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, ryr2, and ryr3, have been identified coding for a skeletal muscle, cardiac muscle, and brain isoform, respectively. Although, the skeletal muscle isoform (RYR1) was shown to be expressed predominantly in skeletal muscle, expression was also detected in the esophagus and brain. To analyze the transcriptional regulation of the RYR1 gene, we have constructed chimeric genes composed of the upstream region of the RYR1 gene and the bacterial chloramphenicol acetyltransferase (CAT) gene and transiently transfected them into primary cultured porcine myoblasts, myotubes, and fibroblasts. A 443-base pair region upstream from the transcription start site was sufficient to direct CAT activity without tissue specificity. Deletion of a 61-base pair fragment from the 5'-end of the promoter resulted in a marked reduction of CAT activity in all three tissue types. A similar reduction of expression was observed when using a construct with the first intron in antisense orientation upstream from the promoter. In contrast, the first intron in sense orientation enhanced expression only in myotubes, while expression was repressed in fibroblasts and myoblasts. Gel retardation analyses showed DNA binding activity in nuclear extracts for two upstream DNA sequence elements. Our data suggest that (i) RYR1 gene expression is regulated by at least two novel transcription factors (designated RYREF-1 and RYREF-2), and (ii) tissue specificity results from a transcriptional repression in nonmuscle cells mediated by the first intron.
Schmoelzl
S
S
Institute of Veterinary Medicine, University of Göttingen, 37073 Göttingen, Federal Republic of Germany.
Leeb
T
T
Brinkmeier
H
H
Brem
G
G
Brenig
B
B
D000595:Amino Acid Sequence; D000818:Animals; D001482:Base Composition; D001483:Base Sequence; D015220:Calcium Channels / Q000096:biosynthesis* / Q000235:genetics*; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D017931:DNA Primers; D005347:Fibroblasts / Q000378:metabolism; D005786:Gene Expression Regulation; D015698:Genomic Library; D007438:Introns; D008099:Liver / Q000378:metabolism; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000096:biosynthesis* / Q000235:genetics*; D018482:Muscle, Skeletal / Q000378:metabolism*; D009928:Organ Specificity; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D015183:Restriction Mapping; D019837:Ryanodine Receptor Calcium Release Channel; D017384:Sequence Deletion; D013552:Swine; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D015220:Calcium Channels; D017931:DNA Primers; D009124:Muscle Proteins; D011993:Recombinant Fusion Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D014157:Transcription Factors; D015500:Chloramphenicol O-Acetyltransferase