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

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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 thevitamin D response element in the distal promoter and sites Band C flank a positioned nucleosome in the proximal promoter.The functional significance of each Cbfa element was addressedby mutating individual or multiple Cbfa sites within the contextof the $-$ 1.1-kb rOC promoter fused to a chloramphenicol acetyltransferasereporter gene. Promoter activity was assayed following transienttransfection and after stable genomic integration in ROS 17/2.8osteoblastic cell lines. We show that all three Cbfa sites arerequired 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 proximalsite C, and paired mutations involving site A (mAB or mAC) resultin a far greater loss of activity than the mBC mutation. Strikingly,mutation of the three Cbfa sites leads to abrogation of responsivenessto vitamin D. Vitamin D-enhanced activity is also not observedwhen sites A and B are mutated. Significantly, related to theselosses in transcriptional activity, mutation of the three Cbfasites results in altered chromatin structure as reflected by lossof DNase I-hypersensitive sites at the vitamin D response elementand over the proximal tissue-specific basal promoter. These findingsstrongly support a multifunctional role for Cbfa factors in regulatinggene expression, not only as simple transcriptional transactivatorsbut also by facilitating modifications in promoter architectureand chromatinorganization.

^* 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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