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首页> 外文期刊>Molecular and Cellular Biology >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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The ecdysone response enhancer of the Fbp1 gene of Drosophila melanogaster is a direct target for the EcR/USP nuclear receptor.

机译：果蝇的Fbp1基因的蜕皮激素响应增强剂是EcR / USP核受体的直接目标。

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摘要

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.
机译：果蝇Fbp1基因的转录是由类固醇激素20-羟基蜕皮激素诱导的，并局限于三龄晚期脂肪体组织。在先前的研究中，我们表明相对于转录起始位点的-68至-138区在转基因测定中充当了蜕皮激素依赖性的第三龄脂肪体特异性增强剂。在这里我们报告说，当从晚期三龄脂肪体中提取核提取物用于凝胶迁移试验时，在该增强子上会在体外形成七个核蛋白复合物。配合物的结合位点的准确作图揭示了一个非常对称的组织。使用特异性抗体，复合物之一被鉴定为由蜕皮激素受体（EcR）和超螺旋体（USP）蛋白组成的异二聚体。通过诱变和甲基化干扰实验确定的异二聚体的结合位点与经典的hsp27蜕皮激素响应元件具有强烈的序列相似性，包括不完善的回文结构。这两个元件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异。使用连接介导的PCR和野生型或蜕皮甾体缺陷型幼虫的体内足迹实验表明，Fbp1 EcR / USP结合位点和邻近区域的占有率取决于蜕皮甾体的高浓度。这些结果提供了强有力的证据，证明果蝇幼虫发育过程中，EcR / USP异二聚体在响应蜕皮激素滴度变化而驱动基因表达中具有直接作用。

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