Abstract 2241 DNA repair factors maintain germline homeostasis after decreasing rRNA synthesis in Caenorhabditis elegans

Germline homeostasis is essential for the accurate transmission of genetic information for reproductive and evolutionary success. Ensuring germline homeostasis in Caenorhabditis elegans involves preserving the mitotic, transition, and meiotic zones along each gonadal arm. This process involves an expected shift as germ cells transition from proliferation to meiosis, moving from a heterochromatic mitotic zone to condensed chromosome formation in the meiotic zone. C. elegans germ cells exhibit a simplistic composition: a nucleus enveloped by a partially open membrane, fostering a shared cytoplasmic space among cells. Considering the nucleolus is a significant structural element within nuclei, and that each germ cell is essentially a single nuclei, we questioned whether disrupting nucleolar integrity would disrupt germline homeostasis. Here, we utilize an auxin inducible degron system (AID) to deplete RPOA-2, a catalytic subunit of RNA polymerase I that is essential for rRNA synthesis. Previous studies have reported that inhibiting rRNA synthesis reduces nucleolar volume, which is attributed to the structure of the nucleolus being governed by RNA-induced liquid-liquid phase separation. Relative to control samples, samples with RPOA-2 depletion observed a significant decrease in the transcription of ribosomal internal transcribed spacers, suggesting a disruption of rRNA synthesis and nucleolar integrity. Using the live SPY555-DNA stain, we concluded that RPOA-2 depleted germ cells appropriately transitioned from the heterochromatic mitotic zone to the meiotic zone, successfully condensing chromosomes in the process. Our findings suggest that germline homeostasis is preserved after RPOA-2 depletion, despite decreased rRNA synthesis. To investigate if stress response pathways are involved in the preservation of germline homeostasis after RPOA-2 depletion, we employed genomic (ATAC-seq) and transcriptomic (RNA-seq) techniques. ATAC-seq analyses unveiled a noteworthy trend, wherein 2%-7% of autosomal peaks exhibited a significant increase in accessibility in RPOA-2-depleted samples, despite the typical repressed germline chromatin state. Utilizing ChIP-Atlas, we examined whether regions exhibiting differential accessibility after RPOA-2 depletion overlapped with sites associated with RNA polymerase II, as documented in other deposited studies. Indeed, preferentially open regions after RPOA-2 depletion were enriched for RNA pol II sites, whereas preferentially closed regions did not exhibit any such enrichment. Consistently, we observed a concurrent upregulation in gene expression for genes proximal to preferentially open peaks. This suggests that there may be an activation of a cellular response aimed at enhancing gene expression, potentially contributing to the maintenance of germline homeostasis following RPOA-2 depletion. Enrichment analysis of gene sets comprising differentially upregulated genes in proximity to differentially open peaks revealed an enrichment of pathways associated with DNA damage response. Additionally, predictions based on the TF binding sites of regions that exhibit a significant increase in accessibility indicate that RPOA-2-depleted germ cells are likely subject to the transcriptional control of efl-2 and efl-1, genes encoding E2F-like proteins essential for DNA damage-induced germ cell apoptosis.