苯并芘暴露经Dnmt1高甲基化Pax3基因致神经管畸形的机制研究

Neural tube defects (NTDs) are among the most common and severe congenital malformations. Epidemiological studies have reported that periconceptional exposure to polycyclic aromatic hydrocarbons was associated with an increased risk of fetal NTDs, however, the underlying mechanisms are unknown. Our previous work successfully established a mouse NTDs model by intraperitoneal injection of benzo[α]pyrene (BaP), a widely studied PAH. We found that the body region of Pax3, a key gene responsible for neural tube closure, in the neural tissues was hypermethylated in BaP exposed embryos. It is known that DNA methylation is regulated by DNA methyltransferase (Dnmts), and histone modification could affect the recruitment of Dnmts. Our preliminary study observed a significant elevated level of Dnmt1 in the neural tissues of embryos exposed to BaP. We searched the ENCODE database and found that histone modifications in the promoter region and body region of Pax3 were different. Thus, we hypothesize that BaP exposure within the developmental window of neural tube closure could up-regulate the expression of Dnmt1, which could then be recruited to the body region of Pax3 by recognition of specific histone modification. Subsequently, the body region of Pax3 could be hypermethylated, and Pax3 gene expression be repressed, leading ultimately to NTDs. The proposed project will be conducted using the established mouse model and additional cell experiments to reveal the critical role of Dnmt1 in the epigenetic mechanism for BaP-induced NTDs, and whether histone modification could mediate the effect of Dnmt1. The results will help elucidate the epigenetic mechanism contributing to the development of NTDs, and provide novel strategies for NTDs prevention.

神经管畸形（NTDs）是一类严重先天缺陷。围孕期环境污染物多环芳烃暴露可增加后代NTDs发生风险，但致病机制不清。申请人采用孕鼠经腹腔注射苯并芘（多环芳烃类物质），成功诱发胎鼠NTDs，并观察到神经管闭合关键基因Pax3基因体区高甲基化改变。已知DNA甲基化水平受DNA甲基转移酶（Dnmt）调控，组蛋白修饰能影响Dnmt特异性募集。前期工作发现在苯并芘致NTDs中，胎鼠神经组织中Dnmt1表达增强，且生物信息分析提示Pax3启动子和基因体区组蛋白标记不同。据此假设：苯并芘暴露后Dnmt1表达上调，并通过识别组蛋白修饰位点使Pax3基因体区高甲基化，下调Pax3表达，最终导致NTDs。本项目拟采用动物模型结合细胞实验，阐明在苯并芘暴露致NTDs中Dnmt1对Pax3甲基化的调控作用，并基于ChIP探讨组蛋白修饰在其中可能的介导作用。预期为NTDs发病机制研究提供线索，为该病防治提供新的靶点。

神经管畸形（NTDs）是一类严重先天缺陷。人群研究表明围孕期多环芳烃暴露与胎儿NTDs发生风险增加有关。我们前期基于小鼠动物模型，已验证孕鼠经腹腔注射苯并芘（多环芳烃类物质），可导致胎鼠NTDs，并观察到神经管闭合关键基因Pax3基因体区高甲基化改变。基于此，本项目拟采用动物模型结合细胞实验，探讨DNA甲基转移酶Dnmts对Pax3甲基化的调控作用，并基于ChIP探讨组蛋白修饰在其中可能的介导作用。结果发现，相较于对照胎鼠和BaP暴露组非NTDs胎鼠，BaP暴露组NTDs胎鼠神经组织中Dnmt1基因和蛋白表达均显著升高，且酶活性增加，而Dnmt3a和Dnmt3b在三组间无明显差异；且采用ChIP-seq实验，发现Dnmt1在BaP暴露组NTDs胎鼠Pax3基因中存在显著增加的差异峰，其中两个峰所在Pax3基因片段呈高甲基化改变，且均位于基因体区。细胞实验发现，当Dnmt1过表达时，Pax3基因甲基化水平增加，基因表达下调，且细胞凋亡水平增加，迁移和活化受到抑制；进一步基于H3K27me3和H3K27ac组蛋白修饰的ChIP实验发现，在BaP暴露组NTDs胎鼠中，H3K27me3可能特异地结合于上述发现的Pax3高甲基化区域。综上所述，我们发现BaP暴露后，Dnmt1可能特异性地调控Pax3基因高甲基化改变，从而导致NTDs的发生，H3K27me3可能与Dnmt1的选择性修饰作用有关。本项目的结果对于揭示NTDs发生的表观遗传机制具有一定的意义。