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。尽管类固醇受体（SR）用于影响基因表达的机制已在内分泌和分子水平上进行了广泛研究，但核细胞生物学水平上的信息却很少。作为核代谢的调节剂，ER必须在核结构的结构/功能组织内工作。由于RNA聚合酶II介导的转录已被证明发生在空间上离散和相对不溶性的“转录工厂”，它是必要的，以了解受体，其辅因子，转录位点，周转机械和核结构之间的机械关系。我们已经确定了一个动态的，亚核池的ER通过高分辨率的免疫荧光和生化分馏，特别是与转录能力的核骨架在激素依赖的方式。有趣的是，ER在空间上仅与核骨架上的小部分pol II转录位点映射。用ER进行的初步突变已经在LDL内鉴定出一种推定的信号，该信号赋予ER与不溶性核亚区室结合的能力，并且也是雌激素介导的反式激活所需的。利用整合的分子形态学方法，我们建议进一步表征ER及其核组织在活细胞和固定细胞中与转录位点、辅因子（SRC 1、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物化学筛选来特异性地鉴定失活点突变，这些突变也将被测试用于亚核分配。这些信号的影响将与辅因子（SRC 1，SMRT）对核组织和反式激活因子功能的影响一起进行检查。与ER作用相关的表征分配机制将直接允许对其在细胞核中的作用位点的功能理解，并为支持核功能高度受核结构影响的新兴范式提供新的启示。

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.