The College of William and Mary (G.M.C.B., L.A.A.), Department of Biology, Williamsburg, Virginia 23187; Institut National de la Santé et de la Recherche Médicale (INSERM) Unité 693-Récepteurs Stéroïdiens, Physiopathologie endocrinienne et métabolique (G.M.C.B., A.G.M.), Faculté de Médecine Paris Sud, 94276 Le Kremlin-Bicetre Cedex France; and Université Paris 7-Denis-Diderot (G.M.C.B.), 75251 Paris Cedex 05, France
Address all correspondence and requests for reprints to: Lizabeth A. Allison, Department of Biology, College of William and Mary, P.O. Box 8795, Millington Hall 116, Williamsburg, Virginia 23187-8795. E-mail: laalli{at}wm.edu.
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ABSTRACT |
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INTRODUCTION |
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In mammalian and avian cancer cells, v-ErbA contributes to tumor formation in part by interfering with the action of liganded and unliganded TR (4, 5, 6, 7). The exact mechanism for transcriptional repression by v-ErbA has not yet been determined; however, two complementary models for the dominant-negative action of v-ErbA are well supported in the literature (8). In the first model, competition for DNA binding accounts for the dominant-negative activity: the oncoprotein binds to a thyroid hormone-responsive element (TRE) and blocks the recruitment of TR
to its TRE (9, 10). In the second model, dominant-negative activity is attributed to competition for TR
auxiliary factors and cofactors such as the retinoid X receptor ß (RXRß) (11, 12, 13). In both models, v-ErbA interferes with TR
and subverts regulation of T3-responsive genes.
Interested in understanding the molecular basis behind the oncogenic conversion of TR into v-ErbA, and the mode of action of dominant-negative transcription factors in general, we studied a relatively unexplored mode of oncogenic action: the effect of altered subcellular localization. TR
has a predominantly nuclear distribution at steady state but shuttles rapidly between the nuclear and cytoplasmic compartments, providing an additional checkpoint in the control of T3-responsive gene expression (14). In contrast, the oncoprotein v-ErbA exhibits a nucleocytoplasmic distribution pattern distinct from TR
. Both cytoplasmic and nuclear populations of v-ErbA are present at steady state in transfected mammalian cells (14, 15) and in avian cells infected with AEV (16). According to the current models of dominant-negative activity of v-ErbA described above, the oncoprotein must enter and be retained in the nucleus to interfere with the action of both liganded and unliganded TR
(4, 7, 17, 18, 19, 20, 21). However, this is inconsistent with the observation that much of v-ErbA remains cytoplasmic (14, 15).
Here we report that v-ErbA dimerizes with both TR and RXRß and sequesters a significant fraction of these two members of the nuclear receptor superfamily in the cytoplasm. Recruitment of TR
to the cytoplasm by v-ErbA can be partially reversed in the presence of ligand and when chromatin is disrupted by the histone deacetylase inhibitor trichostatin A (TSA). These results define a new mode of action of v-ErbA and illustrate the importance of cellular compartmentalization in transcriptional regulation and oncogenesis.
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RESULTS |
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Fluorescent proteins can form dimers or oligomers in some cases (22). Therefore, to ensure that the altered distribution pattern of TR was not an artifact caused by interaction of the tags used, we also analyzed the distribution of GFP-TR
in the presence of untagged v-ErbA (Fig. 2A
). Untagged v-ErbA had the same dramatic effect on TR
localization as its tagged counterparts. In the presence of untagged v-ErbA, GFP-TR
was mislocalized to cytoplasmic foci. Untagged v-ErbA has a distribution pattern comparable to fluorescent protein and epitope-tagged v-ErbA. Indirect immunofluorescence assays using anti-c-ErbA-specific antibodies revealed a distribution pattern ranging from a diffuse nuclear and cytoplasmic localization to cytoplasmic foci (Fig. 2B
). When we increased the ratio between the amount of untagged v-ErbA and GFP-TR
expression vector used for the cotransfections, in a dose-response experiment, there was a positive correlation between the relative increase in untagged v-ErbA and the amount of TR
recruited to the cytoplasm. When cells were transfected with a 19-fold excess of untagged v-ErbA expression plasmid, no cells displayed a primarily nuclear distribution of TR
. In contrast, about 43% of the cells exhibited a primarily nuclear distribution when they were transfected with a 19-fold excess of GFP-TR
expression plasmid (Fig. 2C
).
