In Vivo Function of Neuronal Activity-Induced MeCP2 phosphorylation

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物。X连锁人类MECP2基因突变导致Rett综合征(RTT)，这是一种主要影响女性的自闭症谱系发育障碍。为了了解RTT的分子机制，研究MeCP2是如何动态调节基因转录的，并揭示这种调节的生理意义是很重要的。最近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，而在活动神经元中去磷酸化，而丝氨酸421(S421)在静息神经元中去磷酸化，在活动神经元中去磷酸化。两项体外研究表明，S421的神经元活性诱导的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并随后表达。总之，这些研究提出了这样一种可能性，即响应神经元活动的MeCP2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，导致成人大脑的发育和功能的重要后果。为了在体内验证这一假设，我们产生了几个新的MeCP2敲门等位基因，这些等位基因携带有点突变，这些突变取消或模拟了MeCP2蛋白上S80和S421的磷酸化。作为我们长期目标的一部分，为了了解MeCP2在依赖DNA甲基化的哺乳动物脑发育和功能的表观遗传调控中的动态作用，我们建议：1)研究操纵MeCP2磷酸化对动物行为的影响；2)研究操纵MeCP2磷酸化对成年神经发生的影响；3)研究MeCP2磷酸化如何调节其与BDNF启动子的结合，重塑染色质，从而改变BDNF的表达和神经元活性。总之，在这三个特定目标中提出的实验将为深入了解神经元活动诱导的MeCP2差异磷酸化在调节神经元基因表达中的核心作用及其在神经元发育和动物行为中的功能意义提供见解。这些见解不仅将使我们进一步了解RTT的分子机制，找到RTT的潜在治疗方法，而且有助于对自闭症的总体理解。 公共卫生相关性：这项研究的结果不仅有助于阐明MeCP2在DNA甲基化依赖的表观遗传调节脑功能中的核心作用，而且有助于我们对Rett综合征(RTT)的分子机制的理解。此外，由于RTT和自闭症谱系障碍的临床特征有相当大的重叠，研究RTT所学到的教训也可能有助于对自闭症的一般理解。

DESCRIPTION (provided by applicant): MeCP2 (methyl-CpG binding protein 2) functions as a molecular linker between DNA methylation, chromatin remodeling and transcription regulation. Mutations in the X-linked human MECP2 gene cause of 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, and to reveal the physiological significance of such regulation. Recent 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 serine 421 (S421) is dephosphorylated in resting neurons but phosphorylated in active neurons. Two in vitro studies have shown that neuronal activity- induced phosphorylation at 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. Collectively, those studies raise the possibility that differential phosphorylation of MeCP2 in response to neuronal activity may serve as a molecular switch in dynamically modulating neuronal gene expression, leading to important consequences in development and function of the adult brain. To test this hypothesis in vivo, we have generated several novel Mecp2 knockin alleles carrying point mutations that either abolish or mimic phosphorylation at S80 and S421 on 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) study the effects of manipulating MeCP2 phosphorylation on animal behavior; 2) study the effects of manipulating MeCP2 phosphorylation on adult neurogenesis; 3) study how MeCP2 phosphorylation regulates its binding to the Bdnf promoter, remodels chromatin and subsequently alters BDNF expression and neuronal activity. Together, the experiments proposed in these three specific aims will provide insights into the central role of neuronal activity induced differential phosphorylation of MeCP2 in regulating neuronal gene expression, and its functional significance in neuronal development and animal behavior. These insights will not only bring us closer to understand the molecular mechanism of RTT and find potential treatments for RTT, but also benefit the general understanding of autism. PUBLIC HEALTH RELEVANCE: Results from this study will not only help elucidate the central role of MeCP2 in DNA methylation-dependent epigenetic regulation of brain function, but also advance our understanding of the molecular mechanism of Rett syndrome (RTT). Furthermore, because 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.