Deciphering the Function of Piwi in Selecting Transcription Start Sites

项目总结 受调控的基因表达对生物体的正常发育至关重要。虽然转录 调控已经被广泛研究，选择转录起始点(TSS)的机制只有 部分地理解了。交替转录起始(ATI)，这是来自不同基因的转录 TSSs广泛存在于哺乳动物体内，具有重要的生物学功能。此外，TSS 基因的选择似乎在不同的组织和不同的发育阶段有所不同，表明ATI是受调控的 并得到广泛应用。然而，在特定的细胞类型或发育阶段中，TS是如何被选择的还没有被发现。 经过研究，大多数替代的SSSS仍然具有未知的功能。因此，重要的是要调查 如何监管TSS选择。 使用帽分析基因表达测序(CAGE-SEQ)方法，我们鉴定了编码 失去Piwi后果蝇卵巢中TSS使用改变的mRNA和长非编码RNA。我们的预赛 数据表明，Piwi调控果蝇卵巢中TSs的选择。基于这一令人兴奋的发现，我 建议研究依赖Piwi的TSS选择的分子机制。皮威以沉默著称 转座子和影响基因表达，这些调控是由piRNAs引导的。因此，我假设 Piwi-piRNA途径通过piRNA引导机制调节果蝇卵巢中TSS的选择。 在这个拟议的研究项目中，我将通过以下方式研究piRNAs在TSS使用调控中的作用 计算分析和实验验证。我将敲除野生型苍蝇的特定piRNA 检查目标基因的TSS使用情况。我将探索RNA聚合酶占有率、染色质的变化 在piwi突变体中，通过芯片序列和序列分析改变TSS使用的基因的表观遗传格局 ATAC-SEQ实验。此外，我将鉴定与TSS有关的染色质上与Piwi相互作用的蛋白 常规免疫沉淀结合分馏和质量的选择调节 光谱分析。建议的工作将确定一种新的TSS选择监管机制及其涉及的 各种因素。重要的是，Piwi对于生殖系干细胞的维持和性腺发育是必不可少的。完成 将为Piwi如何调控转录起始和TSS的使用提供新的知识 卵巢发育。此外，它还将揭示一种新的TSS选择机制，具有潜在的广泛意义 对其他生物体中许多生物过程的转录调控的影响。

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.