REGULATION OF TISSUE-SPECIFIC EXPRESSION OF THE SKELETAL-MUSCLE RYANODINE RECEPTOR GENE

The ryanodine receptors (RYR) are a family of calcium release channels that are expressed in a variety of tissues. Three genes, i.e. ryr1, r yr2, and ryr3, have been identified coding for a skeletal muscle, card iac muscle, and brain isoform, respectively. Although, the skeletal mu scle isoform (RYR1) was shown to be expressed predominantly in skeleta l muscle, expression was also detected in the esophagus and brain. To analyze the transcriptional regulation of the RYR1 gene, we have const ructed chimeric genes composed of the upstream region of the RYR1 gene and the bacterial chloramphenicol acetyltransferase (CAT) gene and tr ansiently transfected them into primary cultured porcine myoblasts, my otubes, and fibroblasts. A 443-base pair region upstream from the tran scription start site was sufficient to direct CAT activity without tis sue specificity. Deletion of a 61-base pair fragment from the 5'-end o f the promoter resulted in a marked reduction of CAT activity in all t hree tissue types. A similar reduction of expression was observed when using a construct with the first intron in antisense orientation upst ream hom the promoter. In contrast, the first intron in sense orientat ion enhanced expression only in myotubes, while expression was repress ed in fibroblasts and myoblasts. Gel retardation analyses showed DNA b inding activity in nuclear extracts for two upstream DNA sequence elem ents. Our data suggest that (i) RYR1 gene expression is regulated by a t least two novel transcription factors (designated RYREF-1 and RYREF- 2), and (ii) tissue specificity results from a transcriptional repress ion in nonmuscle cells mediated by the first intron.