Deciphering the Function of Piwi in Selecting Transcription Start Sites

项目摘要 基因表达调控对生物体的正常发育至关重要。虽然转录 调节已被广泛研究，选择转录起始位点（TSS）的机制仅 部分理解。选择性转录起始（ATI），其是来自不同基因的基因的转录。 TSS普遍存在于哺乳动物系统中，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， 并被广泛使用。然而，在特定的细胞类型或发育阶段如何选择TSS还没有被证实。 检查和大多数替代TSS仍然有未知的功能。因此，调查 TSS的选择是如何规定的。 使用帽分析基因表达测序（CAGE-seq）方法，我们鉴定了编码 Piwi缺失后果蝇卵巢中TSS利用率改变的mRNA和长链非编码RNA。我们的初步 数据表明Piwi调节果蝇卵巢中TSS的选择。基于这一令人兴奋的发现，我 研究Piwi依赖的TSS选择的分子机制。皮维以沉默著称 转座子并影响基因表达，这些调控由piRNA指导。因此，我假设 Piwi-piRNA途径通过piRNA引导的机制调节果蝇卵巢中的TSS选择。 在这个拟议的研究项目中，我将研究piRNA在TSS使用调节中的作用， 计算分析和实验验证。我将敲除野生型果蝇中的特定piRNA， 检测靶基因的TSS使用情况。我将探索RNA聚合酶占有率，染色质 通过ChIP-seq，研究了piwi突变体中TSS使用改变的基因的表观遗传格局， ATAC-seq实验。此外，我将确定Piwi相互作用的蛋白质染色质参与TSS 使用常规免疫沉淀结合分级和质谱的选择调节 光谱法这项工作将确定一种新的TSS选择调节机制及其参与的 因素重要的是，Piwi对生殖系干细胞维持和性腺发育至关重要。完成 该项目的研究将提供关于Piwi如何调节转录起始和TSS使用影响 卵巢发育此外，它将揭示一种新的TSS选择机制，具有潜在的广泛应用前景。 在其他生物体中的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.