Multiple Sp1 binding sites in the cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPase gene promoter are required for expression in Sol8 muscle cells.
The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA2) gene encodes a Ca2+ transport pump whose expression is regulated during skeletal and cardiac muscle development and in response to various pathophysiological and hormonal states. Employing transient transfection analyses in Sol8 muscle cells, we have identified two positive regulatory regions, one distal (-1810 base pair (bp) to -1110 bp) and one proximal (-284 bp to -72 bp), within the SERCA2 promoter. The proximal promoter region from -284 bp to -80 bp was shown to confer muscle-specific expression to a heterologous promoter in Sol8 cells. This region is highly GC-rich containing the consensus sequence for four Sp1 elements (GGGCGG) and three Sp1-like elements (GGGAGG). DNase I footprint analysis with Sol8 nuclear extracts and purified Sp1 protein showed the protection of the seven Sp1 binding sites. In addition, site-directed mutagenesis of the Sp1 consensus sites demonstrated that Sp1 sites are essential for the muscle-specific expression of the SERCA2 promoter. Furthermore, we demonstrate that cotransfection of an Sp1 expression vector together with SERCA2-CAT constructs can up-regulate SERCA2 promoter activity. These results imply that the Sp1 transcription factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.
Baker
D L
DL
Section of Molecular Cardiology, Division of Cardiology and Cardiovascular Research Center, Cincinnati, Ohio 45267-0542, USA.
Dave
V
V
Reed
T
T
Periasamy
M
M
D016475:3T3 Cells; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000096:biosynthesis* / Q000235:genetics*; D002460:Cell Line; D002467:Cell Nucleus / Q000378:metabolism; D015500:Chloramphenicol O-Acetyltransferase / Q000096:biosynthesis; D016384:Consensus Sequence; D004247:DNA / Q000737:chemistry / Q000302:isolation & purification; D018983:DNA Footprinting; D015870:Gene Expression; D051379:Mice; D008969:Molecular Sequence Data; D018657:Muscle Fibers, Slow-Twitch / Q000201:enzymology*; D018482:Muscle, Skeletal / Q000201:enzymology*; D016297:Mutagenesis, Site-Directed; D009206:Myocardium / Q000201:enzymology*; D011401:Promoter Regions, Genetic; D051381:Rats; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000378:metabolism; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D016329:Sp1 Transcription Factor / Q000096:biosynthesis / Q000378:metabolism*; D014162:Transfection
D011993:Recombinant Fusion Proteins; D011994:Recombinant Proteins; D016329:Sp1 Transcription Factor; D004247:DNA; D015500:Chloramphenicol O-Acetyltransferase; D000252:Calcium-Transporting ATPases