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 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.
Javed
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Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0106, USA.
Gutierrez
S
S
Montecino
M
M
van Wijnen
A J
AJ
Stein
J L
JL
Stein
G S
GS
Lian
J B
JB
D000818:Animals; D001842:Bone and Bones / Q000166:cytology / Q000502:physiology; D002843:Chromatin / Q000648:ultrastructure*; D005786:Gene Expression Regulation; D009154:Mutation; D009363:Neoplasm Proteins; D010006:Osteoblasts / Q000502:physiology; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D051381:Rats; D020218:Response Elements; D014157:Transcription Factors / Q000378:metabolism*; D014158:Transcription, Genetic; D014807:Vitamin D / Q000494:pharmacology*
D002843:Chromatin; D009363:Neoplasm Proteins; D014157:Transcription Factors; D015675:Osteocalcin; D014807:Vitamin D