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.

The upstream Gγ-globin gene cAMP response element (G-CRE) was previously shown to play a role in drug-mediated fetal hemoglobin induction. This effect is achieved via p38 mitogen activated protein kinase (MAPK)-dependent CREB1 and ATF-2 phosphorylation and G-CRE trans-activation. Since this motif is also a predicted consensus binding site for cJun we extended our analysis to determine the ability of cJun to trans-activate γ-globin through the G-CRE. Using chromatin immunoprecipitation assays we showed comparable in vivo cJun and CREB1 binding to the G-CRE region. Protein-protein interactions were confirmed between cJun/ATF-2 and CREB1/ATF-2 but not between CREB1 and cJun. However, we observed cJun and CREB1 binding to the G-CRE in vitro by electrophoretic mobility shift assay. Promoter pull-down assay followed by sequential western blot analysis confirmed co-localization of cJun, CREB1, and ATF-2 on the G-CRE. To show functional relevance, enforced expression studies with pLen-cJun and a Gγ-promoter (-1500 to +36) luciferase reporter were completed; we observed a concentration-dependent increase in luciferase activity with pLen-cJun similar to that produced by CREB1 enforced expression. Moreover, the G/ A mutation at -1225 in the G-CRE abolished cJun trans-activation. Finally, enforced cJun expression in K562 cells and normal primary erythroid progenitors enhanced endogenous γ-globin gene expression. We concluded from these data indicate that cJun activate the Gγ-globin promoter via the G-CRE in a manner comparable to CREB1 and propose a model for γ-globin activation based on DNA-protein interactions in the G-CRE. List of key wordscJun; CREB1; γ-globin; cAMP response element; ATF-2

Previous studies from our laboratory demonstrated the role of the G-CRE (Gγ-globin cAMP response element) in drug-mediated fetal hemoglobin induction. The G-CRE located at −1222 to −1229 in the promoter of Gγ-globin gene, contains binding site for trans-factors CREB1, ATF-2 and cJun. We previously demonstrated binding of phosphorylated CREB1 and ATF-2 to this element via p38 MAPK signaling triggered by sodium butyrate (NaB) and trichostatin A (TSA). Electrophoretic mobility shift assays with a probe containing the AC → TG mutation in the G-CRE (TGTGGTCA, m2) abolished trans-factor binding to the G-CRE. Furthermore, Gγ promoter activity was abolished in the PGL3 luciferase reporter vector driven by the Gγ promoter (−1500 to +36) carrying the m2 mutation. (Sangerman et al. Blood108:3590–9, 2006). Subsequent studies in our laboratory were aimed at understanding the role of trans-factor cJun, an AP-1 family member, as a regulator of Gγ-globin expression via the G-CRE site. In K562 cells treated with 2mM NaB or 0.3μM TSA for 48 hrs, cJun phosphorylation increased 2.8-fold and 6.4-fold respectively by western blot analysis. Chromatin immunoprecipitation studies showed 16-fold chromatin enrichment in the −1225 Gγ-globin region compared to IgG control studies indicative of significant cJun binding in vivo at steady state. Electrophoretic mobility shift assays using cJun monoclonal antibody demonstrated a supershifted DNA-protein complex confirming binding of cJun to the G-CRE probe. To gain evidence for a functional role of cJun, we performed enforced expression studies using the pLen-cJun vector. In a concentration dependent manner, over-expression of cJun increased luciferase activity up to 350-fold in the luciferase reporter plasmid controlled by the Gγ-promoter (−1500 to +36). As predicted from binding studies, the m2 mutation in this promoter abolished the cJunmediated trans-activation confirming that the G-CRE is required to mediate effects of cJun. We are currently investigating the ability of cJun to trans-activate the endogenous Gγ-globin gene in K562 cells. To achieve this goal, K562 stable lines were established with the expression vectors pLen-cJun and empty vector. A complete analysis of the stable lines is in progress. Future investigations to identify other components of the functional CREB1/ATF2/cJun enhanceosome complex bound to the G-CRE will be performed using affinity chromatography and mass spectrometry. This information will be used to develop strategies for fetal hemoglobin induction.