内源性反转录转座子在神经管畸形基因组中的重排和表观调控机制的初步研究

Endogenous retrotransposon is the largest class of mobile elements, which occupied a large part of mammalian genome. Hypomethylation modification of retrotransposon has been related to lots of biological effects, such as activity of transposition, high frequencies of genomic insertions, genomic instability, and gene expression alternations, and so on. Lots of studies on developmental diseases, including cancer, reported hypomethylaiton of retrotransposon and genomic alternations in somatic genome in pathogenesis. In our previous studies, hypomethylaiton of LINE-1 was also reported in neural tissues of NTDs. Based on this phenomenon, further researches are needed to declare possible biological effects associated with hypomehtyaltion of LINE-1. In this project, genome-wide analysis was used to detect young insertions of LINE-1 and Alu, all insertions will be validated by PCR amplify and sequencing. De novo insertions will be detected by comparing paired-parents genome. Epigenetic modification, including DNA methylaiton and miRNA expression, will also be analyzed in this study in order to analysis the underlying regulation mechanism of activity of transposition. Finally, the relationship among one-carbon metabolism, modification of retrotransposon and activity of transposition will be researched and discussed in vitro, further effects on functional gene expression and signal pathway also will be studied in folate defection/hypomethylat-treatment cell models. With this project will probably allow us to recognize the essential functions and regulation mechanism of retrotransposon and in neurodevelopmental.

内源性反转录转座子占基因组很大比例，是真核生物中数量最大的一类可活动遗传成分。反转录转座子的低甲基化修饰可引起转座活性改变，增加反转录转座序列在基因组内的重排，引起基因组稳定性改变，影响功能基因的表达等一系列生物学效应，目前已证实与包括肿瘤等在内的多种发育性疾病的发生相关。我们前期工作发现NTD中存在LINE-1甲基化的降低，本项目在此基础上，通过高通量测序方法检测LINE-1和Alu两种内源性反转座子在NTD基因组中的分布；通过PCR扩增测序验证，并与亲本基因组比较，寻找NTD发生相关的新发转座插入；通过甲基化修饰和miRNA表达分析，探讨转座调控的机制；通过体外实验，分析一碳代谢和表观修饰对转座活性的调控，并检测转座序列的插入对功能基因和通路的影响，探索NTD中LINE-1低甲基化可能引起的生物学效应，推进理解神经发育中反转座子的重要调控功能和可能机制。

本项目通过对中长散在重复序列LINE-1反转录转座子的研究，发现，神经管畸形患儿的脑组织重复序列LINE-1和基因组总体甲基化水平显著降低，这种变化具有组织特异性，NTDs中LINE-1甲基化的紊乱存在并仅存在在神经组织中；低叶酸营养直接降低基因组LINE-1甲基化的修饰水平；LINE-1启动子的低甲基化促使染色体结构打开，这种开放的结构改变了调控因子的结合，增加了DNA的断裂；由叶酸代谢紊乱诱导基因组发生的双链断裂（DSB） 在核糖体rRNA基因重复序列区域异常显著，断裂热点与H3K4m1修饰位点分布相关；增加叶酸营养通过影响G1期DSB发生从而减少基因组DNA的损伤；低叶酸环境诱导所引起T-box转录因子brachyury甲基化修饰及表达改变，进而扰乱其靶基因和靶通路（FGF通路）的基因表达紊乱。