BBRC Biochemical and Biophysical Research Communications 332 (2005) 1107–1114 www.elsevier.com/locate/ybbrc
Imaging Erg and Jun transcription factor interaction in living cells using fluorescence resonance energy transfer analyses Barbara Camuzeaux a, Corentin Spriet b, Laurent He´liot b, Jean Coll c, Martine Duterque-Coquillaud a,* a
c
UMR 8526CNRS/Institut Pasteur de Lille/Universite´ de Lille2, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille cedex, France b Service d’Imagerie Cellulaire Fonctionnelle, FRC3 CNRS, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille cedex, France UMR 8527CNRS/Institut Pasteur de Lille/Universite´ de Lille2, Institut de Biologie de Lille, BP 447, 1 rue Calmette, 59021 Lille cedex, France Received 3 May 2005 Available online 23 May 2005
Abstract Physical interactions between transcription factors play important roles in modulating gene expression. Previous in vitro studies have shown a transcriptional synergy between Erg protein, an Ets family member, and Jun/Fos heterodimer, members of the bZip family, which requires direct Erg–Jun protein interactions. Visualization of protein interactions in living cells is a new challenge in biology. For this purpose, we generated fusion proteins of Erg, Fos, and Jun with yellow and cyan fluorescent proteins, YFP and CFP, respectively. After transient expression in HeLa cells, interactions of the resulting fusion proteins were explored by fluorescence resonance energy transfer microscopy (FRET) in fixed and living cells. FRET between YFP–Erg and CFP–Jun was monitored by using photobleaching FRET and fluorescence lifetime imaging microscopy. Both techniques revealed the occurrence of intermolecular FRET between YFP–Erg and CFP–Jun. This is stressed by loss of FRET with an YFP–Erg version carrying a point mutation in its ETS domain. These results provide evidence for the interaction of Erg and Jun proteins in living cells as a critical prerequisite of their transcriptional synergy, but also for the essential role of the Y371 residue, conserved in most Ets proteins, in this interaction. 2005 Elsevier Inc. All rights reserved. Keywords: Transcriptional regulation; Protein interactions; Ets and AP1 families; FRET; FLIM
In eukaryotes, networks of protein interactions regulate gene expression in response to environmental stimuli. These interactions must be both versatile as well as selective to enable different members of the same transcription factor family to have differential effects on gene expression. Jun, Fos, and Ets proteins belong to distinct families of transcription factors that target specific DNA elements often found jointly in gene promoters. Previous studies have demonstrated physical and functional interactions between these families [1,2]. Particularly, tran*
Corresponding author. Fax: +33 3 20 87 11 11. E-mail address:
[email protected] (M. Duterque-Coquillaud). 0006-291X/$ - see front matter 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2005.05.057
scriptional synergy between Erg and the Jun/Fos heterodimer requires Erg–Jun physical interactions through the ETS DNA-binding domain of Erg and the bZIP domain of Jun [3]. We have previously identified two conserved amino acids (residues R367 and Y371 of Erg) in the ETS domain required for efficient recruitment of the Jun/Fos heterodimer. Whereas the R367K substitution abolishes DNA binding, interaction with Jun, and consequently transcription synergy, the Y371V mutation abrogates interaction with Jun and synergy without abolishing DNA binding [4]. However, complexes between Erg and Jun transcription factors have not been studied previously in their cellular context. The verification that these protein partners
1108
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
interact in a meaningful way in vivo is difficult to demonstrate. Several methods have been developed to visualize protein complexes in living cells. Fluorescence resonance energy transfer microscopy (FRET) provides a suitable methodology to confirm these protein–protein interactions in intact cells in a non-invasive and nondestructive manner. FRET is the phenomenon by which a donor fluorophore transfers its excited-state energy to an absorbing chromophore (Fig. 3) [5,6]. Fluorophores CFP and YFP, cyan and yellow fluorescent chromophore-mutated variants of green fluorescent protein (GFP), show an excellent spectral overlap and therefore form an appropriate donor–acceptor FRET pair with a distance at which 50% of the excited donor molecules ˚ [7,8]. transfer energy by FRET of 50 A In the present study, we construct YFP–Erg and CFP–Jun fusion proteins and use two FRET measurement techniques to visualize protein interactions in living cells by microscopy: pbFRET technique (photobleaching FRET), based on the measurement of CFP fluorescence increase in case of interaction, after YFP photobleaching [9], and fluorescence lifetime imaging microscopy (FLIM) which measures fluorescence lifetime of the CFP fluorescence decrease in case of interaction [10]. Both techniques, optimized here to observe interactions in nucleus, revealed FRET occurrence in cells co-expressing YFP–Erg and CFP–Jun, a result consistent with their association in the nucleus of these living cells.
