Regulation of sexually dimorphic piRNA pathways in heat-induced infertility

热诱导不孕症中性二态性 piRNA 通路的调节

基本信息

批准号：
10575828
负责人：
Nicole A Kurhanewicz
金额：
$ 7.38万
依托单位：
UNIVERSITY OF OREGON
依托单位国家：
美国
项目类别：
财政年份：
2022
资助国家：
美国
起止时间：
2022-12-05 至 2024-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10575828
关键词：
Acute Affect Caenorhabditis elegans ChIP-seq Chromatin Congenital Abnormality DNA DNA Damage DNA Transposable Elements DNA Transposons Data Deposition Development Elements Exposure to Female Fertility Functional disorder Gene Expression Gene Expression Regulation Gene Silencing Gene Targeting Generations Genes Genome Genomics Germ Cells Heat Stress Disorders Heat-Shock Response Heritability Histone H3 Immunofluorescence Microscopy Impairment Infertility Light Link Lysine Maintenance Male Infertility Mediating Monitor Mutation Oocytes Pathway interactions Prevention Pseudogenes Publishing Regulation Repression Research Role Sex Differences Site Small RNA Spermatocytes Spontaneous abortion Testing Thermogenesis Transposase Work derepression egg follow-up gene repression genetic element genome integrity histone modification insight knock-down male piRNA programs response sex sexual dimorphism sperm cell transcriptome sequencing

项目摘要

PROJECT SUMMARY The maintenance of genomic integrity during sperm and egg development is fundamental for fertility and proper genome inheritance across generations. Germ cells require precise regulation of gene expression to silence deleterious genomic elements, such as transposons, which can cause DNA damage and heritable mutations associated with both infertility and birth defects. During spermatocyte and oocyte development, the conserved PIWI/piRNA small RNA pathway monitors and protects the germ cell genome by repressing gene expression of these deleterious elements. Recent published work from the Libuda lab demonstrates that heat shock produces elevated DNA damage associated with transposon activity in Caenorhabditis elegans spermatocytes, concurrent with reduced fertility in males. Preliminary data suggest a role for the PIWI/piRNA pathway in the production of heat-induced DNA damage specifically in spermatocytes, potentially via impaired regulation of transposon activity. Additional data quantifying the deposition of the repressive chromatin mark H3K9me3 indicates that females produce a robust gene-repressive response to acute heat-stress which is lacking in males. With this research, I will test the hypothesis that sexually dimorphic piRNA pathway responses to heat stress regulate the production of heat-induced DNA damage, associated with transposon activity, in spermatocytes and oocytes. The research proposed herein will define the precise role of altered piRNA pathway function and subsequent transposon de-repression in the production of heat- induced DNA damage in developing sperm and eggs. In Aim 1 I will use small RNA sequencing in conjunction with H3K9me3 chromatin immunoprecipitation sequencing to define sex cell-specific differences in piRNA- directed gene targeting and H3K9me3 induced silencing respectively. In this way I will identify gamete-specific differences in piRNA pathway-mediated gene silencing in response to heat. In Aim 2 I will follow up on my finding that loss of PRG-1, the piRNA pathway master regulator which interacts with piRNAs in the germline to suppress transposons, enhances the production of heat-stress induced DNA damage in spermatocytes. I will use immunofluorescence microscopy and a conditional knockdown for PRG-1 to quantify the production of heat-induced DNA damage and H3K9me3 abundance and localization in spermatocytes and oocytes with and without PRG-1. Further, to better define this link between PRG-1 loss, its impact on chromatin state, and gene expression I will use qPCR to quantify DNA transposon expression in spermatocytes and oocytes with and without PRG-1, as well as with and without heat shock. These studies will provide critical insight into fundamental differences between the oogenic and spermatogenic developmental programs, benefiting our understanding of sexual dimorphism in the maintenance of genomic integrity, as well as shedding light on broader mechanisms underlying the development of heat-sensitive infertility.

项目概要精子和卵子发育过程中基因组完整性的维持对于生育能力和发育至关重要。正确的基因组跨代遗传。生殖细胞需要精确调控基因表达沉默有害的基因组元件，例如转座子，它可能导致 DNA 损伤和遗传与不孕症和出生缺陷相关的突变。在精母细胞和卵母细胞发育过程中，保守的 PIWI/piRNA 小 RNA 通路通过抑制基因来监测和保护生殖细胞基因组这些有害元素的表达。 Libuda 实验室最近发表的研究表明，热电击会导致秀丽隐杆线虫中与转座子活性相关的 DNA 损伤升高精母细胞，同时男性生育能力下降。初步数据表明 PIWI/piRNA 的作用产生热诱导 DNA 损伤的途径，特别是在精母细胞中，可能通过受损转座子活性的调节。量化抑制染色质标记沉积的附加数据 H3K9me3 表明雌性对急性热应激产生强烈的基因抑制反应缺乏男性。通过这项研究，我将检验性二态性 piRNA 途径的假设对热应激的反应调节热诱导的 DNA 损伤的产生，与精母细胞和卵母细胞中的转座子活性。本文提出的研究将定义精确的 piRNA 通路功能改变和随后的转座子去抑制在热产生中的作用诱导发育中的精子和卵子的 DNA 损伤。在目标 1 中，我将结合使用小 RNA 测序使用 H3K9me3 染色质免疫沉淀测序来定义 piRNA 中性细胞特异性差异定向基因靶向和 H3K9me3 分别诱导沉默。通过这种方式，我将识别配子特异性 piRNA 途径介导的基因沉默对热反应的差异。在目标 2 中，我将跟进我的发现 PRG-1 的丢失，PRG-1 是 piRNA 通路的主调节因子，与种系中的 piRNA 相互作用，抑制转座子，增强精母细胞中热应激诱导的 DNA 损伤的产生。我会使用免疫荧光显微镜和 PRG-1 的条件敲低来量化热诱导的 DNA 损伤以及精母细胞和卵母细胞中 H3K9me3 的丰度和定位没有 PRG-1。此外，为了更好地定义 PRG-1 丢失、其对染色质状态的影响和基因之间的联系我将使用 qPCR 来量化精母细胞和卵母细胞中 DNA 转座子的表达，以及没有 PRG-1，以及有和没有热激。这些研究将提供重要的见解卵子发育和精子发生发育程序之间的根本区别，使我们受益了解维持基因组完整性中的性二态性，并阐明热敏性不孕症发展的更广泛机制。