REGULATION OF ECDYSONE RESPONSIVE GENES

这里描述的研究计划涉及基本方面 类固醇激素蜕皮激素的基因调控。蜕皮激素调节 昆虫的发育和诱导变态。功能性蜕皮激素 受体是蛋白质ECR和USP的异源二聚体；这两种蛋白质 多肽是核激素受体家族的成员。ECR是 与甲状腺、维甲酸和维生素D受体关系最密切 和USP与受体RXR结合。ECR/USP异源二聚体与一种 蜕皮激素反应元件(ECRE)。在缺乏荷尔蒙的情况下 相互作用抑制转录；在激素存在的情况下，它刺激转录 这一建议的主要重点是分析相互作用 在ECR和USP两种蛋白质之间，激素蜕皮激素是一种推定的 USP的配基，以及DNA结合位点。在已经被 在蜕皮激素反应基因中被发现，没有明显的联系 它们在体外对ECR/USP的亲和力、它们介导 蜕皮激素在果蝇KC细胞系中的诱导及其对 抑制基础表达。此外，特定的ECRE显示组织- 特定的功能。将使用各种生化技术来 了解ECRE的序列如何改变受体的活性 情结与之捆绑在一起。USP的配体尚不清楚，但最近在我们的 实验室提示，它可能在刺激中发挥重要作用 ECR/USP DNA结合，这是一个将进一步检验的假设。其他 提出了表达ECR和USP功能片段的实验， 研究它们的功能，揭示它们的各个领域的作用 蛋白质，并制备详细结构所需的试剂 学习。ECR同源物在序列上迅速分化，而遥远的同源物 将被回收用于比较结构和功能分析。我们 在果蝇细胞中发现了一种新的基因打靶形式 我们计划用它来创造ECR缺陷和USP缺陷的细胞 LINES；这些将是未来蜕皮激素研究的重要资产 监管。最后，我们对蜕皮激素反应基因Eip71CD的研究已经 揭示了转录因子的作用，该转录因子可能是 脊椎动物淋巴细胞特异性转录因子Lyf-1/Ikaros。我们计划 克隆和研究该转录因子。

The research program described here is concerned with fundamental aspects of gene regulation by the steroid hormone ecdysone. Ecdysone regulates the development of insects and induces metamorphosis. The functional ecdysone receptor is a heterodimer of the proteins EcR and USP; both of these polypeptides are members of the nuclear hormone receptor family. EcR is most closely related to thyroid, retinoic acid, and vitamin D receptors and USP to the receptor RXR. The EcR/USP heterodimer interacts with an ecdysone response element (EcRE). In the absence of hormone this interaction inhibits transcription; in the hormone's presence it stimulates The major emphasis of this proposal is on analysis of the interactions between the two proteins EcR and USP, the hormone ecdysone, a putative ligand for USP, and the DNA binding site. Among EcREs which have been identified in ecdysone-responsive genes, there is no obvious relationship between their affinity for EcR/USP in vitro, their ability to mediate ecdysone induction in the Drosophila Kc cell line, and their ability to inhibit basal expression. Furthermore, particular EcREs display tissue- specific function. A variety of biochemical techniques will be used to understand how the sequence of an EcRE alters the activity of the receptor complex bound to it. The ligand for USP is unknown, but recent work in our laboratory suggests that it may play an important role in stimulating EcR/USP DNA binding, a hypothesis that will be tested further. Other experiments are proposed to express functional fragments of EcR and USP, to study their functions, to reveal the roles of various domains of these proteins, and to prepare the reagents necessary for detailed structural studies. EcR homologs diverge in sequence rapidly and distant homologs will be recovered for comparative structural and functional analysis. We discovered a novel form of gene targeting that occurs in Drosophila cells and we plan to use it to create EcR-deficient and USP-deficient cell lines; these will be important assets in future studies of ecdysone regulation. Finally, our work on the ecdysone-responsive gene Eip71CD has revealed a role for a transcription factor that may be a homolog of the vertebrate lymphocyte-specific transcription factor LyF-1/lkaros. We plan to clone and study this transcription factor.