STEROID RECEPTOR FUNCTION AND NUCLEAR ORGANIZATION

雌激素受体(ER)是核受体超家族的典型成员，是正常和肿瘤生长的重要转录调节因子。虽然类固醇受体(SR)影响基因表达的机制(S)已经在内分泌和分子水平上得到了广泛的研究，但在核细胞生物学水平上的信息很少。作为核代谢的调节者，内质网必须在核结构的结构/功能组织中发挥作用。由于RNA polII介导的转录已被证明发生在空间上不连续且相对难于溶解的“转录工厂”，因此了解受体、它们的辅助因子、转录位置、周转机制和核结构之间的机制关系是非常必要的。我们已经通过高分辨率免疫荧光和生化分级确定了一个动态的亚核内质网池，它以激素依赖的方式与转录能力核骨架特异性地结合。有趣的是，ER在空间上只与核骨架上的一小部分PolII转录位点映射。对ER的初步突变已经在低密度脂蛋白中发现了一个假定的信号，该信号赋予ER与不溶的核亚室结合的能力，也是雌激素介导的反式激活所必需的。利用整合的分子形态学方法，我们建议进一步研究活细胞和固定细胞中ER及其核组织与转录位点、辅助因子(SRC1、SMRT)和周转机制(蛋白酶体)的关系。我们将在酵母中使用一种新的遗传和生化筛查来专门识别失活点突变，这些突变也将进行亚核分区测试。这些信号的影响将与辅助因子(SRC1，SMRT)对核组织和反式激活蛋白功能的影响一起进行研究。与ER作用相关的特征划分机制(S)将直接允许对其在核中的作用部位的功能理解，并为支持核功能的新兴范式提供新的线索，该范式受到核结构的高度影响。

The estrogen receptor (ER) is a prototypical member of the nuclear receptor superfamily and an important transcriptional regulator of normal and neoplastic growth. Whereas mechanism(s) that a steroid receptor (SR) uses to influence gene expression have been extensively studied at the endocrine and molecular level,, little information exists at the nuclear cell biology level. As a regulator of nuclear metabolism, ER must work within the structural/functional organization of nuclear architecture. Since RNA pol II-mediated transcription has been shown to take place at spatially discrete and relatively insoluble 'transcription factories,' it is imperative to understand the mechanistic relationship between receptors, their co-factors, sites of transcription, turnover machinery and nuclear architecture. We have identified a dynamic, sub-nuclear pool of ER through high-resolution immunofluorescence and by biochemical fractionation that specifically associates with the transcription competent nucleoskeleton in a hormone dependent manner. Intriguingly, ER spatially maps only with a minor proportion of pol II transcription sites on the nucleoskeleton. Preliminary mutagenesis with ER has identified a putative signals within the LDL that confers that confers the ability of ER to associate with the insoluble nuclear sub-compartment and is also required for estrogen- mediated transactivation. Utilizing an integrated molecular morphology approach, we propose to further characterize the relationship of ER and its nuclear organization in lived and fixed cells with sites of transcription, co-factors (SRC1, SMRT) and turnover machinery (proteasomes). We will use a novel genetic and biochemical screen in yeast to specifically identify inactivating point mutations that will also be tested for sub-nuclear partitioning. The influence of these signals will be examined in concert with the effects co-factors (SRC1, SMRT) have upon both nuclear organization and transactivator function. Characterization partitioning mechanism(s) associated with ER action will directly allow a functional understanding of its site of action in the nucleus, and shed new light on the burgeoning paradigm that supports nuclear function is highly influenced by nuclear architecture.