Western blotting. Extracts from transfected cells were prepared in RIPA buffer (20 mM Tris/HCl, pH 8, 150 mM NaCl, 1% Triton X100, 0.1% SDS, and 0.5% desoxycholate, protease inhibitors) and quantitated for total protein (Bio-Rad protein assay). Fifty micrograms of protein was resolved by SDS–polyacrylamide gel electrophoresis and transferred to a Hybond-C Extra membrane (Amersham Pharmacia Biotech) with a Bio-Rad dry blotter. Membrane was blocked in PBS with 5%(w/v) dried milk for 2 h and stained with a mouse antibody against all Aequorea Victoria green fluorescent protein (GFP) variants (Clontech) and a secondary goat anti-mouse/peroxidase antibody (Amersham Pharmacia Biotech). Antibody incubations were performed for 2 h in PBS with 5%(w/v) dried milk followed by three 15-min washes in PBS with 0.2% Nonidet P-40. For detection, we used the ECL chemiluminescent peroxidase substrate kit from Amersham Pharmacia Biotech. Confocal laser scanning microscopy of YFP/CFP-protein expressing cells. HeLa cells were plated at 50–60% confluence either on coverglass for localization and acceptor photobleaching experiments (fixed with 4% paraformaldehyde for 20 min and mounted with Mounting Medium Immunofluore ICN) or on Lab-Tek chambered borosilicate coverglass for FLIM experiments. They were transfected the following day with 1 lg of plasmid DNA. Twenty-four hours after transfection, the GFP variant fusion proteins were visualized in intact cells using the Leica SP2 inverted confocal microscope (DMIRE2), incorporating the Acousto-Optical Beam Splitter system and using 100·, NA 1.4, oil immersion, or 63·, NA 1.2, water immersion objectives. For confocal images, CFP was excited at 458 nm (power 13 lW) and observed from 465 to 495 nm. YFP was excited at 514 nm, (power ranging from 3.5 to 13 lW) and observed from 525 to 565 nm. Images were 512 pixels. All power measurements were done at the back pupil of the objective. FRET measurements. For FRET by photobleaching of the acceptor, experiments were performed on fixed cells. First, images of the CFP and the YFP channels were acquired, in appropriate cells, in the same condition as for localization images. YFP was specifically photobleached, at 514 nm, power 4.5 mW. Then, images were acquired in the same condition as before photobleaching experiments. FRET efficiency was calculated on at least 15 cells per experiment, using the following adaptation of the FRET efficiency formula [11]:
Materials and methods Feff ¼ Cell culture. HeLa (Human cervix epitheloid carcinoma) cells and F9 embryonic carcinoma cells were maintained in DulbeccoÕs modified EagleÕs medium supplemented with 10% fetal calf serum. F9 cells are propagated on culture surfaces coated with 0.1% gelatine. HeLa and F9 cells do not express endogenous Erg protein; by contrast only F9 cells lack endogenous AP1 activity. Plasmid constructions. The Erg and the ErgY371V mutant expression vectors have been described previously [3,4]. To construct the YFP–Erg, YFP–ErgYV, YFP–ErgRK-YV, YFP–Fos, and CFP–Jun expression vectors (Fig. 1), the DNA fragments encoding Erg, ErgY371V, ErgR367K-Y371V, Fos, and Jun were obtained by polymerase chain reaction using appropriate primers flanked by convenient restriction sites, then sub-cloned into PCRII (Invitrogen), and finally cloned in-frame into pEYFP-C1 or pECFP-C1 vectors (Clontech). Full details and primer sequences are available on request. All constructs were verified by DNA sequencing. Transfection and luciferase assays. The day before transfection, HeLa and F9 cells were plated at 50–60% confluence in six-well plates (coated with 0.1% gelatin for F9 cells). For transfection, cells were incubated with 1 lg of plasmid DNA and 4 ll of polyethyleneimine (Euromedex, Souffelweyersheim, France) or 4 ll Lipofectamine and Plus Reagent (Invitrogen) for HeLa and F9 cells, respectively, for 6 h in 1 ml of optiMEM and then in fresh complete medium. When necessary, pSG5 plasmid was used as a carrier. Cells were lysed 48 h after transfection and assayed for luciferase activity with a Berthold (Nashua, NH) chemioluminometer. Experiments were repeated at least five times and representative results are shown here.
