Fluorescence resonance energy transfer from cyan to yellow fluorescent protein detected by acceptor photobleaching using confocal microscopy and a single laser.

One manifestation of fluorescence resonance energy transfer (FRET) is an increase in donor fluorescence after photobleaching the acceptor. Published acceptor-photobleaching methods for FRET have mainly used wide-field microscopy. A laser scanning confocal microscope enables faster and targeted bleaching within the field of view, thereby improving speed and accuracy. Here we demonstrate the approach with CFP and YFP, the most versatile fluorescent markers now available for FRET. CFP/YFP FRET imaging has been accomplished with a single laser (argon) available on virtually all laser-scanning confocal microscopes. Accordingly, we also describe the conditions that we developed for dual imaging of CFP and YFP with the 458 and 514 argon lines. We detect FRET in a CFP/YFP fusion and also between signalling molecules (TNF-Receptor-Associated-Factors or TRAFs) that are known to homo- and heterotrimerize. Importantly, we demonstrate that appropriate controls are essential to avoid false positives in FRET by acceptor photobleaching. We use two types of negative control: (a) an internal negative control (non-bleached areas of the cell) and (b) cells with donor in the absence of the acceptor (CFP only). We find that both types of negative control can yield false FRET. Given this false FRET background, we describe a method for distinguishing true positive signals. In summary, we extensively characterize a simple approach to FRET that should be adaptable to most laser-scanning confocal microscopes, and demonstrate its feasibility for detecting FRET between several CFP/YFP partners.

Hormone-dependent, CARM1-directed, arginine-specific methylation of histone H3 on a steroid-regulated promoter.

Activation of gene transcription involves chromatin remodeling by coactivator proteins that are recruited by DNA-bound transcription factors. Local modification of chromatin structure at specific gene promoters by ATP-dependent processes and by posttranslational modifications of histone N-terminal tails provides access to RNA polymerase II and its accompanying transcription initiation complex. While the roles of lysine acetylation, serine phosphorylation, and lysine methylation of histones in chromatin remodeling are beginning to emerge, low levels of arginine methylation of histones have only recently been documented, and its physiological role is unknown. The coactivator CARM1 methylates histone H3 at Arg17 and Arg26 in vitro and cooperates synergistically with p160-type coactivators (e.g., GRIP1, SRC-1, ACTR) and coactivators with histone acetyltransferase activity (e.g., p300, CBP) to enhance gene activation by steroid and nuclear hormone receptors (NR) in transient transfection assays. In the current study, CARM1 cooperated with GRIP1 to enhance steroid hormone-dependent activation of stably integrated mouse mammary tumor virus (MMTV) promoters, and this coactivator function required the methyltransferase activity of CARM1. Chromatin immunoprecipitation assays and immunofluorescence studies indicated that CARM1 and the CARM1-methylated form of histone H3 specifically associated with a large tandem array of MMTV promoters in a hormone-dependent manner. Thus, arginine-specific histone methylation by CARM1 is an important part of the transcriptional activation process.

A glucocorticoid/retinoic acid receptor chimera that displays cytoplasmic/nuclear translocation in response to retinoic acid. A real time sensing assay for nuclear receptor ligands.

Members of the nuclear receptor superfamily play key roles in a host of physiologic and pathologic processes from embryogenesis to cancer. Some members, including the retinoic acid receptor (RAR), are activated by ligand binding but are unaffected in their subcellular distribution, which is predominantly nuclear. In contrast, several members of the steroid receptor family, including the glucocorticoid receptor, are cytoplasmic and only translocate to the nucleus after ligand binding. We have constructed chimeras between RAR and glucocorticoid receptor that selectively respond to RAR agonists but display cytoplasmic localization in the absence of ligand. These chimeric receptors manifest both nuclear translocation and gene activation functions in response to physiological concentrations of RAR ligands. The ability to achieve regulated subcellular trafficking with a heterologous ligand binding domain has implications both for current models of receptor translocation and for structural-functional conservation of ligand binding domains broadly across the receptor superfamily. When coupled to the green fluorescent protein, chimeric receptors offer a powerful new tool to 1) study mechanisms of steroid receptor translocation, 2) detect dynamic and graded distributions of ligands in complex microenvironments such as embryos, and 3) screen for novel ligands of "orphan" receptors in vivo.

The glucocorticoid receptor interacting protein 1 (GRIP1) localizes in discrete nuclear foci that associate with ND10 bodies and are enriched in components of the 26S proteasome.

