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.

项目概要 尽管是转录性的，但受调控的基因表达对于生物体的正常发育至关重要。 调控已被广泛研究，选择转录起始位点（TSS）的机制仅 部分理解替代转录起始（ATI），即来自不同来源的基因的转录。 TSS 广泛存在于哺乳动物系统中，具有重要的生物学功能。 基因的选择似乎因组织和发育阶段而异，表明 ATI 受到调节 然而，如何在特定细胞类型或发育阶段选择 TSS 尚不清楚。 经过检查，大多数替代 TSS 的功能仍然未知，因此，研究很重要。 如何规范 TSS 选择。 使用 cap-analysis 基因表达测序 (CAGE-seq) 方法，我们鉴定了编码 Piwi 丢失后果蝇卵巢中 TSS 使用发生改变的 mRNA 和长链非编码 RNA。 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。 Piwi以沉默着称 转座子并影响基因表达，这些调节是由 piRNA 指导的。 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，我将通过以下方式研究 piRNA 在 TSS 使用调节中的作用： 我将敲除野生型果蝇中的特定 piRNA，并进行实验验证。 检查目标基因的 TSS 使用情况，我将探索 RNA 聚合酶占用率、染色质的变化。 通过 ChIP-seq 和 piwi 突变体中 TSS 使用改变的基因的配置和表观遗传景观 此外，我将鉴定 TSS 染色质上的 Piwi 相互作用蛋白。 使用传统免疫沉淀结合分级和质量进行选择调节 拟议的工作将确定一种新的 TSS 选择调控机制及其相关机制。 重要的是，Piwi 对于生殖干细胞的维持和性腺的发育至关重要。 该项目将提供有关 Piwi 调节转录起始和 TSS 使用如何影响的新知识 此外，它将揭示一种具有潜在广泛用途的新型 TSS 选择机制。 对其他生物体中许多生物过程的转录调控的影响。