ð1 þ abÞðDpost bckpost Þ ðDpret bckpre Þ ; ðDpost bckpost Þ
where ab stands for average level of CFP photobleaching during acquisitions, bck stands for background, D donor channel intensity, and pre and post for before and after photobleaching of the YFP. A defined region in each cell was bleached and the non-bleached area gives an internal reference. For FLIM microscopy and analysis, experiments were performed on living cells. We used a two-photon pulsed excitation, given by the Coherent Mira 900-F laser 5 W (pulse frequency 76 MHz), at a wavelength of 830 nm for CFP excitation. Photon detection was performed in appropriate cells, using the Hamamatsu MCP R3809U-52 PMT. Multi-channel plate detectors are particularly adapted to FLIM imaging with their good temporal resolution [12] (measured FMHW: 36 ps). The omega XF3075 band pass filter allowed specific detection of CFP (465–500 nm). Images were acquired using a 63·, NA 1.2, water immersion objective. The main advantage of two-photon-excited fluorescence, compared to classical laser excitation, is that it only excites a small volume in the focal plane [13], allowing us to work without pinhole. Therefore, the detector was adapted in the non-descanned detector port of the Leica SP2 confocal microscope, minimizing fluorescence photon loss. Knowing that two-photon-excited fluorescence is less invasive than single-photon excitation [14], this system was optimized for living cell imaging. The Becker & Hickl TCSPC 730 card determined the time between fluorophores excitation and photon emission. This card was driven by the SPC software. Fluorescence decay curve fits, allowing lifetime determination, were obtained using the SPCImage software. The choice between
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
1109
Fig. 1. Constructs used for Erg and Jun protein interaction analysis and confirmation of their biological properties. (A) Schematic representation of the Erg, ErgYV, ErgRK-YV, Fos, and Jun proteins fused to YFP (yellow fluorescent protein) or CFP (cyan fluorescent protein). TA: transactivator domain, NTA: N-terminal transactivator domain, CTA: C-terminal transactivator domain, BD: basic domain, LZ: leucine zipper domain. Point mutations are indicated by arrows. (B) Analysis of protein expression by Western blot. HeLa transfected cell extracts were analyzed by SDS–gel electrophoresis, probed with anti-YFP/CFP antibody (1:3000). (C,D) Transient transactivation assays were performed in F9 cells (C) and HeLa cells (D). The sequence of the region of the polyomavirus enhancer, that contains ETS and AP1 recognition elements controlling the TK promoter, is indicated above the graphs. Data are expressed as fold luciferase activity and the synergy fold is indicated at the top of the columns. The synergy fold is defined as Py enhancer activity in the presence of a combination of Erg proteins plus Jun/Fos constructs divided by the sum of the individual fold activations induced by Jun/Fos alone and Erg alone.
1110
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
mono-exponential decay and bi-exponential decay fitting was done using the reduced v2 parameter. The weighted mean lifetime was used to compare lifetime images. Lifetime values were extracted from three regions of interest on a minimum of 15 cells per experiment. Statistical analysis was performed using the Microsoft OfficeÕs Excel software to obtain averages and standard deviations.
