Regulation of sexually dimorphic piRNA pathways in heat-induced infertility

项目总结 在精子和卵子发育过程中保持基因组的完整性是生育和 适当的基因组世代遗传。生殖细胞需要精确的基因表达调控来 沉默有害的基因组成分，如转座子，它可以导致DNA损伤和遗传 突变与不孕症和出生缺陷都有关。在精母细胞和卵母细胞发育过程中， 保守的PIWI/PiRNA小RNA途径通过抑制基因来监控和保护生殖细胞基因组 这些有害元素的表现。利布达实验室最近发表的研究表明，热量 休克引起秀丽线虫转座子活性相关的DNA损伤 精母细胞，同时男性生育力下降。初步数据表明，PIWI/piRNA具有一定的作用 热诱导精母细胞DNA损伤的产生途径，可能是通过损伤 转座子活性的调节。对抑制染色质标记的沉积进行量化的附加数据 H3K9me3表明，女性对急性热应激产生了强大的基因抑制反应，这是 缺少雄性。通过这项研究，我将检验有性二态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 转座子活性，在精母细胞和卵母细胞中。这里提出的研究将定义准确的 改变的piRNA途径功能和随后的转座子抑制在热产生中的作用- 对发育中的精子和卵子造成DNA损伤。在目标1中，我将结合使用小RNA测序 用H3K9me3染色质免疫沉淀测序确定piRNA中性细胞特异性差异 定向基因打靶和H3K9me3分别诱导沉默。通过这种方式，我将识别特定于配子的 热反应中piRNA途径介导的基因沉默的差异。在目标2中，我将跟进我的 发现Prg-1的缺失，它是与生殖系中的piRNA相互作用的piRNA途径主调控子，以 抑制转座子，增强热应激导致精母细胞DNA损伤的产生。这就做 使用免疫荧光显微镜和对PRG-1的条件敲除来量化 热诱导DNA损伤和H3K9me3在精母细胞和卵母细胞中的丰度和定位 没有PRG-1。此外，为了更好地确定PRG-1缺失、其对染色质状态的影响和基因之间的联系 表达I将使用qPCR来定量DNA转座子在精母细胞和卵母细胞中的表达 没有PRG-1，以及有和没有热休克。这些研究将为我们提供对 卵子发育计划和精子发育计划之间的根本区别，有利于我们的 理解性别二型性在维持基因组完整性中的作用，以及揭示 热敏性不孕症发生的更广泛的机制。

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.