Linc-BML在神经分化及自闭症中的功能与机制研究

Neural differentiation to produce abnormal functional neurons is the main cause of many nervous system diseases, including autism.The specific function and molecular mechanism of long non-coding RNAs in neural differentiation and autism are rarely reported. Our foundation work found that knockout Linc-BML seriously impaired the neural differentiation and the adjacent gene Mef2c was downregulated. It has been reported that NSC-conditional knockout Mef2c mice are autistic phenotype, hinting a potential role of BML in ausism. Exogenous overexpression of Mef2c in Rosa26 loci can rescue the phenotype of interfering BML to inhibit the neural differentiation. ChIP results showed that after interference of BML the enrichment of active histone modification H3K4Me3 and RNA polymerase II at Mef2c promoter region were decreased, while inhibitory histone modification H3K27Me3 enrichment was increased. In order to analyze the function and molecular mechanism of BML in neural differentiation and autism, the RIP technology is applied to identify the possible interaction proteins of BML, and ChIP technology is applied to verify the enrichment of these proteins in the promoter region of Mef2c. Blastocyst injection technology was applied to construct BML conditional knockout mice. Animal behavior experiments were applied to explore whether BML knockout mice had autism phenotype. Mef2c expression was detected to demonstrate that knockout BML could produce autism phenotype by regulating Mef2c. This topic provides a new perspective for understanding the mechanism of lncRNAs regulating the development of neural differentiation and the pathogenesis of autism.

神经分化产生功能异常神经元是神经系统疾病包括自闭症的发病主因，lncRNAs在其中功能和机制鲜有报道。前期研究发现敲除Linc-BML神经分化被严重阻碍，邻近基因Mef2c下调。研究报道NSC中条件敲除Mef2c小鼠呈自闭症表型，暗示BML可能参与自闭症发生。Rosa26位点定点整合外源Mef2c可挽回干扰BML抑制神经分化的表型。ChIP结果显示干扰BML后Mef2c启动子区H3K4Me3和RNA聚合酶II富集减少，H3K27Me3富集增多。为了阐明BML在神经分化和自闭症中功能和机制，本课题拟采用RIP技术确定BML互作蛋白，ChIP技术验证这些蛋白在Mef2c启动子区富集变化。构建BML条件敲除小鼠，行为学实验探索BML敲除小鼠是否存在自闭症表型，检测Mef2c表达证明敲除BML通过调节Mef2c产生自闭症表型。本课题为理解lncRNAs调控神经分化发育、自闭症发病机制提供新的视角。

自闭症谱系障碍（ASD）是一种异质性的神经发育性疾病，主要表现为社交能力缺陷、认知障碍以及重复性行为。近年来，二代测序技术飞速发展，但是对于大部分的ASD病人来说，依旧没有良好的分子诊断标记，而且对神经发生异常引发ASD的研究仍不充分。因此，对于在神经细胞分化、神经发育和ASD发生过程中是否还存在其他重要因素参与调控该过程的核心网络，并且如何从分子水平影响神经分化和ASD发生过程还有待更多的研究。LncRNA已被公认在调控ES细胞的命运中具有关键作用，但是到目前为止，绝大多数lncRNA的功能和作用机理仍然是未知的，有待进一步探究。在本项目执行期间，不仅顺利完成了项目任务书既定计划和任务，并在两大方面取得了重要的研究工作进展，具体成果总结如下：（1）首次发现Linc-BML联合表观修饰复合物PRC2，调控其邻近基因Mef2c的表达，而Linc-BML/Mef2c轴能够影响神经分化相关基因的调控网络，并调节与ASD相关基因的表达。更重要的是，在ASD模型小鼠中过表达Linc-BML可以显著改善ASD模型小鼠的自闭样行为；（2）发现特定lincRNA（linc1548）能够与神经诱导重要的转录因子Sox2和Oct6结合，形成一个RNA-蛋白复合物作用于下游靶基因Zfp521的转录表达，进而调控Epiblast向神经前体细胞的转变过程。