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Taken together, our findings show that the oncoprotein v-ErbA mislocalizes TR to the cytoplasm in a dose-dependent manner. Importantly, this mislocalization is neither cell type specific nor is it caused by nonspecific interaction between fluorescent protein tags.
Recruitment of TR to the Cytoplasm by v-ErbA Can Be Partially Reversed in Presence of Ligand
To determine whether liganded or unliganded TR was more sensitive to cytoplasmic mislocalization by v-ErbA, the effect of T3 on subcellular distribution at steady state (46 h incubation) was assessed. When v-ErbA was coexpressed with TR
in the absence of T3, the distribution of TR
shifted significantly (P < 0.0001) from 95 ± 6% of cells with a nuclear distribution (all N or N >C) to only 64 ± 6% of cells with a nuclear distribution of TR
(Fig. 3
). Incubating cells for greater than 6 h in ligand did not alter this steady-state distribution of TR
(data not shown).
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v-ErbA Also Mislocalizes RXRß, an Auxiliary Factor for TR, to the Cytoplasm
TR functions in regulating T3-dependent gene expression as a heterodimer with RXR (23). In addition, RXR has been shown to form heterodimers with v-ErbA in vitro (11, 12, 13, 20, 30, 31). To determine whether an additional mode of action of v-ErbA involves sequestering this important auxiliary factor for TR
in the cytoplasm, we assessed the effect of v-ErbA on the subcellular distribution of RXRß. At steady state, the majority of RXRß is found in the cell nucleus in a diffuse pattern (32) (Fig. 4A
). Here, we show that in cells coexpressing GFP-RXRß and HA-v-ErbA, a subpopulation of RXRß is found in the cytoplasm in a punctate distribution pattern, similar to the pattern seen for TR
(Fig. 4A
). This cytoplasmic mislocalization was also observed using other tag combinations such as GFP/DsRed, CFP/YFP and YFP/CFP (data not shown; see Figs. 56
for CFP/YFP), indicating that the altered distribution pattern of RXRß is not due to interaction between the protein tags themselves. Interestingly, the action of v-ErbA is not limited to genes regulated by TR. It has been shown previously that the oncoprotein interferes with the transcription of other genes, including those regulated by RXR (13, 33, 34) and the retinoic acid (RA) receptor (30, 33). Thus, our observation that v-ErbA also mislocalizes RXR is consistent with the wider action of v-ErbA on genes regulated by other nuclear receptors.
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To determine whether increasing the level of oncoprotein expressed would strengthen the degree of colocalization with TR, a dose response assay was performed. When the ratio between the amount of v-ErbA and TR
expression vector used for transfections was varied, there was a positive correlation between the relative increase in v-ErbA and an increase in TR
colocalization with v-ErbA in the cytoplasm (Table 1
). Up to 94% of cells showed colocalization (partial or total) at a 19-fold excess of v-ErbA expression plasmid, increasing from 45% colocalization when there was a 19-fold excess of TR
expression plasmid.
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These observations correlate with our previous observation that T3, which normally has no effect on TR localization, reduces the amount of the receptor in the cytoplasm when it is coexpressed with v-ErbA. This model is also consistent with previous reports which showed that addition of T3 causes a slight increase in the transcription of genes normally repressed by the oncoprotein and inhibits proliferation of v-ErbA-transformed erythroblasts (6). Furthermore, as we expected, the presence of T3 had no significant effect on the colocalization of RXRß with v-ErbA because neither protein binds this ligand (Fig. 4D
). However, unexpectedly, there was no significant (P = 0.10) change in RXR/v-ErbA colocalization in the presence of 9-cis RA (data not shown). Our findings are consistent with reports that T3 causes a conformational change in TR
that alters its dimerization affinity (35) and, more specifically in this case, its affinity for v-ErbA.
