Taylor H S
Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Connecticut 06520, USA.
Mol Reprod Dev. 1998 Mar;49(3):246-53. doi: 10.1002/(SICI)1098-2795(199803)49:3<246::AID-MRD4>3.0.CO;2-O.
Homeobox genes encode a class of highly evolutionarily conserved transcription factors that control embryonic development. The Drosophila melanogaster empty spiracles gene is the homolog of the two human homeobox genes EMX1 and EMX2. These genes are necessary for central nervous system development. We used a regulatory element of the empty spiracles gene to study the control of homeobox gene expression. The 1.2-kilobase (kb) cis-regulatory element located 3 kb 5' of the transcription start site of the empty spiracles gene was analyzed by evolutionary sequence comparisons, gel mobility shift assays, DNase footprinting, and the generation of transgenic flies. The corresponding element from a related species, Drosophila hydei, was cloned. Three discrete, approximately 100 base pair (bp) regions of sequence homology were identified. Each had two blocks of 10 to 40 bp of near perfect sequence identity. Fusion proteins were produced containing the Abdominal-B homeodomain or the empty spiracles homeodomain, known regulators of empty spiracles gene expression. Gel mobility shift assays showed that each of the three regions is bound by both proteins. DNase footprinting revealed closely linked empty spiracles and Abdominal-B binding sites. We then generated transgenic flies containing a reporter linked to individual conserved regions of the enhancer. Reporter expression was evident only outside of the usual empty spiracles expression domain. These elements are not sufficient alone; a combinatorial model is proposed. Conserved discrete areas within a homeobox gene regulatory element, which function as homeodomain protein transcription factor binding sites, are used in a combinatorial fashion to regulate these developmentally important genes.
同源异型框基因编码一类在进化上高度保守的转录因子，这些转录因子控制胚胎发育。果蝇的空气门基因是两个人类同源异型框基因EMX1和EMX2的同源物。这些基因对中枢神经系统发育是必需的。我们利用空气门基因的一个调控元件来研究同源异型框基因表达的控制。通过进化序列比较、凝胶迁移率变动分析、DNA酶足迹分析以及转基因果蝇的构建，对位于空气门基因转录起始位点上游3 kb处的1.2千碱基（kb）顺式调控元件进行了分析。从相关物种海德氏果蝇中克隆了相应元件。鉴定出三个离散的、大约100碱基对（bp）的序列同源区域。每个区域都有两个10至40 bp的近乎完美序列同一性的片段。产生了包含已知为空气门基因表达调控因子的腹部B同源结构域或空气门同源结构域的融合蛋白。凝胶迁移率变动分析表明，这三个区域都能与这两种蛋白结合。DNA酶足迹分析揭示了紧密相连的空气门和腹部B结合位点。然后我们构建了含有与增强子各个保守区域相连的报告基因的转基因果蝇。报告基因的表达仅在空气门基因通常的表达域之外明显。这些元件单独并不足够；提出了一种组合模型。同源异型框基因调控元件内保守的离散区域，作为同源结构域蛋白转录因子结合位点，以组合方式用于调控这些对发育至关重要的基因。
