Heuchel R, Radtke F, Georgiev O, Stark G, Aguet M, Schaffner W
Institut für Molekularbiologie II, Universität Zürich, Switzerland.
EMBO J. 1994 Jun 15;13(12):2870-5. doi: 10.1002/j.1460-2075.1994.tb06581.x.
We have described and cloned previously a factor (MTF-1) that binds specifically to heavy metal-responsive DNA sequence elements in the enhancer/promoter region of metallothionein genes. MTF-1 is a protein of 72.5 kDa that contains six zinc fingers and multiple domains for transcriptional activation. Here we report the disruption of both alleles of the MTF-1 gene in mouse embryonic stem cells by homologous recombination. The resulting null mutant cell line fails to produce detectable amounts of MTF-1. Moreover, due to the loss of MTF-1, the endogenous metallothionein I and II genes are silent, indicating that MTF-1 is required for both their basal and zinc-induced transcription. In addition to zinc, other heavy metals, including cadmium, copper, nickel and lead, also fail to activate metal-responsive promoters in null mutant cells. However, cotransfection of an MTF-1 expression vector and metal-responsive reporter genes yields strong basal transcription that can be further boosted by zinc treatment of cells. These results demonstrate that MTF-1 is essential for metallothionein gene regulation. Finally, we present evidence that MTF-1 itself is a zinc sensor, which exhibits increased DNA binding activity upon zinc treatment.
我们之前已经描述并克隆了一种因子（金属反应转录因子-1，MTF-1），它能特异性结合金属硫蛋白基因增强子/启动子区域中的重金属反应性DNA序列元件。MTF-1是一种72.5 kDa的蛋白质，含有六个锌指结构和多个转录激活结构域。在此，我们报道通过同源重组在小鼠胚胎干细胞中破坏MTF-1基因的两个等位基因。产生的基因敲除突变细胞系无法产生可检测量的MTF-1。此外，由于MTF-1的缺失，内源性金属硫蛋白I和II基因处于沉默状态，这表明MTF-1对于它们的基础转录和锌诱导转录都是必需的。除了锌之外，其他重金属，包括镉、铜、镍和铅，也无法在基因敲除突变细胞中激活金属反应性启动子。然而，共转染MTF-1表达载体和金属反应性报告基因会产生强烈的基础转录，细胞经锌处理后转录可进一步增强。这些结果表明MTF-1对于金属硫蛋白基因调控至关重要。最后，我们提供证据表明MTF-1本身是一种锌传感器，锌处理后其DNA结合活性会增强。