v-ErbA Does Not Colocalize TR to any of the Major Organelles Studied
The colocalization of TR or RXRß with v-ErbA in cytoplasmic foci was confirmed using confocal microscopy. Visual colocalization was not due merely to occasional, random overlapping of TR
or RXRß with v-ErbA (Fig. 5A
). Foci comprised of both TR
or RXRß and v-ErbA have a similar pattern to cytoplasmic foci present in cells solely transfected with v-ErbA. The punctate patterns containing both v-ErbA and TR
or RXRß were mainly oval shaped (Fig. 5A
) but did not always have the same appearance in every case.
Whether the punctate cytoplasmic distribution of v-ErbA represents localization to specific cytoplasmic subcompartments was investigated by using a panel of compartment-specific probes. To determine whether the punctate cytoplasmic foci represent delivery of misfolded v-ErbA to lysosomes or endosomes for degradation, we examined the colocalization of GFP or YFP-tagged v-ErbA with Lysotracker Red or with the endosome-targeting construct CFP-Endo, respectively. v-ErbA often shows perinuclear and punctate staining, characteristic of the Golgi and the endoplasmic reticulum (ER). To investigate whether or not the oncoprotein colocalizes with these organelles, we analyzed the distribution pattern of YFP-v-ErbA with ER-Tracker Blue-White and the Golgi-specific marker CFP-Golgi. Finally, given that a TR-like protein has been found to be associated with rat liver mitochondria (36), we also tested for aberrant mitochondrial localization of v-ErbA by staining GFP-v-ErbA transfected cells with Mitotracker Red. v-ErbA cytoplasmic foci showed no colocalization with any of these compartment-specific probes (Fig. 5B; and data not shown). There was occasional overlapping of v-ErbA foci with an organelle marker, but the spatial distributions overall were not positively correlated. Thus, the punctate distribution of the oncoprotein does not represent localization to lysosomes, endosomes, Golgi, endoplasmic reticulum, or mitochondria.
To determine whether cytoplasmic foci containing both TR and v-ErbA localized to different cellular subcompartments than foci of v-ErbA alone, we tested for colocalization of v-ErbA/TR
with endosomes and Golgi. No colocalization of YFP-TR
coexpressed with DsRed-v-ErbA was observed with the CFP-Endo or CFP-Golgi probes (Fig. 5C
). This indicates that TR
and v-ErbA are not localized to the endosomes or trapped in the Golgi apparatus. More importantly, these findings provide further evidence that the colocalization observed between TR
and v-ErbA is not an artifact caused by interaction of the fluorescent protein tags. In addition, foci were never observed in cells transfected solely with expression vectors for unfused GFP, YFP, CFP, or DsRed (data not shown).
Histone Deacetylation Enhances the Effect of Ligand on the Disruption of TR and v-ErbA Colocalization
Transcriptional activation by liganded TR requires histone acetylation and remodeling of chromatin (37, 38). To determine whether hyperacetylation of histones would enhance nuclear retention of T3-bound TR, cells coexpressing TR
and v-ErbA were treated with TSA, a histone deacetylase inhibitor (39). In the presence of TSA, the percentage of cells exhibiting partial or total cytoplasmic colocalization of TR
and v-ErbA decreased from 73% to 56%. Concomitant with this disruption of colocalization, the percentage of cells showing primarily nuclear localization of TR
increased from 27% to 44%. Interestingly, there was a striking additive effect of T3 and TSA on the disruption of TR
and v-ErbA colocalization (Table 2
). Together, T3 and TSA reduced the percentage of cells exhibiting partial or total colocalization to only 24% (Table 2
). Associated with this disruption of colocalization, the percentage of cells showing primarily nuclear localization of TR
increased to 76%. Not surprisingly, when cells expressing GFP-TR
alone were treated with T3 and/or TSA the distribution of the nuclear receptor remained unchanged (Fig. 3C
; and data not shown). The sequestration of normal TR
in abnormal cellular compartments is thus partially reversed in the presence of hyperacetylated, active chromatin.