Erg or YFP–Erg proteins in HeLa cells (Fig. 1D, lanes 7 and 9). Taken together, these results showed that the fusion proteins were biologically functional and that the amino-terminal YFP or CFP fluorophore did not alter their functions. Localization of fluorescent proteins
Results and discussion Biological functionality of the fluorescent fusion proteins The major aim of this study was to analyze the Erg and Jun transcription factor interactions in living cells. We chose to use GFP chimeras of these transcription factors for FRET microscopy. We have previously shown that the Erg protein, an Ets family member, is able to bind a specific consensus DNA site and to activate transcription. However, Ets family members are poor transactivators per se [15] but transcriptional activity of Erg toward EBS/AP1 sites carrying promoter is strongly enhanced in the presence of the Jun/Fos dimer, a member of the AP1 family. This transcriptional synergy requires interactions between Erg and Jun proteins as shown by in vitro experiments [3,4]. Nevertheless, this result waited to gain more physiological relevance by visualization in a cellular context. For this purpose, we constructed a series of expression vectors encoding fusion proteins with YFP and CFP fluorophores tethered to the amino-terminal end of Erg, Jun or Fos proteins (Fig. 1A). Expression vectors were transiently transfected in HeLa cells. In each case, Western blotting was used to demonstrate that the expected fusion proteins were synthesized in transfected cells (Fig. 1B). Hence, we challenged the Erg and Jun/ Fos synergy property with the chimeric constructs to evaluate their functionality in transactivation assays. To avoid interference with the endogenous Jun and Fos proteins, we tested the synergistic properties of YFP–Erg and its mutants with the Jun/Fos dimer in F9 cells known for their lack of endogenous AP1 activity [16]. These cells, like HeLa cells, do not express endogenous Erg proteins as well. The results shown in Fig. 1C demonstrated that YFP–Erg induced transcriptional synergy with Jun and Fos proteins (lanes 4 and 6) from a cotransfected Py enhancer element-luciferase reporter gene. As expected, the YFP–ErgYV and YFP–ErgRKYV point mutant proteins, chosen, respectively, for their incapacity to interact with the Jun protein or to interact with DNA and Jun [4], failed to synergize with the Jun/ Fos dimer in transient transfection (Fig. 1C, lanes 6, 8, and 10). Second, we confirmed the synergistic property of YFP–Erg with Jun/Fos in HeLa cells, used hereafter for imaging experiments (Fig. 1D, lanes 6 and 8). Albeit lower than in F9 cells due to endogenous AP1 activity, the synergistic score is similar to the score we previously described [4]. Finally, we also showed that the CFP–Jun and YFP–Fos proteins were able to act in synergy with
The fluorescent proteins allowed subnuclear localization and colocalization of Erg and Jun/Fos proteins. After transient (co)transfection of HeLa cells, intracellular distribution of YFP or CFP-derived fluorescence was imaged by confocal laser scanning microscopy. All experiments were performed between 24 and 48 h post-transfection. Fig. 2A shows that the Erg, Jun, and Fos fusion proteins were in the nucleus, compared with the YFP and CFP wild-type proteins which were observed both in the cytoplasm and the nucleus. The Erg mutant fusion proteins were also detected in the nucleus even though a small amount of the YFP–ErgRK-YV double mutant protein was also detected in the cytoplasm. This last observation could be explained by a recent study concerning the nuclear localization signal (NLS) mapping of Fli-1 protein, closely related to Erg [17]. Actually, the R337 residue of Fli-1 (corresponding to R367 in Erg) present in one of the two NLS defined for this protein is essential for nuclear localization. Thus, in the YFP– ErgRK-YV protein, the R367K mutation combined with the Y371V mutation may reduce the nuclear accumulation of the protein. Otherwise both YFP–Erg and YFP– Fos proteins were excluded from nucleolus while CFP–Jun protein was uniformly distributed in the nucleus. These results obtained for YFP and CFP fusion proteins (Fig. 2A) were consistent with the localization of the Erg protein in HeLa cells transfected by Erg expression vector, or the localization of Jun and Fos endogenous proteins stained by indirect immunofluorescence in fixed cells (data not shown). Thus, fusion of a fluorescent protein at the amino-terminus of Erg, Jun, and Fos proteins did not interfere with their intracellular localization. According to our previous in vitro studies, the Erg protein is able to interact with the Jun/Fos dimer via Jun [3,4]. With the objective to visualize colocalization of Erg–Jun/Fos in living cells, HeLa cells were transiently cotransfected with the fusion expression vectors. Images of the CFP and YFP fluorescence emission were acquired sequentially and overlaid to check colocalization. In Fig. 2B, CFP–Jun, YFP–Erg (wild-type or mutants), and YFP–Fos signals were encoded in pseudocolors as blue and yellow, respectively, whereas a combined detection of both fluorescent proteins is indicated by green signals. In HeLa cells coexpressing CFP–Jun with YFP–Erg or YFP–Erg mutants or YFP–Fos proteins, the merged images revealed areas of overlaps as well as regions where CFP–Jun and YFP–Erg or YFP–Fos are distinct in their localization.
