利用ChIP-seq技术研究转录因子EDAG在全基因组的结合谱  

Genome-wide Research on Transcription Factor EDAG Binding Profiling by Using ChIP-seq

作　　者：董小明[1] 郑巍薇[2] 尹荣华[2] 詹轶群[2] 杨晓明[2] 李长燕[2] 

机构地区：[1]北京工业大学生命科学与生物工程学院,北京100124 [2]军事医学科学院放射与辐射医学研究所,北京100850

出　　处：《中国生物化学与分子生物学报》2013年第6期578-584,共7页Chinese Journal of Biochemistry and Molecular Biology

基　　金：优秀青年科学基金(No.81222005)项目资助~~

摘　　要：为揭示红系分化相关基因(erythroid differentiation-associated gene,EDAG)在造血中的作用机制,利用ChIP-seq分析EDAG的全基因组结合谱.首先从产妇脐带血分离CD34+细胞,EPO诱导CD34+细胞培养5 d.利用EDAG抗体进行染色体免疫共沉淀(chromatin immunoprecipitation,ChIP)实验、Western印迹法检测EDAG抗体的富集情况.将富集到的DNA样品进行高通量测序,最后利用生物信息学分析测序结果.成功富集染色体DNA,经高通量测序和生物信息学分析,共得到1 292个EDAG结合位点的Peaks数目,代表了975个结合的基因且错误发现率(false discovery rate,FDR)小于0.0001.EDAG Peaks主要分布在基因间区和内含子区.进一步利用Q-PCR对ChIP-Seq数据进行了验证,证实EDAG可结合在检测的靶基因调控区上.将EDAG结合的基因进行基因功能(gene ontology,GO)注释,表明EDAG参与了细胞周期、细胞生长、细胞分化、细胞凋亡及信号通路等多种生物学过程.综上,利用ChIP-seq技术在促红细胞生成素(EPO)诱导分化的CD34+细胞中鉴定了1 292个EDAG结合的peaks对应975个基因,并对该结果进行了随机验证,提示EDAG广泛参与了多种生物学过程.该研究为揭示EDAG的功能及作用机制提供了线索.To understand the regulation of erythroid differentiation-associated gene （EDAG） during hematopoietic differentiation, EPO-induced human cord blood CD34＋ cells was used for ChIP-seq analysis. The human cord blood CD34＋ cells were isolated and treated with EPO for 5 days to induce erythroid differentiation. Chromatin DNA was immunoprecipitated with EDAG antibody and detected by Western blotting. The obtained DNA samples were subjected to high-throughput sequencing and bioinformatic analysis. A total of 1292 enrichment peaks corresponding 975 genes was identified with a false discovery rate （FDR） 〈 0. 0001. The mapping of the peaks revealed that they mainly occurred within intergenic and intron. To confirm the reliability of the ChIP-seq data, 11 genes involved in cell growth, cell cycle, apoptosis and hematopoietic differentiation were randomly selected and validated byqPCR for EDAG association. Gene ontology （GO） analysis suggested that EDAG regulated a set of genes involved in various biological processes, including cell proliferation, metabolism, hematopoietic development and cell signaling. These results might provide a useful insight to reveal the function and mechanism of EDAG.

关 键 词：红系分化相关基因(EDAG) 染色体免疫共沉淀-高通量测序(ChIP-seq) CD34+细胞 生物信息学分析 

分 类 号：Q71[生物学—分子生物学]