Role of STAT3 and GATA-1 interactions in γ-globin gene expression
Received 14 September 2008; received in revised form 6 April 2009; accepted 8 May 2009. published online 18 May 2009.
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.
aDepartment of Molecular and Cell Biology, University of Texas at Dallas, Richardson, Tex., USA
bDepartment of Pediatrics, Yale University, New Haven, Conn., USA
cDepartment of Pediatrics, Emory University, Atlanta, Ga., USA
Offprint requests to: Betty S. Pace, M.D., Department of Molecular and Cell Biology, University of Texas at Dallas, FO 3.1, 800 West Campbell Road, Richardson, TX 75080
ABSTRACT