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体。x连锁的人类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如何动态调控基因转录，并揭示这种调控的生理意义十分重要。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，但在活跃神经元中被去磷酸化，而丝氨酸421 （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。两项体外研究表明，神经元活性诱导的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和随后的BDNF表达。总的来说，这些研究提出了一种可能性，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敲入等位基因，这些等位基因携带点突变，可以消除或模拟Mecp2蛋白上S80和S421的磷酸化。为了了解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，我们提出：1)研究操纵MeCP2磷酸化对动物行为的影响；2)研究MeCP2磷酸化调控对成体神经发生的影响；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，进而改变Bdnf的表达和神经元活性。总之，这三个特定目的的实验将提供关于神经元活动诱导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的见解。这些发现不仅将使我们更接近于了解RTT的分子机制和寻找RTT的潜在治疗方法，而且有利于对自闭症的普遍认识。

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.