Deciphering the Function of Piwi in Selecting Transcription Start Sites

解读 Piwi 在选择转录起始位点中的功能

• 批准号: 10560467
• 负责人: Jiaying Chen
• 金额: $ 3.26万
• 依托单位: YALE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2022
• 资助国家: 美国
• 起止时间: 2022-02-01 至 2025-01-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10560467
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; ATAC-seq; Affect; Bioinformatics; Biological Assay; Biological Process; Caenorhabditis elegans; Cell Maintenance; Cell Nucleus; Cells; ChIP-seq; Chromatin; Code; Complex; Computer Analysis; Congenital Abnormality; DNA Polymerase II; DNA Transposable Elements; DNA-Directed RNA Polymerase; Data; Databases; Defect; Development; Disease; Drosophila genus; Embryo; Enhancers; Epigenetic Process; Fractionation; Gene Expression; Gene Expression Profiling; Gene Expression Regulation; Genes; Genetic; Genetic Transcription; Genomic Segment; Goals; HDAC3 gene; Histones; Human; Immunofluorescence Microscopy; Immunoprecipitation; Knock-out; Knowledge; Literature; Malignant Neoplasms; Mass Spectrum Analysis; Mediating; Medical; Messenger RNA; Methods; Mission; Molecular; Mutate; National Institute of Child Health and Human Development; Organism; Ovarian; Ovary; Pathway interactions; Polymerase; Proteins; RNA; Regulation; Repression; Research Project Grants; Role; Schizophrenia; Site; Small Interfering RNA; Specific qualifier value; System; Tissues; Transcript; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Transposase; Untranslated RNA; Validation; Work; cell type; chromatin protein; domain mapping; epigenetic regulation; experimental study; fly; germline stem cells; gonad development; insight; knock-down; mutant; novel; piRNA; protein function; recruit; response; stem cell self renewal

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.

项目摘要 受调节的基因表达对于生物的正常发育至关重要。虽然转录 调节已广泛研究，选择转录起始位点（TSS）的机制仅为 部分理解。替代转录计划（ATI），即来自不同基因的转录 TSSS，在哺乳动物系统中很普遍，具有重要的生物学功能。此外，TSS 基因的选择似乎在组织和跨发育阶段之间有所不同，表明ATI受到调节 并广泛使用。但是，如何在特定的细胞类型或发育阶段中选择TSSS 检查和大多数替代TSSS仍然具有未知的功能。因此，调查很重要 如何调节TSS选择。 使用Cap-Analses基因表达测序（CAGE-SEQ）方法，我们确定了编码的基因 Piwi丢失后，mRNA和长的非编码RNA在果蝇卵巢中使用TSS使用变化。我们的初步 数据表明PIWI调节果蝇卵巢中TSS的选择。基于这个令人兴奋的发现，我 提出研究PIWI依赖性TSS选择的分子机制。众所周知PIWI沉默 转座子和影响基因表达，这些法规由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使用中的作用 计算分析和实验验证。我会在野生型苍蝇中敲除特定的pirnas和 检查靶基因的TSS使用情况。我将探索RNA聚合酶占用率，染色质的变化 构型，以及通过chip-seq和 ATAC-SEQ实验。此外，我将在参与TSS的染色质上鉴定PIWI相互作用的蛋白 使用常规免疫沉淀与分馏和质量结合的选择调节 光谱法。拟议的工作将确定一种新颖的TSS选择调节机制及其所涉及的机制 因素。重要的是，PIWI对于种系干细胞维持和性腺发育至关重要。完成 该项目将提供有关PIWI监管转录计划和TSS使用情况如何影响的新知识 卵巢发展。此外，它将揭示一种具有潜在范围的新型TSS选择机制 在其他生物体中许多生物过程的转录调节中的含义。