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. MATERIALS AND 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. CONCLUSION: These data provide evidence that GATA-1 can reverse STAT3-mediated gamma-globin gene silencing in erythroid cells.
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. MATERIALS AND 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 mouseerythroleukemia 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 STAT3expression augmented its binding at the gamma-globin 5'UTR in vivo and silenced gamma-promoter-driven luciferase activity. Stable enforced STAT3expression in K562 cells reduced endogenous gamma-globin messenger RNA level. This effect was reversed by GATA-1. CONCLUSION: These data provide evidence that GATA-1 can reverse STAT3-mediated gamma-globin gene silencing in erythroid cells.
Authors: Osamu Tanabe; Fumiki Katsuoka; Andrew D Campbell; Weimin Song; Masayuki Yamamoto; Keiji Tanimoto; James Douglas Engel Journal: EMBO J Date: 2002-07-01 Impact factor: 11.598
Authors: Koichi R Katsumura; Andrew W DeVilbiss; Nathaniel J Pope; Kirby D Johnson; Emery H Bresnick Journal: Cold Spring Harb Perspect Med Date: 2013-09-01 Impact factor: 6.915