Vitamin D-mediated modifications in protein-DNA interactions at two promoter elements of the osteocalcin gene.
By the combined use of DNase I footprinting, electrophoretic mobility-shift assay, and methylation interference analysis, we have identified a series of sequence-specific protein-DNA interactions in the 5' flanking region of the rat osteocalcin gene. Stimulation of osteocalcin gene expression by 1,25-dihydroxyvitamin D3, a physiologic mediator of this bone-specific gene in vitro and in vivo, is associated with modifications in the binding of ROS 17/2.8 cell nuclear factors to two promoter segments that up-regulate transcription. One segment located between -462 and -437 exhibits a vitamin D-dependent increase in sequence-specific binding of nuclear factors. This element (CTGGGTGAATGAGGACATTACTGACC), identified at single nucleotide resolution, contains a region of hyphenated dyad symmetry and shares sequence homology with consensus steroid-responsive elements and with the sequence that has been identified as the vitamin D receptor binding site in the human osteocalcin gene. We have also observed that vitamin D stimulation of osteocalcin gene expression results in a 5-fold increase in protein binding to the region of the osteocalcin box, a 24-nucleotide segment in the proximal promoter with a CCAAT motif as the central core. Our results demonstrate protein-DNA interactions in a vitamin D-responsive element and in a second sequence, the osteocalcin box, both of which are involved in the physiologic regulation of the osteocalcin gene in response to 1,25-dihydroxyvitamin D3.
Markose
E R
ER
Department of Cell Biology, University of Massachusetts Medical Center, Worcester 01655.
Stein
J L
JL
Stein
G S
GS
Lian
J B
JB
D000818:Animals; D001483:Base Sequence; D002117:Calcitriol / Q000494:pharmacology*; D002460:Cell Line; D004247:DNA / Q000235:genetics / Q000378:metabolism*; D015342:DNA Probes; D003850:Deoxyribonuclease I; D015870:Gene Expression / Q000187:drug effects; D005796:Genes / Q000187:drug effects*; D008969:Molecular Sequence Data; D009687:Nuclear Proteins / Q000378:metabolism*; D009710:Nucleotide Mapping; D015675:Osteocalcin / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000187:drug effects*; D011485:Protein Binding; D051381:Rats; D014157:Transcription Factors / Q000378:metabolism
D015342:DNA Probes; D009687:Nuclear Proteins; D014157:Transcription Factors; D015675:Osteocalcin; D004247:DNA; D003850:Deoxyribonuclease I; D002117:Calcitriol