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FRET microscopy detects the result of a nonradiative transfer of energy from a donor fluorophore to a nearby acceptor that can only occur over a distance less than about 100 Å (Fig. 6C) (41, 42, 43, 44, 45). When FRET occurs between a pair of fluorophores, the donors emission signal is quenched, whereas a sensitized light is emitted by the acceptor above the spectral background signal. In the present study, CFP and YFP were used, respectively, as the donor and acceptor pair for FRET. To record FRET, there are a number of well-established approaches (46). Among these techniques, FRET can be recorded through the sensitization of the donor or through the dequenching of the donor after photobleaching of the acceptor (pbFRET). Traditionally, these approaches have used one-photon (1p) excitation (41, 43, 44, 47, 48, 49, 50). More recently, however, two-photon (2p) excitation has become the method of choice for some applications (45, 51, 52, 53). Here, we assessed FRET via 2p sensitization of YFP and confirmed it with 1p acceptor photobleaching.
To analyze FRET through sensitization of the donor, we first collected 12 images of unfused CFP or YFP alone under the same settings. These images were used to measure the coefficients of spectral cross talk from CFP or YFP in the FRET channel, and to correct the images of cells coexpressing both donor and acceptor. Although, the cross-section of YFP and CFP is broader for 2p excitation than for 1p (54) at 820 nm, the CFP/YFP cross-section ratio is approximately 4050 (55). This is important because, in FRET imaging, the higher the CFP/YFP cross-section ratio is at the wavelength used to image CFP, the smaller will be the YFP signal monitored in the FRET channel (less cross talk). We found that 2p excitation for CFP/YFP FRET has similar cross talk compared with 1p (data not shown). Nevertheless, it is important to keep in mind when using other fluorophores that the large cross-section of many fluorophores with 2p excitation may make this technique unsuitable for other FRET pairs traditionally used with 1p excitation. Thus, it is imperative to optimize the 2p excitation wavelength for each FRET pair used, especially to minimize the excitation of the donor when exciting the acceptor.
Second, 12 images of cells coexpressing both unfused CFP and YFP (CFP+YFP) together were collected as a negative control for FRET and 12 images of cells expressing the CFP-YFP fusion construct (44) were collected as a positive control for FRET. The positive control exhibits a 30% FRET efficiency (Fig. 6D). This value was used to quantify precisely the efficiency of FRET between TR
or RXRß and v-ErbA. Finally, without prior knowledge of the treatment, we recorded images of cells displaying obvious colocalization of TR
or RXRß with v-ErbA. At least 18 images each of cells coexpressing YFP-TR
or YFP-RXRß with CFP-v-ErbA in the presence or absence of ligand were collected from three different transfection experiments. A significantly higher FRET signal (P < 0.001) was measured between both TR
+v-ErbA and RXRß+v-ErbA compared with that of the negative control CFP+YFP. These data confirm that both RXR and TR colocalize with v-ErbA. On average, the FRET efficiency between TR
or RXRß and v-ErbA was 19% (Fig. 6D
). With the widely used orientation factor of 2/3 (45), we calculated a distance of approximately 67±2 Å between the fluorescent tags, which is well below the 100 Å limit for FRET detection (Fig. 6C
). We did not do any direct measurements of the dipole orientation of the heterodimers, so this value is only an approximation. However, the distance measured is similar to that of other proteins interacting in complexes, such as the vacuolar H+-ATPase-a (VHA-a) and vacuolar H+-ATPase-c (VHA-c) subunits in the vacuolar H+-ATPase complex in plants (50). Because the DNA binding domain of RXR alone is 38 x 74 x 25 Å (56), the relatively small distance measured between CFP and YFP provides further evidence that there is direct interaction of the receptors with the oncoprotein. In the presence of ligand (T3 or 9-cis RA), the efficiency of FRET was slightly reduced between v-ErbA and TR
or RXRß (Table 3
); however, this decrease was not statistically significant (P = 0.017 and P = 0.022, respectively). Finally, FRET signals greater than the positive control CFP-YFP (>30%), sometimes occurred within cytoplasmic foci and/or nuclei of cells coexpressing v-ErbA and either TR
or RXRß (Fig. 6D
).
