Mechanism of Regulation of Sodium Pump Gane Expressions in Muscle Differentiation

我们用瞬时转染法在L6大鼠骨骼肌成肌细胞中鉴定了大鼠Na， k - atp酶α 2亚基基因（Atpla2） 5'侧区域的顺式元件。通过5'缺失突变分析，鉴定-175和-108位点之间的区域为正调控区。在该区域，远端E盒（-144 ~ -139）作为负调控元件，Spl共识序列（-123 ~ -118）和GGGAGG序列（-114 ~ -109）作为正调控元件。凝胶阻滞分析显示结合因子为E-box结合蛋白和Spl。dna酶I足迹和甲基化干扰分析显示，Spl结合在-122至-101区域，E-box结合蛋白在-144至-136区域。T4 DNA聚合酶足迹显示该区域存在三个Spl结合位点，并且Spl以互斥的方式与三个位点中的一个结合。结合位点的冗余性可以保证Spl的结合更加稳定。此外，每个Spl的结合可能导致转录起始的动力学协同作用。我们使用电泳迁移位移法检测DNA弯曲，以确定Spl是否诱导DNA的结构变化。结果表明，Spl在结合其识别序列时使DNA弯曲。这种由Spl引起的DNA结构变化可能导致协同激活，使Spl更有利于进入转录装置的其他组分。

We identified cis elements in the 5'-flanking region of rat Na, K-ATPase alpha2 subunit gene(Atpla2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5' deletion mutation analysis, the region between positions -175 and -108 was identified as a positive regulatory region. In the region, the distal E box (-144 to -139) acts as a negative regulatory element, and the Spl consensus sequence (-123 to -118) and the GGGAGG sequence (-114 to -109) act as positive regulatory elements. Gel retardation analysis revealed that binding factors are an E-box binding protein and Spl. DNase I footprinting and methylation interference analyzes revealed that Spl binds to the region from -122 to -101 and the E-box binding protein from -144 to -136. T4 DNA polymerase footprinting revealed that there are three Spl binding sites in the region and that Spl binds to one of the three sites in a mutually exclusive manner. The redundancy of binding sites may ensure more stable binding of Spl. Furthermore, the binding of each Spl may result in kinetic synergism in transcription initiation.We examined DNA bending using an electrophoretic mobility shift assay to determine whether Spl induces structural changes in DNA.The results indicated that Spl bends DNA upon binding to its recognition sequence. This structural change DNA by Spl may result in synergistic activation by making access of Spl to other components of the transcriptional apparatus more favorable.