The glucocorticoid receptor interacting protein-1 (GRIP1) is a member of the steroid receptor coactivator (SRC) family of transcriptional regulators. Green fluorescent protein (GFP) fusions were made to full-length GRIP1, and a series of GRIP1 mutants lacking the defined regulatory regions and the intracellular distribution of these proteins was studied in HeLa cells. The distribution of GRIP1 was complex, ranging from diffuse nucleoplasmic to discrete intranuclear foci. Formation of these foci was dependent on the C-terminal region of GRIP1, which contains the two characterized transcriptional activation domains, AD1 and AD2. A subpopulation of GRIP1 foci associate with ND10s, small nuclear bodies that contain several proteins including PML, SP100, DAXX, and CREB-binding protein (CBP). Association with the ND10s is dependent on the AD1 of GRIP1, a region of the protein previously described as a CBP-interacting domain. The GRIP1 foci are enriched in components of the 26S proteasome, including the core 20S proteasome, PA28alpha, and ubiquitin. In addition, the irreversible proteasome inhibitor lactacystin induced an increase in the total fluorescence intensity of the GFP-GRIP1 expressing cells, demonstrating that GRIP1 is degraded by the proteasome. These findings suggest the intriguing possibility that degradation of GRIP1 by the 26S proteasome may be a key component of its regulation.

Nuclear cytoplasmic shuttling by thyroid hormone receptors. multiple protein interactions are required for nuclear retention.

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

Dynamics of gene targeting and chromatin remodelling by nuclear receptors.

Activation of the murine-mammary-tumour virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). We have reconstituted this nucleoprotein transition with chromatin assembled on MMTV LTR DNA with Drosophila embryo extracts, purified GR, and HeLa nuclear extract. Chromatin remodelling in vitro is ATP-dependent and maps to a region identical with that found in vivo. We demonstrate specific, glucocorticoid response element dependent, binding of purified GR to a large, multi-nucleosome MMTV chromatin array and show that GR-dependent chromatin remodelling is a multistep process. In the absence of ATP, GR binds to multiple sites on the chromatin array and inhibits nuclease access to GR recognition sites. On the addition of ATP, GR induces remodelling resulting in a large increase in access of enzymes to their sites within the transition region. These findings are complemented by studies in living cells; using a tandem array of MMTV-Ras reporter elements and a form of GR labelled with the green fluorescent protein, we have observed direct targeting of the receptor to response elements in live mouse cells. Whereas the ligand-activated receptor is associated with the MMTV promoter for observable periods, photobleaching experiments provide direct evidence that the hormone-occupied receptor undergoes rapid exchange between chromatin and the nucleoplasmic compartment. The results both in vitro and in vivo are consistent with a dynamic model ('hit and run') in which GR first binds to chromatin after ligand activation, recruits a remodelling activity and is then lost from the template.

Structure and dynamic properties of a glucocorticoid receptor-induced chromatin transition.

Activation of the mouse mammary tumor virus (MMTV) promoter by the glucocorticoid receptor (GR) is associated with a chromatin structural transition in the B nucleosome region of the viral long terminal repeat (LTR). Recent evidence indicates that this transition extends upstream of the B nucleosome, encompassing a region larger than a single nucleosome (G. Fragoso, W. D. Pennie, S. John, and G. L. Hager, Mol. Cell. Biol. 18:3633-3644). We have reconstituted MMTV LTR DNA into a polynucleosome array using Drosophila embryo extracts. We show binding of purified GR to specific GR elements within a large, multinucleosome array and describe a GR-induced nucleoprotein transition that is dependent on ATP and a HeLa nuclear extract. Previously uncharacterized GR binding sites in the upstream C nucleosome region are involved in the extended region of chromatin remodeling. We also show that GR-dependent chromatin remodeling is a multistep process; in the absence of ATP, GR binds to multiple sites on the chromatin array and prevents restriction enzyme access to recognition sites. Upon addition of ATP, GR induces remodeling and a large increase in access to enzymes sites within the transition region. These findings suggest a dynamic model in which GR first binds to chromatin after ligand activation, recruits a remodeling activity, and is then lost from the template. This model is consistent with the recent description of a "hit-and-run" mechanism for GR action in living cells (J. G. McNally, W. G. Müller, D. Walker, and G. L. Hager, Science 287:1262-1264, 2000).

Trafficking of nuclear receptors in living cells.

