Saturday, December 21, 2024
12/21/2024
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.
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