DNA羟甲基化表观修饰调控神经干细胞向神经元分化的分子机制研究

DNA 5-hydroxymethylcytosine (5-hmC), also known as the sixth base, is a new manner of DNA epigenetic modification. We have previous established, for the first time, the genome-wide 5-hmC epigenomic maps of mouse brain cortex and striatal regions, and compared the 5-hmC epigenomic maps between normal and Huntington’s disease (HD) brains. Our results suggest that the abnormal 5-hmC modification in HD may cause the deficiency in neuronal differentiation of neural stem cells. However, one key question has not been answered. The dynamic 5-mC/5-hmC epigenomic modification during neuronal differentiation and how this 5-mC/5-hmC regulates the nueonal determination are far from clear. Therefore the present proposal is going to be focused on the dynamic, whole genome-wide 5-mC/5-hmC modification during the process of neuronal differentiation, complemented with RNA-seq profiles, to elucidate how the gene expression and neuronal determination are regulated by 5-mC/5-hmC modifications. The 5-mC/5-hmC epigenomic maps of HD neural stem cells during differentiation will also be established and compared with that of normal neural stem cells. The expected outcomes will not only elucidate the molecular mechanisms of 5-mC/5-hmC in regulating the neuronal differentiation and neuronal determination of neural stem cell, but also provide new insights into the dysregulation of 5-mC/5-hmC epigenome in neurological disease, and into development of new therapies targeting on epigenomic modifications.

5-羟甲基胞嘧啶(5hmC)是近几年新发现的一种DNA表观修饰方式，被认为是第六个碱基。我们的前期工作率先在国际上构建了小鼠大脑皮层和纹状体区的5hmC修饰图谱，并系统比较了亨廷顿神经退行性疾病模型鼠与正常鼠大脑5hmC表观修饰谱式。结果提示，5hmC修饰异常可能造成疾病小鼠神经干细胞的神经分化缺陷。然而5mC/5hmC修饰如何调控神经干细胞向神经元分化目前还不清楚。因此本项目将聚焦在神经干细胞定向分化过程中5mC/5hmC修饰的动态图谱，以及5mC/5hmC修饰的动态变化如何调节基因表达进而调控神经干细胞的命运决定。疾病神经干细胞5mC/5hmC的动态修饰图谱也将被构建，并与正常神经干细胞的修饰图谱进行对应分析。预期结果不仅可以揭示5mC/5hmC修饰调控神经干细胞神经分化决定的分子机制，而且对于搞清神经疾病病理过程导致的5hmC表观修饰紊乱和开发以表观修饰为靶标的新治疗手段有重要意义。

DNA的5-羟甲基胞嘧啶(5hmC)是近几年新发现的一种DNA表观修饰方式。DNA羟甲基化修饰异常可能造成神经疾病神经干细胞的神经分化缺陷。然而DNA的甲基化和羟甲基化修饰如何调控神经干细胞向神经元分化目前还不清楚。项目实施一年来，已建立并完善了钙信号刺激依赖的神经干细胞向神经细胞定向分化的体外诱导方法。通过优化生长因子浓度和钙信号通路刺激强度，可使体外分化为MAP2阳性神经细胞的分化效率提高到70%-80%。我们还对影响神经干细胞增殖、分化能力的表观因素进行了研究，发现来自线粒体的超氧信号也可显著增加神经干细胞的增殖和分化，并且是钙信号依赖的。在舞蹈病细胞中，由于钙信号的异常增强，导致超氧信号增强，进而导致神经干细胞的增殖和分化速率增加，在疾病小鼠则表现为海马区神经干细胞过早耗竭。推测这可能是舞蹈病神经退行的一个重要机制。钙信号诱导的超氧可快速升高DNA的甲基化，与此对应，羟甲基化水平快速降低，这与我们观察到的舞蹈病神经组织DNA羟甲基化水平大大降低是一致的。表明钙信号依赖的超氧信号对DNA的甲基化/羟甲基化修饰有一个快速建立/消除的调控过程，具体机制正在研究中。发现一种调控钙信号的新型钙通道CLAC，并建立了CLAC突变的CFT疾病模型。该疾病模型细胞表现为钙信号异常升高，小鼠则表现为海马、皮层纹和状体区的显著萎缩和认知障碍。推测异常增高的钙信号和超氧信号导致的神经发生异常参与了CFT疾病的神经方面的表型。表观因素(DNA甲基化的改变)参与的具体机制正在进一步研究中。这些发现对于揭示DNA表观修饰紊乱参与神经疾病病理过程的机制和开发以表观修饰为靶标的新治疗手段有重要意义。