Latinkić Branko V, Cooper Brian, Towers Norma, Sparrow Duncan, Kotecha Surendra, Mohun Timothy J
Division of Developmental Biology, National Institute for Medical Research, The Ridgeway, London NW7 1AA, UK.
Dev Biol. 2002 May 1;245(1):57-70. doi: 10.1006/dbio.2002.0639.
During vertebrate embryonic development, cardiac and skeletal muscle originates from distinct precursor populations. Despite the profound structural and functional differences in the striated muscle tissue they eventually form, such progenitors share many features such as components of contractile apparatus. In vertebrate embryos, the alpha-cardiac actin gene encodes a major component of the myofibril in both skeletal and cardiac muscle. Here, we show that expression of Xenopus cardiac alpha-actin in the myotomes and developing heart tube of the tadpole requires distinct enhancers within its proximal promoter. Using transgenic embryos, we find that mutations in the promoter-proximal CArG box and 5 bp downstream of it specifically eliminate expression of a GFP transgene within the developing heart, while high levels of expression in somitic muscle are maintained. This sequence is insufficient on its own to limit expression solely to the myocardium, such restriction requiring multiple elements within the proximal promoter. Two additional enhancers are active in skeletal muscle of the embryo, either one of which has to interact with the proximal CArG box for correct expression to be established. Transgenic reporters containing multimerised copies of CArG box 1 faithfully detect most sites of SRF expression in the developing embryo as do equivalent reporters containing the SRF binding site from the c-fos promoter. Significantly, while these motifs possess a different A/T core within the CC(A/T)(6)GG consensus and show no similarity in flanking sequence, each can interact with a myotome-specific distal enhancer of cardiac alpha-actin promoter, to confer appropriate cardiac alpha-actin-specific regulation of transgene expression. Together, these results suggest that the role of CArG box 1 in the cardiac alpha-actin gene promoter is to act solely as a high-affinity SRF binding site.
在脊椎动物胚胎发育过程中，心肌和骨骼肌起源于不同的前体细胞群。尽管它们最终形成的横纹肌组织在结构和功能上存在显著差异，但这些祖细胞具有许多共同特征，如收缩装置的组成成分。在脊椎动物胚胎中，α-心肌肌动蛋白基因编码骨骼肌和心肌中肌原纤维的主要成分。在此，我们表明非洲爪蟾心肌α-肌动蛋白在蝌蚪的肌节和发育中的心脏管中的表达需要其近端启动子内的不同增强子。利用转基因胚胎，我们发现启动子近端的CArG框及其下游5个碱基对中的突变特异性地消除了发育中心脏内绿色荧光蛋白转基因的表达，而体节肌中的高水平表达得以维持。该序列本身不足以将表达仅限制在心肌，这种限制需要近端启动子内的多个元件。另外两个增强子在胚胎的骨骼肌中具有活性，其中任何一个都必须与近端CArG框相互作用才能建立正确的表达。含有CArG框1多聚体拷贝的转基因报告基因能如实地检测发育中胚胎中SRF表达的大多数位点，含有来自c-fos启动子的SRF结合位点的等效报告基因也是如此。值得注意的是，虽然这些基序在CC(A/T)(6)GG共有序列中具有不同的A/T核心，并且在侧翼序列上没有相似性，但它们每一个都可以与心肌α-肌动蛋白启动子的肌节特异性远端增强子相互作用，以赋予转基因表达适当的心肌α-肌动蛋白特异性调控。总之，这些结果表明CArG框1在心肌α-肌动蛋白基因启动子中的作用仅仅是作为一个高亲和力的SRF结合位点。
