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.

项目总结 生殖系永生对于物种的生存至关重要。从植物到动物的证据表明生殖系 永生依赖于促进基因组稳定的小RNA途径。在秀丽隐杆线虫种系中， 与PIWI ArgAerte蛋白PRG-1相互作用的小RNA-称为piRNAs-启动外源基因的沉默 然后由蠕虫宇航员(WAGO)维护的序列。核WAGO-9与核小体结合 通过组蛋白脱乙酰酶(HDA-1)启动异染色质的重塑和脱乙酰酶(NuRD)复合体 在piRNA靶标上形成。PiRNA途径缺陷激活外源序列的表达并扰乱细菌 细胞发育和生育能力。PiRNA途径和异染色质机制之间的合作如何 目前尚不清楚是否有促进生殖系永生的规定。 在线虫生殖系中，HDA-1组装到NuRD复合体中依赖于类泛素的小分子 修改器(相扑)。在表达不能SUMO化的突变体HDA-1的蠕虫中，piRNA介导的沉默， 生殖系异染色质形成和生育能力丧失。有趣的是，Hda-1的SUMO化不是其必需的 在体细胞胚胎中组装成NuRD复合体。综上所述，这些发现表明，piRNA途径 依靠相扑在种系中增选NuRD复合体组装，以沉默piRNA靶标。 这项建议试图探索种系NuRD复合体的组装及其通过piRNA途径的招募 都是受监管的。目标1将探索SUMOylated和生殖系特异性因子在防止违约中的作用 (与相扑无关)在生殖系中组装NuRD复合体。高度保守的MRG-1染色域 蛋白质是piRNA介导的沉默所必需的，并且是SUMO化的。初步数据显示Sumoylate 生殖系中的MRG-1使HDA-1的功能依赖于相扑。免疫共沉淀分析将是 用于确定SUMOylated MRG-1是否阻止未经修饰的HDA-1组装到生殖系中 努德情结。由于Sumoylated MRG-1在胚胎组织中含量丰富，因此这一目标也将使用质量 光谱分析和免疫荧光分析确定潜在的种系特异性因子与 SUMOylated MRG-1使种系HDA-1功能相扑依赖。目标2将决定HDA是否- 1 SUMO化促进与WAGO-9的相互作用。WAGO-9的假定相扑互动主题将是 突变，并将确定对piRNA介导的沉默和HDA-1相互作用的影响。这一目标还将 通过计算分析已发表的表征piRNA-靶相互作用强度的数据，以探索 PiRNA途径可能利用其对转录本的靶向，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促进生殖系 永生，并为该研究员提供遗传学、显微镜、生物化学和生物信息学方面的培训，以 为他们未来的职业生涯做好准备，成为一名独立调查员。