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, Soraya Gutierrez, Martin Montecino, Andre J van Wijnen, Janet L. Stein, Gary S. Stein, Jane B. Lian

Research output: Contribution to journalArticle

117 Citations (Scopus)

Abstract

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.

Original languageEnglish (US)
Pages (from-to)7491-7500
Number of pages10
JournalMolecular and Cellular Biology
Volume19
Issue number11
StatePublished - Nov 1999
Externally publishedYes

Fingerprint

Osteocalcin
Vitamin D
Chromatin
Vitamin D Response Element
Mutation
Vitamin B Complex
Chloramphenicol O-Acetyltransferase
Trans-Activators
Nucleosomes
Deoxyribonuclease I
Reporter Genes
Transfection
Gene Expression
Bone and Bones
Cell Line

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics
  • Cell Biology

Cite this

Javed, A., Gutierrez, S., Montecino, M., van Wijnen, A. J., Stein, J. L., Stein, G. S., & Lian, J. B. (1999). Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization. Molecular and Cellular Biology, 19(11), 7491-7500.

Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization. / Javed, Amjad; Gutierrez, Soraya; Montecino, Martin; van Wijnen, Andre J; Stein, Janet L.; Stein, Gary S.; Lian, Jane B.

In: Molecular and Cellular Biology, Vol. 19, No. 11, 11.1999, p. 7491-7500.

Research output: Contribution to journalArticle

Javed, A, Gutierrez, S, Montecino, M, van Wijnen, AJ, Stein, JL, Stein, GS & Lian, JB 1999, 'Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization', Molecular and Cellular Biology, vol. 19, no. 11, pp. 7491-7500.
Javed, Amjad ; Gutierrez, Soraya ; Montecino, Martin ; van Wijnen, Andre J ; Stein, Janet L. ; Stein, Gary S. ; Lian, Jane B. / Multiple Cbfa/AML sites in the rat osteocalcin promoter are required for basal and vitamin D-responsive transcription and contribute to chromatin organization. In: Molecular and Cellular Biology. 1999 ; Vol. 19, No. 11. pp. 7491-7500.
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