Calvo S, Venepally P, Cheng J, Buonanno A
Unit on Molecular Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1999 Jan;19(1):515-25. doi: 10.1128/MCB.19.1.515.
The regulatory elements that restrict transcription of genes encoding contractile proteins specifically to either slow- or fast-twitch skeletal muscles are unknown. As an initial step towards understanding the mechanisms that generate muscle diversity during development, we have identified a 128-bp troponin I slow upstream element (SURE) and a 144-bp troponin I fast intronic element (FIRE) that confer fiber type specificity in transgenic mice (M. Nakayama et al., Mol. Cell. Biol. 16:2408-2417, 1996). SURE and FIRE have maintained the spatial organization of four conserved motifs (3' to 5'): an E box, an AT-rich site (A/T2) that binds MEF-2, a CACC site, and a novel CAGG motif. Troponin I slow (TnIs) constructs harboring mutations in these motifs were analyzed in transiently and stably transfected Sol8 myocytes and in transgenic mice to assess their function. Mutations of the E-box, A/T2, and CAGG motifs completely abolish transcription from the TnI SURE. In contrast, mutation of the CACC motif had no significant effect in transfected myocytes or on the slow-specific transcription of the TnI SURE in transgenic mice. To assess the role of E boxes in fiber type specificity, a chimeric enhancer was constructed in which the E box of SURE was replaced with the E box from FIRE. This TnI E box chimera, which lacks the SURE NFAT site, confers essentially the same levels of transcription in transgenic mice as those conferred by wild-type SURE and is specifically expressed in slow-twitch muscles, indicating that the E box on its own cannot determine the fiber-type-specific expression of the TnI promoter. The importance of the 5' half of SURE, which bears little homology to the TnI FIRE, in muscle-specific expression was analyzed by deletion and linker scanning analyses. Removal of the 5' half of SURE (-846 to -811) results in the loss of expression in stably transfected but not in transiently expressing myocytes. Linker scanning mutations identified sequences in this region that are necessary for the function of SURE when integrated into chromatin. One of these sites (GTTAATCCG), which is highly homologous to a bicoid consensus site, binds to nuclear proteins from several mesodermal cells. These results show that multiple elements are involved in the muscle-specific activity of the TnIs promoter and that interactions between upstream and downstream regions of SURE are important for transcription in the context of native chromatin.
将编码收缩蛋白的基因转录特异性限制在慢肌或快肌骨骼肌中的调控元件尚不清楚。作为理解发育过程中产生肌肉多样性机制的第一步，我们鉴定出了一个128 bp的肌钙蛋白I慢肌上游元件（SURE）和一个144 bp的肌钙蛋白I快肌内含子元件（FIRE），它们在转基因小鼠中赋予纤维类型特异性（M. 中山等，《分子与细胞生物学》16:2408 - 2417，1996）。SURE和FIRE保持了四个保守基序（从3'到5'）的空间组织：一个E框、一个结合MEF - 2的富含AT的位点（A/T2）、一个CACC位点和一个新的CAGG基序。在瞬时和稳定转染的Sol8肌细胞以及转基因小鼠中分析了这些基序发生突变的肌钙蛋白I慢肌（TnIs）构建体以评估其功能。E框、A/T2和CAGG基序的突变完全消除了TnI SURE的转录。相反，CACC基序的突变在转染的肌细胞中或对转基因小鼠中TnI SURE的慢肌特异性转录没有显著影响。为了评估E框在纤维类型特异性中的作用，构建了一个嵌合增强子，其中SURE的E框被FIRE的E框取代。这个缺乏SURE NFAT位点的TnI E框嵌合体在转基因小鼠中赋予的转录水平与野生型SURE赋予的基本相同，并且在慢肌中特异性表达，这表明E框自身不能决定TnI启动子的纤维类型特异性表达。通过缺失和连接子扫描分析研究了与TnI FIRE几乎没有同源性的SURE 5'端在肌肉特异性表达中的重要性。去除SURE的5'端（-846至-811）导致在稳定转染而非瞬时表达的肌细胞中表达丧失。连接子扫描突变鉴定出了该区域中整合到染色质时对SURE功能必需的序列。其中一个位点（GTTAATCCG）与双尾蝇共有位点高度同源，能与几种中胚层细胞的核蛋白结合。这些结果表明多个元件参与了TnIs启动子的肌肉特异性活性，并且SURE上下游区域之间的相互作用对于天然染色质环境中的转录很重要。
