MULTIPLE SP1 BINDING-SITES IN THE CARDIAC SLOW-TWITCH MUSCLE SARCOPLAMSIC RETICULUM CA2-ATPASE GENE PROMOTER ARE REQUIRED FOR EXPRESSION INSOL8 MUSCLE-CELLS()

The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum Ca2+-ATPa se (SERCA2) gene encodes a Ca2+ transport pump whose expression is reg ulated 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 po sitive regulatory regions, one distal (-1810 base pair (bp) to -1110 b p) and one proximal (-284 bp to -72 bp), within the SERCA2 promoter. T he proximal promoter region hom -284 bp to -80 bp was shown to confer muscle-specific expression to a heterologous promoter in So18 cells. T his region is highly CC-rich containing the consensus sequence for fou r Spl 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, si te-directed mutagenesis of the Sp1 consensus sites demonstrated that S p1 sites are essential for the muscle-specific expression of the SERCA 2 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 transcripti on factor plays an important role in the transcriptional regulation of SERCA2 within muscle cells.