The vitamin D-responsive element in the rat bone Gla protein gene is an imperfect direct repeat that cooperates with other cis-elements in 1,25-dihydroxyvitamin D3- mediated transcriptional activation.
The gene for rat bone gla protein (BGP) was isolated and 1250 basepairs (bp), including 1100 bp of 5' flanking DNA, were placed up-stream of the human GH reporter gene. After transient transfection into the osteoblast-like rat osteosarcoma cell line ROS 17/2.8, the BGP promoter demonstrated a low level of basal activity that was increased approximately 10-fold by the addition of 10(-8) M 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3]. A single 250-bp fragment (-523 to -274) was sufficient to confer hormone inducibility upon both heterologous and homologous promoters. Deletion studies, complemented by evaluation with synthetic oligomers, enabled localization of the 1,25-(OH)2D3 response element to within 19 bp (-456 to -438), containing an element with an imperfect direct repeat [GGTGA(N4)GGACA] and homology to other steroid-responsive elements. Gel retardation assays demonstrated that partially purified chick intestinal 1,25-(OH)2D3 receptor bound specifically and with high affinity to a DNA fragment containing the putative 1,25-(OH)2D3 response element, and this binding was perturbed by monoclonal antibodies to the 1,25-(OH)2D3 receptor. Surprisingly, the 250-bp fragment, when linked in an antisense orientation with respect to the BGP promoter, blocked basal and hormone-dependent gene expression. However, a 246-bp fragment 5' to the 250-bp element (-1100 to -855) restored 20-fold inducibility when linked to the first fragment in the same orientation, suggesting cooperativity between at least two elements to achieve the hormonal regulation observed in this gene.
Terpening
C M
CM
Department of Biochemistry, College of Medicine, University of Arizona, Tucson 85724.
Haussler
C A
CA
Jurutka
P W
PW
Galligan
M A
MA
Komm
B S
BS
Haussler
M R
MR
D000818:Animals; D001483:Base Sequence; D001842:Bone and Bones / Q000187:drug effects / Q000378:metabolism*; D002117:Calcitriol / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000235:genetics*; D002645:Chickens; D004247:DNA / Q000737:chemistry; D004742:Enhancer Elements, Genetic; D016326:Extracellular Matrix Proteins; D005786:Gene Expression Regulation; D006801:Humans; D007422:Intestines / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D051381:Rats; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid / Q000235:genetics; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D000097568:Matrix Gla Protein
D002117:Calcitriol; D002135:Calcium-Binding Proteins; D004247:DNA; D016326:Extracellular Matrix Proteins; D018167:Receptors, Calcitriol; D011987:Receptors, Steroid; D000097568:Matrix Gla Protein