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.