SYNERGISTIC ACTIVATION OF TRANSCRIPTION BY UNC-86 AND MEC-3 IN CAENORHABDITIS-ELEGANS EMBRYO EXTRACTS
SYNERGISTIC ACTIVATION OF TRANSCRIPTION BY UNC-86 AND MEC-3 IN CAENORHABDITIS-ELEGANS EMBRYO EXTRACTS
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DOI：
10.1002/j.1460-2075.1995.tb00065.x
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发表时间：
1995-08-15
期刊：
影响因子：
11.4
通讯作者：
TJIAN, R
中科院分区：
文献类型：
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作者：
LICHTSTEINER, S;TJIAN, R
线虫秀丽隐杆线虫一直是使用经典和分子遗传学方法研究发育调控的选择生物体，因此，许多遗传定义的途径已被描述，并且已鉴定出许多调节基因。然而，这些假定转录因子的生化和功能特性仍未表征，部分原因是尚未开发出秀丽隐杆线虫无细胞转录反应。在这里，我们描述了两种秀丽隐杆线虫同源域蛋白的体外转录激活特性，线虫胚胎核提取物中的 UNC-86 和 MEC-3，虽然 POU 同源域蛋白 UNC-86 单独能够在体外激活 mec-3 启动子的转录，但 LIM 同源域蛋白 MEC-3 无法结合 DNA 或自行激活转录。然而，在UNC-86和MEC-3同时存在的情况下，我们在体外观察到与mec-3启动子的协同结合和转录的协同激活，蛋白质-蛋白质相互作用测定表明UNC-86可以直接与MEC-3结合，体外转录研究表明这两种蛋白质都含有功能性激活结构域，因此，含有两个激活结构域的异聚复合物的形成会产生高效的激活剂，这些研究为两种线虫协调转录激活提供了直接的功能证据。 DNA 结合蛋白在遗传学上被定义为胚胎发生过程中基因表达的调节因子。
The nematode Caenorhabditis elegans has been a choice organism for the study of developmental regulation using classical and molecular genetic approaches, Consequently, many genetically defined pathways have been described and numerous regulatory genes have been identified, However, the biochemical and functional properties of these putative transcription factors have remained uncharacterized, partly because C.elegans cell-free transcription reactions have not been developed, Here we describe the in vitro transcriptional activation properties of two C.elegans homeodomain proteins, UNC-86 and MEC-3, in nuclear extracts derived from C.elegans embryos, Whereas the POU homeodomain protein, UNC-86, alone was able to activate transcription of the mec-3 promoter in vitro, the LIM homeodomain protein, MEC-3, failed to bind DNA or activate transcription on its own. However, in the presence of both UNC-86 and MEC-3, we observed cooperative binding to the mec-3 promoter and synergistic activation of transcription in vitro, Protein-protein interaction assays revealed that UNC-86 can bind directly to MEC-3, and in vitro transcription studies indicate that both proteins contain a functional activation domain, Thus, formation of a heteromeric complex containing two activation domains results in a highly potent activator, These studies provide direct functional evidence for coordinated transcriptional activation by two C.elegans DNA binding proteins that have been defined genetically as regulators of gene expression during embryogenesis.