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Mapping Protein/DNA Interactions by Cross-Linking [Internet]. Paris: Institut national de la santé et de la recherche médicale; 2001.

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Mapping Protein/DNA Interactions by Cross-Linking [Internet].

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Direct Visualization of Transcription Factor Binding to Regulatory Elements in Living Cells

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Introduction

Transcription factors bind to regulatory elements in eukaryotic chromosomes and modify the rate of promoter utilization for target genes. These regulatory processes are among the most important, and most intensively studied, mechanisms in modern biology. Methodology to characterize gene regulatory events has largely fallen into two general categories. One avenue is to introduce genetically modified copies of a given promoter into cells and ascertain information regarding the function of a given factor binding site through the functional consequences of mutations introduced in that site. A second general approach is to isolate the factors and the template and attempt to reconstruct regulatory events with purified components in cell-free systems.

Each of these approaches is susceptible to unique shortcomings. In vivo transfection methods provide information from the actual transcriptional environment of the living cell, but serious problems are encountered in controlling the dose level and structure of introduced templates. A large component of regulation involves modification of the chromatin template, and transiently introduced templates are not appropriately organized at the nucleoprotein level (Smith and Hager 1997). Furthermore, it is difficult to reduce and control the complexity of the transcriptional apparatus in this approach, although minimal efforts can be made in this direction (gene knockouts, antisense nucleic acids, antibody microinjection, etc.)

Cell-free systems offer the considerable advantage of reduced complexity and control over constituents, but these systems are notoriously susceptible to artifact. It is often difficult to discern in the in vitro approach whether the result of an experimental manipulation arises from the identification of a mechanism that functions in the living cell or simply an "effect" in the cell-free environment.

We have developed an alternate methodology that permits the direct analysis of factor binding to regulatory elements in living cells in real time. This approach derives, first, from the ability to tag protein factors with a fluorescent label, express the tagged protein at physiological levels in target cells, and observe direct localization of the protein. The tags are provided by the rapidly growing family of fluorescent proteins isolated from naturally luminescent organisms, including the green fluorescent protein (GFP) family from the jellyfish, Aequorea victoria (Cubitt et al. 1995), and more recent isolates from other organisms, such as the red protein (DsRed). Secondly, we have devised an amplified gene target system that permits real-time localization of the fluorescent receptors on actual regulatory sites in living cells.

Nuclear receptors form one major group of transcription factors and are unique in that their transcriptional regulatory activity is triggered by the binding of a cognate ligand. These proteins are therefore particularly useful in studies of subcellular localization and gene targeting, because activity of the tagged factors can be rapidly manipulated by addition of ligand to living cells. Thus, the discussion here will focus on the nuclear receptor superfamily; the methods and techniques, however, are equally useful for any group of transcription factors. We have now studied fluorescent derivatives of most of the major nuclear receptors and find that these reagents are highly effective in studies on intracellular localization and movement for this family of proteins. Furthermore, the transactivation potential and ligand specificity for the substituted molecules are very similar to that of the normal receptors, indicating that the GFP fusions are valid reagents for the study of intracellular receptor gene targeting.

The glucocorticoid receptor (GR) is located in the cytoplasm in the absence of ligand and translocates to the nucleus when occupied by ligand (Mendel et al. 1990; Pratt 1990). GR seems to be unique in its exclusive cytoplasmic compartmentalization. A fraction of the progesterone receptor (PR) is also present in the cytoplasm, but most of this receptor is found in the nucleus under ligand-free conditions (Guiochon-Mantel et al. 1994; Savouret et al. 1993). The thyroid receptor (TR) and retinoic acid receptor (RAR) subfamily (Song et al. 1995), as well as the estrogen receptor (ER) (Gorski et al. 1993; Greene and Press 1986; Yamashita 1998), are usually described as bound to the nucleus both in the presence and absence of hormone.

