PROJECT SUMMARY Regulated gene expression is crucial for normal development of an organism. Although transcriptional regulation has been extensively studied, mechanisms that select the transcription start site (TSS) are only partially understood. Alternative transcription initiation (ATI), which is the transcription of a gene from different TSSs, is found prevalent in mammalian systems and has important biological functions. Furthermore, TSS choices of genes seem to vary among tissues and across developmental stages, indicating that ATI is regulated and widely used. However, how TSSs are selected in specific cell types or developmental stages has not been examined and majority of alternative TSSs still have unknown functions. Therefore, it is important to investigate how TSS selection is regulated. Using the cap-analysis gene expression sequencing (CAGE-seq) method, we identified genes coding for mRNA and long non-coding RNA with altered TSS usage in Drosophila ovaries upon loss of Piwi. Our preliminary data indicate that Piwi regulates the selection of TSSs in Drosophila ovaries. Based on this exciting finding, I propose to investigate the molecular mechanism of Piwi-dependent TSS selection. Piwi is known to silence transposons and affect gene expression and these regulations are guided by piRNAs. Therefore, I hypothesize that the Piwi-piRNA pathway regulates TSS selection in Drosophila ovaries via a piRNA-guided mechanism. In this proposed research project, I will investigate the role of piRNAs in TSS usage regulation by computational analysis and experimental validation. I will knockdown specific piRNAs in wildtype flies and examine target genes’ TSS usage. I will explore changes in RNA polymerase occupancy, chromatin configuration, and the epigenetic landscape in genes with altered TSS usage in piwi mutants via ChIP-seq and ATAC-seq experiments. Furthermore, I will identify Piwi-interacting proteins on chromatin involved in TSS selection regulation using the conventional immunoprecipitation in combination with fractionation and mass spectrometry. The proposed work will identify a novel TSS selection regulatory mechanism and its involved factors. Importantly, Piwi is essential for germline stem cell maintenance and gonadal development. Completion of this project will provide new knowledge on how Piwi regulated transcription initiation and TSS usage affect ovarian development. Moreover, it will reveal a novel TSS selection mechanism with potentially broad implications in transcriptional regulation of many biological processes in other organisms.