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Understanding transcriptional mechanisms critical for neural development

了解对神经发育至关重要的转录机制

基本信息

批准号：
9769521
负责人：
Adam Winfield Clemens
金额：
$ 3.09万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2018
资助国家：
美国
起止时间：
2018-09-01 至 2020-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/9769521
关键词：
Address Affect Binding Brain Cerebral cortex ChIP-seq Characteristics Chromatin Remodeling Factor Clustered Regularly Interspaced Short Palindromic Repeats Complex CpG dinucleotide Cytosine DNA DNA Methylation DNA-Directed RNA Polymerase Development Disease EP300 gene Enhancers Etiology Gene Duplication Gene Expression Gene Expression Regulation Genes Genetic Transcription Genetic study Genome Genomic approach Genomics Goals HDAC3 gene Histone Acetylation Human Genetics Knock-out Knockout Mice Laboratories Lead Measures Mediating Methods Methyl-CpG-Binding Protein 2 Methylation Modification Molecular Molecular Neurobiology Mutant Strains Mice Mutation Nervous system structure Neurodevelopmental Disorder Neurons Pathology Polymerase Proteins RNA Regulation Regulatory Element Research Resolution Rett Syndrome Role Site Syndrome Technology Testing Therapeutic Transcription Elongation Transcription Initiation Transcription Regulatory Protein Transcription Repressor/Corepressor Transcriptional Regulation Up-Regulation Wild Type Mouse Work autism spectrum disorder base chromatin modification epigenome gene repression genome-wide genome-wide analysis global run on sequencing insight mRNA Expression mRNA Precursor molecular modeling nervous system disorder neurodevelopment novel overexpression protein function relating to nervous system transcription factor transcriptome

项目摘要

Project Summary: Human genetic studies have revealed that mutations in transcriptional components such as transcription factors and chromatin modifiers underlie autism spectrum disorders and related neurodevelopmental disorders. Therefore, an important new challenge in molecular neurobiology is to understand normal functions of these proteins in neural development and determine how their disruption alters gene expression to trigger disease. Disruption of methyl-CpG binding protein 2 (MeCP2), a transcriptional regulator with global gene-expression effects, is the primary monogenic cause of Rett syndrome, a severe neurodevelopmental disorder. MeCP2 has been shown to preferentially bind both methylated CG dinucleotides (mCG) as well as neuron-enriched methylation that occurs at cytosines in non-CpG contexts (mCH, where H=C, T, A). Additionally, MeCP2 interacts with transcriptional co-repressor complexes, including NCoR/HDAC3, suggesting it can function as a transcriptional repressor. Depletion of MeCP2 results in an upregulation of long neuronal genes (>100kb) that are enriched for neuron-specific mCH sites within their gene body, suggesting MeCP2 mediates a novel mechanism of neuronal transcriptional control. However, the direct mechanisms of MeCP2-mediated regulation are largely unknown. Our long-term goal is to elucidate the functional importance of MeCP2 and mCA as components of the neuronal-specific epigenome and how their disruption leads to neurological disorders. I will begin to address this by analyzing the mechanism of MeCP2 and mCH in transcriptional regulation of long, highly methylated genes within the cerebral cortex. In this proposal, I will determine how binding of MeCP2 to DNA methylation within gene bodies of very long, highly methylated genes impacts transcription. These analyses will examine how MeCP2 may control transcription through inhibition of transcriptional initiation. In Aim 1, I will test the hypothesis that binding of MeCP2 to mCH within genes leads to repression of transcription. Using ChIP-seq and GRO-seq analysis in MeCP2 knockout mice, I will measure direct changes in transcription at the level of RNA polymerase binding and transcription of pre-mRNA. In Aim 2, I will further dissect how this regulation takes place by determining how MeCP2 engages transcriptional co-regulatory complexes such as NCoR and CBP to exert its effects. This analysis will provide a high resolution read-out of exactly how transcription is affected when MeCP2 is lost, and build a molecular model for how MeCP2 exerts these effects. In Aim 3, I will directly dissect the mechanisms by which mCH and MeCP2 within gene bodies can affect gene regulation. Using CRISPR/dCas9 technology to target mCH to distinct sites in the genome, I will test how the presence of mCH at genic regions affects MeCP2 binding and gene expression. This analysis will provide insight into how MeCP2 and mCH regulate transcription within neurons for proper brain development and how disruption of these components leads to dysregulation that occurs during Rett syndrome and other neurological disorders.

项目摘要：人类基因研究揭示了转录成分的突变，如转录因子和染色质修饰物是自闭症谱系障碍及其相关因素的基础神经发育障碍。因此，分子神经生物学面临的一个重要新挑战是了解这些蛋白质在神经发育中的正常功能，并确定它们的破坏是如何改变的引发疾病的基因表达。转录因子甲基-CpG结合蛋白2(MeCP2)的干扰具有全局基因表达效应的调节子是Rett综合征的主要单基因原因，严重神经发育障碍。MeCP2已被证明优先结合两个甲基化的CG二核苷酸 (MCG)以及在非CpG环境中发生在胞嘧啶(MCH，其中H=C， T，A)。此外，MeCP2还与转录共抑制物相互作用，包括NCoR/HDAC3，这表明它可以作为转录抑制因子发挥作用。MeCP2的耗尽导致Long的上调神经元基因(&gt；100kb)在其基因体内富含神经元特异性的MCH位点，这表明 MeCP2介导了一种新的神经元转录调控机制。然而，其直接机制是 MeCP2介导的调控在很大程度上是未知的。我们的长期目标是阐明 MeCP2和MCA作为神经元特异性表观基因组成分的重要性及其如何分裂会导致神经紊乱。我将通过分析MeCP2的机制来开始解决这个问题和MCH在大脑皮层内高度甲基化的长基因转录调控中的作用。在这个提案中，我将确定MeCP2与DNA甲基化的结合如何在基因体内非常长的、高度甲基化的基因会影响转录。这些分析将考察MeCP2如何通过抑制转录启动来控制转录。在目标1中，我将测试假设在基因内，MeCP2与MCH结合导致转录抑制。使用CHIP-SEQ和GRO-SEQ 在MeCP2基因敲除小鼠中进行分析，我将在RNA聚合酶水平上测量转录的直接变化 Pre-mRNA的结合和转录。在目标2中，我将进一步剖析这一规定是如何通过确定MeCP2如何参与转录共调控复合体，如NCoR和CBP，以发挥其效果。这项分析将提供一个高分辨率的读数，准确地显示当MeCP2 并为MeCP2如何发挥这些作用建立了一个分子模型。在《目标3》中，我将直接剖析基因体内的MCH和MeCP2影响基因调控的机制。使用CRISPR/dCas9 将MCH定位于基因组中的不同位置的技术，我将测试MCH在基因区域的存在影响MeCP2结合和基因表达。这一分析将提供对MeCP2和MCH如何调节神经元内的转录以促进大脑的正常发育，以及这些成分如何被破坏导致在Rett综合征和其他神经疾病期间发生的调节失调。