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 CBFalpha/AML/PEBP2alpha (
core binding factor alpha/
acute myelogenous leukemia/polyoma enhancer
binding protein 2alpha)
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 CBFalpha
proteins. We analyzed binding of bacterially produced Cbfalpha2Alpha and Cbfalpha2B, two splice variants of the human CBFalpha2 gene, and determined the effect of heterodimerization with the Cbfbeta subunit on binding activity. Our results indicate that binding of the truncated Cbfalpha2A
protein to naked
DNA is independent of Cbfbeta whereas Cbfalpha2A binding to nucleosomal
DNA was enhanced by Cbfbeta. In contrast, the Cbfalpha2B interaction with either naked or nucleosomal
DNA was strongly dependent on heterodimerization with the Cbfbeta subunit. Additionally, our results demonstrate that both Cbfalpha2A alone and Cbfalpha2B complexed with Cbfbeta 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 CBFalpha site. This situation was achieved with
a DNA segment of 182 bp from the rat OC promoter that preferentially positions mononucleosomes upstream of the CBFalpha 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 CBFalpha factors to nucleosomal
DNA.