Haerry T E, Gehring W J
Biozentrum, University of Basel, Switzerland.
Dev Biol. 1997 Jun 1;186(1):1-15. doi: 10.1006/dbio.1997.8582.
The evolutionary conservation of the homeodomains suggests that their in vivo DNA binding sites may also be conserved between vertebrates and invertebrates. The regulatory function of the mouse Hoxa-4 and Hoxb-4 introns were analyzed in Drosophila since they both contain a cluster of three homeodomain binding sites, the HB1 element, which was also found in the introns of other Hox genes ranging from fish to humans as well as in the Ultrabithorax (Ubx) and decapentaplegic (dpp) genes of Drosophila. The enhancer of the Hoxa-4 intron was found to respond to several homeobox genes activating a lacZ reporter gene in particular cells of the epidermis in Drosophila embryos. The enhancer activity was found to be similar to previously described autoregulatory elements of Deformed (Dfd), the Drosophila homolog of Hoxa-4, but additional expression was observed in more posterior segments activated by Ubx and repressed by abdominal-A (abd-A). Point mutations in the homeodomain binding sites in HB1 abolished the enhancer activity. A second site suppression experiment showed that UBX interacts directly with the HB1 element. When the HB1 element in the Hoxa-4 intron was replaced by that of the mesodermal enhancer of dpp, which was previously shown to be directly controlled by Ubx, Ubx-dependent activation was retained, but repression by abd-A was lost. The same result was obtained when the third binding site of HB1 was altered, suggesting that this site is responsible for abd-A-dependent repression. Finally, deletion of potential cofactor binding sites flanking the HB1 element that are also conserved in the medaka, chicken, and mouse genes revealed that they are important for enhancer function in Drosophila and that the Dfd-dependent and the Ubx-dependent expression requires different sites. The evolutionary and functional conservation of the HB1 elements indicates that not only the homeodomains but also some of their in vivo binding sites are conserved between vertebrates and invertebrates.
同源结构域的进化保守性表明，它们在体内的DNA结合位点在脊椎动物和无脊椎动物之间可能也是保守的。对小鼠Hoxa-4和Hoxb-4内含子的调控功能在果蝇中进行了分析，因为它们都包含一组三个同源结构域结合位点，即HB1元件，该元件在从鱼类到人类的其他Hox基因的内含子中也有发现，以及在果蝇的超双胸（Ubx）和背腹分化蛋白（dpp）基因中也有发现。发现Hoxa-4内含子的增强子对几个同源异型框基因有反应，在果蝇胚胎表皮的特定细胞中激活一个lacZ报告基因。发现增强子活性与先前描述的变形（Dfd）的自调控元件相似，Dfd是Hoxa-4的果蝇同源物，但在由Ubx激活并被腹A（abd-A）抑制的更后部节段中观察到额外的表达。HB1中同源结构域结合位点的点突变消除了增强子活性。第二个位点抑制实验表明UBX直接与HB1元件相互作用。当Hoxa-4内含子中的HB1元件被dpp的中胚层增强子的元件取代时，先前已证明该增强子直接受Ubx控制，Ubx依赖性激活得以保留，但abd-A的抑制作用丧失。当改变HB1的第三个结合位点时也得到了相同的结果，表明该位点负责abd-A依赖性抑制。最后，删除HB1元件侧翼的潜在辅因子结合位点，这些位点在青鳉、鸡和小鼠基因中也保守，结果表明它们对果蝇中的增强子功能很重要，并且Dfd依赖性和Ubx依赖性表达需要不同的位点。HB1元件的进化和功能保守性表明，不仅同源结构域，而且它们在体内的一些结合位点在脊椎动物和无脊椎动物之间也是保守的。
