Functional assessment of two vitamin D-responsive elements in the rat 25-hydroxyvitamin D3 24-hydroxylase gene.
Two vitamin D-responsive elements (VDRE-1 and VDRE-2) were recently identified in the 5'-upstream region of the rat 25-hydroxyvitamin D3 24-hydroxylase gene at -151/-137 and -259/-245, respectively. We studied the transcriptional regulation of this gene by vitamin D by means of mutational analysis. Introducing mutations into VDRE-1 and VDRE-2 in the native promoter -291/+9 reduced vitamin D-dependent chloramphenicol acetyltransferase activity by 86 and 41%, respectively. Mutation of the direct repeat -169/-155 located at 3 base pairs upstream of VDRE-1 also caused 50% decrease of chloramphenicol acetyltransferase activity. Connection of the element -169/-155 to VDRE-1 enhanced the vitamin D responsiveness of VDRE-1 5-fold through the heterologous beta-globin promoter. The fragment -291/-102 containing the two VDREs showed two shifted bands in the presence of the vitamin D receptor and retinoid X receptor in gel retardation analysis, and the appearance of the slower migrating band indicates that two sets of receptor complexes bind to this fragment simultaneously. These results demonstrate that VDRE-1 is a stronger mediator of vitamin D function than VDRE-2 due to the presence of the accessory element -169/-155 located adjacent to VDRE-1, although VDRE-2 exhibits a smaller dissociation constant for the vitamin D receptor-retinoid X receptor complex than VDRE-1.
Ohyama
Y
Y
Graduate Department of Gene Science, Faculty of Science, Hiroshima University, 1-3-1 Kagamiyama, Higashi-Hiroshima 739, Japan.
Ozono
K
K
Uchida
M
M
Yoshimura
M
M
Shinki
T
T
Suda
T
T
Yamamoto
O
O
D000818:Animals; D001665:Binding Sites; D002117:Calcitriol / Q000502:physiology*; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic; D005796:Genes; D046911:Macromolecular Substances; D011401:Promoter Regions, Genetic; D051381:Rats; D018167:Receptors, Calcitriol / Q000502:physiology*; D018168:Receptors, Retinoic Acid / Q000502:physiology*; D047488:Retinoid X Receptors; D015398:Signal Transduction; D013250:Steroid Hydroxylases / Q000235:genetics*; D014157:Transcription Factors / Q000502:physiology*; D065668:Vitamin D3 24-Hydroxylase
D004268:DNA-Binding Proteins; D046911:Macromolecular Substances; D018167:Receptors, Calcitriol; D018168:Receptors, Retinoic Acid; D047488:Retinoid X Receptors; D014157:Transcription Factors; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; D065668:Vitamin D3 24-Hydroxylase; D002117:Calcitriol