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.

项目摘要 种系永生对于物种生存至关重要。从动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（称为piRNA）相互作用的小rNA - 外国的沉默 然后由蠕虫（Wagos）维护的序列。核Wago-9结合核小体 通过组蛋白脱乙酰基酶（HDA-1）的重塑和脱乙酰基酶（NURD）复合物，启动异染色质 piRNA靶标的形成。 piRNA途径缺陷激活异物的表达并破坏胚芽 细胞发育和生育能力。 Pirna途径与异染色质机械之间的协作如何 受监管以促进种系永生尚不清楚。 在秀丽隐杆线虫种系中，HDA-1在NURD复合体中的组装取决于小泛素样 修饰符（Sumo）。在表达不可掺入的突变体HDA-1的蠕虫中，piRNA介导的沉默， 种系异染色质形成和生育能力丧失。有趣的是，HDA-1 sumoylation不需要 在体细胞胚胎中组装到NURD综合体中。这些发现一起表明PIRNA途径 依靠Sumo在种系中的同采用复合物组件来沉默PIRNA靶标。 该提议旨在探讨培养培养基大会及其在皮尔纳途径的招募如何 AIM 1将探讨Sumoylated和种系特定因素在防止违约方面的作用 （Sumo独立）在种系中的NURD复合物的组装。高度保存的MRG-1染色域 蛋白质对于PIRNA介导的沉默至关重要，并被掺入蛋白质。初步数据建议 种系中的MRG-1使HDA-1函数依赖于SUMO。共免疫沉淀测定将是 用于确定sumoy的MRG-1是否阻止未修饰的HDA-1组装到种系中 努尔德综合体。由于sumoy的MRG-1在胚胎组织中很丰富，因此此目标也将使用质量 光谱法和免疫荧光测定法，以识别潜在的种系特异性因素与 Sumoypated MRG-1以渲染种系HDA-1功能相关。 AIM 2将确定HDA-是否是否 1 sumoylation促进与Wago-9的相互作用。 Wago-9的推定相互作用图案将是 将确定对PIRNA介导的沉默和HDA-1互动的突变和影响。这个目标也将 计算分析已发布的数据表征了pirna-target相互作用的强度，以探索如何 PIRNA途径可以利用其对转录本的靶向通过异染色质进行下游沉默。这 工作将揭示高度保守的小RNA和染色质因子如何辅助促进种系 永生，并为研究员提供遗传学，显微镜，生物化学和生物信息学的培训 为他们作为独立调查员的未来职业做好准备。