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 CBF alpha /AML/PEBP2 alpha (core binding factor alpha /acute myelogenous leukemia/ polyoma enhancer binding protein 2 alpha) 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 CBF alpha proteins. We analyzed binding of bacterially produced Cbf alpha 2A and Cbf alpha 2B, two splice variants of the human CBF alpha2 gene, and determined the effect of heterodimerization with the Cbf beta subunit on binding activity. Our results indicate that binding of the truncated Cbf alpha 2A protein to naked DNA is independent of Cbf beta whereas Cbf alpha 2A binding to nucleosomal DNA was enhanced by Cbf beta. In contrast, the Cbf alpha 2B interaction with either naked or nucleosomal DNA was strongly dependent on heterodimerization with the Cbf beta subunit, Additionally, our results demonstrate that both Cbf alpha 2A alone and Cbf alpha 2B complexed with Cbf beta 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 CBF alpha: site. This situation was achieved with a DNA segment of 182 bp from the rat OC promoter that preferentially positions mononucleosomes upstream of the CBF alpha 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 CBF alpha factors to nucleosomal DNA.