Characterization of Stat3 Binding in the γ-Globin Promoter: The Role of DNA-Protein Interactions in Gene Silencing.

We previously demonstrated that signal transducer and activator of transcription 3β (Stat3β) acts as a negative regulator of γ-globin gene expression. We speculate that Stat3β participates in the γ to β-globin switch recapitulated during erythroid maturation. Regulatory elements located between nucleotide −52 and +36 relative to the γ-globin cap site may define a silencing domain where Stat3β exerts its negative effect. We analyzed DNA-protein interactions in a putative Stat3 binding site at +9γ and an overlapping Stat3/GATA-1 consensus sequence at +26γ. The +9γ motif was required for Stat3β mediated γ-gene silencing in K562 cells and normal erythroid progenitors. Mutating the +26γ site disrupted both Stat3 and GATA-1 binding which was confirmed by electrophoretic mobility shift assay. We demonstrated for the first time direct interaction between Stat3 and GATA-1 using immunoprecipitation techniques and protein extracts isolated from K562 and mouse erythroleukemia cells. We speculate that developmental stage specific DNA-protein interactions in the −52 to +36 region are required for γ-gene silencing. To investigate this hypothesis, we tested the ability of Stat3β to bind in vivo using chromatin immunoprecipitation (ChIP) assay. K562 cells were treated with Interleukin-6 (100ng/ml) to activate Stat3β and then used for ChIP analysis with phosphorylated-Stat3β antibody. Specific PCR primers were designed to amplify the region between nucleotide −52 and +36 relative to the γ-globin cap site. We observed increased Stat3β binding to the γ-globin silencing domain in vivo after Interleukin-6 treatment. To compliment this approach, we modified our ChIP assay as follows. A trypsin digest step was added after cross-linking proteins to the γ-globin silencing domain in vitro. The samples will be analyzed by nanospray LC/MS/MS mass spectrometry to characterize the transcription factors that bind the silencing domain and interact with Stat3β in this region. Understanding the DNA-protein interactions required for γ-globin regulation will provide insights into mechanisms for globin gene silencing.