Involvement of Sp1/Sp3 in the activation of the GATA-1 erythroid promoter in K562 cells.
GATA-1 is a hematopoietic transcription factor that is essential for the terminal maturation of proerythroblasts, megakaryocytic cells and mast cells. The erythroid-specific promoter of the human GATA-1 gene directs the high expression of a reporter gene in K562 cells. Multiple putative transcription factor binding sites were identified in the promoter from the -860 to the -1 base pair (bp). For a better understanding of the transcriptional control of human GATA-1 gene expression, we tested the transcriptional activity of a series of deletions from the 5' end of the 860-bp promoter. A region between -221 and -128 bp retains most of the transcriptional activity of the full-length promoter. Deletion of the CGCCC box at -195 bp reduced reporter gene activity to 60.4%. Further deletion of the CACCC box at -173 bp nearly abolished reporter gene expression, indicating that the CACCC box is more critical. In vitro experiments of electrophoretic mobility shifts and in vivo studies using chromatin immuno-precipitation (ChIP) assays show that the Sp1/Sp3 proteins bind the CACCC site in the nuclei of K562 cells. Coincidently, hyperacetylation of histones in the GATA-1 erythroid promoter was also shown by ChIP assay. Co-transfection of Sp1 expression plasmids and plasmids with a wild-type promoter showed enhanced reporter gene activity in a dose-dependent manner. The combined data demonstrate that Sp1/Sp3, but not EKLF, is involved in the activation of the GATA-1 erythroid promoter, and that histones H3 and H4 are highly acetylated in this promoter region for an actively transcribed GATA-1 gene in K562 cells in which EKLF is barely detectable.
Hou
Chun Hui
CH
Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, 320 Yueyang Road, Shanghai 200031, China.
Huang
Jian
J
He
Qi Ye
QY
Zhang
Cai Ning
CN
Zhang
Xue Jun
XJ
Qian
Ruo Lan
RL
D000107:Acetylation; D001483:Base Sequence / Q000235:genetics; D050982:GATA1 Transcription Factor / Q000096:biosynthesis* / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D006657:Histones / Q000235:genetics / Q000378:metabolism; D006801:Humans; D020014:K562 Cells; D051741:Kruppel-Like Transcription Factors / Q000235:genetics / Q000378:metabolism; D020218:Response Elements / Q000502:physiology*; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D014158:Transcription, Genetic / Q000502:physiology*
D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; D006657:Histones; D051741:Kruppel-Like Transcription Factors; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; C080774:erythroid Kruppel-like factor; D051705:Sp3 Transcription Factor