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首页> 外文期刊>Molecular and Cellular Biology >Quantitative Proteomic Identification of Six4 as the Trex-Binding Factor in the Muscle Creatine Kinase Enhancer
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Quantitative Proteomic Identification of Six4 as the Trex-Binding Factor in the Muscle Creatine Kinase Enhancer

机译：蛋白质组学定量鉴定Six4作为肌肉肌酸激酶增强子中的Trex结合因子。

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Transcriptional regulatory element X (Trex) is a positive control site within the Muscle creatine kinase (MCK) enhancer. Cell culture and transgenic studies indicate that the Trex site is important for MCK expression in skeletal and cardiac muscle. After selectively enriching for the Trex-binding factor (TrexBF) using magnetic beads coupled to oligonucleotides containing either wild-type or mutant Trex sites, quantitative proteomics was used to identify TrexBF as Six4, a homeodomain transcription factor of the Six/sine oculis family, from a background of ～900 copurifying proteins. Using gel shift assays and Six-specific antisera, we demonstrated that Six4 is TrexBF in mouse skeletal myocytes and embryonic day 10 chick skeletal and cardiac muscle, while Six5 is the major TrexBF in adult mouse heart. In cotransfection studies, Six4 transactivates the MCK enhancer as well as muscle-specific regulatory regions of Aldolase A and Cardiac troponin C via Trex/MEF3 sites. Our results are consistent with Six4 being a key regulator of muscle gene expression in adult skeletal muscle and in developing striated muscle. The Trex/MEF3 composite sequence ([C/A]ACC[C/T]GA) allowed us to identify novel putative Six-binding sites in six other muscle genes. Our proteomics strategy will be useful for identifying transcription factors from complex mixtures using only defined DNA fragments for purification.
机译：转录调节因子X（Trex）是肌肌酸激酶（MCK ）增强子中的阳性对照位点。细胞培养和转基因研究表明，Trex位点对于骨骼肌和心肌中 MCK 表达很重要。在使用磁珠与含有野生型或突变Trex位点的寡核苷酸偶联的磁珠选择性富集Trex结合因子（TrexBF）之后，使用定量蛋白质组学将TrexBF识别为Six4，Six4 /正弦球菌家族的同源域转录因子，来自〜900种共纯化蛋白的背景。使用凝胶迁移测定法和六特异性抗血清，我们证明Six4是小鼠骨骼肌细胞和胚胎第10天雏鸡骨骼肌和心肌中的TrexBF，而Six5是成年小鼠心脏中的主要TrexBF。在共转染研究中，Six4通过Trex / MEF3位点激活 MCK 增强子以及 Aldolase A 和心肌肌钙蛋白C 的肌肉特定调节区域。 。我们的结果与Six4是成年骨骼肌和发育中的横纹肌中肌肉基因表达的关键调节剂一致。 Trex / MEF3复合序列（[C / A] ACC [C / T] GA）使我们能够鉴定其他六个肌肉基因中新的推定的六结合位点。我们的蛋白质组学策略对于仅使用定义的DNA片段进行纯化即可从复杂混合物中识别转录因子，将非常有用。

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