RETINOIC ACID REPRESSION OF CELL-SPECIFIC HELIX-LOOP-HELIX OCTAMER ACTIVATION OF THE CALCITONIN CALCITONIN-GENE-RELATED PEPTIDE ENHANCER

We have investigated the mechanism underlying repression of calcitonin /calcitonin gene-related peptide (CT/CGRP) gene expression by retinoic acid. Retinoic acid treatment of the CA77 thyroid C-cell line decreas ed CT/CGRP promoter activity two- to threefold, which correlates well with the decrease in calcitonin and CGRP mRNA levels. Repression is me diated through the nuclear retinoic acid receptors (RAR) on the basis of the retinoid specificity, the sensitivity of repression (half-maxim al repression at 0.2 nM), and the additional repression caused by cotr ansfection of an alpha-RAR expression vector. The sequences required f or retinoic acid repression were localized to an 18-bp element contain ing cell-specific enhancer activity. The enhancer binds helix-loop-hel ix (HLH) and octamer transcription factors that act synergistically to activate transcription. Retinoic acid repression requires both these factors since mutations in either motif resulted in the loss of repres sion. Furthermore, repression was observed only in cell lines containi ng enhancer activity. We have used electrophoretic mobility shift assa ys to show that repression does not involve direct DNA binding of RAR or RAR-retinoid X receptor heterodimers. Instead, repression appears t o involve interactions with the stimulatory enhancer factors. Followin g retinoic acid treatment, there was a specific decrease in an enhance r complex containing both HLH and octamer proteins. Formation of the H LH-octamer complex was also specifically blocked by the addition of ex ogenous RAR-retinoid X receptor protein. These results demonstrate tha t RAR can repress CT/CGRP gene transcription by interfering with combi natorial activation by cell-specific HLH and octamer proteins.