TY - JOUR
T1 - Role of STAT3 and GATA-1 interactions in γ-globin gene expression
AU - Yao, Xiao
AU - Kodeboyina, Sirisha
AU - Liu, Li
AU - Dzandu, James
AU - Sangerman, Jose
AU - Ofori-Acquah, Solomon F.
AU - Pace, Betty S.
N1 - Funding Information:
This work was supported by grant #HL073447 from the National Institutes of Health (Bethesda, MD, USA) to the University of Texas at Dallas (Richardson, TX, USA) on behalf of Betty S. Pace. Special thanks to Inderdeep Kalra for technical assistance with the ChIP assay.
PY - 2009/8
Y1 - 2009/8
N2 - Objective: We previously demonstrated a silencing role for signal transducers and activators of transcription 3 (STAT3) in γ-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 γ-globin gene expression. Materials and Methods: Chromatin immunoprecipitation assay was used to ascertain in vivo protein binding at the γ-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 γ-globin promoter reporter and levels of γ-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 γ-globin gene expression. Results: We observed interaction between STAT3 and GATA-1 in K562 and mouse erythroleukemia cells in vivo at the γ-globin 5′UTR by chromatin immunoprecipitation assay. Electrophoretic mobility shift assay performed with a 41-base pair γ-globin DNA probe (γ41) demonstrated the presence of STAT3 and GATA-1 proteins in complexes assembled at the γ-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 γ-globin 5′UTR in vivo and silenced γ-promoter-driven luciferase activity. Stable enforced STAT3 expression in K562 cells reduced endogenous γ-globin messenger RNA level. This effect was reversed by GATA-1. Conclusion: These data provide evidence that GATA-1 can reverse STAT3-mediated γ-globin gene silencing in erythroid cells.
AB - Objective: We previously demonstrated a silencing role for signal transducers and activators of transcription 3 (STAT3) in γ-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 γ-globin gene expression. Materials and Methods: Chromatin immunoprecipitation assay was used to ascertain in vivo protein binding at the γ-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 γ-globin promoter reporter and levels of γ-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 γ-globin gene expression. Results: We observed interaction between STAT3 and GATA-1 in K562 and mouse erythroleukemia cells in vivo at the γ-globin 5′UTR by chromatin immunoprecipitation assay. Electrophoretic mobility shift assay performed with a 41-base pair γ-globin DNA probe (γ41) demonstrated the presence of STAT3 and GATA-1 proteins in complexes assembled at the γ-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 γ-globin 5′UTR in vivo and silenced γ-promoter-driven luciferase activity. Stable enforced STAT3 expression in K562 cells reduced endogenous γ-globin messenger RNA level. This effect was reversed by GATA-1. Conclusion: These data provide evidence that GATA-1 can reverse STAT3-mediated γ-globin gene silencing in erythroid cells.
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U2 - 10.1016/j.exphem.2009.05.004
DO - 10.1016/j.exphem.2009.05.004
M3 - Article
C2 - 19447160
AN - SCOPUS:67650088333
SN - 0301-472X
VL - 37
SP - 889
EP - 900
JO - Experimental Hematology
JF - Experimental Hematology
IS - 8
ER -