Sukovich D A, Shabbeer J, Periasamy M
Department of Physiology and Biophysics, University of Vermont, College of Medicine, Burlington 05405.
Nucleic Acids Res. 1993 Jun 11;21(11):2723-8. doi: 10.1093/nar/21.11.2723.
The rabbit cardiac/slow twitch muscle sarcoplasmic reticulum (SR) Ca2+ ATPase (SERCA2) gene encodes a Ca2+ transport pump whose expression is regulated during skeletal/cardiac muscle development and by different pathophysiological states of the heart. This study was designed to delineate cis-acting regulatory elements involved in SERCA2 gene expression. A series of unidirectionally deleted fragments of the upstream 1,460 bp SERCA2 promoter were linked to the chloramphenicol acetyltransferase (CAT) reporter gene. Transient DNA transfection experiments performed with these constructs in C2C12 muscle cells and NIH3T3 fibroblasts revealed a 17 bp upstream promoter element (UPE) important for transcription of the SERCA2 gene in skeletal muscle cells. These studies have also identified a strong (muscle specific) negative regulatory region located upstream of nucleotide -658. Gel mobility shift and southwestern analyses using the 17 bp UPE have revealed a specific DNA binding complex referred to as Ca2+ ATPase promoter factor -1 (CaPF1). The binding factor has an approximate M(r) of 43 kDa. Comparison of CaPF1 with known transcription factors suggests that the CaPF1 complex may be a novel DNA-binding transcription factor which plays a role in SERCA2 gene regulation in vivo.
兔心肌/慢肌肌浆网（SR）Ca2+ATP酶（SERCA2）基因编码一种Ca2+转运泵，其表达在骨骼肌/心肌发育过程中以及心脏的不同病理生理状态下受到调控。本研究旨在阐明参与SERCA2基因表达的顺式作用调控元件。将上游1460bp SERCA2启动子的一系列单向缺失片段与氯霉素乙酰转移酶（CAT）报告基因相连。用这些构建体在C2C12肌细胞和NIH3T3成纤维细胞中进行的瞬时DNA转染实验揭示了一个17bp的上游启动子元件（UPE），它对骨骼肌细胞中SERCA2基因的转录很重要。这些研究还确定了一个位于核苷酸-658上游的强（肌肉特异性）负调控区。使用17bp UPE进行的凝胶迁移率变动分析和蛋白质印迹分析揭示了一种特定的DNA结合复合物，称为Ca2+ATP酶启动子因子-1（CaPF1）。该结合因子的近似分子量为43kDa。将CaPF1与已知转录因子进行比较表明，CaPF1复合物可能是一种新型的DNA结合转录因子，在体内SERCA2基因调控中发挥作用。
