Banerjee-Basu S, Buonanno A
Unit on Molecular Neurobiology, National Institute of Child Health and Human Development, Bethesda, Maryland 20892.
Mol Cell Biol. 1993 Nov;13(11):7019-28. doi: 10.1128/mcb.13.11.7019-7028.1993.
Transcription of the genes coding for troponin I slow (TnIslow) and other contractile proteins is activated during skeletal muscle differentiation, and their expression is later restricted to specific fiber types during maturation. We have isolated and characterized the rat TnIslow gene in order to begin elucidating its regulation during myogenesis. Transcriptional regulatory regions were delineated by using constructs, containing TnIslow gene sequences driving the expression of the chloramphenicol acetyltransferase (CAT) reporter gene, that were transiently transfected into undifferentiated and differentiated C2C12 cells. TnIslow 5'-flanking sequences directed transcription specifically in differentiated cells. However, transcription rates were approximately 10-fold higher in myotubes transfected with constructs containing the 5'-flanking sequences plus the intragenic region residing upstream of the translation initiation site (introns 1 and 2), indicative of interactions between elements residing upstream and in the introns of the gene. Deletion analysis of the 5' region of the TnIslow gene showed that the 200 bp upstream of the transcription initiation site is sufficient to confer differentiation-specific transcription in C2C12 myocytes. MyoD consensus binding sites were found both in the upstream 200-bp region and in a region residing in the second intron that is highly homologous to the quail TnIfast enhancer. Transactivation experiments using transfected NIH 3T3 fibroblasts with TnI-CAT constructs containing intragenic and/or upstream sequences and with the myogenic factors MyoD, myogenin, and MRF4 showed different potentials of these factors to induce transcription. Transgenic mice harboring the rat TnI-CAT fusion gene expressed the reporter specifically in the skeletal muscle. Furthermore, CAT levels were approximately 50-fold higher in the soleus than in the extensor digitorum longus, gastrocnemius, or tibialis muscle, indicating that the regulatory elements that restrict TnI transcription to slow-twitch myofibers reside in the sequences we have analyzed.
编码肌钙蛋白I慢型（TnIslow）及其他收缩蛋白的基因转录在骨骼肌分化过程中被激活，其表达随后在成熟过程中局限于特定的纤维类型。我们已分离并鉴定了大鼠TnIslow基因，以便开始阐明其在肌生成过程中的调控机制。通过使用含有驱动氯霉素乙酰转移酶（CAT）报告基因表达的TnIslow基因序列的构建体，将其瞬时转染到未分化和分化的C2C12细胞中，从而划定转录调控区域。TnIslow 5'侧翼序列特异性地指导分化细胞中的转录。然而，在用含有5'侧翼序列加上位于翻译起始位点上游的基因内区域（内含子1和2）的构建体转染的肌管中，转录率大约高10倍，这表明基因上游和内含子中的元件之间存在相互作用。对TnIslow基因5'区域的缺失分析表明，转录起始位点上游200 bp足以在C2C12肌细胞中赋予分化特异性转录。在转录起始位点上游200 bp区域以及位于第二个内含子中的一个与鹌鹑TnIfast增强子高度同源的区域中都发现了MyoD共有结合位点。使用转染了含有基因内和/或上游序列的TnI-CAT构建体以及肌生成因子MyoD、肌细胞生成素和MRF4的NIH 3T3成纤维细胞进行的反式激活实验表明，这些因子诱导转录的潜力不同。携带大鼠TnI-CAT融合基因的转基因小鼠在骨骼肌中特异性表达报告基因。此外，比目鱼肌中的CAT水平比趾长伸肌、腓肠肌或胫骨前肌中的CAT水平高约50倍，这表明将TnI转录限制在慢肌纤维中的调控元件存在于我们分析的序列中。
