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

Abstract

Objective-We previously demonstrated a silencing role for 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 the activity of Stat3 in vivo. Therefore we completed studies to determine if interactions between these two factors influence γ-globin gene expression.Methods-Chromatin immunoprecipitation (ChIP) assay was used to ascertain in vivo protein binding at the γ-globin 5'untranslated region (5'UTR); protein-protein interactions were examined by co-immunoprecipitation analysis. In vitro protein-DNA binding were completed using surface plasmon resonance and electrophoretic mobility shift assay (EMSA). Activity of a luciferase γ-globin promoter reporter, and levels of γ-globin mRNA 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 ChIP assay. EMSA performed with a 41 base pair DNA probe (γ41) demonstrated the presence of Stat3 and GATA-1 proteins in complexes assembled at the γ-globin 5'UTR. A consensus Stat3 binding DNA probe inhibited GATA-1 binding in a concentrationdependent 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 mRNA 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.

Original Research: Stable expression of miR-34a mediates fetal hemoglobin induction in K562 cells

“…60 Furthermore, as erythroid differentiation progresses, erythropoietin has the ability to activate STAT3 through serine 727 phosphorylation 62 as g-globin gene silencing occurs. Consistent with these observations, miR-34a overexpression induced HbF and reduced totaland phosphorylated-STAT3 levels in K562 stable clones.…”

Protein tyrosine phosphatase PTPN9 regulates erythroid cell development through STAT3 dephosphorylation in zebrafish

“…S4D). Previous studies have shown that formation of the STAT3-GATA1 complex leads to the inhibition of the expression of c-globin (Ezoe et al, 2005;Foley et al, 2002;Kirito et al, 1998;Yao et al, 2009). Furthermore, the Krüppel-type zinc finger transcription factor ZBP-89 has been previously reported to interact with GATA1 and regulate expression of the globin genes and erythroid differentiation during hematopoiesis in zebrafish (Li et al, 2006;Ohneda et al, 2009;Woo et al, 2011;Woo et al, 2008).…”

“…Previous studies have elucidated that phosphorylated STAT3 inhibits GATA1 through a direct protein-protein interaction (Ezoe et al, 2005;Yao et al, 2009). The ability of phosphorylated STAT3 to suppress GATA1 transactivation has been attributed to the alteration of GATA1 binding stability.…”

“…Owing to the inhibitory effect of phosphorylated STAT3 on erythroid cell maturation (Foley et al, 2002;Yao et al, 2009), we assumed that knockdown of PTPN9 might increase STAT3 phosphorylation and formation of the STAT3-GATA1-ZBP-89 complex, which prevents binding of GATA1 and ZBP-89 to the promoters of their target genes. We found that increased STAT3 phosphorylation, by overexpression of dominantnegative N9CS, led to the recruitment of more GATA1 and ZBP-89 into the complex (Fig.…”

Cell signaling pathways involved in drug-mediated fetal hemoglobin induction: Strategies to treat sickle cell disease

“…69 In subsequent studies, we observed in vivo binding of STAT3 and GATA-1 to this region by chromatin immunoprecipitation assay in K562 and mouse erythroleukemia cells. 70 DNA- 75 Lou et al, 77 Ikuta et al, 93 Tang et al 96 Butyrate HDAC inhibitor p38, cGMP, cAMP Witt et al, 71 Pace et al, 78 Sangerman et al 81 Trichostatin protein interaction studies confirmed that a STAT3/GATA-1 complex is assembled in this region. STAT3b enforced expression in K562 cells reduced g-globin expression which was reversed by GATA-1 over-expression.…”