THE ECDYSONE RESPONSE ENHANCER OF THE FBP1 GENE OF DROSOPHILA-MELANOGASTER IS A DIRECT TARGET FOR THE ECR/USP NUCLEAR RECEPTOR
THE ECDYSONE RESPONSE ENHANCER OF THE FBP1 GENE OF DROSOPHILA-MELANOGASTER IS A DIRECT TARGET FOR THE ECR/USP NUCLEAR RECEPTOR
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DOI：
10.1128/mcb.14.7.4465
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发表时间：
1994-07-01
影响因子：
5.3
通讯作者：
LEPESANT, JA
中科院分区：
文献类型：
--
作者：
ANTONIEWSKI, C;LAVAL, M;LEPESANT, JA
果蝇Fbp 1基因的转录是由类固醇激素20-羟基蜕皮激素诱导的，并仅限于三龄后期的脂肪体组织。在以前的研究中，我们表明，-68至-138区域相对于转录起始位点作为蜕皮激素依赖的三龄脂肪体特异性增强子在转基因检测。在这里，我们报告说，7核蛋白复合物在体外形成的增强剂时，从三龄后期脂肪体的核提取物被用于凝胶位移测定。精确映射的复合物的结合位点揭示了一个非常对称的组织。使用特异性抗体，其中一个复合物被鉴定为由蜕皮激素受体（EcR）和超气门（USP）蛋白组成的异源二聚体。通过诱变和甲基化干扰实验所定义的异二聚体的结合位点与典型的HSP 27蜕皮激素反应元件具有很强的序列相似性，包括不完美的回文结构。这两个元素在两个半位点的三个位置处发散，表明活性EcR/USP结合位点的结构允许相当大的序列变化。在体内足迹实验中使用连接介导的PCR和野生型或蜕皮类固醇缺乏的幼虫显示，占用Fbp 1 EcR/USP结合位点和相邻区域依赖于高浓度的蜕皮类固醇。这些结果提供了强有力的证据，直接作用的EcR/USP异源二聚体在驱动基因表达的蜕皮激素滴度的变化在果蝇幼虫发育过程中。
The transcription of the Drosophila melanogaster Fbp1 gene is induced by the steroid hormone 20-hydroxyecdysone and restricted to the late-third-instar fat body tissue. In a previous study we showed that the -68 to -138 region relative to the transcription start site acts as an ecdysone-dependent third-instar fat body-specific enhancer in a transgenic assay. Here we report that seven nucleoprotein complexes are formed in vitro on this enhancer when a nuclear extract from late-third-instar fat body is used in a gel shift assay. Accurate mapping of the binding sites of the complexes revealed a remarkably symmetrical organization. Using specific antibodies, one of the complexes was identified as a heterodimer consisting of the ecdysone receptor (EcR) and Ultraspiracle (USP) proteins. The binding site of the heterodimer as defined by mutagenesis and methylation interference experiments bears strong sequence similarity to the canonical hsp27 ecdysone response element, including an imperfect palindromic structure. The two elements diverge at three positions in both half-sites, indicating that the structure of an active EcR/USP binding site allows considerable sequence variations. In vivo footprinting experiments using ligation-mediated PCR and wild-type or ecdysteroid-deficient larvae show that occupancy of the Fbp1 EcR/USP binding site and adjacent region is dependent on a high concentration of ecdysteroids. These results provide strong evidence for a direct role of the EcR/USP heterodimer in driving gene expression in response to changes of the ecdysteroid titer during Drosophila larval development.