Tagging Nuclear Receptors with Fluorescent Proteins

GFP--fusion chimeras for several of the major nuclear receptor groups have now been reported, including GR (Htun et al. 1996; Carey et al. 1996), MR (Fejes-Toth et al. 1998) PR_B and PR_A (Lim et al. 1999), AR (Georget et al. 1997), ER (Htun et al. 1999), TRβ1 (Zhu et al. 1998), and aromatic hydrocarbon receptor (AHR) (Chang and Puga 1998). Although C-terminal GFP chimeras for several other cellular proteins have been described, most of the nuclear receptors have been prepared as N-terminal additions. One exception is a C-terminal GR--GFP fusion described by Macara and colleagues (Carey et al. 1996). Using this reagent, these authors studied the involvement of Ran-ATPase in receptor translocation. A second C-terminal example is provided by the work of Puga and colleagues (Chang and Puga 1998), who utilized a C-terminal fusion to characterize intracellular distribution of the AHR, and identify constitutive nuclear localization signals. Although ligand binding and transactivation features of these receptors were not completely characterized, these examples suggest that C-terminal labeling also yields a functional receptor.

A critical point concerns the fusion between GFP and a target receptor. The crystal structure of GFP (Ormo et al. 1996) indicates that the protein has a rather rigid globular domain. The potential exists that fusion of GFP directly into the open reading frame of a given protein may disrupt an essential feature of the target structure. Isolation of the GFP moiety from the candidate protein with a peptide linker could minimize the impact of the GFP globular domain. This feature was first incorporated in GFP--GR (Htun et al. 1996) and subsequently included in the design of several tagged receptors (Chang and Puga 1998; Htun et al. 1999; Lim et al. 1999; Zhu et al. 1998). A comprehensive study to determine whether inclusion of this linker serves to preserve native structure of the target protein has not been performed, but inclusion of such a linker is a relatively simple modification and can easily be included in the chimeric design.

Characterization of the Fusion Chimera

Prior to use of a given receptor--GFP fusion for studies on intracellular distribution, it is imperative that the basic parameters of the chimeric receptor be verified. The size of the fusion should conform to the predicted molecule weight and should be confirmed by Western blot analysis. Many of the chimeric proteins described to date include an epitope tag, such as the HA antigen, that facilitate discrimination between the fusion chimera and the endogenous, native receptor (Chang and Puga 1998; Htun et al. 1999; Lim et al. 1999; Zhu et al. 1998). To be considered a useful reagent, the fusion chimera should obviously be expressed as a relatively stable protein of the predicted size, with little evidence of breakdown products.

The GFP chimera should also be characterized for its transactivation potential with an appropriate hormone-responsive promoter and reporter. This analysis will demonstrate that the basic transactivation domains of the fusion protein are intact and functional. It is useful in this analysis to evaluate the chimeric receptor with both agonists and antagonist to insure, insofar as possible, that the tagged receptor retains proper ligand dependency.

The fusion chimera should be tested for ligand affinity by carrying out a dose--response analysis in an in vivo response system. The chimeric receptor should manifest a Kd for ligand affinity similar to that measured for the wild-type receptor. Finally, electrophoretic mobility shift analysis (EMSA) with the cognate DNA response element will demonstrate that the fusion maintains native DNA recognition specificity.

Mode of Expression

Almost all experimentation to date with nuclear receptor GFP chimeras has been performed by transient introduction of the GFP receptor. Although this is the mode most readily available, there are potentially serious drawbacks to this method of expression. First, it is now clear that transiently expressed steroid receptors often function abberantly in gene activation. Smith, Hager, and colleagues have described pronounced differences between the activation potential of the transient and endogenous progesterone receptor (Hager et al. 1993; Smith et al. 1993; Smith and Hager 1997). Secondly, transient transfection often produces very high levels of expression per cell, and these levels may induce inappropriate localization of the receptor chimera or improper interaction with other molecules.

For these reasons, stable introduction of a receptor chimera into a cell line of choice is a much preferred method for study of receptor behavior in that cell. This will likely have three positive effects: the levels of expression per cell are more likely to be in the normal physiological range; all cells in the population will have a similar complement of chimeric receptor; and the stably expressed receptor has a better chance of forming appropriate associations with other components of multi-protein complexes in which receptors usually reside.

Well-characterized systems for the controlled expression of introduced chimeras are also now available. The tetracycline-inducible expression system (Gossen and Bujard 1992) has been widely used and has been found to work well with GFP--GR (Walker et al. 1999). This approach provides several additional advantages. Levels of receptor expression can be regulated to whatever level is desired, and subcellular trafficking can be observed after expression of the receptor is induced.

