# Deciphering the Function of Piwi in Selecting Transcription Start Sites

> **NIH NIH F31** · YALE UNIVERSITY · 2023 · $32,579

## Abstract

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.

## Key facts

- **NIH application ID:** 10560467
- **Project number:** 5F31HD104415-02
- **Recipient organization:** YALE UNIVERSITY
- **Principal Investigator:** Jiaying Chen
- **Activity code:** F31 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2023
- **Award amount:** $32,579
- **Award type:** 5
- **Project period:** 2022-02-01 → 2025-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/10560467

## Citation

> US National Institutes of Health, RePORTER application 10560467, Deciphering the Function of Piwi in Selecting Transcription Start Sites (5F31HD104415-02). Retrieved via AI Analytics 2026-05-24 from https://api.ai-analytics.org/grant/nih/10560467. Licensed CC0.

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