DOWN-REGULATION OF CALCITONIN-GENE TRANSCRIPTION BY VITAMIN-D REQUIRES 2 WIDELY SEPARATED ENHANCER SEQUENCES

Transcription of the calcitonin (CT) gene is down-regulated by vitamin D in normal and transformed thyroid C cells. DNA transfer techniques have been previously used to map and characterize a cAMP-induced enhan cer at nucleotides -255 to -129 and an enhancer of basal transcription at -1060 to -905 in the CT 5' flanking DNA. The same methods were use d to identify a negative response element for vitamin D. Deletion muta nts of a genomic fragment of CT extending from nucleotides -1460 to +9 0 were attached to a promoterless GH gene and transfected individually into the medullary thyroid carcinoma cell line TT. CT nucleotides -14 60 to -129 induced significant basal transcription of the GH reporter gene in TT cells. Basal transcription was elevated 3-fold to 4-fold by treatment with cAMP analog. The biologically active metabolite of vit amin D3, 1,25-dihydroxyvitamin D3, had a minor (20%) inhibitory effect on basal transcription but inhibited more than 60% of the cAMP-induce d transcription. We further investigated the cAMP-induced response and found that transcriptional activity of the downstream cAMP-induced en hancer was greatly synergized in the presence of the upstream enhancer of basal transcription. The latter enhancer contained three functiona l CANNTG sequences designated E1 (nucleotides -1060 to -1030), E2 (nuc leotides -940 to -920), and E3 (nucleotides -920 to -900). E2 and E3 w ere essential, for maximal cAMP-induced transcription. Detailed mappin g of the vitamin D response showed that a minimum requirement for inhi bition of the cAMP-induced enhancer by vitamin D was a sequence overla pping E3 (nucleotides -920 to -829). We conclude that a negative respo nse element to vitamin D is located between nucleotides -920 and -829 in the CT 5' flanking DNA. It is possible that vitamin D inhibits tran scription by interfering with the synergistic interaction between the cAMP-induced enhancer and the enhancer of basal transcription.