Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization

Three Cbfa motifs are strategically positioned in the bone-specific rat ost eocalcin (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 r eporter gene. Promoter activity was assayed following transient transfectio n and after stable genomic integration in ROS 17/2.8 osteoblastic cell line s. We show that all three Cbfa sites are required for maximal basal express ion 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 genom ic context, sites A and B can largely compensate for a mutation at the prox imal 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 o f 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 mut ated. Significantly, related to these losses in transcriptional activity, m utation of the three Cbfa sites results in altered chromatin structure as r eflected by loss of DNase I-hypersensitive sites at the vitamin D response element and over the proximal tissue-specific basal promoter. These finding s strongly support a multifunctional role for Cbfa factors in regulating ge ne expression, not only as simple transcriptional transactivators but also by facilitating modifications in promoter architecture and chromatin organi zation.