Role of STAT3 and GATA-1 interactions in gamma-globin gene expression.
OBJECTIVE
We previously demonstrated a silencing role for signal transducers and activators of transcription 3 (STAT3) in gamma-globin gene regulation in primary erythroid cells. Recently, GATA-1, a key transcription factor involved in hematopoietic cell development, was shown to directly inhibit STAT3 activity in vivo. Therefore, we completed studies to determine if interactions between these two factors influence gamma-globin gene expression.


METHODS
Chromatin immunoprecipitation assay was used to ascertain in vivo protein binding at the gamma-globin 5' untranslated region (5'UTR); protein-protein interactions were examined by coimmunoprecipitation analysis. In vitro protein-DNA binding were completed using surface plasmon resonance and electrophoretic mobility shift assay. Activity of a luciferase gamma-globin promoter reporter and levels of gamma-globin messenger RNA and fetal hemoglobin in stable K562 cell lines overexpressing STAT3 and GATA-1, were used to determine the influence of the STAT3/GATA-1 interaction on gamma-globin gene expression.


RESULTS
We observed interaction between STAT3 and GATA-1 in K562 and mouse erythroleukemia cells in vivo at the gamma-globin 5'UTR by chromatin immunoprecipitation assay. Electrophoretic mobility shift assay performed with a 41-base pair gamma-globin DNA probe (gamma41) demonstrated the presence of STAT3 and GATA-1 proteins in complexes assembled at the gamma-globin 5'UTR. A consensus STAT3 DNA probe inhibited GATA-1-binding in a concentration-dependent manner, and the converse was also true. Enforced STAT3 expression augmented its binding at the gamma-globin 5'UTR in vivo and silenced gamma-promoter-driven luciferase activity. Stable enforced STAT3 expression in K562 cells reduced endogenous gamma-globin messenger RNA level. This effect was reversed by GATA-1.


CONCLUSIONS
These data provide evidence that GATA-1 can reverse STAT3-mediated gamma-globin gene silencing in erythroid cells.
Yao
Xiao
X
Department of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Tex. 75080, USA.
Kodeboyina
Sirisha
S
Liu
Li
L
Dzandu
James
J
Sangerman
Jose
J
Ofori-Acquah
Solomon F
SF
Pace
Betty S
BS
D020121:5' Untranslated Regions; D000818:Animals; D041905:Erythroid Cells / Q000378:metabolism; D050982:GATA1 Transcription Factor / Q000378:metabolism*; D005786:Gene Expression Regulation; D020868:Gene Silencing; D006801:Humans; D020014:K562 Cells; D051379:Mice; D011485:Protein Binding; D012333:RNA, Messenger / Q000032:analysis; D050796:STAT3 Transcription Factor / Q000378:metabolism*; D055545:gamma-Globins / Q000235:genetics*
D020121:5' Untranslated Regions; D050982:GATA1 Transcription Factor; C494688:GATA1 protein, human; C494689:Gata1 protein, mouse; D012333:RNA, Messenger; D050796:STAT3 Transcription Factor; C494086:STAT3 protein, human; C494087:Stat3 protein, mouse; D055545:gamma-Globins