利用RNA转录组数据库挖掘肿瘤细胞EZH2基因候选转录因子

The RNA Transcriptome Database Was Used to Mine the Candidate Transcription Factors of EZH2 Gene in Tumor Cells

转录因子的筛选是基因转录调控研究的重要环节。通常人们通过候选转录因子基序(motif)结构域的DNA结合序列是否存在于靶基因启动子而进行筛选。基因表达相关性是发现基因间相互作用的一种有效手段。利用已有的多种人类基因组转录组公共数据库,通过对已知转录因子与靶基因共转录关系分析,本研究发现,肿瘤细胞系大百科全书(CCLE)基因共转录相关系数可作为筛选候选转录(抑制和激活)因子的新方法。对所挖掘出的7个与EZH2基因转录高度相关的候选转录因子(TCF7L2、PML、TBP、PHF8、RBBP5、MYBL2、NRF1)进行实验验证,发现PHF8和NRF1过表达(或敲降)确实促进(或抑制)EZH2基因转录;染色质免疫共沉淀实验和荧光素酶报告基因结果亦证实,PHF8和NRF1能够结合EZH2基因启动子DNA,提示PHF8和NRF1可能是EZH2基因的转录因子。

Selection of transcription factor(TF)is an important step in the study of regulation of gene transcription.Traditionally,a TF candidate is selected based on whether the candidate contains a motif that can bind the promoter DNA sequences of target genes.Gene co-expression is an effective method for discovering inter-gene interactions.In this study,using a set of publically available databases of the human transcriptomes,we found that the expression levels of transcription activator(or repressor)genes were significantly and positively(or inversely)correlated with those of target genes in the cancer cell line encyclopedia(CCLE)databases,suggesting gene co-transcription coefficients could be used to screen TF candidates.Using the co-transcription coefficients,7 not reported and mostly EZH2-coexpressed TF candidates(TCF7L2,PML,TBP,PHF8,RBBP5,MYBL2,NRF1)for EZH2 gene were selected for experiment validation.Finally,we found that enforced overexpression(or siRNA knockdown)of PHF8 and NRF1 genes could upregulate(or downregulate)EZH2 transcription in quantitative RT-PCR analyses,and that PHF8 and NRF1 proteins could increase EZH2 promoter activity and bind EZH2 promoter DNA in luciferase reporter and chromatin immunoprecipitation analyses.In conclusion,these results indicated that PHF8 and NRF1 may be TFs for the EZH2 gene.