TRANSCRIPTIONAL REGULATION OF THE NEURONAL L-TYPE CALCIUM-CHANNEL ALPHA(1D) SUBUNIT GENE

1. The transcriptional regulation of the rat brain L-type calcium channel alpha(1D) subunit (RB alpha(1D)) gene was investigated using NG108-15 neuroblastoma-glioma cells. 2. Differentiation of NG108-15 cells in the presence of prostaglandin E(1) or retinoic acid resulted in the appearance of mRNA encoding the RB alpha(1D) subunit detected using Northern blot analysis. 3. A rat genomic DNA library was screened, and a 15.2-kb clone was isolated and partially sequenced which included part of the 5' upstream sequence through the initial part of intron 2 of the RB alpha(1D) gene. 4. Deletion analysis, using a CAT reporter gene and transfected NG108-15 cells, revealed that the 1.2-kb 5'-upstream sequence from the RB alpha(1D) gene contains cis-acting positive and negative regulatory elements. A deletion of the 3' end of exon 1 also suggested the presence of regulatory elements in the first exon. 5. DNase footprinting of exon 1 of the RB alpha(1D) gene revealed two regions protected from digestion by specific protein binding, and the second region included an (ATG)(7) trinucleotide repeat sequence. Electrophoretic mobility shift assays confirmed nuclear protein(s) binding to the (ATG)(7) sequence. 6. The (ATG)(7) sequence functions as a enhancer when linked to a thymidine kinase promoter and a CAT reporter gene. 7. These results provide the initial description of the transcriptional regulation of the RB alpha(1D) gene and identify a novel enhancer that consists of an (ATG), trinucleotide repeat sequence.