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
Fig. 2. Distribution of fluorescent fusion proteins in living cells detected by confocal laser scanning microscopy. (A) Confocal fluorescence images of HeLa cells transiently transfected with plasmids expressing the indicated YFP or CFP fusion protein alone. CFP fluorescent emission of transfected cells is shown in blue (excitation 458 nm, emission 465–495 nm) and YFP-fluorescent emission is shown in yellow (excitation 514 nm, emission 525–565 nm). (B) Localization of the indicated proteins in cells transfected with pairs of CFP–Jun with different YFP fusion proteins. Colocalization is illustrated by green signals in the merge column. Scale bars are 5 lm. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
Remarkably, coexpression of these transcription factors did not induce their nuclear distribution changes. However, a direct demonstration of physical interactions between the proteins is needed since the colocalization studies are limited by the optical resolution of the light microscope and can only indicate proximity on the scale of approximately 0.25 lm. Thus, colocalization of two different color fluorescent proteins only tells us that they are in the same vicinity in the cell nucleus and provides little information regarding their potential to physically interact. Analysis of molecular interactions by pbFRET and FLIM To examine physical interactions between Erg and Jun proteins, fluorescent fusion proteins were used to
1111
Fig. 3. Principle and measurements of fluorescent resonant energy transfer (FRET). (A,B) FRET is a quantum mechanical process involving the radiationless transfer of energy from a donor fluorophore to an appropriately positioned acceptor fluorophore. When two fluorescent proteins interact, the excitation of the donor induces an energy transfer from the donor to the acceptor resulting in the quenching of the donor and a rise of acceptor fluorescence. (C,D) FRET measurements by photobleaching of the acceptor (pbFRET). The selective photobleaching of the acceptor fluorophore abolishes FRET, and in the regions of the cell where FRET occurred, there is an enhancement in the donor emission because of dequenching. The schematic curves in (D) show the intensity of fluorescence, represented in gray level (in arbitrary units) of a region of interest at indicated wavelengths, before (in green) and after (in purple) photobleaching. After photobleaching of the acceptor fluorophore, there is a decrease of the intensity of fluorescence in the acceptor emission area (red arrow), and an increase of the intensity of fluorescence in the donor emission area (blue arrow). By measuring donorÕs intensity before and after photobleaching of the acceptor, FRET occurrences are measured. (E,F) FRET measurements by lifetime imaging (FLIM). The fluorescence lifetime imaging (FLIM) was performed by time correlated single photon counting, providing a spatial lifetime map of this probe within the cells. FRET from a donor fluorophore to acceptor molecules in the local environment substantially influences the donor fluorescence lifetime. (E) Graph represents in full curve the fluorescence lifetime of CFP fusion proteins when they are alone and in punctuated curve the lifetime of two populations of CFP fusion proteins, alone (a) and interacting with YFP fusion proteins (b). The lifetime of CFP is deduced from the slopes of the photon emission time [12]. (F) The left cell picture represents the CFP fluorescence lifetime image calculated on a pixel-by-pixel basis, and pseudocolored according to the lifetime scale indicated in the horizontal axis of the 2D histogram on the right. Red-orange colors in the lifetime image depict low fluorescence lifetime (FRET) and the blue colors in the lifetime image depict higher fluorescence lifetimes (no FRET). The same representation is used in Fig. 5. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
1112
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
perform FRET experiments with CFP as a donor and YFP as an acceptor (Figs. 3A and B). FRET, described by Fo¨rster, consists in a non-radiative energy transfer between two appropriate fluorophores [8]. The FRET efficiency depends on several parameters [12]. Especially, ˚ is the Fo¨rster distance for the CFP– the distance of 50 A YFP FRET pair, where the half-maximal FRET effect occurs (FRET efficiency of 50%). Given that FRET declines with the 6th power of the distance, it can usually ˚ [8]. Consequently, it is be detected up to about 100 A reasonable to associate the FRET measurement to interaction level [9,12]. Several techniques exist to measure FRET efficiency [11]. We have chosen two complementary approaches to do those measurements: a photobleach dequenching FRET assay (pbFRET) in fixed cells and a fluorescence lifetime imaging microscopy technique (FLIM) in living cells [11]. pbFRET measurements When FRET occurs, the non-radiative transfer quenches the donor fluorescence while exalting the acceptor (Fig. 3B). Specific photobleaching of the acceptor results in the dequenching of the CFP fluorophore. This property is used in the FRET by acceptor photobleaching method or pbFRET (Figs. 3C and D). Comparing images of the CFP channel before and after photobleaching of the YFP gives the variation of the CFP dequenching. This experiment was performed with several transcription factor pairs in fixed HeLa cells. The mean pbFRET efficiencies, calculated in 15 cells for the different transfected pairs, are reported in Fig. 4. Since the Jun protein is known to interact with the Fos protein, CFP–Jun/YFP–Fos di-
Fig. 4. PbFRET measured on HeLa cells transiently transfected with CFP and YFP fusion proteins. HeLa cells were cotransfected with CFP–Jun and YFP–Fos (as a positive interacting control) or YFP–Erg wild-type or mutant. The FRET efficiency is given by comparison of the CFP obtained before and after photobleaching. Scale bars are 5 lm. The graph represents the mean of FRET efficiency calculated with at least 15 observed cells.
