Transcriptional regulation of the human calcitonin/alpha-CGRP gene

The human calcitonin/α-CGRP gene is expressed in a tissue-specific manner; the C cells of the thyroid produce calcitonin, whilst specific subsets of neurons in the central and peripheral nervous system make CGRP. In certain tumours, in particular those of the lung, this tissue-specificity is lost and the gene is ectopically expressed. The mechanism of ectopic expression is of particular interest as it may provide insights into the processes regulating gene expression in the normal and transformed state. Sequencing of 1.8 kb of 5' flanking region of the human calcitonin/a-CGRP gene revealed many potential cis-acting control sequences. In order to test the activity of these sequences, a series of deletion vectors were constructed with portions of these 5' flanking sequences driving expression of the bacterial reporter gene, chloramphenicol acetyl transferase (CAT). These calgcat constructs were transfected into both BEN cells, a lung carcinoma cell line that ectopically expresses the calcitonin/α-CGRP gene, and HeLa cells, a cervical carcinoma cell line that does not express the gene. Analysis of the resulting CAT activity revealed that there is a sequence upstream of the transcription start site that is able to mediate the repression of the gene in HeLa cells. This repression could be abolished by co-transfection with an excess of the identical sequence, indicating that it is probably mediated by the binding of a repressor protein. This upstream region did not mediate repression in BEN cells, suggesting that the repressor is not active in BEN cells, and presenting one possible explanation for ectopic expression of the gene in these cells. The repressor binding site was localised to a 53 bp sequence, situated between -1.51 kb and -1.46 kb upstream of the transcription start site. This 53bp fragment was unable to repress transcription from an HSV tk promoter-CAT construct in HeLa cells, indicating that repression may operate by interaction with other proteins which specifically bind to the calcitonin/α-CGRP promoter region. Stimulation of the calcitonin/a-CGRP gene by forskolin and phorbol esters in BEN cells was also examined. Both agents increased the level of endogenous calcitonin mRNA and of transcription from the calgcat constructs. Phorbol ester stimulation was three fold higher than that of forskolin for the largest calgcat construct. Interestingly, phorbol esters were unable to stimulate expression of any of the calgcat constructs in HeLa cells. The 5' flanking region of the human calcitonin/α-CGRP gene contains a higher than expected frequency of CpG dinucleotides, which are unmethylated in all tissues, characteristic of a CpG island, A region in the second intron exhibits variable methylation with expression, being undermethylated in expressing tissues. This suggests that regulatory sequences are located here. Various fragments from this region were inserted into an HSV tk promoter-CAT plasmid, and transfected into both HeLa and BEN cells. Sequences from this region were shown to enhance and repress CAT activity relative to the parent vector, demonstrating that sequences in intron 2 do possess regulatory activity. A model for the mechanism of ectopic expression is proposed on the basis of these results in which inactivation of repressor activity is a primary event in inducing expression of the gene in neoplasia