Interaction of CBFα/AML/PEBP2α transcription factors with nucleosomes containing promoter sequences requires flexibility in the translational positioning of the histone octamer and exposure of the CBFα site

Chromatin remodeling at eukaryotic gene promoter sequences accompanies transcriptional activation. Both molecular events rely on specific protein-DNA interactions that occur within these promoter sequences. Binding of CBFα/AML/PEBP2α (core binding factor α/acute myelogenous leukemia/polyoma enhancer binding protein 2α) proteins is a key event in both tissue-specific and developmentally regulated osteocalcin (OC) promoter activity. To address linkage between chromatin organization and transcription factor binding, we reconstituted segments of the rat OC gene proximal promoter into mononucleosomes and studied binding of CBFα proteins. We analyzed binding of bacterially produced Cbfα2A and Cbfα2B, two splice variants of the human CBFα2 gene, and determined the effect of heterodimerization with the Cbfβ subunit on binding activity. Our results indicate that binding of the truncated Cbfα2A protein to naked DNA is independent of Cbfβ whereas Cbfα2A binding to nucleosomal DNA was enhanced by Cbfβ. In contrast, the Cbfα2B interaction with either naked or nucleosomal DNA was strongly dependent on heterodimerization with the Cbfβ subunit. Additionally, our results demonstrate that both Cbfα2A alone and Cbfα2B complexed with Cbfβ can interact with nucleosomal DNA only if there is a degree of flexibility in the positioning of the histone octamer on the DNA fragment and exposure of the CBFα site. This situation was achieved with a DNA segment of 182 bp from the rat OC promoter that preferentially positions mononucleosomes upstream of the CBFα binding site and leaves this element partially exposed. Taken together, these results suggest that nucleosomal translational positioning is a major determinant of the binding of CBFα factors to nucleosomal DNA.