The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx.
The DAF-3 Smad binds DNA and represses gene expression in the Caenorhabditis elegans pharynx.
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发表时间：
1999
期刊：
影响因子：
4.6
通讯作者：
J. Thatcher;Christina Haun;P. Okkema
中科院分区：
文献类型：
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作者：
J. Thatcher;Christina Haun;P. Okkema
秀丽隐杆线虫咽部肌肉中的基因表达部分受器官特异性信号的控制，在myo-2基因中，这些信号针对一个被称为C亚元件的短DNA序列。为了确定与这些信号有关的基因，我们进行了酵母单杂交筛选编码结合C亚基的因子的cDNA。其中一个克隆来自daf-3，它编码一个与脊椎动物Smad4关系最密切的Smad。我们用凝胶迁移率改变和DNase1保护实验证明了DAF-3与C亚基DNA直接和特异结合。GTCTG序列中任何碱基的突变都干扰了凝胶迁移率变化分析中的结合，表明该五核苷酸是DAF-3结合的核心识别序列。已知DAF-3促进Dauer幼虫的形成，这种活性受TGFbeta样信号的负调控。为了确定daf-3如何影响体内C亚基增强子的活性，我们检测了由串联的C亚基寡核苷酸控制的GFP报告在daf-3突变体和其他突变体中的表达，该突变体影响控制Dauer形成的TGFbeta样信号通路。我们的结果表明，野生型DAF-3可以抑制幼虫发育过程中C亚基增强子的活性，并且与其促进Dauer的活性一样，DAF-3‘S抑制物的活性也受到TGFβ样信号的负调控。我们已经检测了这个GFP报告在达尔幼虫中的表达，没有观察到依赖daf-3的C活性抑制。这些结果表明，daf-3在非Dauer发育过程中直接调控咽部基因的表达。
Gene expression in the pharyngeal muscles of Caenorhabditis elegans is controlled in part by organ-specific signals, which in the myo-2 gene target a short DNA sequence termed the C subelement. To identify genes contributing to these signals, we performed a yeast one-hybrid screen for cDNAs encoding factors that bind the C subelement. One clone recovered was from daf-3, which encodes a Smad most closely related to vertebrate Smad4. We demonstrated that DAF-3 binds C subelement DNA directly and specifically using gel mobility shift and DNase1 protection assays. Mutation of any base in the sequence GTCTG interfered with binding in the gel mobility shift assay, demonstrating that this pentanucleotide is a core recognition sequence for DAF-3 binding. daf-3 is known to promote formation of dauer larvae and this activity is negatively regulated by TGFbeta-like signaling. To determine how daf-3 affects C subelement enhancer activity in vivo, we examined expression a gfp reporter controlled by a concatenated C subelement oligonucleotide in daf-3 mutants and other mutants affecting the TGFbeta-like signaling pathway controlling dauer formation. Our results demonstrate that wild-type daf-3 can repress C subelement enhancer activity during larval development and, like its dauer-promoting activity, daf-3's repressor activity is negatively regulated by TGFbeta-like signaling. We have examined expression of this gfp reporter in dauer larvae and have observed no daf-3-dependent repression of C activity. These results suggest daf-3 directly regulates pharyngeal gene expression during non-dauer development.