Coordinate occupancy of AP-1 sites in the vitamin D-responsive and CCAAT box elements by Fos-Jun in the osteocalcin gene: model for phenotype suppression of transcription.
Osteocalcin, a bone-specific protein and marker of the mature osteoblast, is expressed only in nonproliferating osteoblasts in a mineralizing extracellular matrix, while type I collagen is expressed in proliferating cells. The nuclear proteins encoded by the c-fos and c-jun protooncogenes are expressed during the proliferation period of osteoblast phenotype development. We present evidence that AP-1 (HeLa cell-activating protein 1) sites residing within two promoter elements of the osteocalcin gene bind the Fos-Jun protein complex: the osteocalcin box (OC box; nucleotides -99 to -76), which contains a CCAAT motif as a central element and influences tissue-specific basal levels of osteocalcin gene transcription, and the vitamin D-responsive element (VDRE; nucleotides -462 to -440), which mediates enhancement of osteocalcin gene transcription. Gel electrophoretic mobility-shift analysis demonstrated high AP-1 binding activity in proliferating osteoblasts and dramatic changes in this activity after the down-regulation of proliferation and the initiation of extracellular-matrix mineralization in primary cultures of normal diploid osteoblasts. Methylation interference analysis established at single nucleotide resolution that purified recombinant Fos and Jun proteins bind in a sequence-specific manner to the AP-1 sites within the VDRE and OC box. Similarly, an AP-1 motif within a putative VDRE of the alkaline phosphatase gene, which is also expressed after the completion of proliferation, binds the Fos-Jun complex. These results support a model in which coordinate occupancy of the AP-1 sites in the VDRE and OC box in proliferating osteoblasts may suppress both basal level and vitamin D-enhanced osteocalcin gene transcription as well as transcription of other genes associated with osteoblast differentiation--a phenomenon we describe as phenotype suppression. This model is further supported by binding of the Fos-Jun complex at an AP-1 site in the type alpha I collagen promoter that is contiguous with, but not overlapping, the VDRE. Such a sequence organization in the collagen VDRE motif is compatible with vitamin D modulation of collagen but not with osteocalcin and alkaline phosphatase expression in proliferating osteoblasts.
Owen
T A
TA
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
Bortell
R
R
Yocum
S A
SA
Smock
S L
SL
Zhang
M
M
Abate
C
C
Shalhoub
V
V
Aronin
N
N
Wright
K L
KL
van Wijnen
A J
AJ
D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002454:Cell Differentiation; D002455:Cell Division; D002460:Cell Line; D002478:Cells, Cultured; D004247:DNA; D004268:DNA-Binding Proteins / Q000235:genetics*; D005796:Genes / Q000187:drug effects; D005809:Genes, Regulator; D006367:HeLa Cells / Q000378:metabolism; D006801:Humans; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D015675:Osteocalcin / Q000235:genetics*; D012516:Osteosarcoma; D010641:Phenotype; D011505:Protein-Tyrosine Kinases / Q000235:genetics; D011518:Proto-Oncogene Proteins / Q000235:genetics*; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D011519:Proto-Oncogenes / Q000187:drug effects; D051381:Rats; D012045:Regulatory Sequences, Nucleic Acid; D013489:Suppression, Genetic; D014157:Transcription Factors / Q000235:genetics*; D014158:Transcription, Genetic
D004268:DNA-Binding Proteins; D011518:Proto-Oncogene Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA; D011505:Protein-Tyrosine Kinases; D002117:Calcitriol