转录激活因子样效应蛋白（TALEs）介导TET3蛋白靶向胃癌PCDH17基因mCpG去甲基化的研究

CpG hypermethylation in promoter is an important mechanism of tumor suppressor gene inactivation. However, the targeted CpG site demethylation means is extremely lacking. Latest research reported that Ten-eleven translocation proteins(TET) can oxidize 5-mCpG into 5-hmCpG and initiate demethylation. Transcription activator-like effector proteins(TALEs) can specifically identify the nucleotides and mediate modification enzyme to target gene editing. We hypothesized that TALEs could mediate TET3 to target demethylation and become the mean for targeted demethylation. Our previous studies have shown that the hypermethylation of -84 and-74 CpG sites in PCDH17 promoter leads to gene inactivation in gastric cancer, suggesting that PCDH17 gene could be the gene model of targeted demethylation study. In this study, we will use Golden Gate assembly, fluorescence reporter plasmid identification, Tab-seq sequencing and other methods to build PCDH17 gene specific TALEs and hydroxylase TET3 protein, and perform demethylation in -84 and -74CpG sites in PCDH17 gene. We will also evaluate the gene activation as well as the biological characteristics of gastric cancer cells after targeted demethylation by using bisulfite sequence and real time cell analysis and other methods. This study will provide theoretical and experimental evidence for the application of gene targeting epigenetic modification.

启动子CpG位点的异常高甲基化是抑癌基因失活的重要机制，但靶向CpG位点去甲基化恢复基因功能的手段极为缺乏。新近发现TET3蛋白可氧化mCpG去甲基化，转录激活因子样效应蛋白（TALE）能特异识别核苷酸并介导基因靶向编辑，我们推测TALE能介导TET3蛋白成为靶向CpG位点去甲基化的有效手段。我们前期发现胃癌PCDH17基因启动子-84、-74CpG位点异常高甲基化导致基因沉默，提示其可作为靶向去甲基化研究的基因模型。本项目拟构建特异识别PCDH17基因启动子双链序列的TALE蛋白（TALE-F和TALE-R），结合TET3蛋白组装成靶向去甲基化工具TALE-F-TET3和TALE-R-TET3，并鉴定其靶向操作的特异性和有效性；在此基础上，研究胃癌PCDH17基因靶向去甲基化后基因表达和胃癌细胞生物学特征的改变。本课题将为基因靶向表观遗传修饰手段的开发应用提供依据。