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Taken together, these data provide further confirmation that TR and RXRß can physically interact with v-ErbA in situ and show that DNA binding is not required for heterodimer formation or stability.
v-ErbA/TR Heterodimers Follow a Chromosome Region Maintenance 1 (CRM1)-Mediated Nuclear Export Pathway
Because TR and v-ErbA are both shuttling proteins, the sequestration of TR
/v-ErbA heterodimers in the cytoplasm could be explained by at least two mechanisms. One possibility is that v-ErbA, being mostly a cytoplasmic protein, interacts with TR
during its nucleocytoplasmic shuttling. Heterodimers would then be sequestered in the cytoplasm because v-ErbA itself is retained in the cytoplasm. Alternatively, the two proteins could interact in the nucleus. Heterodimers would then be actively and rapidly exported to the cytoplasm by a CRM1-mediated pathway, because v-ErbA exits the nucleus by such a pathway, in contrast to TR
(14, 15). We thus sought to ascertain whether coexpressing v-ErbA with TR
would confer to TR
the ability to follow a CRM1-mediated export pathway. To explore this model, we used heterokaryon assays (Fig. 7A
) to determine whether TR
nuclear export becomes sensitive to leptomycin B (LMB) in the presence of v-ErbA. LMB is a specific inhibitor of CRM1-mediated export (39). Heterokaryons (or, in some cases, monokaryons where only one of the mouse nuclei of the fused cells was transfected) were left to shuttle for 6 h to ensure a sufficient time for TR
shuttling, which normally occurs within 1.5 h (14). In the presence of v-ErbA, TR
nuclear export exhibited sensitivity to LMB because there was no detectable shuttling even after 6 h (Fig. 7B
). Under the same conditions (presence of LMB) and incubation time, but in the absence of v-ErbA, TR
was able to shuttle. In contrast v-ErbA remains trapped within the mouse nucleus in the presence of LMB (Fig. 7B
). These findings, in conjunction with our findings that TR
forms dimers in vivo with v-ErbA, indicate that a CRM1-mediated export pathway is most likely followed by v-ErbA/TR
heterodimers (Fig. 7B
). These observations also suggest that TR
may normally enter and exit the nucleus as a homodimer or heterodimer. Even if not all TR
becomes LMB sensitive by virtue of its interaction with the oncoprotein, its export kinetics or nuclear retention are clearly greatly altered in the presence of v-ErbA. Whether there is, in addition, active retention or anchoring of TR
in the cytoplasm by v-ErbA and other associated factors remains to be determined.
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DISCUSSION |
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Evidence from other studies supports the role of altered nuclear export, and cytoplasmic and/or nuclear mislocalization in transcriptional deregulation and oncogenesis. For example, mislocalization of INI1/hSNF5, a component of the SWI/SNF chromatin remodeling complex, blocks its normal tumor suppression function (58), p53 is hyperactively exported from the nucleus in some transformed cells (59), and ectopic expression of the hepatitis B virus X protein sequesters CRM1 in the cytoplasm, suggesting that the inactivation of the CRM1-mediated pathway may be an early step during viral-mediated liver carcinogenesis (60). Moreover, a recent report suggests that the hepatitis C virus core protein modulates the retinoid signaling pathway by sequestering Sp110b, a corepressor of the RA receptor , to the endoplasmic reticulum (61). Finally, a mutant of the androgen receptor, which is aberrantly localized in nuclear foci and subsequently mislocalizes steroid receptor coactivator 1, has been linked to human prostate cancer (62). Oncogenic conversion of TR
into v-ErbA thus not only involves changes in DNA binding specificity and ligand binding properties, but also the acquisition of altered nuclear export capabilities and subcellular localization (Fig. 8
).
