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Molecular and Cellular Biology, November 1999, p. 7491-7500, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.

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

Amjad Javed,1 Soraya Gutierrez,1 Martin Montecino,2 André J. van Wijnen,1 Janet L. Stein,1 Gary S. Stein,1 and Jane B. Lian1,*

Department of Cell Biology, University of Massachusetts Medical School, Worcester, Massachusetts 01655-0106,1 and Departamento de Biologia Molecular, Facultad de Ciencias Biologicas, Universidad de Concepcion, Concepcion, Chile2

Received 25 March 1999/Returned for modification 12 May 1999/Accepted 5 August 1999

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.

* Corresponding author. Mailing address: Department of Cell Biology, University of Massachusetts Medical School, 55 Lake Ave. North, Worcester, MA 01655-0106. Phone: (508) 856-5625. Fax: (508) 856-6800. E-mail: Jane.Lian{at}umassmed.edu.

Molecular and Cellular Biology, November 1999, p. 7491-7500, Vol. 19, No. 11
0270-7306/99/$04.00+0
Copyright © 1999, American Society for Microbiology. All rights reserved.


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