Sox10蛋白在内耳发育过程中的表观修饰及调控机制研究

Inner ear developmental malformation is a common cause of congenital hearing loss. Recent studies found that Sox10 gene plays an important role in inner ear development. Our research team has successfully developed an inner ear malformation model based on Sox10 mutation in domestic miniature pigs. Based on this model, we have identified several related genes which are critical in inner ear development using transcriptomics analysis and reporter assay, We also found the mutation of SOX10 can cause fail of SOX10 protein transcription. Our preliminary data suggest that Sox10 gene has controlled the transcriptional activation of the target gene promoter region, although the detail mechanism is still not clear. Based on our pilot data, we hypothesize that Epigenetic as an existence of an unknown regulatory mechanism underlies SOX10 protein expression in inner ear development. To test this hypothesis, we plan to use mass spectrometry technology to screen the SOX10 protein in the inner ear of miniature pigs to analyze the post-translational modification, especially on the methylation of No.109 arginine. Then we will detect the apparent enzyme of Epigenetic modification species during the SOX10 translation using cutting edge technologies, including pull-down, Co-IP, isotopically labeled and truncating mutation, etc. In summary, the project will use a new novel concept in apparent biology to reveal how SOX10 selectively regulate the transcription of the downstream target genes. The success of the project will provide guidance on clinical diagnosis and treatment in inner ear developmental malformation.

内耳畸形是常见感觉功能疾病。研究发现Sox10与内耳发育密切相关，在已建立Sox10突变致内耳畸形小型猪模型基础上，借助比较转录组学鉴定出部分内耳发育关键靶基因，但同时发现该突变仅导致Sox10蛋白部分转录功能丧失, 表明Sox10可能存在对特定靶基因启动子区的选择性转录激活效应，但具体机制不明。因此申请者创新性提出“Sox10蛋白在内耳发育过程中可能受到表观机制调控”的科学假设。为验证该假设，本研究拟通过质谱技术分析耳发育过程SOX10蛋白的翻译后修饰情况，并重点关注第109位精氨酸的甲基化修饰；随后借助pull-down、Co-IP、同位素标记和截断突变等技术手段，鉴定介导Sox10蛋白发生翻译后修饰的表观酶种类、两者互作结构域以及对Sox10相关功能转录组学的影响。通过这些研究，本课题将以表观生物学这一全新视角解析内耳发育中Sox10蛋白对下游目的基因选择性转录调控的分子机理。

SOX10是决定胚胎细胞发育命运的关键核质穿梭转录因子，突变可导致Waardenburg综合征，表现为内耳畸形及听功能障碍，目前SOX10调控耳蜗发育分子机制仍存在较大空白。本研究借助Sox10 p106A to G基因突变猪模型，成功模拟了与人类高度相似的瓦氏综合征疾病特征，借助胚胎猪耳蜗全转录组分析，获得差异表达的miRNA, lncRNA及mRNA多组学关联网络，最终明确DNA表观修饰酶TET2为lncRNA-miRNA-mRNA分子网络中的关键节点，该分子介导基因组DNA 5羟甲基胞嘧啶修饰（5hmC），本研究进一步对同窝野生型及SOX10突变胚胎猪耳蜗进行5hmC组学分析，发现近90%的差异5hmC主要分布于基因间甚至异染色质区域（位于基因区的差异5hmC占比不足10%），这意味着SOX10突变介导5hmC修饰变化多通过远端调控方式影响相关基因表达，后续关联分析进一步证明包括EPHB3,ILS1，MAP1B, NRP1, NTN1, RTN4, SEMA3D, SEMA4D, Wnt5a等一系列存在远端5hmC调控的差异基因均被注释到“轴突及神经系统发育”，“细胞迁移”，“神经投射”等神经发育相关的条目之中。经q-RT-PCR, WB，IF验证表明部分差异基因的确在SOX10突变猪耳蜗中存在显著表达变化。本研究原创性发现DNA表观修饰酶TET2受到SOX10的直接调控，进而间接影响全基因组5hmC修饰水平，借助表观遗传学视角，开启了“表观修饰调控大动物耳蜗及听神经发育”这一全新研究领域，同时为瓦氏综合征临床药物研发提供了表观修饰酶相关的新型分子靶标，具有极其重要的科学意义及潜在应用价值！