mer was used as a positive control [18]. As expected (Fig. 4), cells cotransfected by both CFP–Jun and YFP–Fos exhibited significant CFP fluorescence emission after photobleaching. In this case, the calculated pbFRET efficiency was 7.5% in average and provided strong evidence for interaction of these proteins. This result is in agreement with the current knowledge concerning the Jun/Fos dimer formation in vitro and in vivo [18,19]. Similarly, in CFP–Jun and YFP–Erg cotransfected cells, an increase of CFP fluorescence emission was observed in the same conditions. By contrast, cotransfection of CFP–Jun with YFP–ErgYV failed to increase fluorescence after photobleaching. Actually, it was 7% in average for CFP–Jun/YFP–Erg and dropped to 0.1% when YFP–ErgYV protein was transfected with CFP–Jun (Fig. 4). These results suggest direct interactions between CFP–Jun and YFP– Erg in the nucleus of cells. This observation is specific since YFP–ErgYV, a mutation known to abolish interaction with Jun in vitro, failed to make FRET in pbFRET measurements. This absence of FRET between CFP–Jun and YFP–ErgYV proteins proves the involvement of the residue Y371 in Erg/Jun protein interaction. Since crosslinking and subsequent treatments of fixed cells may have induced changes in nucleus structures, FLIM experiments were performed in living cells. FLIM Fluorescence lifetime imaging microscopy (FLIM) is a powerful method to detect FRET in biological samples because the lifetime decay of donor fluorescence is characteristically accelerated by FRET (Figs. 3A–F). To go further in our study, we measured FRET efficiency by lifetime imaging in living cells transfected by CFP and YFP fusion proteins. Fig. 5A (left panel) exemplified the lifetime images of CFP–Jun and the distribution of this lifetime from 1 to 3.2 ns in representative cells. The mean lifetime measurements from 15 cells were reported in Fig. 5B-white columns. The mean lifetime of CFP–Jun alone, used as negative interaction control, was 2500 ± 50 ps (blue) while that of CFP–Jun/ YFP–Fos, positive control of interaction, was shortened to 1500 ± 100 ps (orange). The mean lifetime measured for CFP–Jun and YFP–Erg was similar to CFP–Jun/ YFP–Fos mean lifetime measurements, suggesting that FRET occurrence occurs between these proteins in the nucleus of the observed living cells. Moreover, in order to measure the influence of the specific DNA target of the Erg and Jun/Fos proteins on the FRET occurrence, the FLIM measurements were also obtained in living HeLa cells transfected with the fluorescent fusion proteins in the presence (Fig. 5A, right panel) of the polyoma enhancer (Py), known to be activated in synergy by the CFP–Jun and YFP–Erg fluorescent proteins (Fig. 1C). The mean lifetime measurements
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
1113
Fig. 5. FLIM analysis of the CFP fluorescence in living HeLa cells coexpressing CFP–Jun protein with either YFP–Fos or YFP–Erg and mutant fusion proteins. Only the CFP fluorescence (lifetime) of the fusion proteins is monitored by use of a cyan fluorescent protein-specific emission filter. (A) HeLa cells were cotransfected by indicated fusion protein expressing vectors without (left column) or with (right column) the Py reporter. Low fluorescence lifetime is represented in red-orange whereas high fluorescence lifetime is in blue. An HeLa cell for each transfection is shown. Scale bars are 5 lm. (B) Graph represents the mean lifetime (ps: picoseconds) for each cotransfection of fusion fluorescent proteins with or without Py reporter plasmid. The mean lifetime was calculated with the lifetime values extracted from three regions of interest on a minimum of 15 cells per experiment. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this paper.)
