Ohno C K, Ueda H, Petkovich M
Cancer Research Laboratories, Queen's University, Kingston, Ontario, Canada.
Mol Cell Biol. 1994 May;14(5):3166-75. doi: 10.1128/mcb.14.5.3166-3175.1994.
The striped pattern of fushi tarazu (ftz) expression found in the blastoderm of the Drosophila melanogaster embryo is generated largely through complex interactions between multiple transcription factors that bind to the zebra element of the ftz gene. A motif in the zebra element, the FTZ-F1 recognition element (F1RE), has been shown to bind a transcription factor, FTZ-F1 alpha, that is a member of the nuclear receptor family. We recently identified a second, related member of this family, FTZ-F1 beta, that also binds to this motif. To investigate the possibility that FTZ-F1 alpha and FTZ-F1 beta coregulate ftz transcription through the F1RE, we have studied the DNA binding properties of FTZ-F1 alpha and FTZ-F1 beta. We demonstrate that recombinant FTZ-F1 alpha and FTZ-F1 beta proteins produce similar in vitro DNase I footprint patterns on a 14-nucleotide region of the zebra element and bind to this site with similar affinities and sequence specificities. Using wild-type and N-terminally truncated receptors, we have determined that FTZ-F1 alpha and FTZ-F1 beta both bind as monomers to the 9-bp F1RE in the zebra element, as well as to an imperfect inverted F1RE repeat present in the Drosophila alcohol dehydrogenase gene. A polyclonal antibody raised against FTZ-F1 beta identifies a predominant F1RE-binding component in embryonic nuclear extracts. Although FTZ-F1 alpha is also present in these extracts, FTZ-F1 alpha and FTZ-F1 beta do not appear to form heterodimers with each other. Cotransfection assays in mammalian cell culture indicate that both receptors contribute to the net transcriptional activity of a reporter gene through their direct interaction with the F1RE. These data suggest that FTZ-F1 alpha and FTZ-F1 beta likely coregulate common target genes by competition for binding to a 9-bp recognition element.
在黑腹果蝇胚胎胚盘细胞中发现的腹节基因（ftz）表达的条纹模式，很大程度上是由多个转录因子之间复杂的相互作用产生的，这些转录因子与ftz基因的斑马元件结合。斑马元件中的一个基序，即FTZ-F1识别元件（F1RE），已被证明能结合一种转录因子FTZ-F1α，它是核受体家族的成员。我们最近鉴定出该家族的第二个相关成员FTZ-F1β，它也能结合这个基序。为了研究FTZ-F1α和FTZ-F1β通过F1RE共同调节ftz转录的可能性，我们研究了FTZ-F1α和FTZ-F1β的DNA结合特性。我们证明，重组的FTZ-F1α和FTZ-F1β蛋白在斑马元件的一个14核苷酸区域上产生相似的体外DNA酶I足迹模式，并以相似的亲和力和序列特异性结合到该位点。使用野生型和N端截短的受体，我们确定FTZ-F1α和FTZ-F1β都以单体形式结合到斑马元件中的9碱基对F1RE上，以及果蝇乙醇脱氢酶基因中存在的一个不完全反向F1RE重复序列上。针对FTZ-F1β产生的多克隆抗体识别胚胎核提取物中一种主要的F1RE结合成分。尽管FTZ-F1α也存在于这些提取物中，但FTZ-F1α和FTZ-F1β似乎不会相互形成异二聚体。哺乳动物细胞培养中的共转染实验表明，两种受体都通过与F1RE的直接相互作用对报告基因的净转录活性有贡献。这些数据表明，FTZ-F1α和FTZ-F1β可能通过竞争结合一个9碱基对识别元件来共同调节共同的靶基因。
