The glucocorticoid receptor (GR) contains several activation domains, tau1 (AF-1), tau2, and AF-2, which were initially defined using transiently transfected reporter constructs. Using domain mutations in the context of full-length GR, this study defines those domains required for activation of the mouse mammary tumor virus (MMTV) promoter in two distinct nucleoprotein configurations. A transiently transfected MMTV template with a disorganized, accessible chromatin structure was largely dependent on the AF-2 domain for activation. In contrast, activation of an MMTV template in organized, replicated chromatin requires both domains but has a relatively larger dependence on the tau1 domain. Domain requirements for GR-induced chromatin remodeling of the latter template were also investigated. Mutation of the AF-2 helix 12 domain partially inhibits the induction of nuclease hypersensitivity, but the inhibition was relieved in the absence of tau1, suggesting the occurrence of an important interaction between the two domains. Further mutational analysis indicates that GR-induced chromatin remodeling requires the ligand-binding domain in the region of helix 3. Our study shows that the GR activation surfaces required for transcriptional modulation of a target promoter were determined in part by its chromatin structure. Within a particular cellular environment the GR appears to possess a significant degree of versatility in the mechanism by which it activates a target promoter.

Chromatin remodeling by the glucocorticoid receptor (GR) is associated with activation of transcription at the mouse mammary tumor virus (MMTV) promoter. We reconstituted this nucleoprotein transition with chromatin assembled on MMTV DNA. The remodeling event was ATP dependent and required either a nuclear extract from HeLa cells or purified human Swi/Snf. Through the use of a direct interaction assay (magnetic bead pull-down), we demonstrated recruitment of human Swi/Snf to MMTV chromatin by GR. Unexpectedly, we found that GR is actively displaced from the chromatin template during the remodeling process. ATP-dependent GR displacement was reversed by the addition of apyrase and was specific to chromatin templates. The disengagement reaction could also be induced with purified human Swi/Snf. Although GR apparently dissociated during chromatin remodeling by Swi/Snf, it participated in binding of the secondary transcription factor, nuclear factor 1. These results are paralleled by a recent discovery that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment in living cells. Both the in vitro and in vivo results are consistent with a dynamic model (hit and run) in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, facilitates transcription factor binding, and is simultaneously lost from the template.

In this report, we have studied the intracellular dynamics and distribution of the thyroid hormone receptor-β (TRβ) in living cells, utilizing fusions to the green fluorescent protein. Wild-type TRβ was mostly nuclear in both the absence and presence of triiodothyronine; however, triiodothyronine induced a nuclear reorganization of TRβ. By mutating defined regions of TRβ, we found that both nuclear corepressor and retinoid X receptor are involved in maintaining the unliganded receptor within the nucleus. A TRβ mutant defective in DNA binding had only a slightly altered nuclear/cytoplasmic distribution compared with wild-type TRβ; thus, site-specific DNA binding is not essential for maintaining TRβ within the nucleus. Both ATP depletion studies and heterokaryon analysis demonstrated that TRβ rapidly shuttles between the nuclear and the cytoplasmic compartments. Cotransfection of nuclear corepressor and retinoid X receptor markedly decreased the shuttling by maintaining unliganded TRβ within the nucleus. In summary, our findings demonstrate that TRβ rapidly shuttles between the nucleus and the cytoplasm and that protein-protein interactions of TRβ with various cofactors, rather than specific DNA interactions, play the predominant role in determining the intracellular distribution of the receptor.

In this report, we have studied the intracellular dynamics and distribution of the thyroid hormone receptor-beta (TR beta) in living cells, utilizing fusions to the green fluorescent protein. Wild-type TB beta was mostly nuclear in both the absence and presence of triiodothyronine; however, triiodothyronine induced a nuclear reorganization of TR beta. By mutating defined regions of TR beta, we found that both nuclear corepressor and retinoid X receptor are involved in maintaining the unliganded receptor within the nucleus. A TR beta mutant defective in DNA binding had only a slightly altered nuclear/cytoplasmic distribution compared with wild-type TR beta; thus, site-specific DNA binding is not essential for maintaining TR beta within the nucleus. Both AT beta depletion studies and heterokaryon analysis demonstrated that TR beta rapidly shuttles between the nuclear and the cytoplasmic compartments. Cotransfection of nuclear corepressor and retinoid X receptor markedly decreased the shuttling by maintaining unliganded TR beta within the nucleus. In summary, our findings demonstrate that TR beta rapidly shuttles between the nucleus and the cytoplasm and that protein-protein interactions of TR beta with various cofactors, rather than specific DNA interactions, play the predominant role in determining the intracellular distribution of the receptor.

Transcription regulatory proteins are an integral component of the cell nucleus and a great deal of work has been done to characterize the subnuclear distribution of these proteins. Much of the early work on this subject was done with immunofluorescence. The development of the green fluorescent protein (GFP) as a marker for intracellular protein localization has allowed for the real time study of protein localization and dynamics in living cells. In this review, an overview of the way in which GFP can be utilized to study protein localization is presented.