Characterization of Combinatorial Stat3/GATA-1 Complexes in the γ-Globin 5′UTR.

Regulation of human γ-globin expression depends on localized combinatorial interactions of trans-factors with cis-elements in the proximal promoter however, the full complement of proteins involved in γ-gene regulation remains obscure. We previously demonstrated the ability of ^Stat3 α to bind a +9 site in the ^Aγ-globin 5′untranslated region (^Aγ5′UTR) to activate transcription. To expand on this observation we identified an overlapping Stat3/GATA-1 site (GAGATTATCAA) at nucleotide +21γ. Recently Stat3 and GATA-1 were shown to participate in protein-protein interactions to regulate cell differentiation. Therefore we hypothesized that GATA-1 and Stat3 may regulate γ-gene transcription through a similar mechanism. Initial chromatin immunoprecipitation (ChIP) assays were performed to demonstrate in vivo GATA-1 and Stat3 binding to the γ-promoter. ChIP assay for the ^Aγ-globin region between −122 to +65 produced 9-fold and 4-fold chromatin enrichment in K562 cells with GATA-1 and Stat3α antibodies respectively compared to no enrichment with IgG. Treatment with Interleukin-6 mediated a 12-fold increase in bound Stat3α and a concomitant 2-fold decrease in GATA-1 interactions. TFIID binding increased from 25-fold to 40-fold supporting transcriptional activation. To provide in vitro evidence for competitive binding in the +21γsite, electrophoretic mobility shift assays (EMSA) were performed with K562 nuclear extracts and ³²P- labeled γ41 (+1 to +41), +21γ, and Stat3 and GATA-1 consensus probes. Four specific DNA-protein complexes were established with γ41. Stat3 and GATA-1 antibody studies with the four probes demonstrated that Stat3 dimers and GATA-1 dimers were bound in the Band 1 and Band 4 complexes respectively; in contrast Stat3/GATA-1 heterodimers were present in the Band 3 complex. Studies with the +21γ probe confirmed our findings with γ41. The data are consistent with our hypothesis that GATA-1 and Stat3 compete for binding to the +21γ cis-element. To further our understanding of the in vivo consequences of GATA-1/Stat3 interactions, preliminary transient transfections were performed in K562 cells. The wild type -210γLuc reporter containing the −210 to +36 γ-promoter (10μg) and this mutant −201γ(+21mStat3)Luc reporter were co-transfected individually with β-galactosidase. The luciferase activity of −201γLuc increased 110-fold after enforced pZeoStat3α (30μg) expression which dropped 75-fold when tested with the mutant Stat3 reporter. Residual activity was attributed to the intact +9γ Stat3 site. Enforced pGFP-GATA-1 expression activated the −201γLuc reporter 35-fold which increased further to 70-fold for the mutant reporter. Additional cotransfection studies will be completed to determine whether competitive interactions of Stat3 and GATA-1 occur to regulate γ-globin transcription in vivo. To confirm and verify these observations, vitro competitive binding utilizing purified Stat3 and GATA-1 proteins will be performed. 2-Dimensional EMSA coupled with mass spectroscopy will be used for comprehensive identification of the protein components of complexes bound to the ^Aγ5′UTR.