The molecular regulation mechanism in the cardiac calcium signaling proteins

为了检测和鉴定负责RYR2和磷蛋白转录调控的序列，我们分离了基因的5'-侧翼区域，并鉴定了基因5'-侧翼区域的潜在调控元件，将基因5'-侧翼区域的一系列5'-缺失构建物融合到荧光素酶基因中，然后测定了它们在大鼠新生心肌细胞中的启动子活性。对于RYR2基因，结果显示在5'侧区存在一个含有正调控元件的区域。对引入GC盒和调控区的替换突变的分析表明，除了位于56 -51的GC盒外，两个调控元件（RYR2P1和RYR2P2）对启动子活性至关重要。这些结果表明，Sp1和结合RYR2P1和RYR2P2的转录因子共同增强了RYR2基因的表达。通过对启动子荧光素酶基因在骨骼肌细胞中的瞬时转染实验，我们发现在-209和-90之间存在一个抑制RYR2基因在骨骼肌细胞中的表达的负调控区。另一方面，荧光素酶基因构建包括-2080 bp的磷蛋白基因上游区域，以及高达96 bp的渐进5'缺失显示出高水平的荧光素酶活性。相反，从-78 bp到-4 bp的进一步缺失导致荧光素酶活性显著降低。这些结果表明，phospholambn基因的96 ~ 78区含有主要的正调控元件。序列分析显示，在初步转录起始位点的第93位有一个典型的CCAAT序列，在第10位有一个TATA盒。为了证实CAAT box对磷蛋白基因转录的功能意义，我们在磷蛋白基因5'侧区产生了内部缺失突变体。去除CAAT盒使荧光素酶活性降低到对照组的40%。CAAT盒和C/EBP β元件（位于113）的内部缺失将活性降低至对照的5%。凝胶移位实验表明，心脏核蛋白与CAAT盒子结合。这些结果表明，CAAT盒子与C/EBP β元件对磷蛋白基因的转录活性起着关键作用。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