Most of the steroid receptor family, with the exception of the estrogen receptor, are classically viewed as 'translocating receptors'. That is, they move from an exclusively, or principally, cytoplasmic distribution in the absence of hormone to a predominately nuclear localization in hormone stimulated cells. The estrogen receptor and the nuclear receptor family are found exclusively in the nucleus, both in hormone stimulated and hormone free cells. This behavior has now been studied with GFP-fusions in living cells, and has in general been confirmed. However, there are important exceptions, and new findings, particularly with regard to sub-nuclear localization. We propose that the intracellular distribution of both receptor classes is dependent not only on subcellular localization signals directly encoded in the receptors, but also on the nature and composition of the large, macromolecular complexes formed by each receptor. Furthermore, we find that most members of the receptor superfamily form focal accumulations within the nucleus in response to ligand, and suggest that these structures may participate in the biological life cycle of the receptors. Finally, we propose that receptor movement in the nucleus is highly dynamic, with the receptors undergoing constant exchange between genomic regulatory elements, multi-protein complexes with other transcription factor partners, and subnuclear structures that are as yet poorly defined.

Tracking components of the transcription apparatus in living cells.

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.

Differential localization and activity of the A- and B-forms of the human progesterone receptor using green fluorescent protein chimeras.

Subcellular localization and transcriptional activity of green fluorescent protein-progesterone receptor A and B chimeras (GFP-PRA and GFP-PRB) were examined in living mammalian cells. Both GFP-PRA and B chimeras were found to be similar in transcriptional activity compared with their non-GFP counterparts. GFP-PRA and PRA were both weakly active, while GFP-PRB and PRB gave a 20- to 40-fold induction using a reporter gene containing the full-length mouse mammary tumor virus long-terminal repeat linked to the luciferase gene (pLTRluc). Using fluorescence microscopy, nuclear/cytoplasmic distributions for the unliganded and hormone activated forms of GFP-PRA and GFP-PRB were characterized. The two forms of the receptor were found to have distinct intracellular distributions; GFP-PRA was found to be more nuclear than GFP-PRB in four cell lines examined. The causes for and implications of this differential localization of the A and B forms of the human PR are discussed.

The glucocorticoid receptor is tethered to DNA-bound Oct-1 at the mouse gonadotropin-releasing hormone distal negative glucocorticoid response element.

An element required for glucocorticoid repression of mouse gonadotropin-releasing hormone (GnRH) gene transcription, the distal negative glucocorticoid response element (nGRE), is not bound directly by glucocorticoid receptors (GRs) but is recognized by Oct-1 present in GT1-7 cell nuclear extracts or by Oct-1 purified from HeLa cells. Furthermore, purified full-length GRs interact directly with purified Oct-1 bound to the distal nGRE. Increasing the extent of distal nGRE match to an Oct-1 consensus site not only increases the affinity of Oct-1 binding, but also alters the conformation of DNA-bound Oct-1 and the pattern of protein DNA complexes formed in vitro with GT1-7 cell nuclear extracts. In addition, the interaction of purified GR with DNA-bound Oct-1 is altered when Oct-1 is bound to the consensus Oct-1 site. Mutation of the distal nGRE to a consensus Oct-1 site is also associated with reduced glucocorticoid repression in transfected GT1-7 cells. Furthermore, repression of GnRH gene transcription by 12-O-tetradecanoylphorbol-13-acetate, which utilizes sequences that overlap with the nGRE, is reversed by this distal nGRE mutation leading to activation of GnRH gene transcription. Thus, changes in the assembly of multi-protein complexes at the distal nGRE can influence the regulation of GnRH gene transcription.

Simultaneous visualization of the yellow and green forms of the green fluorescent protein in living cells.

In this study we sought to develop a method for the co-localization of proteins in living cells utilizing the enhanced green fluorescent protein (EGFP) and a red-shifted EGFP variant, EYFP (enhanced yellow fluorescent protein). EYFP was expressed as an unsubstituted molecule while EGFP was fused to NF1 (EGFP-NF1), a transcription factor found exclusively in the nucleus. The Leica TCS SP laser scanning confocal microscope was used. This microscope allows the user to monitor the emitted light at defined wavelengths owing to the presence of a monochrometer in the emission light path. pEGFP-NF1 and pEYFP were co-expressed in the same cell and excited with the 476-nm and 488-nm argon laser lines. To separate the EYFP and EGFP fluorescence, EGFP-NF1 emission was recorded between 496 and 505 nm. These wavelengths are on the left shoulder of the EGFP emission peak and exclude most of the EYFP fluorescence. The EYFP emission was followed between 670 and 754 nm, utilizing the tail of EYFP emission that extends well beyond that for EGFP. Under these conditions we obtained excellent discrimination between EYFP fluorescence and EGFP-NF1 emission. These observations demonstrate that EYFP- and EGFP-substituted chimeras can be used for simultaneous detection in living cells.