Rett氏综合症蛋白MeCP2磷酸化位点S421A;S424A双突变体小鼠蛋白质复合物及MeCP2靶基因的研究

MeCP2 mutations cause Rett syndrome(RTT), an X-linked Autism Spectrum Disorders(ASDs) exclusively for girls.It has been reported that phosphorylation in MeCP2 plays critical role in regulating function of MeCP2 in neurons and furthermore affects global gene-binding patterns in mutant mice. We discovered that MeCP2 phosphorylation and mapped as more as 8 phosphorylation sites in different cell lines and brain tissues ranging from mouse, rat and human in coupling with immunoprecipitation (IP) and mass spectrometory (MS). In collaborating with Jaenisch lab at Whitehead(MIT), they developed 9 MeCP2 mutants mice after we provided those phosphorylation mapping information. We speculated that MeCP2 mutant mouse is changed in its protein complex and target gene-binding parten based on the previous research. Here we will focus on MeCP2 complex and its global gene-binding pattern in MeCP2 S421/424 double mutational knock-in mouse based on our previous experience in MeCP2 research. Alternatively, we will explore how MeCP2 S421/424 mouse phenotype rescue in employing AAV-MeCP2 wide type into test double mutant animals. We believe this project will reveal how phosphorylation site mutation in MeCP2 changing its protein complex and global gene-bending pattern and futher demonstrate phosphorylation machineary in regulating MeCP2 function. We expect that those researches will help us to more understand how MeCP2 mutations causeing RTT and ASDs. On the other hand, we hope this project will find a possible strategy to rescue phenotype in MeCP2 mutant mice, which will be good to us to pipleline a possible approach for gene therapy in RTT.

瑞氏综合症(Rett syndrome, RTT)是由于甲基化ＤＮＡ结合蛋白(MeCP2)突变所致。RTT与X-染色体连锁，发病于6－18月女婴，属自闭症谱系障碍症.MeCP2磷酸化在调节MeCP2功能上起重要作用。利用抗体免疫沉淀和质谱技术,我们发现MeCP2磷酸化现象并定位8个磷酸化位点。其中S421和S424的磷酸化只发生在脑神经元处于去极化或动物处于癫痫状态下。本研究将探索MeCP2 S421/424磷酸化双突变体小鼠脑组织蛋白质复合物结构和性质以及对靶基因调节的作用，并利用基因治疗技术探讨以AAV病毒导入正常MeCP2对突变体小鼠病理状况的修复作用,力争为RTT的基因治寻找可行的方法。本项目将揭示癫痫导致MeCP2磷酸化是如何改变蛋白质复合物及及靶基因的调节,为阐明MeCP2与RTT的发病机理提供科学实验依据，有助于更好地理解包括自闭症在内的人类重大疾病发生发展的表观遗传学机制。

背景.因原题目所饲养小鼠从美国引进后出现繁育障碍故改变研究内容但仍紧扣MeCP2重要点展开.主要进展.1）MeCP2在系统生物进化中功能研究，以斑马鱼为脊推动物的低级代表，研究MeCP2在斑马鱼发育尤其是神经系统发育中的功能.2）MeCP2与DNA结合的特异结构motif.3）MeCP2的RNA结合蛋白性质研究.4）MeCP2与线粒体ATP产能复合物的关系研究.重要结果.1）MeCP2基因沉默后，斑马鱼个体发育异常，表明MeCP2在斑马鱼个体发育中起重要作用.2）MeCP2在斑马鱼脑部表达明显高于其它部位，MeCP2在斑马鱼细胞中定位为核蛋白，提示MeCP2在斑马鱼神经发育中起重要作用.3）MeCP2调控神经前体细胞成熟过程.4）敲低MeCP2导致Notch信号通路活性提高.5）敲低MeCP2的胚胎中her2的表达明显增加.6）MeCP2通过ID1介导与her家族蛋白的相互作用（相关工作以申请人单位为第1作者和通讯作者于2015年发表于Journal of Cell Science）.7）通过IP-MS技术获得2个与MeCP2形成复合物的RNA结合蛋白，从而表明MeCP2亦是一个RNA结合蛋白，这一工作正在进行中，并在后其发表..8）发现数个MeCP2与DNA结合的特异元件motif.9）发现MeCP2表达水平明显变化,在线粒体产能蛋白IDH2基因敲除后，表明MeCP2与线粒体功能相关，印证了早期Jaenisch lab在MeCP2 KO小鼠中的发现..以上发现表明MeCP2在动物发育生长中起重要作用.更精密的研究表明MeCP2是因ID1介导her家族的相互作用.MeCP2与DNA新的motif发现将为本领域带来振动人心的事.MeCP2与线粒体能量产生的蛋白相互作用,表明MeCP2参与细胞中多种重要生命活动