PROJECT ABSTRACT Small RNAs – short, noncoding RNAs – are critical regulators in animal physiology and disease that silence gene expression by complementary base pairing interactions to control mRNA stability/translation and epigenetic modifications. Small RNA-mediated silencing pathways are evolutionarily conserved, and the largest class of small RNAs comprise Piwi-interacting RNAs (piRNAs). Extensive studies in fly established that piRNAs silence transposons and are expressed in a sex-specific manner; piRNAs are essential for genome integrity and germline development. However, most piRNAs in mammals and worm do not map to transposons, and instead are predicted to regulate germline-expressed genes. Although novel associations of individual piRNAs with endogenous genes have been reported, endogenous gene targets of piRNAs remain largely unknown. In C. elegans, each of the ~15,000 piRNAs is autonomously transcribed and contains an upstream cis- regulatory element, the Ruby motif. We found piRNAs are differentially expressed during spermatogenesis and oogenesis, and male piRNAs have a strong bias for the 5’C nucleotide in the Ruby motif. Furthermore, we identified SNPC-1.3 as a sex-specific transcription factor critical for male fertility. SNPC-1.3 depends on a known core piRNA biogenesis factor, SNPC-4, to drive male piRNA expression during spermatogenesis. Loss of snpc- 1.3 during spermatogenesis results in global depletion of male piRNAs and sperm maturation defects. Other piRNA biogenesis factors have been identified, but how these trans-acting factors interact with each other and the Ruby motif is poorly understood. In this proposed research, I hypothesize that sex-specific regulatory mechanisms underlie piRNA expression to regulate endogenous genes critical for proper germline development. To test my hypothesis in the context of spermatogenesis, in Aim 1 I will use a recently developed strategy Cleavage Under Targets and Release Using Nuclease (CUT&RUN) to characterize protein-DNA binding profiles of 5 piRNA biogenesis trans- acting factors at the Ruby motif cis-regulatory element. Furthermore, I will determine if the 5’ C nucleotide in the Ruby motif acts as a male specific element for SNPC-1.3. I will also use computational tools to identify putative, novel cis-regulatory elements for male and female piRNAs. In Aim 2, I will computationally identify endogenous gene targets of male piRNAs from small RNA-seq and mRNA-seq in wild-type and snpc-1.3(-) animals as well as piRTarBase, a database of computationally predicted and experimentally identified piRNA targeting sites. I will experimentally validate male piRNAs and their predicted endogenous gene targets using reporter assays for endogenous and synthetic piRNAs and applying CRISPR/Cas9-mediated mutagenesis of piRNAs or their targets. Collectively, this research will strengthen our understanding of how sex-specific piRNA expression is transcriptionally regulated and provide new insights in...