胚胎干细胞分化为表皮样干细胞基因启动子区差异甲化研究

 目的： 用全基因组甲基化芯片探讨可能参与小鼠胚胎干细胞(ESCs)分化为表皮样细胞(ELCs)的基因DNA甲基化调控机制。方法： 利用人羊膜建立体外诱导体系，将小鼠ESCs诱导定向分化为ELCs，分别取未分化的ESCs和诱导分化后的ELCs进行芯片分析，利用甲基化免疫共沉淀技术将每组染色质DNA的甲基化片段共沉淀下来，和本底对照 (input)分别标记Cy3和Cy5荧光，一同上样于Roche NimbleGen高密度(2.1M，芯片覆盖22 425个启动子)甲基化芯片，启动子区采用UCSC数据库进行注释，启动子覆盖转录起始位点上游8 200 bp、下游3 000 bp，通过对这些启动子区甲基化谱的分析，筛选出甲基化调控可能与ESCs向ELCs定向分化相关的基因。结果： 小鼠ESCs和ELCs 两组细胞在基因组水平上有17 500个启动子存在甲基化，其中有3 435个启动子发生甲基化差异变化，高CpG岛的启动子有894个发生差异甲基化，中度CpG岛的启动子有974个发生差异甲基化，低CpG岛的启动子有1 567个发生差异甲基化。结论： 在ESCs向ELCs定向分化的过程中，众多的基因启动子区发生了甲基化程度的变化，说明细胞分化是一个复杂的表观遗传学事件。这些基因启动子的差异甲基化在ESCs向ELCs定向分化过程中所起的作用及其机制尚有待进一步的功能学研究阐明。

Genome-wide screen of promoter methylation Analysis of ES Cells and ES Derived Epidermal-like Cells

Genome-wide screen of promoter methylation Analysis of ES Cells and ES Derived Epidermal-like Cells ZHANG Ren-li1△ , LIU Cai-xia1，MENG Jin-xiu2,Zhang Lili1, HAN Dong1, Wen An-min1 (1.Reproductive Medicine Center; 2. Medical Research Center, Guangdong General Hospital//Guangdong General Hospital, Guangzhou 215006, China) Abstract] OBJECTIVE: To investigate differentially methylated promoters may be involved in process of differentiation from embryonic stem cells (ESC) into epidermal-like cells (ELC) with DNA methylation chip. METHODS: Mouse ESC was induced to differentiate into ELC in vitro by using human amnion, and whole-genome DNA differential methylation between undifferentiated ESC and ELC were analyzed with DNA methylation chip. Immunoprecipitation of methylated DNA was performed using BiomagTM magnetic beads coupled mouse monoclonal antibody against 5-methylcytidine. The total input and immunoprecipitated DNA were labeled with Cy3- and Cy5-labeled random 9-mers, respectively, and hybridized to NimbleGen MM9 CpG Promoter arrays, a 2.1M format array containing all known CpG Islands annotated by UCSC and all well-characterized RefSeq promoter regions (from about -8200 to +3000bp of the TSSs) totally covered by 210,000 probes. DNA methylation of promoter regions was analyzed to discover the potentially related genes. RESULTS: Among all the 17,500 DNA mathylation regions of gene promoters in both ESC and ELC, there were 3,435 gene promotors showed to be differentially methylated, involving 894 HCP, 974 ICP and 1567 LCP, which enriched peaks in high, intermediate, and low CpG–containing promoter of the 2 groups. CONCLUSION: Great change of methylation of a large amount of genes promoter occurs during the differentiation of ESC to ELC, indicating that the differentiation is a complex pcocess regulated by many genes, and how these gene worked and co worked to regulate the differentiation procedure need further investigations. KEY WORDS] DNA methylation chip; ES cells; epidermal-like cells; promoter; Immunoprecipitation of methylated DNA