Mechanisms of Transcriptional Silencing by the Nuclear Piwi Protein in Drosophila Germ Cells

Citation

Rogers, Alicia Kathryn (2018) Mechanisms of Transcriptional Silencing by the Nuclear Piwi Protein in Drosophila Germ Cells. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/Z9KK991X. https://resolver.caltech.edu/CaltechTHESIS:04092018-141017685

Abstract

An important characteristic for life is the ability to persist – to reproduce and defend oneself against different stresses. The ability of a species to persist from one generation to the next heavily depends on the integrity of the genetic material being passed down, and thus organisms have developed strategies to ensure the integrity of their genomes remain in tact. In Metazoan germlines, piwi proteins and their associated piwi-interacting RNAs (piRNAs) provide a RNA-interference (RNAi) based defense system against the expression of transposable elements (TEs). TE expression is detrimental to an organism’s genome – resulting in disruption of genes, double-stranded DNA breaks, and germ cell death – ultimately leading to the sterility of the organism. In Drosophila melanogaster, the piRNA pathway is composed of two cytoplasmic piwi clade Argonuate proteins, Aubergine (Aub) and Argonaute3 (Ago3), and a single nuclear piwi clade Argonuate protein, Piwi. The piwi clade Argonaute proteins bind piRNAs to form effector complexes that repress TE sequences.

The work presented in this thesis examines the role of the nuclear piwi clade Argonaute – Piwi – and the mechanisms by which Piwi accomplishes its functions. Chapter Two presents how Piwi/piRNA complexes identify genomic loci expressing TEs and direct the establishment of a repressive chromatin state to transcriptionally silence the loci. In Chapter Three, we explore the piRNA-induced transcriptional silencing (piRITS) pathway using a heterologous reporter based tethering system in vivo. We discuss how the recruitment of Piwi alone to a locus is not sufficient to induce repression, and establish a model for the connection bridging the Piwi/piRNA complex and effector silencing complex in the piRITS pathway. In Chapter Four, we employ our heterologous reporter based tethering system to explore the mechanism of piRNA precursor selection in the two cell types that make up Drosophila ovaries. We uncover a common mechanism of piRNA biogenesis in the two cell types and establish a unifying model of piRNA substrate selection. Finally, in Chapter Five, as essential step to understanding how Piwi achieves its nuclear function, we developed a heterologous two-hybrid system to identify factors that directly interact with Piwi. Overall, the work presented in this thesis provides a piece of the groundwork in understanding the mechanisms of transcriptional silencing of TEs in germ cells by Piwi. The work proposes that Piwi has dual functions in the nucleus. First, upon target recognition, Piwi recruits the piRITS complex to target loci to accomplish Piwi- mediated transcriptional silencing by deposition of H3K9me3. Then, Piwi recruits the RDC complex to specifically bind H3K9me3 at target loci to allow piRNA-production from the locus.

Thesis Files