利用染色质免疫共沉淀测序研究Twist1结合的靶DNA序列

目的:利用染色质免疫共沉淀测序研究Twist1结合的靶DNA序列,比较过表达Bcl-2和正常状态下Twist1结合靶DNA序列的变化。方法:筛选常态表达Twist1的肝癌细胞系,分别转染Bcl-2表达质粒和对照空载体,收集转染后细胞以甲醛交联DNA,收集样品进行染色质免疫共沉淀,获得与Twist1蛋白结合的DNA片段进行测序分析。结果:通过对5种肝癌细胞系进行筛选,观察到HepG2可表达Twist1,其与DNA结合序列在空载体对照组和Bcl-2过表达组具有很大的差别,结合DNA数量分别为68条和212条,其中共享序列154条。结论:Twist1在不同环境下与DNA结合位点多达212个,涉及粘附分子、细胞骨架、信号转导、代谢和转录调节等多种生物学功能。

Using chromatin immunoprecipitation to study the target DNA binding sequences of Twist1

Objective: To compare the difference of the target DNA binding sequences of Twist1 in cells that overexpress Bcl-2 or not using chromatin immunoprecipitation.Methods: The hepatocellular carcinoma cell lines was screened and the cell line with normal twist expression was selected.Cells were transfected with Bcl-2 expression plasmid and control empty vector.After formaldehyde cross-linking,chromatin immunoprecipitation was performed to get the target DNA binding sequences of Twist1 to be sequenced and analyzed.Results: After screening of 5 hepatocellular carcinoma cell lines,HepG2 was observed to express twist.There was a significant difference of the target DNA binding sequences of Twist1 in cells that overexpress Bcl-2 or not.The amount of binding-DNA was 68 and 212,respectively.At the same time,they shared 154 sequences.Conclusion: There are 212 DNA binding sequences of Twist1 in different microenvironment,involving adhesion molecules,cytoskeleton,apoptosis,cell motility and migration and other biological functions.