组蛋白甲基化修饰在神经管畸形发生中的作用及分子机制研究

Neural tube defects (NTDs) are common birth defects, which caused by the impediment of neural tube closure. The previous studies showed that Hox genes are important for the neural tube closure, which dysregulation would lead to NTDs, however the mechanism was not clear. We intend to select some Hox genes significantly differentially expressed in NTDs mouse by RNA-seq, and investigate the temporal and spatial expression pattern of Hox genes in NTDs. Then in order to understand what influence the Hox genes expression, we detect the regulation of histone methylation modification on Hox genes using ChIP assay, and also study the regulation of special histone methyltransferase enzymes on histone methylation at the cellular or tissue level. In addition, the influence of the supplementation of folic acid on histone methylation modification and Hox genes expression will confirm the hypothesis that Hox genes lead to NTDs through histone methylation modification. Furthermore, we will make the further validation on the function and mechanisms of histone methylation modifications in the human NTDs samples. This project will provide the new mechanisms of NTDs caused by environment intervention from the epigenetic perspective and provide the important basic data for the early prevention of NTDs, which is of great significance to improve the quality of the population under two-child policy.

神经管畸形（Neural tube defects, NTDs）是由于胚胎发育过程中神经管闭合障碍引起的先天性出生缺陷疾病。研究表明Hox基因是参与神经管闭合的关键基因，表达紊乱可致NTDs发生，但其紊乱机制尚不清楚。本项目拟在前期建立的NTDs小鼠模型中，采用RNA-seq寻找多个差异表达的Hox基因并揭示其在NTDs中的时空表达规律；利用ChIP assay在细胞和组织水平研究多种组蛋白甲基化修饰对Hox基因表达的影响，并通过特异性修饰酶的检测探讨组蛋白甲基化修饰改变的原因，在补充甲基供体叶酸后进一步确认Hox基因表达紊乱引起NTDs受到组蛋白甲基化修饰的调控；并将此表观遗传调控通路在临床NTDs样本中进行验证。本项目从表观遗传学角度，揭示组蛋白甲基化修饰调控下游Hox基因表达引起NTDs发生的分子机制，为NTDs早期预防提供重要的基础数据，对于当前我国二孩政策下促进生殖健康意义重大。

Hox是参与神经管闭合的关键基因，其表达异常与NTDs发生密切相关，但其调控机制尚不清楚。本研究前期采用转录组测序研究发现：在RA诱导的NTDs鼠胚中10个Hox基因表达异常升高，并经RT-qPCR得到验证。在RA干预的鼠ESCs中也观察到类似结果。而在RA干预的鼠ESCs及NTDs鼠胚中H3K27me3表达却显著降低。进一步采用ChIP技术发现：RA处理后10个Hox基因与H3K27me3结合减弱，提示Hox基因表达紊乱由H3K27me3调控所致。接着，我们研究发现UTX酶活性升高和Suz12表达下降可导致H3K27me3表达下降及Hox表达升高。最后，我们在临床无脑畸形胚胎中验证了Hox表达均显著升高和H3K27me3表达下调，相关性分析显示HOXB4、HOXC4和HOXD1与H3K27me3表达呈显著负相关。以上结果提示，RA可促进UTX酶活性升高和Suz12表达下降，引起H3K27me3水平下降，导致Hox基因表达升高，参与NTDs的发生，从表观遗传学角度为NTDs的发生提出了新的机制，为NTDs早期预防提供了基础数据。此外，我们采用表达谱芯片筛选发现：低叶酸处理的ESCs中胶质细胞缺失因子（Glial cell missing 1，Gcm1）表达异常升高。进一步实验证明其表达升高受到Nanog转录因子调节。接着我们研究发现低叶酸条件下Gcm1与Wnt/β-catenin信号通路中的关键分子β-catenin和TCF4结合增加，三者形成的复合物与下游靶基因结合激活其表达，并激活该通路。最后，我们在低叶酸联合甲氨蝶呤诱导的NTDs小鼠模型以及临床低叶酸NTDs病例中对上述结果进行了验证。以上研究结果首次揭示了Gcm1调控Wnt/β-catenin信号通路参与低叶酸致NTDs发生的新的分子机制，为明确叶酸补充预防NTDs这一“未解”课题提供了相应的理论基础，也为Wnt/β-catenin信号通路在调控其它早期出生发育缺陷疾病的机制研究提供了研究切入点。.