Dlk1-Dio3印记区域内母本甲基化差异甲基化区Meg8-DMR的功能研究

The Dlk1-Dio3 imprinted domain plays crucial roles in embryonic development, cell reprogramming and genetic diseases. As the fist detected maternal methylated region located in Dlk1-Dio3 imprinted domain, Meg8-DMR shows the dynamic process with dymethylation and de novo methylation, but its biological functions still remain unclear. Based on the CRISPR/Cas9 genome editing technique, Meg8-DMR deletion cell line and knockout mouse model were constructed. Through the genome-wide expression profiling and DNA methylation profiling analysis of cell lines and different embryonic developmental stages by high-throughput sequencing, as well as ChIP experiments and the CTCF-dependent insulator activity studies, combined with the analysis of cell behavior and mouse phenotype, detect the formation and the regulatory mechanism of the Dlk1-Dio3 imprinted domain which the differentially methylated region Meg8-DMR participate in, and uncover the biological functions of the differentially methylated region Meg8-DMR during the embryonic development. This study will further clarify the transcriptional regulation mechanism of the Dlk1-Dio3 imprinted domain, and provide new molecular targets for the future developmental biology research, cell reprogramming, the regulation of organ development and the evaluation of genetic diseases.

Dlk1-Dio3印记区域在胚胎发育、细胞重编程和遗传疾病过程中起重要作用。Meg8-DMR是Dlk1-Dio3印记区域内第一个被发现的母本甲基化的差异甲基化区，在着床前后的胚胎发育中呈现去甲基化和重新甲基化的动态变化过程，其生物学功能尚不清楚。本课题基于CRISPR/Cas9基因编辑技术，构建Meg8-DMR缺失细胞系和敲除小鼠模型。通过细胞系和不同发育阶段胚胎高通量的表达谱分析和全基因组范围内DNA甲基化谱分析，以及ChIP实验和依赖于CTCF的绝缘子活性研究，结合细胞行为和小鼠表型分析，探索差异甲基化区Meg8-DMR参与Dlk1-Dio3印记区域内的印记形成和调控的分子机制，揭示Meg8-DMR在胚胎发育过程中的生物学功能。本课题将进一步阐明Dlk1-Dio3印记区域的转录调控机制，并为今后发育生物学研究、细胞重编程、器官发育的调控及遗传性疾病的评估诊断提供新的分子靶标。

Dlk1-Dio3印记区域在胚胎发育、细胞重编程和遗传疾病过程中起重要作用。Meg8-DMR是Dlk1-Dio3印记区域内第一个被发现的母本甲基化的差异甲基化区，在着床前后的胚胎发育中呈现去甲基化和重新甲基化的动态变化过程。本研究基于CRISPR/Cas9基因编辑技术，构建Meg8-DMR敲除小鼠模型和缺失细胞系。形态学研究发现，Meg8-DMR敲除小鼠胚胎期的发育（体重、遗传比例、胎盘形态及分层等）无影响，小鼠能正常出生并发育成个体。Dlk1-dio3区间内印记基因的状态和差异甲基化区的甲基化模式未发生变化。高通量测序及定量PCR分析显示Meg8-DMR敲除影响Dlk1-Dio3区间内Rian的表达，并对小鼠的行为有一定的影响。在小鼠睾丸间充质细胞中的研究显示, Meg8-DMR具有较强的绝缘子活性，其绝缘子活性依赖于CTCF结合基序，并且串联重复序列(GGCG)n对于Meg8-DMR的绝缘子活性是不可缺少的。同时Meg8-DMR敲除不改变Dlk1-Dio3区间内差异甲基化区的甲基化模式；Meg8-DMR敲除抑制了MLTC-1细胞的增殖，促进侵袭和迁移。上述研究结果为更好的阐明Meg8-DMR在胚胎发育及肿瘤发生过程中的生物学功能及其转录调控机制奠定良好的基础。