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。虽然类固醇受体影响基因表达的机制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研究却很少。作为核代谢的调节者，内质网必须在核结构的结构/功能组织中起作用。由于RNA pol ii介导的转录已被证明发生在空间离散且相对不溶的“转录工厂”中，因此有必要了解受体、它们的辅助因子、转录位点、周转机制和核结构之间的机制关系。我们通过高分辨率免疫荧光和生化分离鉴定了一个动态的亚核内质网库，它以激素依赖的方式特异性地与转录核骨架相关。有趣的是，内质网在空间上仅与核骨架上一小部分pol II转录位点相对应。内质网的初步诱变已经在LDL中发现了一个假定的信号，该信号赋予内质网与不溶性核亚室相结合的能力，并且也是雌激素介导的交易激活所必需的。利用综合分子形态学方法，我们建议进一步表征活细胞和固定细胞中内质网及其核组织与转录位点、辅助因子（SRC1、SMRT）和转换机制（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选来特异性地识别失活点突变，这些突变也将用于亚核分配测试。这些信号的影响将与辅助因子（SRC1， SMRT）对核组织和反激活子功能的影响一起进行研究。表征与内质网作用相关的分配机制将直接使我们对其在细胞核中的作用部位有一个功能性的了解，并为支持核功能受核结构高度影响的新兴范式提供新的视角。

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.