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: 10313267
• 负责人: Jiaying Chen
• 金额: $ 4.45万
• 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/10313267
• 关键词: 3' Untranslated Regions; 5' Untranslated Regions; Affect; Bioinformatics; Biological Assay; Biological Process; Caenorhabditis elegans; Cell Maintenance; Cell Nucleus; Cells; ChIP-seq; Chromatin; Co-Immunoprecipitations; Code; Complex; Computer Analysis; Congenital Abnormality; DNA Polymerase II; DNA Transposable Elements; DNA-Directed RNA Polymerase; Data; 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; Guide RNA; HDAC3 gene; Histones; Human; Immunofluorescence Microscopy; Immunoprecipitation; Knock-out; Knowledge; Lead; 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; RNA Databases; Regulation; Research Project Grants; Role; Schizophrenia; Site; Small Interfering RNA; System; Tissues; Transcript; Transcription Initiation; Transcription Initiation Site; Transcriptional Regulation; Transposase; Untranslated RNA; Validation; Work; base; cell type; chromatin protein; domain mapping; epigenetic regulation; experimental study; fly; germline stem cells; gonad development; insight; knock-down; mutant; novel; 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 常规基因表达对于生物体的正常发育至关重要。 调节已经进行了广泛的研究，选择转录起始位点（TSS）的机制仅为 部分理解的替代转录开始（ATI） TSS在哺乳动物系统中盛行，并且具有重要的生物学功能 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是规则的 但是，如何在特定的单元格类型或发育阶段中选择TSS 检查和大多数替代TSS仍然具有未知的功能。 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 mRNA和长期非编码RNA在果蝇卵巢中使用的TSS使用变化 数据表明PIWI调节果蝇卵巢中TSS的选择。 建议研究PIWI依赖性TSS选择的分子机制。 转座子和影响基因表达，这些法规由PIRNA指导。 Piwi-PiRNA途径通过PIRNA引导的机制来实现果蝇卵巢中的TSS选择。 在此支撑研究项目中，我将通过 计算分析和实验验证。 检查靶基因的TSS使用情况。 构型，以及通过chip-seq和 ATAC-SEQ实验。 使用常规免疫沉淀与分馏和质量结合的选择调节 光谱法。 重要的是。 在该项目中，提供了有关PIWI常规转录启动和TSS USS的新知识的新知识 此外，它将揭示出具有潜在广泛的新型TSS选择机制 在其他生物体中许多生物学过程的转录调节中的含义。