from 15 cells were reported in Fig. 5B-grey columns. The same significant mean lifetime differences were obtained with and without cotransfection of Py except for YFP– ErgYV transfected with CFP–Jun. In this case, we observed an intermediate lifetime value when Py vector was cotransfected with these proteins (Fig. 5B). This result could be due to the closeness of the YFP–ErgYV and CFP–Jun bound on their specific DNA sequence since we have previously shown that the ErgYV mutant protein retains the capacity to bind to DNA in vitro without interacting with Jun [4]. Therefore, we tested this hypothesis using a double mutant Erg protein, YFP–ErgRKYV, since the RK mutation abolishes DNA-binding properties [4]. In this case, cotransfection of CFP–Jun and YFP–ErgRK-YV with or without Py showed lifetime measurements similar to the CFP–Jun alone, the negative control in these FRET experiments. This result confirms that DNA specific target can bring together these factors without interactions but allowing few FRET occurrences. This seems reminiscent of some examples describing FRET induction between fluorescent proteins brought together by a bridging partner in a ternary complex [20]. The fact that there was no difference in FRET values between Jun and Fos or Jun and Erg partners in the presence of their DNA target could
be regarded, at first glance, as disappointing. Indeed, one can envision that DNA, as a true player of the transcriptional complex, should stabilize the complex as was already suggested for Fos/Jun/NFAT [21] or Fos/Jun/ Ets-2 [22,23]. In this respect, the result obtained with the YFP–ErgYV mutant is an indirect proof of the role of DNA that can be readily demonstrated in case of engagement of poor interacting players. This is more difficult to visualize in the conditions used here for already strong interactors like wild-type proteins. However, and as illustrated in Fig. 5A, lifetime measurements in some single cells seem to suggest that such stabilization may occur in the cell. Further investigation will be needed to address precisely this point. In conclusion, ‘‘catching the actors in the act’’ in the living cells through exploitation of imaging resources is one of the major challenging issues to be addressed in biology. In this respect, FRET microscopy is one of the developing approaches to visualize true interactions in vivo [5]. Nevertheless, among the numerous transcription factor interactions described in vitro, only a small number of these interactions have been actually visualized in living cells. For example, various experiments have been successfully used to show the Pit-1 protein homodimerization, the interactions between Pit-1 and
1114
B. Camuzeaux et al. / Biochemical and Biophysical Research Communications 332 (2005) 1107–1114
Ets-1 [24], Jun and Fos proteins, NFjB proteins or the formation of the Maf and Sox complex in living cells nucleus [19,25]. In this context, the findings presented in this study represent the first report on physical interaction of Erg and Jun as fusion proteins with YFP and CFP in living cells and the physiological relevance of a signal mutation in the DNA binding domain of Erg to impair binding with Jun. The results of this FRET microscopy study not only confirm our in vitro experiments but also extend these observations by providing evidence for the localization of physical interactions in the nucleus of the living cells. FRET approaches give a powerful tool to detect interactions within nuclear complexes in their natural environment in living cells. Acknowledgments We thank Anne Flourens, Nathalie Tomavo, and Didier Desle´e for technical assistance, Yves Rouille´ and Bernard Vandendunder for helpful discussions, and Alexis Verger for critically reading the manuscript. B. Camuzeaux was supported by Ministe`re de la Recherche, C. Spriet by Re´gion Nord-Pas-de-Calais. This work was partly supported by CNRS, Association pour la Recherche sur le Cancer (ARC), Re´gion Nord-Pas-deCalais (FEDER No. 02070317). References [1] A.G. Bassuk, J.M. Leiden, A direct physical association between ETS and AP-1 transcription factors in normal human T cells, Immunity 3 (1995) 223–237. [2] G. Buttice, M. Duterque-Coquillaud, J.P. Basuyaux, S. Carrere, M. Kurkinen, D. Stehelin, Erg, an Ets-family member, differentially regulates human collagenase1 (MMP1) and stromelysin1 (MMP3) gene expression by physically interacting with the Fos/ Jun complex, Oncogene 13 (1996) 2297–2306. [3] S. Carrere, A. Verger, A. Flourens, D. Stehelin, M. DuterqueCoquillaud, Erg proteins, transcription factors of the Ets family, form homo, heterodimers and ternary complexes via two distinct domains, Oncogene 16 (1998) 3261–3268. [4] A. Verger, E. Buisine, S. Carrere, R. Wintjens, A. Flourens, J. Coll, D. Stehelin, M. Duterque-Coquillaud, Identification of amino acid residues in the ETS transcription factor Erg that mediate Erg-Jun/Fos-DNA ternary complex formation, J. Biol. Chem. 276 (2001) 17181–17189. [5] R.N. Day, S.K. Nordeen, Y. Wan, Visualizing protein–protein interactions in the nucleus of the living cell, Mol. Endocrinol. 13 (1999) 517–526. [6] R.N. Day, A. Periasamy, F. Schaufele, Fluorescence resonance energy transfer microscopy of localized protein interactions in the living cell nucleus, Methods 25 (2001) 4–18.
