MeCP2蛋白保守结构域AT-hook1的破坏导致行为学异常及其分子机制研究

Neuronal enriched nuclear protein MeCP2 plays an important role in chromatin construction and gene transcription by binding to methylated DNA. Mutations in MECP2 cause Rett syndrome and autism related disorders. In addition, MeCP2 involves in the progress of seizure, pain and drug addiction. Since the identification of the conserved domain AT-hook1, there is no report on the function study. We and others identified de novo novel missense mutations of MECP2 in the AT-hook1 domain, which were responsible for Rett syndrome or intellectual disability. Moreover, we generated a mouse line carrying deletion of eight conserved amino acids in AT-hook1 domain. Mutant mice exhibited low locomotor activity, motor incoordination and cognitive deficit. In addition, these mutant mice presented with increased anxiety. Moreover, pain insensitivity was noted in the mutant males. In the present study, we will generate another mouse line bearing missense mutation in the AT-hook1 domain. Among the wide type mice, mice with missense mutant in AT-hook1 and mice with AT-hook1 deletion, the difference will be compared on the behavioral and pathological changes, DNA methylation and transcriptomics. The histone 3 modification and its regulation on the transcription of the down-stream genes will be assessed on the effect of neuronal development. This study will provide evidence for the role of AT-hook1 in MeCP2 protein.

神经元内高表达的核蛋白MeCP2在DNA甲基化、染色体重构及基因转录调控中起桥梁作用，为成熟神经元功能所必需。MECP2基因突变导致了Rett综合征（RTT）等孤独症谱系病，同时MeCP2也在癫痫、药物成瘾、疼痛等病程中起重要作用。AT-hook1是MeCP2的高度保守结构域，但其生物学功能尚未见报道。我们及其他学者均发现了AT-hook1的错义突变可导致RTT或智力障碍。我们前期建立的携带AT-hook1结构域缺失的基因敲入（KI）小鼠也出现了运动和学习记忆功能障碍、焦虑及痛觉迟钝等多种行为异常。本项目拟另构建携带有AT-hook1的错义突变KI小鼠，通过比较野生型、AT-hook1错义突变及缺失KI小鼠之间的行为学、病理及分子水平，特别是甲基化组学和转录组学上的差异，并观察组蛋白甲基化修饰及一些关键下游靶基因对神经发育的影响，从而阐明AT-hook1结构域介导的MeCP2的功能。

背景:MECP2基因突变导致Rett综合征等孤独症谱系病。核蛋白MeCP2在神经元内高表达，是重要表观遗传调控因子。其DNA结合域、转录抑制功能域等对MeCP2功能起重要作用，而保守的AT-hook1结构域（aa184-195）的作用尚不清楚。.研究内容: 1. 对发生于AT-hook1内变异的临床病例或文献资料进行分析，阐明患者R190H变异的致病性；2. 通过构建AT-hook1缺失（△AT）小鼠模型，观察动物行为学、病理及分子水平变化，阐明AT-hook1变异的致病性；3. 在体外培养的细胞中过表达△AT和R190H变异对细胞生物学功能的影响；4. 通过基因编辑构建携带△AT和R190H两种变异的人源诱导性多能干细胞（iPSCs）株，进行神经元分化，观察神经元功能的影响，并通过基因治疗药物进行干预，观察神经元功能恢复情况。.结果：1. 明确诊断一例携带有AT-hook1-R190H新变异的Rett综合征患者，阐明了新变异的致病性。2. 建立了首个Mecp2-AT-hook1缺失疾病模型鼠，发现△AT模型鼠出现类似RTT的行为学异常表现：运动减少、运动协调功能受损、学习记忆功能障碍、焦虑及痛觉迟钝等；疾病鼠海马CA1区变薄；组蛋白修饰水平变化、抑制性神经元相关基因及G蛋白耦联受体家族基因表达明显差异可能是动物行为学异常及病理变化的分子机制。3. 建立了△AT和R190H的稳定表达神经细胞株，阐明AT-hook1的缺失及点突变均表现为MeCP2功能的缺失, 导致细胞活力下降和凋亡，进一步验证了△AT小鼠大脑转录组学差异。4. 构建了携带有R190H和∆AT突变的人源iPS细胞株，后续的神经元分化及生物学功能、分子机制验证和基因治疗尚在完善中。.科学意义：本项目阐明了MeCP2-AT-hook1功能域的重要性，其变异会导致MeCP2生物学功能障碍，出现大脑转录组学差异，导致疾病的发生。.