Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle - Isolation of a cDNA clone for repressor protein Nished
Two distinct factor-binding DNA elements in cardiac myosin light chain 2 gene are essential for repression of its expression in skeletal muscle - Isolation of a cDNA clone for repressor protein Nished
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DOI：
10.1074/jbc.272.29.18490
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发表时间：
1997-07-18
影响因子：
4.8
通讯作者：
Siddiqui, MAQ
中科院分区：
文献类型：
--
作者：
Dhar, M;Mascareno, EM;Siddiqui, MAQ
心肌肌球蛋白轻链2（MLCS）基因的表达在骨骼肌中受到抑制，这是由于其转录的负调控。两个调控元件，位于上游（~ 360个碱基对）的心脏特异性序列（CSS）和下游负调节序列（NMS），它们彼此协同作用，是抑制骨骼肌中MLC 2启动子活性所必需的。单独使用CSS和NMS没有效果。重组质粒的瞬时转染分析表明，CSS和NMS介导的转录抑制是位置和方向依赖性的，可转移到异源启动子。CSS和NMS中都存在一个最小的保守基序GAAG/CTTC，它负责抑制，因为核心CTTC序列中的突变本身就足以消除其抑制剂活性。凝胶迁移率变动分析的DNA结合试验表明，两个复合物之一，CSSBP 2，是显着丰富的胚胎骨骼肌相对于心肌。在来自成年骨骼肌的提取物中，其中心脏MLC 2表达被抑制，CSSBP 1和CSSBP 2两种复合物都存在，而心肌提取物仅含有CSSBP 1，这表明CSSBP 2复合物中的蛋白质解释了骨骼肌中心脏MLC 2的负调节。以多聚化CSS-DNA为探针，从骨骼肌cDNA文库中筛选到一个特异性的候选阻遏因子的部分cDNA克隆（Nished）。重组Nished蛋白与CSS-DNA结合，但不与核心CTTC序列突变的Delta CSS-DNA结合。Nished的氨基酸序列显示出与转录因子“runt”序列的显著结构相似性，该转录因子“runt”是果蝇中已知的间隙和配对规则基因表达的阻遏物。
The expression of the cardiac myosin light chain 2 (MLCS) gene is repressed in skeletal muscle as a result of the negative regulation of its transcription. Two regulatory elements, the cardiac specific sequence (CSS) located upstream (-360 base pairs) and a downstream negative modulatory sequence (NMS), which function in concert with each other, are required for repression of the MLC2 promoter activity in skeletal muscle. Individually, CSS and NMS have no effect. Transient transfection analysis with recombinant plasmids indicated that CSS- and NMS-mediated repression of transcription is position- and orientation-dependent and is transferable to heterologous promoters. A minimal conserved motif, GAAG/CTTC, present in both CSS and NMS, is responsible for repression as the mutation in the core CTTC sequence alone was sufficient to abrogate its repressor activity. The DNA binding assay by gel mobility shift analysis revealed that one of the two complexes, CSSBP2, is significantly enriched in embryonic skeletal muscle relative to cardiac muscle. In extracts from adult skeletal muscle, where the cardiac MLC2 expression is suppressed, both complexes, CSSBP1 and CSSBP2, were present, whereas the cardiac muscle extracts contained CSSBP1 alone, suggesting that the protein(s) in the CSSBP2 complex accounts for the negative regulation of cardiac MLC2 in skeletal muscle. A partial cDNA clone (Nished) specific for the candidate repressor factor was isolated by expression screening of the skeletal muscle cDNA library by multimerized CSS-DNA as probe. The recombinant Nished protein binds to the CSS-DNA, but not to Delta CSS-DNA where the core CTTC sequence was mutated. The amino acid sequence of Nished showed a significant structural similarity to the sequence of transcription factor ''runt,'' a known repressor of gap and pair-rule gene expression in Drosophila.