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 生殖细胞的永生对物种的生存至关重要。从植物到动物的证据表明 永生依赖于促进基因组稳定性的小RNA途径。在秀丽隐杆线虫生殖系中， 与PIWI Argonaute蛋白PRG-1相互作用的小RNA-称为piRNA-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 重塑和脱乙酰酶（NuRD）复合物通过组蛋白脱乙酰酶（HDA-1），启动异染色质 形成皮尔纳靶点。皮尔纳途径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。皮尔纳通路和异染色质机制之间的协作是如何进行的？ 促进种系永生的监管尚不清楚。 在C.在线虫种系中，HDA-1组装成NuRD复合物依赖于小的泛素样 修饰符（SUMO）。在表达不能SUMO化的突变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化并不需要， 在体细胞胚中组装成NuRD复合体。总之，这些发现表明皮尔纳途径 依赖于SUMO在种系中协同NuRD复合物组装以沉默皮尔纳靶标。 该提案旨在探索生殖系NuRD复合物的组装及其通过皮尔纳途径的募集 是有规律的。目的1将探讨SUMO化和生殖系特异性因子在预防违约中的作用 (SUMO在种系中的NuRD复合物的独立）组装。高度保守的MRG-1染色体结构域 蛋白质是piRNA介导的沉默所必需的，并且是SUMO化的。初步数据表明SUMO化 生殖细胞中的MRG-1使HDA-1的功能具有SUMO依赖性。免疫共沉淀试验将 用于确定SUMO化的MRG-1是否阻止未修饰的HDA-1组装成种系 NuRD复合物。由于SUMO化的MRG-1在胚胎组织中丰富，因此该目标也将使用质量 光谱和免疫荧光测定，以确定潜在的种系特异性因子相互作用， SUMO化的MRG-1使种系HDA-1功能具有SUMO依赖性。目标2将决定HDA是否- 1 SUMO化促进与WAGO-9的相互作用。推测的WAGO-9的SUMO相互作用基序将是 突变，并确定对piRNA介导的沉默和HDA-1相互作用的影响。这一目标还将 计算分析表征piRNA-靶标相互作用强度的已发表数据，以探索 皮尔纳途径可以利用其对转录物的靶向通过异染色质来实施下游沉默。这 这项工作将揭示高度保守的小RNA和染色质因子是如何协调促进生殖细胞的生长的。 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方面的培训， 为他们将来成为独立调查员做好准备。