Dlk-Gtl2印记基因网络调控在叶酸缺乏干扰胚胎发育健康中的作用研究

Disordered imprintings establishment have been suggested to be involved in birth defects with folic acid deficiency. Our previously studies have suggested a significant loss of methylation in Dlk1-Gtl2 imprinting cluster with folate deficiency, however, the mechanism underlied about how imprinting alternations works in unhealthy development is still unclear. New trials of this project is trying to use rat and ESC models with FA deficiency to analysis changes of histone modifications and transfer factors in Dlk1-Gtl2 DMRs; and to analysis expression levels of imprinting genes and miRNAs associated with Dlk1- Gtl2 imprinting alternations; to analysis the interactions of miR-370 and DNMT3a and roles in imprinting establishment; to draw an imprinted gene network regulated by DLK1-Gtl2 imprinting; to detect activity of signal pathway in embryogenesis affected by DLK1-Gtl2 imprinted gene network; and to discuss the mechanisms underlying how parental folate deficient affect DLK1-Gtl2 imprinted gene network and healthy development, then by using anaplerotic folate metabolic pathway in ESCs and mutation cell lines, to further confirm the roles of Dlk-Gtl2 imprinted gene network regulation with folic acid deficiency in embryonic development; by using IG-DMR knock out mice model to confirm the role of Dlk-Gtl2 imprinted gene regulation with folic acid during embrogenesis. At the same time, neural tube defects (NTDs) with low folic acid level were picked up to analysis the association between folate metabolism and DLK1-Gtl2 imprinted gene network regulation. In summary, an imteresting epigenetic mechanisms behind how folate acid regulated DLK1-Gtl2 imprinted gene network through interactions of miR-370 and DNMT3a during embryogenesis will be studied and powerful experimental proof will be provided with the completion of this project.

叶酸缺乏影响印记建立是导致出生缺陷的机制之一。我们前期研究证实围孕期低叶酸导致Dlk1-Gtl2印记簇印记丢失，但Dlk1-Gtl2印记丢失致发育缺陷的调控机制还不清楚。本研究拟采用叶酸缺乏的杂合小鼠模型结合低叶酸细胞模型，比较不同亲本叶酸缺乏对发育中Dlk1-Gtl2印记建立的影响，结合印记区组蛋白修饰和调控因子变化研究印记基因的表观修饰交互调控；进而结合低叶酸条件下编码和非编码RNA表达改变，分析Dlk1-Gtl2印记调控的miR-370抑制靶基因DNMT3a对Dlk1- Gtl2印记基因网络的影响，及对发育通路活性的调控；并利用印记中心IG-DMR敲除的小鼠模型验证Dlk1-Gtl2印记簇基因在叶酸缺乏致发育缺陷中的作用机制；最后在低叶酸神经管畸形病例中探讨Dlk1-Gtl2印记基因网络在低叶酸增加NTD风险中的表观调控机制，推动对早期叶酸影响印记建立干扰多系统发育健康的理解。

叶酸缺乏影响发育早期印记建立是导致出生缺陷的机制之一。我们前期研究证实围孕期低叶酸导致Dlk1-Gtl2印记簇印记丢失，但Dlk1-Gtl2印记丢失致发育缺陷的调控机制还不清楚。本研究通过采用叶酸缺乏的小鼠模型结合叶酸干扰细胞模型,结合人类低叶酸神经管畸形标本，比较围孕期叶酸缺乏对胚胎发育中Dlk1-Gtl2印记建立的影响，分析叶酸敏感的表观调控作用位点，结合印记区miRNA的变化研究叶酸在NTD发生中的表观调控机制。同时，将印记调控的作用拓展到更多的发育缺陷中。通过项目实施，总结主要结果如下：.1 人神经管畸形中样本中，检测发现Dlk1-Gtl2印记簇中甲基化调控区域IG-gDMR区域存在印记丢失，Dlk1- Glt2印记基因簇内gDMR区域的甲基化水平受到叶酸的调控,叶酸缺乏引起的印记丢失发生在配子期，延续到胚胎发育晚期。叶酸缺乏诱导的Dlk1- Glt2印记改变，激活其内编码的miR-370-3p表达升高，其靶基因DNMT3a的表达量降低，甲基化酶DNMT3a的减少，与DNMT3a结合的基因数量和片段大小显著降低，改变的主要基因富集在细胞链接和催化酶活基因等；.2 围孕期叶酸缺乏通过改变甲基化水平调控hsa-let-7g表达，hsa-let-7g通过调节靶基因SMOX的表达水平在叶酸缺乏的NTDs中发挥其生物学功能的；.3叶酸缺乏的脑膨出中FGF通路受到干扰，敲除双向潜能的T-Box转录因子Brachyury的缺陷鼠中，Brachyury靶向调控的FGF通路激活子Fgf8表达下调，而抑制子Dusp6表达上升，同时发现FGF通路其他关键基因表达降低，提示在叶酸不足的条件下，通过高甲基化抑制Brachyury基因表达，引起FGF通路活性降低，最终导致神经发育异常。.4 miR-324-5p的表达受叶酸梯度调控，甲基化修饰在其中其重要作用。miR-324-5p梯度过表达引起由重到轻的不同神经管闭合缺陷。miR-324-5p通过抑制HH-Gli1通路增加NTD的发生风险，回补叶酸HH-Gli1通路活性回复，NTD风险降低。.5在复杂畸形的表观调控机制研究中发现，异常增高的SNRPN、ZAC1甲基化水平和降低的INPP5F的甲基化水平可能通过改变其表达水平，从而增加CHD合并EM的患病风险。