[7] R. Heim, R.Y. Tsien, Engineering green fluorescent protein for improved brightness, longer wavelengths and fluorescence resonance energy transfer, Curr. Biol. 6 (1996) 178–182. [8] T. Forster, Zwischenmolekulare Energiewanderung und Fluoreszenz, Ann. Phys. (Leipzig) (1948) 55–75. [9] T.W. Gadella Jr., T.M. Jovin, Oligomerization of epidermal growth factor receptors on A431 cells studied by time-resolved fluorescence imaging microscopy. A stereochemical model for tyrosine kinase receptor activation, J. Cell Biol. 129 (1995) 1543– 1558. [10] V. Calleja, S.M. Ameer-Beg, B. Vojnovic, R. Woscholski, J. Downward, B. Larijani, Monitoring conformational changes of proteins in cells by fluorescence lifetime imaging microscopy, Biochem. J. 372 (2003) 33–40. [11] E.A. Jares-Erijman, T.M. Jovin, FRET imaging, Nat. Biotechnol. 21 (2003) 1387–1395. [12] J. Lakowicz, Principle of Fluorescence Spectroscopy, second ed., 1999. [13] W. Denk, J.H. Strickler, W.W. Webb, Two-photon laser scanning fluorescence microscopy, Science 248 (1990) 73–76. [14] J.M. Squirrell, D.L. Wokosin, J.G. White, B.D. Bavister, Longterm two-photon fluorescence imaging of mammalian embryos without compromising viability, Nat. Biotechnol. 17 (1999) 763– 767. [15] P. Crepieux, J. Coll, D. Stehelin, The Ets family of proteins: weak modulators of gene expression in quest for transcriptional partners, Crit. Rev. Oncog. 5 (1994) 615–638. [16] R. Chiu, W.J. Boyle, J. Meek, T. Smeal, T. Hunter, M. Karin, The c-Fos protein interacts with c-Jun/AP-1 to stimulate transcription of AP-1 responsive genes, Cell 54 (1988) 541–552. [17] W. Hu, A.S. Philips, J.C. Kwok, M. Eisbacher, B.H. Chong, Identification of nuclear import and export signals within Fli-1: roles of the nuclear import signals in Fli-1-dependent activation of megakaryocyte-specific promoters, Mol. Cell Biol. 25 (2005) 3087–3108. [18] P. Sassone-Corsi, L.J. Ransone, W.W. Lamph, I.M. Verma, Direct interaction between fos and jun nuclear oncoproteins: role of the Ôleucine zipperÕ domain, Nature 336 (1988) 692–695. [19] C.D. Hu, Y. Chinenov, T.K. Kerppola, Visualization of interactions among bZIP and Rel family proteins in living cells using bimolecular fluorescence complementation, Mol. Cell 9 (2002) 789–798. [20] S.F. Oliveria, L.L. Gomez, M.L. DellÕAcqua, Imaging kinase— AKAP79—phosphatase scaffold complexes at the plasma membrane in living cells using FRET microscopy, J. Cell Biol. 160 (2003) 101–112. [21] V.R. Ramirez-Carrozzi, T.K. Kerppola, Dynamics of Fos-JunNFAT1 complexes, Proc. Natl. Acad. Sci. USA 98 (2001) 4893– 4898. [22] J.P. Basuyaux, E. Ferreira, D. Stehelin, G. Buttice, The Ets transcription factors interact with each other and with the c-Fos/ c-Jun complex via distinct protein domains in a DNA-dependent and -independent manner, J. Biol. Chem. 272 (1997) 26188–26195. [23] A. Verger, M. Duterque-Coquillaud, When Ets transcription factors meet their partners, Bioessays 24 (2002) 362–370. [24] R.N. Day, Visualization of Pit-1 transcription factor interactions in the living cell nucleus by fluorescence resonance energy transfer microscopy, Mol. Endocrinol. 12 (1998) 1410–1419. [25] N. Rajaram, T.K. Kerppola, Synergistic transcription activation by Maf and Sox and their subnuclear localization are disrupted by a mutation in Maf that causes cataract, Mol. Cell. Biol. 24 (2004) 5694–5709.
