Experience-Dependent Redeployment of MeCP2 Across the Mouse Genome

MeCP2 在小鼠基因组中的经验依赖性重新部署

• 批准号: 8133342
• 负责人: Qiang Chang
• 金额: $ 17.82万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-08-25 至 2012-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8133342
• 关键词: Affect; Alleles; Am 80; Autistic Disorder; Binding; Biochemical; Brain; Brain-Derived Neurotrophic Factor; Chromatin; Clinical; DNA; DNA Methylation; Development; Embryo; Embryonic Development; Epigenetic Process; Female; Foundations; Gene Expression; Gene Targeting; Genes; Genetic Transcription; Genome; Goals; Human; In Vitro; Knock-in Mouse; Learning; Life; Link; Mediating; Methyl-CpG-Binding Protein 2; Methylation; Molecular; Mus; Mutation; Nervous system structure; Neurites; Neurons; Oligonucleotides; Pathogenesis; Patients; Phase; Phosphorylation; Phosphorylation Site; Plastics; Play; Point Mutation; Process; Proteins; Regulation; Research; Rest; Rett Syndrome; Role; Sensory; Serine; Site; Staging; Stimulus; Synapses; System; Transcriptional Regulation; autism spectrum disorder; axonal pathfinding; base; chromatin immunoprecipitation; chromatin remodeling; developmental disease; experience; mouse genome; novel; postnatal; promoter; public health relevance; research study; response; synaptogenesis

DESCRIPTION (provided by applicant): MeCP2 (methyl-CpG binding protein 2) functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Several lines of evidence exist to support the possibility that differential phosphorylation of MeCP2 in response to neuronal activity may serve as a molecular switch in dynamically modulating neuronal gene expression, which underlies the activity-dependent phase of mammalian brain development. First, MeCP2 expression is dramatically up-regulated in mature neurons during the period of synaptic refinement. Second, Rett Syndrome (RTT, an autism spectrum developmental disorder caused by mutations in the MECP2 gene) patients are born normal (suggesting MeCP2 is not required for activity-independent embryonic brain development), but become symptomatic during the period of synaptic refinement (suggesting MeCP2 is required for activity-dependent postnatal brain development). Third, two in vitro studies showed that neuronal activity-induced phosphorylation at serine 421 (S421) precedes the release of MeCP2 from the neuronal specific promoter of the brain-derived neurotrophic factor (BDNF) gene and the subsequent expression of BDNF. Finally, comprehensive biochemical analysis has identified 8 phosphorylation sites on the MeCP2 protein. Among these, serine 80 (S80) is phosphorylated in resting neurons but dephosphorylated in active neurons, whereas S421 is dephosphorylated in resting neurons but phosphorylated in active neurons. To determine how neuronal activity induced differential phosphorylation of MeCP2 fine-tunes the promoter occupancy of MeCP2 across the genome and induces corresponding changes in chromatin marks, we have generated several novel Mecp2 knock-in alleles carrying point mutations that either abolish or mimic phosphorylation at S80 and S421 on the MeCP2 protein, as well as a FLAG tag at the carboxyl terminal of the MeCP2 protein. As a part of our long-term goal to understand the dynamic role of MeCP2 in DNA methylation-dependent epigenetic regulation of mammalian brain development and functions, we propose to: 1) perform ChIP-chip (chromatin immunoprecipitation followed by hybridization onto a DNA oligo array) experiments to reveal how changes in the phosphorylation status of MeCP2 cause changes in its ability to bind to gene promoters across the entire genome; 2) perform ChIP-chip experiments to reveal how changes in the phosphorylation status of MeCP2 induce corresponding changes in chromatin marks at its target gene promoters across the entire genome. PUBLIC HEALTH RELEVANCE: Mutations in the X-linked human MECP2 gene (methyl-CpG binding protein 2) cause Rett syndrome (RTT), an autism spectrum developmental disorder that predominantly affects females. To understand the molecular mechanism of RTT, it is important to study how MeCP2 dynamically regulates gene transcription. Results from this study will advance our understanding of the molecular mechanism of Rett syndrome (RTT). Furthermore, because of the considerable overlap in clinical features between RTT and autistic spectrum disorders, the lessons learned studying RTT might also benefit the general understanding of autism.

描述（由申请人提供）：MECP2（甲基-CPG结合蛋白2）在DNA甲基化，染色质重塑和转录调控之间起分子接头。存在几种证据，以支持MECP2对神经元活性的差异磷酸化的可能性，可以作为动态调节神经元基因表达的分子转换，这是哺乳动物脑发育的活性依赖性阶段的基础。首先，在突触改进期间，在成熟神经元中MECP2表达显着上调。其次，RETT综合征（RTT，一种由MECP2基因突变引起的自闭症谱系发育障碍）患者天生正常（表明与活动无关的胚胎脑发育不需要MECP2），但在突触改进期间（表明MECP2在活动依赖于活动后依赖于肾上腺大脑脑发育需要MECP2）。第三，两项体外研究表明，神经元活性诱导的丝氨酸421（S421）的磷酸化之前是MECP2从脑衍生的神经营养因子（BDNF）基因的神经元特异性启动子中释放出来的，并且随后的BDNF表达。最后，全面的生化分析已经确定了MECP2蛋白上的8个磷酸化位点。其中，丝氨酸80（S80）在静息神经元中被磷酸化，但在活性神经元中被脱磷酸化，而S421在静息神经元中脱磷酸化，但在活性神经元中磷酸化。为了确定神经元活性如何诱导MECP2微调的差异磷酸化，MECP2跨基因组的启动子占用者占用子占用者的占用者，并诱导染色质标记的相应变化，我们已经在S80和S421 ON s421 arty As421 On and Mimic phoberal probertation Mecp2敲击等位基因携带的MECP2敲点突变产生MECP2蛋白的末端。作为我们长期目标的一部分，是了解MECP2在DNA甲基化依赖性表观遗传学调节中的动态作用，对哺乳动物脑发育和功能，我们建议：1）进行CHIP-CHIP（染色质蛋白免疫沉淀（染色质蛋白免疫沉淀）（通过杂交，然后通过杂交进行杂交到DNA寡头阵列中的启动者跨度跨度的跨度跨度跨度2的启动器，该跨度跨度的变化是MEC的变化。整个基因组； 2）执行芯片芯片实验，以揭示MECP2的磷酸化状态的变化如何引起整个基因组的靶基因启动子处染色质标记的相应变化。 公共卫生相关性：X连锁人MECP2基因（甲基-CPG结合蛋白2）的突变引起RETT综合征（RTT），这是一种自闭症谱系发育障碍，主要影响女性。为了了解RTT的分子机制，重要的是研究MECP2如何动态调节基因转录。这项研究的结果将提高我们对RETT综合征（RTT）分子机制的理解。此外，由于RTT和自闭症谱系障碍之间的临床特征有很大的重叠，因此学习RTT的经验教训也可能有益于自闭症的一般理解。