Cell type-specific regulation of transcription by cyclic adenosine 3,'5'-monophosphate-responsive elements within the calcitonin promoter.
A cAMP-induced enhancer was previously mapped to nucleotides -255 to -85 of the calcitonin (CT) gene 5'-flanking DNA. To determine the functional cis-acting elements within this region, we transfected medullary thyroid carcinoma (MTC) cells with CT 5'-flanking DNA/GH fusion genes containing potential cAMP-responsive elements and assessed their transcriptional activities with and without cAMP. In CT-expressing MTC cells (the TT line), we identified by deletions and point mutations three transcriptionally active motifs: a cAMP-responsive element (CRE), TGACGTCA, at -253 to -246, and a hybrid site containing a CRE-like element (CREL; TGACCTCA, -169 to -162) adjacent to an equally transcriptionally active octamer (O) sequence (ATG-CAAAT, -161 to -154). These three motifs acted synergistically and their transcriptional activity was completely dependent on cAMP. In HeLa cells their synergistic activity was more constitutive than cAMP induced, whereas in CT-negative MTC cells (the RO-D81-1 line) these motifs were inactive. Gel mobility shift assays with antibodies against CRE-binding protein (CREB) and activating transcription factor 1 (ATF-1) showed that both CREB and ATF-1 interacted with the CRE in MTC cells, whereas in HeLa cells only ATF-1 bound to the CRE. Specific binding to the CREL/O motif was detectable in extracts from tumors induced by injection of TT cells but not in extracts from any of the three cultured cell lines. We conclude that cAMP-induced transcription of the CT gene is modulated in a cell-specific manner by the CRE and the CREL/O elements.
D001483:Base Sequence; D002116:Calcitonin / Q000235:genetics*; D002277:Carcinoma / Q000378:metabolism; D003001:Cloning, Molecular; D000242:Cyclic AMP / Q000378:metabolism*; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D018014:Gene Transfer Techniques; D006367:HeLa Cells; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D011401:Promoter Regions, Genetic; D013964:Thyroid Neoplasms / Q000378:metabolism; D015533:Transcriptional Activation; D014407:Tumor Cells, Cultured
D017362:Cyclic AMP Response Element-Binding Protein; D002116:Calcitonin; D000242:Cyclic AMP