The molecular regulation mechanism in the cardiac calcium signaling proteins

为了检测和鉴定RYR2和磷蛋白的转录调控序列，我们分离了RYR2和磷蛋白基因的5‘侧翼区，并在基因的5’侧翼区确定了潜在的调控元件，将基因5‘侧翼区的一系列5’-缺失结构与荧光素酶基因融合，然后测定了它们在新生大鼠心肌细胞中的启动子活性。对GC盒和调控区的替换突变分析表明，除了GC盒位于-56到-51之间外，还有两个调控元件(RYR2P1和RYR2P2)对启动子的活性是必不可少的。这些结果表明，Sp1和与RYR2P1和RYR2P2结合的转录因子协同增强了RYR2基因的表达。在瞬时转染实验中inv…进一步解析骨骼肌细胞中的启动子-荧光素酶基因结构，我们在-209到-90之间发现了一个负调控区域，它抑制了RYR2基因在骨骼肌细胞中的表达。另一方面，荧光素酶基因结构包括荧光素酶基因上游的-2080个碱基和5‘端长达-96个碱基的渐进性缺失，显示出高水平的荧光素酶活性。反之，-78~-4个碱基的进一步缺失导致荧光素酶活性显著降低。序列分析表明，磷蛋白基因-96~-78区域含有主要的正调控元件。序列分析显示，在转录起始点-93处有一个规范的CCAAT序列，在-10处有一个TATA盒。为了证实CAAT盒在磷蛋白基因转录中的功能意义，在磷蛋白基因5‘侧翼区构建了内部缺失突变体。去除CAAT盒使荧光素酶活性降低到对照的40%。CAAT box和位于-113的C/EBPβ元件的内部缺失使活性降低到对照的5%。凝胶漂移实验表明，心脏核蛋白与CAAT盒结合。这些结果表明，CAAT盒与C/EBPβ元件一起对磷蛋白基因的转录活性起着关键作用。较少

In order to examine and identify sequenses responsible for transcriptional regulation of the RYR2 and phospholamban, we have isolated the 5'-flanking region of the genes, and to identify potential regulatory elements in the 5'-flanking region of the genes, a series of 5'-deletion constructs in the 5'-flanking region of the genes were fused to the luciferase gene, and then their promoter activity in rat neonatal cardiac myocytes was determined.As to the RYR2 gene, the results revealed the presence of a region containing positive regulatory elements in the 5'-flanking region. Analyzes of substitutional mutations introduced into the GC boxes and the regulatory region indicated that besides the GC box located at-56 to-51, two regulatory elements (RYR2P1 and RYR2P2) and 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 inv … More olving the promoter-luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between-209 and-90 which represses the expression of the RYR2 gene in skeletal muscle cells.In another hand, luciferase gene constucts including-2080 bp of the upstream region of phopholamban gene and progressive 5' deletions up to-96 bp showed a high level of luciferase activity. In contrust, further deletions from -78 to -4 bp resulted in remarkable decrease of luciferase activity. These results indicated that the region from-96 to-78 of the phospholambn gene contains the major positive regulatory element.Sequence analysis revealed a canonial CCAAT sequence at position-93 from the tentative transcription initiation site and a TATA box at-10.To confirm the functional significance of CAAT box for the transcription of phospholamban gene, internal deletion mutants were generated in the 5' flanking region of the phospholamban gene. Removal of the CAAT box reduced the luciferase activity to 40% that of the control. Internal deletion of both CAAT box and C/EBP beta element, which is located at-113, reduced the activty to 5% of the control. Gel shift assay indicated that nuclear proteins from heart bind to the CAAT box. These results suggested that the CAAT box together with the C/EBP beta element has critical role for the transcriptional activity of the phospholamban gene. Less