We pioneered the study of steroid/nuclear receptor subcellular trafficking in living cells. Our research in this area led to the surprising finding that receptors and several of their coregulators exchange rapidly with regulatory elements in living cells. These findings in turn have led to significant controversy in the field, and an impending major paradigm shift. We have articulated a """"""""hit-and-run"""""""" model for the interaction of nuclear receptors with their regulatory elements, and are exploring molecular mechanisms involved in this fundamentally important behavior of nuclear receptors and their cofactors.1) We observed Pol II dependent transcription for the first time in living cells. Using the MMTV tandem array, we detected GFP-Pol II recruitment to the MMTV promoter, and measured the resident time for the polymerase. We also demonstrated that Pol II recruitment was reversible in the continued presence of hormone, indicating a sequential progression in the activity state of promoter.2) We have extended the findings on nuclear receptor subcellular trafficking and mobility to several other systems, including the thyroid hormone receptor TR the peroxisomal proliferator activated receptor PPAR, the progesterone receptor PR, the androgen receptor AR, and the dioxin receptor AhR. For TR, we discovered that nuclear retention of the receptor requires protein/protein interactions between TR and its corepressor, NCoR, while disruption of the DNA binding activity of receptor does not prevent nuclear localization. These findings challenge the view that constitutive DNA binding by the type II nuclear receptors is responsible for their nuclear sequestration, and support our alternative hypothesis that template location for these receptors is also transient and dynamic.3) We studied the interaction of the progesterone receptor with the MMTV promoter in living cells, and found a pronounced ligand effect on mobility of the receptor. We compared the in vivo ligand effects with the ability of PR to carry out chromatin remodeling at the MMTV promoter in vitro. We found that ZK98229, a complete antagonist of PR, increases PR mobility in living cells, while the agonist R5020 decreases its rate of exchange on gene targets. In vitro, ZK98229 liganded receptor was unable to catalyze the SWI/SNF dependent displacement reaction, while R5020 occupied receptor was effective in the reaction. These findings suggest a direct role for chromatin remodeling in receptor movement in the nucleus.4) We developed an in vitro system to study nuclear receptor mobility. After permeablization of cells, we showed that receptor mobility was lost (for both PR and GR), but could be restored by the addition of highly purified molecular chaperones. In contrast, the equally mobile HP1a was refractory to addition of chaperones. These findings introduce a new paradigm for chaperone involvement in nuclear protein mobility. 5) We showed that the aryl hydrocarbon receptor (AhR) and its heterodimer partner, ARNT, are recruited to subnuclear focal structures in response to receptor activation with ligand (dioxin). These nuclear foci correspond to active sites of transcription, and do not form in cells that do not express the ARNT cofactor. These results indicate that AhR/ARNT complexes are recruited to specific subnuclear compartments in a ligand-dependent fashion and that these foci represent the sites of AhR target genes.
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