Experience-Dependent Redeployment of MeCP2 Across the Mouse Genome

描述（申请人提供）：MeCP2（甲基- cpg结合蛋白2）是DNA甲基化、染色质重塑和转录调控之间的分子连接体。一些证据支持MeCP2在神经元活动响应中的差异磷酸化可能作为动态调节神经元基因表达的分子开关的可能性，这是哺乳动物大脑发育活动依赖阶段的基础。首先，MeCP2的表达在成熟神经元突触完善期显著上调。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）患者出生时正常（表明MECP2不需要活动独立的胚胎大脑发育），但在突触完善期间出现症状（表明MECP2是活动依赖的出生后大脑发育所需要的）。第三，两项体外研究表明，神经元活性诱导的丝氨酸421 （S421）磷酸化先于脑源性神经营养因子（BDNF）基因的神经元特异性启动子释放MeCP2以及随后的BDNF表达。最后，综合生化分析鉴定出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，但在活跃神经元中被去磷酸化，而S421在静息神经元中被去磷酸化，但在活跃神经元中被磷酸化。为了确定神经元活动如何诱导MeCP2的差异磷酸化微调MeCP2在基因组中的启动子占用并诱导染色质标记的相应变化，我们产生了几个新的MeCP2敲入等位基因，这些等位基因携带点突变，这些突变可以消除或模拟MeCP2蛋白上S80和S421的磷酸化，以及MeCP2蛋白羧基端的FLAG标签。作为我们了解MeCP2在哺乳动物大脑发育和功能的DNA甲基化依赖表观遗传调控中的动态作用的长期目标的一部分，我们建议：1)进行ChIP-chip（染色质免疫沉淀后杂交到DNA寡核苷酸阵列）实验，以揭示MeCP2磷酸化状态的变化如何导致其与整个基因组中基因启动子结合能力的变化；2)通过ChIP-chip实验揭示MeCP2磷酸化状态的变化如何在整个基因组中诱导其靶基因启动子染色质标记的相应变化。

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.