Instrumentation

Much of the preliminary characterization for subcellular distribution of a new receptor chimera can be carried out with a standard epifluorescence microscope. These instruments are now widely available and, when equipped with a high-quality oil emersion lens (60--100X), provide excellent images with sufficient resolution for initial experimentation. For higher resolution studies, however, particularly in attempts to define intranuclear receptor trafficking patterns, a state-of-the-art laser scanning confocal microscope is indispensable. Furthermore, when attempting to discriminate between multiple chromophores, the excitation wavelength selections available on these modern instruments are invaluable.

Newer technology promises to bring more sophistication to the analysis of receptor function with GFP chimeras. Collection of three-dimensional images through "optical deconvolution" or "extensive reassignment of light" is rapidly emerging as an alternative to confocal imaging. Deconvolution offers the significant advantage of utilizing essentially all of the fluorescence emitted from a sample, whereas confocal imaging is only about 1% efficient. This is a major advantage when using chromophores excited in the UV band of the spectrum, because these wavelengths can be quite damaging to cells. The even more recent development of "two-photon" imaging also promises to expand the boundaries of possible experimentation.

Gene Targets

It is unlikely that direct interaction of a fluorescent transcription factor with a single gene target in a eukaryotic cell will be possible using known technologies. We have adopted the strategy of using cell lines with amplified copies of a specific promoter structure. In particular, we utilized a cell line (3134) that contains 200 copies of the mouse mammary tumor virus promoter (Kramer et al. 1999). Because each promoter contains six GR binding sites, the aggregate structure will potentially bind 1,000 receptors. It is relatively straightforward to observe binding of the GFP--GR and GFP--PR receptors to this structure in living cells (McNally et al. 2000). One large locus on chromosome 4 of the target cell is readily visualized after hormone stimulation of the cells.

Dynamic Movement of Receptors on Gene Targets

The clear observation of receptors on genomic regulatory sites permits the application of a variety of photobleaching techniques to the study of receptor interaction with gene targets. Several types of photobleaching analysis have been developed in recent years. Under the classic method, fluorescence recovery after photobleaching (or FRAP), fluorescent receptor molecules bound to the gene array are rapidly bleached with an intense, focused, laser beam. If these molecules are statically bound to the regulatory elements, the region occupied by the array will remain bleached after laser irradiation is discontinued. Conversely, if the receptors are exchanging with their cognate regulatory sites, the bleached molecules will be rapidly replaced by unbleached molecules from the nucleoplasmic space. The rate of replacement will reflect the rate of exchange. The latter result was observed (McNally et al. 2000), i.e., there is rapid and constant exchange between GFP--GR and the MMTV regulatory elements, even in the continued presence of ligand.

Two more recent variations of the photobleaching technique offer considerable advantages with respect to FRAP. In one version, FLIP, the target itself is not irradiated. Rather, fluorescent molecules in the target compartment (in this case, the nucleoplasm) are continuously bleached with a pulsitile irradiation. The overall concentration of fluorescent receptors in the compartment is gradually depleted, and the target array is observed during this process. Again, if molecules on the target are static, there will be no observed change in the target, but if the molecules are mobile, fluorescence intensity associated with the target will diminish. This approach has the considerable advantage that neither the target nor the molecules on the target are subject to irradiation, eliminating the possibility of artifacts associated with such irradiation.

A third photobleaching procedure has not been widely utilized. This technique, referred to in our laboratory as inverse FRAP, or I-FRAP, is similar to FLIP, except the entire non-target region of the nuclear compartment is instantly bleached, rather than slow bleaching over a time period. This approach allows the direct observation of molecules disengaging from the target (off-rate) rather than the composite behavior, involving both binding and release, observed in FRAP and FLIP.

It is now clear that these fluorescent reagents will provide a major new tool in exploring the architecture of the interphase nucleus, which is poorly understood. The potential exists to detect direct molecular interaction between receptors and interacting factors by the use of fluorescence resonance energy transfer (FRET), or fluorescence correlation spectroscopy. FRET analysis for energy transfer between GFP variants has been reported (Heim and Tsien 1996). It is not yet clear whether these sophisticated approaches for the detection of direct molecular interactions will emerge as a major tool in the study of nuclear receptors and other transcription factors. Finally, the ability to monitor receptor function in living cells in response to ligand stimulation provides new opportunities to characterize ligand effects on compartmentalization and to conduct searches for new classes of agonists and antagonists.

Reference List

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Copyright © 2001, Institut national de la santé et de la recherche médicale (INSERM)
Bookshelf ID: NBK7113PMID: 21413372

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