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[摘要]：Calsequestrin is the main calcium-binding protein inside the sarcoplasmic reticulum of striated muscle. In mammals, the cardiac calsequestrin gene (casq2) mainly expresses in cardiac muscle and to a minor extent in slow-twitch skeletal muscle and it is not expressed in non-muscle tissues. This work is the first study on the transcriptional regulation of the casq2 gene in cardiac and skeletal muscle cells. The sequence of the casq2 genes proximal promoter ( 180 bp) of mammals and avians is highly conserved and contains one TATA box, one CArG box, one E-box, and one myocyte enhancer factor 2 (MEF-2) site. We cloned the human casq2 gene 5 '-regulatory region into aluciferase reporter expression vector. By functional assays we showed that a construct containing the first 288 bp of promoter was up-regulated during myogenic differentiation of Sol8 cells and had higher transcriptional activity compared with longer constructs. In neonatal rat cardiac myocytes, the larger construct containing 3.2 kb showed the highest transcriptional activity, demonstrating that the first 288 bp are sufficient to confer muscle specificity, whereas distal sequences may act as a cardiac-specific enhancer. Electrophoretic mobility shift assay studies demonstrated that the proximal MEF-2 and CArG box sequences were capable of binding MEF-2 and serum response factor, respectively, whereas the E-box did not show binding properties. Functional studies demonstrated that site-directed mutagenesis of the proximal MEF-2 and CArG box sites significantly decreased the transcription of the gene in cardiac and skeletal muscle cells, indicating that they are important to drive cardiac and skeletal muscle-specific transcription of the casq2 gene. |
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