Deciphering the germline-specific mechanisms regulating piRNA gene expression from large genomic domains

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Project Summary/Abstract The Piwi-interacting RNA (piRNA) pathway is a conserved small RNA pathway that protects germ cells from consequences arising from active foreign genetic elements such as transposons. In C. elegans, >10,000 sequence-diverse piRNA genes cluster in two distinct megabase-scale regions in the genome. piRNA clustering is conserved across nematode species, implying that it is important for piRNA expression. Despite being clustered within distinct genomic regions, piRNA genes are individually transcribed by RNA Polymerase II (RNA pol II) and the resulting short RNAs are suggested to be produced when RNA pol II is in its “paused” state. The goal of this proposal is to understand how over 10,000 piRNAs are coordinately upregulated from these large genomic domains in a germline-specific manner. Our lab and others identified the transcription factors SNPC-4 and PRDE-1, which form a complex that spreads across piRNA gene clusters specifically in the germ line to promote piRNA production. However, the mechanism by which SNPC-4/PRDE-1 coordinates piRNA gene expression is unknown. I hypothesize that SNPC-4/PRDE-1 spreading mediates piRNA biogenesis by affecting chromatin organization and/or controlling transcriptional activity. Recently, our lab developed a reliable technique to isolate germ nuclei (IGN) at quantities for large scale genomic assays, which I will use to define at high resolution and specificity the germline-specific patterns of chromatin organization and transcriptional machinery of piRNA gene clusters. To date, I have isolated germ nuclei from wildtype and prde-1 mutants and investigated three candidate histone modifications using ChIP-seq, and observed a global change in repressive histone modifications. By combining the IGN technique with a variety of genomic approaches, I aim to investigate whether SNPC-4/PRDE-1 coordinate piRNA expression by influencing chromatin organization (Aim 1), and transcriptional events (Aim 2). In Aim 1, I will investigate whether SNPC-4/PRDE-1 affect chromatin accessibility across the piRNA gene clusters and whether the local chromatin environment affects SNPC-4/PRDE-1 binding. In Aim 2, I will investigate whether SNPC-4/PRDE-1 aids in RNA Pol II recruitment and whether SNPC-4/PRDE-1 interact with factors that control the paused state of RNA Pol II. In addition, I will determine if the RNA Pol II paused state affects SNPC-4/PRDE-1 binding at piRNA gene clusters. Completion of these aims will advance our understanding of piRNA biogenesis by deciphering the mechanisms that control chromatin organization and transcriptional machinery of the piRNA gene clusters, which is essential for germline maintenance and function. Ultimately, this work is likely to be relevant to understanding the mechanisms that underlie regulation of complex gene regulatory loci in many different genomes across species.