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

Abstract

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.

Chromatin Remodeling and Transcriptional Activity of the Bone-specific Osteocalcin Gene Require CCAAT/Enhancer-binding Protein β-dependent Recruitment of SWI/SNF Activity

“…This indicates that interaction of both factors with the OC promoter is independent of SWI/SNF activity and therefore prior to the formation of the proximal DNase I-hypersensitive site. In support of this conclusion, we have previously demonstrated that Runx factors are capable of recognizing their cognate elements within the context of a nucleosomal organization (27,39) and independent of the level of core histone acetylation. 4 Together these results indicate that binding of Runx2 and C/EBP␤ is one of the early events during chromatin remodeling and transcriptional activation of the OC gene.…”

Granulocyte-Macrophage Colony-Stimulating Factor Enhancer Activation Requires Cooperation between NFAT and AP-1 Elements and Is Associated with Extensive Nucleosome Reorganization

“…Furthermore, the GM330 and GM420 elements defined two clusters of sites located within adjacent nucleosomes, where binding of NFAT and AP-1 appeared necessary before closely linked Sp1 and AML1 sites could form stable complexes at sites located more centrally within these nucleosomes. It is known that Sp1 and AML1 can bind to nucleosomes but at greatly reduced efficiencies compared to that of free DNA; therefore, they are likely to rely on additional factors for stable binding (3,24,37,38). However, AML1 appears to be able to bind independently to the osteocalcin promoter if its binding site is located close to the end of nucleosomal DNA (24).…”

“…For this purpose, we bred the previously described VOL. 24,2004 NFAT FUNCTION AND CHROMATIN REMODELLING 7915 transgenic mouse line M268 (13) to homozygosity to create a line carrying approximately 100 copies of the human GM-CSF gene. mRNA analyses.…”

“…The next stage in defining mechanisms of NFAT-dependent enhancer activation was to determine which elements were actually required for enhancer function and the extent to which different elements acted syn-VOL. 24,2004 NFAT FUNCTION AND CHROMATIN REMODELLING 7917…”

Bone-Specific Transcription Factor Runx2 Interacts with the 1α,25-Dihydroxyvitamin D3 Receptor To Up-Regulate Rat Osteocalcin Gene Expression in Osteoblastic Cells

“…The fusion proteins glutathione S-transferase (GST)-Runx2, GST-Runx2⌬376, GST-Runx2⌬361, GST-Runx2⌬230, GST-Runx2(209-361), GST-VDR, GST-VDR⌬1-111, GST-VDR⌬1-21, GST-VDR⌬22, GST-VDR⌬165, GST-VDR⌬111, and GST-VDRS51G were obtained by expression in Escherichia coli BL21 as previously reported (5,18,24). GST-free VDR proteins were obtained by cleaving GST-VDR with 1.0 U of factor Xa (New England Biolabs, Beverly, Mass.)…”