Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization
Biochemistry & Molecular Pharmacology
Graduate School of Biomedical Sciences; Department of Cell Biology
Medical Subject Headings
Animals; Bone and Bones; Chromatin; Gene Expression Regulation; Mutation; *Neoplasm Proteins; Osteoblasts; Osteocalcin; *Promoter Regions (Genetics); Protein Binding; Rats; *Response Elements; Transcription Factors; Transcription, Genetic; Vitamin D
Life Sciences | Medicine and Health Sciences
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.
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Citation: Mol Cell Biol. 1999 Nov;19(11):7491-500.