Cloning and characterization of the 5'-upstream regulatory region of the Ca(2+)-release channel gene of cardiac sarcoplasmic reticulum.
To elucidate the transcriptional regulation mechanism for the Ca(2+)-release channel gene of the cardiac sarcoplasmic reticulum (RYR2), we isolated and analyzed the 5'-upstream flanking region of the gene. Sequence analysis indicated that the core promoter region lacks canonical TATA and CAAT boxes, but contains three overlapping GC boxes. A gel shift assay indicated that Sp1 binds to the region containing the GC boxes. Different 5'-deletion constructs in the 5'-flanking region of the RYR2 gene were fused to the luciferase gene, and their promoter activity in rat neonatal cardiac myocytes was subsequently determined. The results revealed the presence of a region containing positive regulatory elements in the 5'-flanking region. Analyses of substitutional mutations introduced into the GC boxes and the regulatory region indicated that in addition to the GC box located at -56 to -51, two regulatory elements (RYR2P1 and RYR2P2) are essential for the promoter activity. These results indicated that Sp1 and transcription factors that bind to RYR2P1 and RYR2P2 cooperatively enhance the expression of the RYR2 gene. In a transient transfection experiment involving promoter-luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between positions -209 and -90 that represses the expression of the RYR2 gene in skeletal muscle cells.
Nishida
K
K
First Department of Medicine, Osaka University Medical School, Japan.
Otsu
K
K
Hori
M
M
Kuzuya
T
T
Tada
M
M
D000818:Animals; D001483:Base Sequence; D015220:Calcium Channels / Q000737:chemistry / Q000235:genetics*; D002460:Cell Line; D003001:Cloning, Molecular; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D008969:Molecular Sequence Data; D009124:Muscle Proteins / Q000737:chemistry / Q000235:genetics*; D018482:Muscle, Skeletal / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000378:metabolism*; D011401:Promoter Regions, Genetic; D011817:Rabbits; D015183:Restriction Mapping; D019837:Ryanodine Receptor Calcium Release Channel; D012519:Sarcoplasmic Reticulum / Q000378:metabolism*; D017384:Sequence Deletion; D016329:Sp1 Transcription Factor / Q000378:metabolism; D014158:Transcription, Genetic; D014162:Transfection
D015220:Calcium Channels; D009124:Muscle Proteins; D019837:Ryanodine Receptor Calcium Release Channel; D016329:Sp1 Transcription Factor