Buskin J N, Hauschka S D
Department of Biochemistry, University of Washington, Seattle 98195.
Mol Cell Biol. 1989 Jun;9(6):2627-40. doi: 10.1128/mcb.9.6.2627-2640.1989.
The muscle creatine kinase (MCK) gene is transcriptionally induced when skeletal muscle myoblasts differentiate into myocytes. The gene contains two muscle-specific enhancer elements, one located 1,100 nucleotides (nt)5' of the transcriptional start site and one located in the first intron. We have used gel mobility shift assays to characterize the trans-acting factors that interact with a region of the MCK gene containing the 5' enhancer. MM14 and C2C12 myocyte nuclear extracts contain a sequence-specific DNA-binding factor which recognizes a site within a 110-nt fragment of the MCK enhancer region shown to be sufficient for enhancer function. Preparative mobility shift gels were combined with DNase I footprinting to determine the site of binding within the 110-nt fragment. Site-directed mutagenesis within the footprinted region produced a 110-nt fragment which did not bind the myocyte factor in vitro. The mutant fragment had about 25-fold-less activity as a transcriptional enhancer in myocytes than did the wild-type fragment. Complementary oligomers containing 21 base pairs spanning the region protected from DNase degradation were also specifically bound by MM14 and C2C12 myocyte nuclear factors. The oligomer-binding activity was not found in nuclear extracts from the corresponding myoblasts, in nuclear extracts from a variety of nonmuscle cell types (including differentiation-defective MM14-DD1 cells and 10T1/2 mesodermal stem cells), or in cytoplasmic extracts. Both the 5' and intron 1 enhancer-containing fragments competed for factors that bind the oligomer probe, while total mouse genomic DNA and several DNA fragments containing viral and cellular enhancers did not. Interestingly, a 5' MCK proximal promoter fragment that also contains muscle-specific positive regulatory elements did not compete for factor binding to the oligomer. We have designated the factor which interacts with the two MCK enhancers myocyte-specific enhancer-binding nuclear factor 1 (MEF 1). A consensus for binding sites in muscle-specific regulatory regions is proposed.
当骨骼肌成肌细胞分化为肌细胞时，肌肉肌酸激酶（MCK）基因会在转录水平上被诱导。该基因包含两个肌肉特异性增强子元件，一个位于转录起始位点上游1100个核苷酸（nt）处，另一个位于第一个内含子中。我们利用凝胶迁移率变动分析来鉴定与包含5'增强子的MCK基因区域相互作用的反式作用因子。MM14和C2C12肌细胞核提取物含有一种序列特异性DNA结合因子，该因子可识别MCK增强子区域110 nt片段内的一个位点，该片段已被证明足以发挥增强子功能。制备性迁移率变动凝胶与DNase I足迹法相结合，以确定110 nt片段内的结合位点。在足迹区域内进行的定点诱变产生了一个110 nt的片段，该片段在体外不与肌细胞因子结合。与野生型片段相比，突变片段在肌细胞中作为转录增强子的活性降低了约25倍。包含跨越免受DNase降解区域的21个碱基对的互补寡聚体也被MM14和C2C12肌细胞核因子特异性结合。在相应成肌细胞的核提取物、多种非肌肉细胞类型（包括分化缺陷的MM14-DD1细胞和10T1/2中胚层干细胞）的核提取物或细胞质提取物中未发现寡聚体结合活性。包含5'和内含子1增强子的片段都能竞争与寡聚体探针结合的因子，而小鼠总基因组DNA和几个包含病毒和细胞增强子的DNA片段则不能。有趣的是，一个同样包含肌肉特异性正调控元件的5' MCK近端启动子片段不能竞争与寡聚体的因子结合。我们将与两个MCK增强子相互作用的因子命名为肌细胞特异性增强子结合核因子1（MEF 1）。本文提出了肌肉特异性调控区域中结合位点的共有序列。
