叶酸缺乏引起的甲基化差异区域的印记改变在胚胎发育中的作用研究

Epigenetic alternations have been suggested to be involved in developmental abnormalities caused by folic acid deficiency, methylation modification alternations of imprinting genes is regarded as one of key mechanisms of epigenetic regulation in developmental programming. Differentially methylated regions (DMRs) are core regulatory regions of genomic imprinting, the mechanism of folate deficiency altered DMRs methylations in earlier development is still unclear. New trials of this project is trying to use rat and ESC models with FA deficiency to analysis imprinting alternations of DMRs associated with periconceptional folic acid deficiency, to study rules and characteristics of imprinting alternations with FA deficiency, to analysis alternations of chromosome structure and imprinting regular factors with FA deficiency, to analysis histone modifications and miRNA level in imprinting regions, to discuss the mechanism underlying folate affecting imprinting modifications, to detect the expression levels of imprinting genes with methylation alternations and function abnormal related to expression alternations, and in the end, to explore the mechanism of DMRs imprinting abnormal with developmental defection. At the same time, neural tube defects (NTDs) was selected to analysis the association between folate level and imprinting modifications of DMRs in disease cases, as it was reported to connected with folate defection. In summary, a scientific epigenetic mechanism behind how folate acid regulated healthy development will be studied and powerful experimental proof will be provided with the completion of this project.

叶酸缺乏引起的重大发育异常可能与表观修饰的改变相关,发育编程中印记基因甲基化修饰是表观调控的关键机制之一。甲基化差异区域(DMR)作为基因组印记调控的核心区域,发育早期叶酸缺乏对其甲基化修饰改变的作用及机制尚未完全阐明。本研究拟采用叶酸缺乏小鼠模型，结合叶酸缺乏的胚胎干细胞系，比较甲基化差异区域(DMR)甲基化修饰的改变；研究叶酸代谢异常对印记形成影响的规律及特征；分析叶酸缺乏对印记区域染色体结构和印记调控因子的影响；分析印记变化区域组蛋白甲基化修饰和miRNA变化；探讨叶酸代谢影响印记形成的机制；并探索异常DMRs甲基化状态对发育相关印记基因表达及表达产物功能的影响；进一步分析和验证DMRs异常印记状态与发育异常的可能机制；进而在低叶酸营养的神经管畸形标本中分析并验证叶酸代谢与印记修饰的关联。本课题的研究为叶酸缺乏影响发育的详细表观遗传机制的进一步阐明提供了实验依据。

叶酸作为重要的一碳营养素，不仅提供DNA合成的重要原料，同时也为细胞和机体提供DNA和蛋白质修饰所需的一碳基团。为研究叶酸在发育早期对生命健康的调控机制，本项目通过围孕期低叶酸营养的小鼠模型，结合低叶酸神经管畸形标本、先心病标本和流产标本，以及低叶酸的胚胎干细胞模型，研究发现了发育早期叶酸代谢异常影响生殖细胞甲基化差异区域（gDMRs）印记修饰的建立，确认印记基因是围孕期叶酸代谢影响的一类靶基因。通过对胚胎停育、神经管畸形、先天性心脏病等重大发育缺陷疾病的研究，发现存在印记修饰的异常可能是叶酸影响重大发育缺陷发生的重要机制；通过建立围孕期低叶酸饮食营养的小鼠模型，确认了围孕期不同亲本叶酸缺乏对胎鼠发育的影响的亲本特异性；发现了低叶酸营养影响GNAS印记基因簇的印记建立，发育早期低叶酸营养早在配子期干扰了GNAS印记建立，GNAS印记建立失败影响细胞cAMP信号，干扰发育健康；发现了围孕期低叶酸营养可能通过甲基化修饰调控miRNA let-7g的表达水平，影响其靶基因的功能，参与NTD的发生，证实低叶酸营养影响甲基化与miRNA的交互作用；发现了低叶酸营养通过干扰DLK印记簇内印记建立，影响miR-370的水平进而干扰其靶基因DNMT3a的活性，调控了基因组内更多区域的甲基化建立。通过本项目实施，证实多种表观修饰调控的交互作用参与了围孕期低叶酸增加出生缺陷的发生，为围孕期补充叶酸的理论提供了实验数据。