Catala F, Wanner R, Barton P, Cohen A, Wright W, Buckingham M
Centre National de la Recherche Scientifique, URA 1947, Department of Molecular Biology, Pasteur Institute, Paris, France.
Mol Cell Biol. 1995 Aug;15(8):4585-96. doi: 10.1128/MCB.15.8.4585.
The mouse myosin light-chain 1A (MLC1A) gene, expressed in the atria of the adult heart, is one of the first muscle genes to be activated when skeletal as well as cardiac muscles form in the embryo. It is also transcribed in skeletal muscle cell lines at the onset of differentiation. Transient transfection assays of mouse skeletal muscle cell lines with DNA constructs containing MLC1A promoter fragments fused to the chloramphenicol acetyltransferase (CAT) gene show that the first 630 bp of the promoter is sufficient to direct expression of the reporter gene during myotube formation. Two E boxes located at bp -76 and -519 are necessary for this regulation. MyoD and myogenin proteins bind to them as heterodimers with E12 protein and, moreover, transactivate them in cotransfection experiments with the MLC1A promoter in nonmuscle cells. Interestingly, the effect of mutating each E box is less striking in primary cultures than in the C2 or Sol8 muscle cell line. A DNA fragment from bp -36 to -597 confers tissue- and stage-specific activity to the herpes simplex virus thymidine kinase promoter in both orientations, showing that the skeletal muscle-specific regulation of the MLC1A gene is under the control of a muscle-specific enhancer which extends into the proximal promoter region. At bp -89 is a diverged CArG box, CC(A/T)6AG, which binds the serum response factor (SRF) in myotube nuclear extracts, as does the wild-type sequence, CC(A/T)6GG. Both types of CArG box also bind a novel myotube-enriched complex which has contact points with the AT-rich part of the CArG box and adjacent 3' nucleotides. Mutations within the CArG box distinguish between the binding of this complex and binding of SRF; only SRF binding is directly involved in the specific regulation of the MLC1A gene in skeletal muscle cell lines.
小鼠肌球蛋白轻链1A（MLC1A）基因在成年心脏的心房中表达，是胚胎期骨骼肌和心肌形成时最早被激活的肌肉基因之一。在骨骼肌细胞系分化开始时也会转录该基因。用含有与氯霉素乙酰转移酶（CAT）基因融合的MLC1A启动子片段的DNA构建体对小鼠骨骼肌细胞系进行瞬时转染分析表明，启动子的前630 bp足以在肌管形成过程中指导报告基因的表达。位于-76和-519 bp处的两个E盒对于这种调控是必需的。MyoD和肌细胞生成素蛋白与E12蛋白以异二聚体形式结合到它们上面，此外，在非肌肉细胞中与MLC1A启动子进行共转染实验时，它们能够反式激活这些E盒。有趣的是，在原代培养物中，单个E盒发生突变的影响不如在C2或Sol8肌肉细胞系中那么显著。从-36到-597 bp的DNA片段在两个方向上都赋予单纯疱疹病毒胸苷激酶启动子组织和阶段特异性活性，这表明MLC1A基因的骨骼肌特异性调控受一个延伸到近端启动子区域的肌肉特异性增强子的控制。在-89 bp处有一个变异的CArG盒，CC(A/T)6AG，它在肌管核提取物中与血清反应因子（SRF）结合，野生型序列CC(A/T)6GG也是如此。这两种类型的CArG盒还与一种新的富含肌管的复合物结合，该复合物与CArG盒富含AT的部分和相邻的3'核苷酸有接触点。CArG盒内的突变区分了这种复合物的结合和SRF的结合；只有SRF的结合直接参与骨骼肌细胞系中MLC1A基因的特异性调控。
