Multiple Cbfa/AML Sites in the Rat Osteocalcin Promoter Are Required for Basal and Vitamin D-Responsive Transcription and Contribute to Chromatin Organization

Abstract

Three Cbfa motifs are strategically positioned in the bone-specific rat osteocalcin (rOC) promoter. Sites A and B flank the vitamin D response element in the distal promoter and sites B and C flank a positioned nucleosome in the proximal promoter. The functional significance of each Cbfa element was addressed by mutating individual or multiple Cbfa sites within the context of the ؊1.1-kb rOC promoter fused to a chloramphenicol acetyltransferase reporter gene. Promoter activity was assayed following transient transfection and after stable genomic integration in ROS 17/2.8 osteoblastic cell lines. We show that all three Cbfa sites are required for maximal basal expression of the rOC promoter. However, the distal sites A and B each contribute significantly more (P < 0.001) to promoter activity than site C. In a genomic context, sites A and B can largely compensate for a mutation at the proximal site C, and paired mutations involving site A (mAB or mAC) result in a far greater loss of activity than the mBC mutation. Strikingly, mutation of the three Cbfa sites leads to abrogation of responsiveness to vitamin D. Vitamin D-enhanced activity is also not observed when sites A and B are mutated. Significantly, related to these losses in transcriptional activity, mutation of the three Cbfa sites results in altered chromatin structure as reflected by loss of DNase I-hypersensitive sites at the vitamin D response element and over the proximal tissue-specific basal promoter. These findings strongly support a multifunctional role for Cbfa factors in regulating gene expression, not only as simple transcriptional transactivators but also by facilitating modifications in promoter architecture and chromatin organization.Bone tissue-restricted expression of the osteocalcin (OC) gene during development of the osteoblast phenotype requires a multiplicity of transactivating factors. Among the key regulators of OC expression are transcription factors that play essential roles in embryonic formation of the skeleton and osteoblast differentiation. These include the Cbfa (core binding factors ␣)/AML (acute myelogenous leukemia) family of runt homology domain (rhd) DNA binding proteins (reviewed in reference 33), the Msx and Dlx homeodomain proteins (28, 51, 63), AP-1 proteins (41), and steroid hormone receptors (reviewed in reference 40).The Cbfa/AML family of transcriptional activators are critical factors for the development of hematopoietic and skeletal tissues. Each of three known genes, Cbfa1 (human AML-3/mouse Pebp2a [hAML-3/mPebp2a]), Cbfa2 (hAML-1/ mPebp2b), and Cbfa3 (hAML-2/mPebp2c), encodes several mRNA splice variants (1, 37, 56). The tissue-specific transcriptional properties of the Cbfa proteins are in part accounted for by their selective representation in distinct cellular phenotypes. Cbfa2/AML-1 primarily regulates expression of genes related to the development of thymus and hematopoietic tissues, and a null mutant of this gene results in embryonic lethality due to the absence of definitive hematopoiesis. Several isoform...

An architectural perspective of vitamin D responsiveness

“…The rat OC gene promoter contains three recognition sites for Runx2 interactions, site A (−605 to −595), site B (−438 to −430), and site C (−138 to −130). Mutation of all three Runx2 sites results in significantly reduced OC expression in bone-derived cells [39]. The retention of a nucleosome between the proximal and upstream enhancer domains reduces the distance between the basal regulatory elements and the VDRE and supports a promoter configuration that is conducive to proteinprotein interactions between VDR-associated proteins and components of the RNA polymerase II-bound complex ( Figure 2B).…”

Integration of Runx and Smad regulatory signals at transcriptionally active subnuclear sites

“…Here, we examine subnuclear distribution of Smads (BMP-regulated Smad1 and Smad5 and TGF-␤-regulated Smad2 and Smad3) by using cell lines expressing (ROS 17͞2.8) or lacking (HeLa) endogenous Runx factors (31,32). Fig.…”

Impaired intranuclear trafficking of Runx2 (AML3/CBFA1) transcription factors in breast cancer cells inhibits osteolysis in vivo

“…The role of Runx proteins in both gene expression and nuclear structure is a reflection of their modular organization. The N terminus binds specific DNA sequences, whereas the C-terminal domain interacts with coregulatory factors and associates with the nuclear matrix (10)(11)(12)(13). During tissue differentiation, Runx proteins form stage-specific complexes with subsets of coactivators (14)(15)(16), corepressors (17)(18)(19), and factors that are end points of key signaling cascades (20,21).…”