Serrano N, Brock H W, Demeret C, Dura J M, Randsholt N B, Kornberg T B, Maschat F
Département de Biologie du Développement, Institut Jacques Monod, CNRS et Université Paris 7, France.
Development. 1995 Jun;121(6):1691-703. doi: 10.1242/dev.121.6.1691.
In Drosophila, Engrailed is a nuclear regulatory protein with essential roles in embryonic segmentation and in normal development of posterior compartments. One of its regulatory targets appears to be polyhomeotic (ph), a Polycomb group gene. We observed, by immunostaining, that Engrailed protein binds to the site of the polyhomeotic locus in region 2D of polytene chromosomes. The same analysis carried out on a transgenic line containing one copy of a P(ph-lacZ) construct shows an additional Engrailed-binding site at the location of the insert. In vivo, polyhomeotic depends on engrailed function in germ-band-elongated embryos, when engrailed and polyhomeotic genes are expressed in similar patterns. By in vitro immunoprecipitations and gel shift assays, we identified two classes of high affinity Engrailed-binding sites upstream of each of the two polyhomeotic transcription units. DNA fragments containing these sites were also immunoprecipitated from embryonic UV crosslinked chromatin in presence of anti-Engrailed antibody. These results suggest that polyhomeotic activation in germ-band-elongated embryos could be mediated by Engrailed-binding to these sites.
在果蝇中，Engrailed是一种核调节蛋白，在胚胎分割和后区室的正常发育中起关键作用。其调控靶点之一似乎是多同源异形蛋白（ph），一种多梳家族基因。我们通过免疫染色观察到，Engrailed蛋白与多线染色体2D区域的多同源异形蛋白基因座位点结合。对含有一份P（ph-lacZ）构建体的转基因品系进行的相同分析表明，在插入位置有一个额外的Engrailed结合位点。在体内，当Engrailed和多同源异形蛋白基因以相似模式表达时，多同源异形蛋白在胚带伸长的胚胎中依赖于Engrailed的功能。通过体外免疫沉淀和凝胶迁移试验，我们在两个多同源异形蛋白转录单元的每个上游鉴定出两类高亲和力的Engrailed结合位点。含有这些位点的DNA片段在抗Engrailed抗体存在的情况下也从胚胎紫外线交联染色质中被免疫沉淀。这些结果表明，胚带伸长的胚胎中多同源异形蛋白的激活可能由Engrailed与这些位点的结合介导。
