转录因子GATA-2对iASPP基因的转录调控研究  

Effects of transcription factor GATA-2 on transcriptive regulation of iASPP gene

作　　者：邢海燕 贾玉娇 唐克晶 田征 陈一瑞 饶青 王敏[1] 王建祥 

机构地区：[1]中国医学科学院、北京协和医学院血液学研究所、血液病医院,实验血液学国家重点实验室,天津300020

出　　处：《中国实验血液学杂志》2013年第3期550-555,共6页Journal of Experimental Hematology

基　　金：国家自然科学基金资助项目(编号81270635,81070389,81070427)

摘　　要：癌基因iASPP促进细胞增殖、抑制细胞凋亡,其转录起始位点上游序列有转录因子GATA-2的假定结合位点,GATA-2在造血干/祖细胞的增殖和分化中起重要作用。本研究旨在探讨GATA-2对iASPP的转录调控作用。首先检测了部分白血病细胞系中iASPP和GATA-2蛋白的表达,然后构建带有GATA-2开放读码框的真核表达载体与带有iASPP转录起始位点上游3000 bp至下游500 bp序列中GATA-2假定结合位点的荧光素酶报告载体,共转染HEK293和CV-1细胞,检测荧光素酶活性,并对荧光素酶活性上调的结合位点区进行染色质免疫共沉淀实验。结果显示,白血病细胞系中iASPP高表达的细胞大多存在GATA-2的高表达;GATA-2对iASPP报告基因荧光素酶活性有显著影响,并呈现明显的"剂量-效应"关系。当pCMV5-GATA2转染剂量上升至100 ng时,iASPP荧光素酶活性在HEK293细胞中上调到2倍;此转录激活作用在CV-1细胞中尤为显著,荧光素酶活性上调可达6.7。染色质免疫共沉淀结果证实GATA-2与iASPP转录起始区nt-361～-334有特异性结合。结论:转录因子GATA-2可以与iASPP的转录调控区结合,并促进iASPP基因转录。iASPP can prompt the cell proliferation and inhibit the apoptosis of many cells. There are putative binding sites of transcription factor GATA-2 upstream of iASPP transcription start site. GATA-2 plays an important role in the proliferation and differentiation of hematopoietic stem cells （HSC） and progenitors. This study was aimed to explore the role of GATA-2 protein in iASPP gene transcription. Firstly, the expression of iASPP and GATA-2 protein in some leu kemia cell lines was detected by Western blot. Second, The expressive vector of pCMV5-GATA2 and the luciferase re-porter vectors containing possible binding sites of GATA-2 were constructed and co-transfected into HEK293 and CV-1 cells. Then the luciferase activity was assayed by luminometer. Also, ChIP assays were performed to further confirm the specific binding of GATA-2 to iASPP promoter. The results showed that GATA-2 was overexpressed in most cell lines with high level of iASPP. GATA-2 exhibited a significant effect on luciferase activity of reporter gene iASPP and in a dose-dependant manner. The relative luciferase activity was up-regulated to about two-fold of the empty vector control when the transfection dose of pCMVS-GATA2 plasmid was increased to 100 ng. While the effect was more significant in CV-1 cells and showed a 6.7-fold increase. The ChIP assay demonstrated the in vivo specific binding of GATA-2 to iASPP. The binding sites of GATA2 were located between nt -361 - -334 in upstream of iASPP gene transcription start site. It is concluded that transcription factor GATA-2 can bind with the cis-regulatory region of the iASPP promoter and up-regulate iASPP expression.

关 键 词：GATA-2 IASPP 转录调控 荧光素酶活性 

分 类 号：R733.7[医药卫生—肿瘤]