STEROID RECEPTOR FUNCTION AND NUCLEAR ORGANIZATION

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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.