Role of STAT3 and GATA-1 interactions in γ-globin gene expression

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.