Regulation of the calcitonin/calcitonin gene-related peptide gene by cell-specific synergy between helix-loop-helix and octamer-binding transcription factors

Abstract

The calcitonin/calcitonin gene-related peptide (CGRP) gene is transcribed in thyroid C-cells and a subset of neurons. We have localized sequences required for cell-specific enhancement of calcitonin/CGRP transcription in rat thyroid C-cell lines. An 18-base pair element approximately 1 kilobase pair upstream of the transcriptional start site stimulated expression of a luciferase reporter gene 50-fold in 44-2C C-cells. There was less than 2-fold stimulation in HeLa and Rat-1 cells, which do not express the endogenous calcitonin/CGRP gene. The enhancer contains potential binding sites for helix-loop-helix (HLH) and octamer transcription factors based on sequence homologies. The functional significance of these sites was shown by point mutations in the HLH and octamer motifs and by separation of the two motifs, all of which decreased enhancer activity greater than 10-fold. The involvement of HLH proteins was further shown by co-expression of the mammalian achaete-scute homologue-1 HLH protein, which activated the enhancer severalfold in HeLa cells. Electrophoretic mobility shift analyses revealed several DNA-protein complexes containing HLH and octamer-binding proteins. One octamer-binding complex (OB1) most likely contains the ubiquitous Oct-1 protein, whereas a second complex (OB2) was cell-specific. In contrast to OB1, OB2 had lower affinity for a consensus octamer motif, and its DNA binding was not affected by addition of antiserum that recognizes Oct-1 and Oct-2 proteins. In addition, we observed a large complex that appears to contain both an HLH protein and OB2. These results demonstrate that calcitonin/CGRP enhancer activity is controlled by a cell-specific synergistic activation involving HLH and octamer-binding factors.