MOLECULAR MECHANISMS OF GLUCOCORTICOID HORMONE ACTION

了解基因表达的调控机制是至关重要的。 对发育和分化的理解，因此，癌症和 当这些过程出错时可能出现的其他障碍。类固醇 激素调控的基因表达提供了一个很好的模型系统 以此来研究监管机制。小鼠乳腺肿瘤病毒 (MMTV)启动子已成为研究开发 糖皮质激素分子机制的概念框架 行动。尽管糖皮质激素调节元件(GRE)共识 序列已被提出，功能反应元件不能 根据序列数据可靠地预测。另外，更多的应对要素， 尤其是那些调解负面监管的人，正在被绘制成地图， 不符合共识。我们的研究集中在一个系统的 用突变方法确定糖皮质激素反应元件 MMTV发起人。在之前的授权期内，我们开发了 确定了GRE点突变文库的特征。的量化问题 60多个突变体的活动使我们能够完善我们的 GRE的概念。我们建议补充生物活性 与突变的HREs结合的受体的体外分析数据。这个 约束性研究的结果将与功能 构建受体关键残基图谱的活性分析 结合和荷尔蒙反应。这些映射数据将产生如下见解 受体-DNA相互作用的分子细节，特别是 确定受体进行氢键接触的可能位置 使其能够区分HRE序列。因为MMTV的发起人现在已经 被证明对至少4种不同类别的类固醇有反应 通过将这些分析扩展到 调节这个启动子的其他类固醇受体系统。是这样的 比较分析将揭示不同的受体如何区分 识别序列的不同特征。这些研究的重点是 典型的积极GRE将得到类似分析的补充 增殖蛋白基因A受体识别位点GRE阴性 与MMTV GRE只有微弱的同源性。最后，提出了解决这些问题的途径。 研究体内受体DNA的相互作用，不仅是与GRE突变体， 但在与类固醇无反应相关的情况下也是如此 例如与细胞相关的类固醇拮抗剂或类固醇耐药性 转型。

Understanding the mechanisms of regulation of gene expression is essential to a comprehension of development and differentiation, and thus, cancer and the other disorders that may arise when these processes go awry. Steroid hormone regulated gene expression provides an excellent model system with which to investigate regulatory mechanisms. The mouse mammary tumor virus (MMTV) promoter has served as the prototype for studies developing the conceptual framework of the molecular mechanisms of glucocorticoid hormone action. Although glucocorticoid regulatory element (GRE) consensus sequences have been proposed, functional response elements cannot be reliably predicted from sequence data. In addition, more response elements, especially those mediating negative regulation, are being mapped which do not conform to the consensus. Our studies have focused on a systematic mutagenesis approach to defining the glucocorticoid response element of the MMTV promoter. During the previous grant period we developed and characterized a library of GRE point mutants. The quantitation of the activity of more than 60 of the mutants has allowed us to refine our conception of the GRE. We propose to complement the biological activity data with in vitro analyses of receptor binding to the mutant HREs. The results of the binding studies will be combined with the functional activity analyses to assemble a map of residues critical for receptor binding and for hormone response. These mapping data will yield insights as to molecular details of the receptor-DNA interaction particularly to identify the likely sites where receptors make hydrogen bond contacts that allow it to discriminate HRE sequences. Because the MMTV promoter has now been shown to respond to at least 4 different classes of steroids, the mutant library can be further exploited by extending these analyses to the other steroid-receptor systems that regulate this promoter. Such comparative analyses will reveal how the different receptors distinguish different features of the recognition sequence. The studies focusing on the prototypical positive GRE will be complemented by similar analyses on the negative GRE of the proliferin gene a receptor recognition site that bears only weak homology to the MMTV GRE. Finally, approaches are proposed to investigate receptor DNA interaction in vivo, not only with GRE mutants, but also under circumstances associated with steroid non-responsiveness e.g. steroid antagonists or steroid resistance associated with cell transformation.