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

基本信息

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
项目类别：
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

项目摘要

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.

项目概要从植物到动物的证据表明，种系永生对于物种生存至关重要。在秀丽隐杆线虫种系中，永生取决于促进基因组稳定性的小 RNA 途径。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蠕虫 Argonautes (WAGO) 维持的序列与核小体结合。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 靶标上的形成缺陷会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方式。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在表达突变体 HDA-1 的蠕虫中，该突变体不能被 SUMO 化，piRNA 介导的沉默，种系异染色质形成，并且生育能力丧失，表明其不需要 HDA-1 SUMO 化。总之，这些发现表明 piRNA 途径在体细胞胚胎中组装成 NuRD 复合体。 SUMO 依靠种系中的 Co-opt NuRD 复合体组装来沉默 piRNA 靶标。该提案旨在探索种系 NuRD 复合物如何组装及其通过 piRNA 途径的招募目标 1 将探讨 SUMO 化和种系特异性因素在预防违约方面的作用。种系中 NuRD 复合物的（SUMO 独立）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，并且是 SUMO 化的，初步数据表明是 SUMO 化的。种系中的 MRG-1 使 HDA-1 功能成为 SUMO 依赖性的。用于确定 SUMOylated MRG-1 是否阻止未修饰的 HDA-1 组装到种系中 NuRD 复合物由于 SUMO 化的 MRG-1 在胚胎组织中含量丰富，因此该目标也将使用质量。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SUMO 目标2将确定HDA-1是否依赖。 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 相互作用的影响也将被确定。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。