Marín María-Cruz, Rodríguez José-Rodrigo, Ferrús Alberto
Instituto Cajal, Consejo Superior de Investigaciones Cientificas, Madrid 28002, Spain.
Mol Biol Cell. 2004 Mar;15(3):1185-96. doi: 10.1091/mbc.e03-09-0663. Epub 2004 Jan 12.
The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, we show that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects.
果蝇的翅上A基因编码肌钙蛋白I。位于转录起始位点上游的两个区域(上游调控元件)和第一个内含子中的区域(内含子调控元件),在特定的发育和肌肉类型结构域中调节基因表达。基于转基因品系中LacZ报告基因的表达,上游调控元件和内含子调控元件产生相同的表达模式。定量逆转录-聚合酶链反应分析表明,这两个元件对于体内的完全表达水平都是必需的。在每个调控元件中,已经对三个肌细胞增强因子-2结合位点进行了功能表征。使用外显子特异性探针,我们表明顺式效应是基于同源染色体上的转录变化,并且Zeste和Zeste抑制因子3基因产物作为翅上A基因的阻遏物。在转录起始位点附近确定了顺式效应和Zeste效应的关键区域。在对昆虫(按蚊和果蝇)和脊椎动物(大鼠和鹌鹑)进行电子分析后,果蝇的调控组织似乎是保守的。肌钙蛋白I(TnI)在肌肉祖细胞开始融合之前表达,并且肌节形态发生受到TnI缺失的影响,因为Z盘无法形成,这揭示了该蛋白质或其转录本的一种新的发育作用。此外,TnI同工型之间的异常化学计量比,而不是它们的绝对水平,似乎导致了功能性肌肉缺陷。