Nakayama M, Stauffer J, Cheng J, Banerjee-Basu S, Wawrousek E, Buonanno A
Unit on Molecular Neurobiology, National Institute of Child Health and Human Development, Bethesda, Maryland 20892, USA.
Mol Cell Biol. 1996 May;16(5):2408-17. doi: 10.1128/MCB.16.5.2408.
The molecular mechanisms generating muscle diversity during development are unknown. The phenotypic properties of slow- and fast-twitch myofibers are determined by the selective transcription of genes coding for contractile proteins and metabolic enzymes in these muscles, properties that fail to develop in cultured muscle. Using transgenic mice, we have identified regulatory elements in the evolutionarily related troponin slow (TnIs) and fast (TnIf) genes that confer specific transcription in either slow or fast muscles. Analysis of serial deletions of the rat TnIs upstream region revealed that sequences between kb -0.95 and -0.5 are necessary to confer slow-fiber-specific transcription; the -0.5-kb fragment containing the basal promoter was inactive in five transgenic mouse lines tested. We identified a 128-bp regulatory element residing at kb -0.8 that, when linked to the -0.5-kb TnIs promoter, specifically confers transcription to slow-twitch muscles. To identify sequences directing fast-fiber-specific transcription, we generated transgenic mice harboring a construct containing the TnIs kb -0.5 promoter fused to a 144-bp enhancer derived from the quail TnIf gene. Mice harboring the TnIf/TnIs chimera construct expressed the transgene in fast but not in slow muscles, indicating that these regulatory elements are sufficient to confer fiber-type-specific transcription. Alignment of rat TnIs and quail TnIf regulatory sequences indicates that there is a conserved spatial organization of core elements, namely, an E box, a CCAC box, a MEF-2-like sequence, and a previously uncharacterized motif. The core elements were shown to bind their cognate factors by electrophoretic mobility shift assays, and their mutation demonstrated that the TnIs CCAC and E boxes are necessary for transgene expression. Our results suggest that the interaction of closely related transcriptional protein-DNA complexes is utilized to specify fiber type diversity.
发育过程中产生肌肉多样性的分子机制尚不清楚。慢肌纤维和快肌纤维的表型特性由这些肌肉中编码收缩蛋白和代谢酶的基因的选择性转录决定，而这些特性在培养的肌肉中无法发育。利用转基因小鼠，我们在进化相关的肌钙蛋白慢型（TnIs）和快型（TnIf）基因中鉴定出了调控元件，这些元件可在慢肌或快肌中进行特异性转录。对大鼠TnIs上游区域的系列缺失分析表明，-0.95 kb至-0.5 kb之间的序列对于赋予慢纤维特异性转录是必需的；包含基础启动子的-0.5 kb片段在测试的五个转基因小鼠品系中无活性。我们在-0.8 kb处鉴定出一个128 bp的调控元件，当它与-0.5 kb的TnIs启动子相连时，可特异性地赋予慢肌纤维转录。为了鉴定指导快纤维特异性转录的序列，我们构建了转基因小鼠，其携带一个包含与源自鹌鹑TnIf基因的144 bp增强子融合的TnIs -0.5 kb启动子的构建体。携带TnIf/TnIs嵌合构建体的小鼠在快肌而非慢肌中表达转基因，表明这些调控元件足以赋予纤维类型特异性转录。大鼠TnIs和鹌鹑TnIf调控序列的比对表明，核心元件存在保守的空间组织，即一个E盒、一个CCAC盒、一个MEF-2样序列和一个以前未鉴定出的基序。通过电泳迁移率变动分析表明核心元件可结合其同源因子，并且它们的突变表明TnIs的CCAC盒和E盒对于转基因表达是必需的。我们的结果表明，利用密切相关的转录蛋白-DNA复合物的相互作用来确定纤维类型的多样性。
