止未修饰的 HDA-1 组装到种系中 NuRD复合体。由于 SUMOylated MRG-1 在胚胎组织中含量丰富，因此该目标还将使用质量 光谱测定法和免疫荧光测定法来识别潜在的种系特异性因子相互作用 SUMO 化的 MRG-1 使种系 HDA-1 功能依赖于 SUMO。目标 2 将确定 HDA- 1 SUMO 化促进与 WAGO-9 的相互作用。 WAGO-9 的假定 SUMO 相互作用基序将是 突变以及对 piRNA 介导的沉默和 HDA-1 相互作用的影响将被确定。这一目标也将 通过计算分析已发表的表征 piRNA-靶标相互作用强度的数据，以探索如何 piRNA 途径可能利用其转录本的靶向性，通过异染色质实现下游沉默。这 这项工作将揭示高度保守的小RNA和染色质因子如何协调以促进种系 永生，并为研究员提供遗传学、显微镜学、生物化学和生物信息学方面的培训 为他们未来作为独立调查员的职业生涯做好准备。