Understanding transcriptional mechanisms critical for neural development

项目概述：人类遗传学研究表明，转录成分的突变，如 转录因子和染色质修饰剂是自闭症谱系障碍及相关疾病的基础 神经发育障碍因此，分子神经生物学的一个重要的新挑战是， 了解这些蛋白质在神经发育中的正常功能，并确定它们的破坏如何改变 基因表达引发疾病甲基-CpG结合蛋白2（MeCP 2）的破坏， 具有全局基因表达效应的调节因子，是Rett综合征的主要单基因原因， 神经发育障碍MeCP 2已显示优先结合甲基化CG二核苷酸 (mCG)以及在非CpG背景下发生在胞嘧啶处的神经元富集甲基化（mCH，其中H=C， T，A）。此外，MeCP 2与转录辅阻遏物复合物相互作用，包括NCoR/HDAC 3， 这表明它可以作为转录抑制因子发挥作用。MeCP 2的消耗导致长链RNA的上调。 神经元基因（> 100 kb）在其基因体内富集神经元特异性mCH位点，表明 MeCP 2介导神经元转录控制的新机制。然而， MeCP 2介导的调节在很大程度上是未知的。我们的长期目标是阐明 MeCP 2和mCA作为神经元特异性表观基因组组分的重要性以及它们如何 分裂会导致神经紊乱我将开始通过分析MeCP 2的机制来解决这个问题 和mCH在大脑皮层内长的高度甲基化基因的转录调节中的作用。 在这个提议中，我将确定MeCP 2如何结合到基因体内的DNA甲基化， 非常长的高度甲基化的基因影响转录。这些分析将研究MeCP 2如何 通过抑制转录起始来控制转录。在目标1中，我将检验以下假设： MeCP 2与基因内mCH的结合导致转录抑制。使用ChIP-seq和GRO-seq 在MeCP 2基因敲除小鼠的分析中，我将在RNA聚合酶水平上测量转录的直接变化 前体mRNA的结合和转录。在目标2中，我将通过以下方式进一步剖析这种调节是如何发生的： 确定MeCP 2如何与转录共调控复合物如NCoR和CBP结合以发挥其作用， 方面的影响.该分析将提供当MeCP 2表达时，转录究竟如何受到影响的高分辨率读数。 并建立一个分子模型来研究MeCP 2如何发挥这些作用。在目标3中，我将直接剖析 基因体内的mCH和MeCP 2可以影响基因调控的机制。使用CRISPR/dCas 9 技术靶向mCH在基因组中的不同位点，我将测试如何在基因区域mCH的存在 影响MeCP 2结合和基因表达。该分析将提供关于MeCP 2和mCH 调节神经元内的转录，以促进大脑的正常发育，以及这些成分的破坏是如何发生的。 导致雷特综合症和其他神经系统疾病期间发生的调节失调。

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.