Transcription of the human calcitonin gene is mediated by a c cell-specific enhancer containing E-box-like elements

The calcitonin (CT) gene is expressed normally in thyroidal C-cells and in a restricted population of cells in the central and peripheral nerve system. To define the cis-elements within the 5′-flanking DNA of the human CT gene which mediate this cell-specific expression, we used DNA transfer techniques and a transient transfection approach. We found that a DNA sequence located between -1290 and -820 of the CT 5′-flanking DNA functioned as an enhancer of basal transcription in C-cells (from medullary thyroid carcinoma) but not in rat glioma (C6), hamster insulinoma (HIT), fibroblasts (3T3), or epithelial cells (HeLa and CV1). Further mapping revealed the presence of at least two elements within the enhancer region; an upstream element (USE, located between -1060 and -1030) which could not function independently but its removal caused 70-80% loss of enhancer activity and a downstream element (DSE, located at -1033 to -920) which functioned independently as a cellspecific enhancer but with reduced activity. The binding pattern of nuclear proteins from C-cells to the enhancer elements was studied by an electrophoretic mobility shift assay. A protein-DNA complex was formed with the USE which could be competed, specifically, by an oligonucleotide containing the μE2 motif of the immunoglobulin gene enhancer. A similar complex was formed with the DSE fragment. Nuclear proteins from HeLa cells failed to form complexes with USE. Moreover, the binding pattern of proteins derived from HeLa cells to DSE was different from that of C-cells. We conclude that a region spanning 1060 to 905 base pairs upstream from the start of transcription is responsible for cell-specific expression of the CT gene. We hypothesize that augmentation of this cell-specific transcription is likely to involve interaction of nuclear factors with E boxes on USE and DSE.