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首页> 外文期刊>Molecular and Cellular Biology >Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2
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Activation of a Delayed-Early Gene Encoding MHR3 by the Ecdysone Receptor Heterodimer EcR-B1–USP-1 but Not by EcR-B1–USP-2

机译：蜕皮激素受体异二聚体EcR-B1-USP-1激活了编码MHR3的延迟早期基因，但没有激活EcR-B1-USP-2

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MHR3, a homolog of the retinoid orphan receptor (ROR), is a transcription factor in the nuclear hormone receptor family that is induced by 20-hydroxyecdysone (20E) in the epidermis of the tobacco hornworm, Manduca sexta. Its 2.7-kb 5′ flanking region was found to contain four putative ecdysone receptor response elements (EcREs) and a monomeric (GGGTCA) nuclear receptor binding site. Activation of this promoter fused to a chloramphenicol acetyltransferase (CAT) reporter by 2 μg of 20E per ml inManduca GV1 cells was similar to that of endogenous MHR3, with detectable CAT by 3 h. When the ecdysone receptor B1 (EcR-B1) and Ultraspiracle 1 (USP-1) were expressed at high levels under the control of a constitutive promoter, CAT levels after a 3-h exposure to 20E increased two- to sixfold. In contrast, high expression of EcR-B1 and USP-2 caused little increase in CAT levels in response to 20E. Moreover, expression of USP-2 prevented activation by EcR-B1–USP-1. Deletion experiments showed that the upstream region, including the three most proximal putative EcREs, was responsible for most of the 20E activation, with the EcRE3 at ?671 and the adjacent GGGTCA being most critical. The EcRE1 at ?342 was necessary but not sufficient for the activational response but was the only one of the three putative EcREs to bind the EcR-B1–USP-1 complex in gel mobility shift assays and was responsible for the silencing action of EcR-B1–USP-1 in the absence of hormone. EcRE2 and EcRE3 each specifically bound other protein(s) in the cell extract, but not EcR and USP, and so are not EcREs in this cellular context. When cell extracts were used, the EcR-B1–USP-2 heterodimer showed no binding to EcRE1, and the presence of excess USP-2 prevented the binding of EcR-B1–USP-1 to this element. In contrast, in vitro-transcribed-translated USP-1 and USP-2 both formed heterodimeric complexes with EcR-B1 that bound ponasterone A with the sameKd (7 × 10?10 M) and bound to both EcRE1 and heat shock protein 27 EcRE. Thus, factors present in the cell extract appear to modulate the differential actions of the two USP isoforms.
机译：MHR3是类维生素A孤儿受体（ROR）的同系物，是烟草角虫表皮中的20-羟基蜕皮激素（20E）诱导的核激素受体家族中的转录因子， Manduca sexta 。发现其2.7kb 5'侧翼区含有四个推定的蜕皮激素受体反应元件（EcRE）和一个单体（GGGTCA）核受体结合位点。融合于氯霉素乙酰转移酶（CAT）报告基因的该启动子在每毫升Gandman GV1细胞中每毫升2μg20E的活化与内源性MHR3相似，在3 h时可检测到CAT。当蜕皮激素受体B1（EcR-B1）和Ultraspiracle 1（USP-1）在组成型启动子的控制下以高水平表达时，暴露于20E 3小时后的CAT水平增加了2到6倍。相反，EcR-B1和USP-2的高表达导致响应20E的CAT水平几乎没有增加。此外，USP-2的表达阻止了EcR-B1-USP-1的激活。删除实验表明，上游区域，包括三个最接近的假定的EcRE，负责大部分20E激活，其中EcRE3在671℃，而相邻的GGGTCA最关键。 342处的EcRE1是必需的，但不足以引起激活反应，但在凝胶迁移率迁移测定中是三个推定的EcRE与EcR-B1-USP-1复合物结合的唯一原因之一，并且对EcR-在没有激素的情况下为B1-USP-1。 EcRE2和EcRE3各自特异性结合细胞提取物中的其他蛋白质，但不结合EcR和USP，因此在此细胞环境中不是EcRE。当使用细胞提取物时，EcR-B1-USP-2异二聚体未显示与EcRE1的结合，过量的USP-2的存在阻止了EcR-B1-USP-1与该元素的结合。相反，体外转录翻译的USP-1和USP-2均与EcR-B1形成异二聚体复合物，该复合物以相同的 K d （7× 10 ？10 M）并与EcRE1和热激蛋白27 EcRE结合。因此，细胞提取物中存在的因子似乎可调节两种USP亚型的差异作用。

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