Mutants of the Rous sarcoma virus Gag polypeptide, as well as defective endogenous retroviruses have been shown to localize to the secretory pathway in host cells (63). Here we show that the portion of Gag fused to the N terminus of v-ErbA does not target v-ErbA to either the ER or the Golgi apparatus. Interestingly, this sequence of v-ErbA encompasses the matrix domain of Rous sarcoma virus Gag (Fig. 1), known to be responsible for membrane targeting (53, 63) and which may also be responsible for the formation of cytoplasmic complexes. Other viral Gag proteins have been reported to interact with various structures in the cytoplasm. For example, the Mason-Pfizer monkey virus matrix association domain of Gag interacts with the dynein/dynactin molecular motor and targets Gag to the pericentriolar region of the cell (64, 65). We recently reported that the altered nuclear export properties of v-ErbA are mainly attributed to a CRM1-dependent nuclear export sequence in the C-terminal portion of the p10 domain of Gag (15). Whether interaction and colocalization of Gag with subcellular components contributes to formation of foci is under investigation. Although the Gag domain may play a role in the formation of these cytoplasmic foci, it is unlikely that Gag is the sole contributor to the punctate distribution. We have observed a similar distribution in a DNA binding mutant of TR
(14). Likewise, a TR
mutant in which the entire D domain is deleted was shown by immunostaining to localize to the cytoplasm, and then over time to become localized to the perinuclear region or in cytoplasmic patches at the border of the nuclei (66). These observations suggest that cytoplasmic localization of TR
mutants and v-ErbA may have more to do with decreased nuclear retention, or a shift in the balance of nuclear import vs. nuclear export, than with Gag-mediated targeting to subcellular compartments.
The nature of v-ErbA cytoplasmic foci remains to be determined, but results presented here suggest that v-ErbA is not associated with a single organelle or other subcellular compartment. Furthermore, it is unlikely that these foci represent nonspecific aggregation of misfolded proteins because other studies suggest that, in cells, protein aggregation is highly specific (67). Cytoplasmic foci were not simply the result of aggregation of the DsRed tag, because a similar pattern of distribution was observed with CFP, YFP, and HA-tagged receptors, and when cells were cotransfected with expression vectors for untagged, native v-ErbA. Furthermore, in the presence of LMB, v-ErbA foci disperse over time and v-ErbA becomes trapped in the nucleus with a diffuse distribution pattern (Allison, L., unpublished observations), suggesting that these foci represent dynamic structures. Finally, cytoplasmic foci were not observed in cells transfected with expression vectors for unfused GFP, YFP, CFP, or DsRed.
The acquired oncogenic characteristics of v-ErbA, including a viral NES, lead to sequestration of normal TR and auxiliary factors in the cytoplasm. This mislocalization can be partially reversed in the presence of ligand and active, hyperacetylated chromatin. Our findings, along with previous reports (10, 37, 38), emphasize the importance of chromatin structure for TR binding and transcriptional regulation. Although previous reports showed that the ligand for RXR, 9-cis RA, disrupts RXR/v-ErbA dimers and, moreover, allows the recovery of RXR transcriptional activity (13), we did not record a significant effect of ligand on the cytoplasmic colocalization of RXR and v-ErbA. However, the modest decrease in FRET efficiency suggests that 9-cis RA may partially disrupt RXR/v-ErbA dimers. It has been shown that the responsiveness of RXR to its ligand is greatly increased by the presence of TR (68). Therefore, it is possible that disruption of colocalization between RXR and v-ErbA, and concomitant restoration of RXR to the nucleus may require other factors, such as TR.
Our data show that increasing the amount of v-ErbA relative to TR increases the degree of receptor mislocalization and colocalization with the oncoprotein in the cytoplasm. In some instances, inappropriate cytoplasmic accumulation of nuclear proteins labeled with GFP has been reported. For example, overexpression of GFP-tagged SMN (survival of motor neuron proteins) leads to aberrant cytoplasmic accumulation of SMN-complex proteins and core snRNP proteins in transiently transfected cells (69). However, cytoplasmic localization of v-ErbA is not a result of overexpression or tagging with GFP because we observed cytoplasmic accumulation over a range of different expression levels, and whether v-ErbA was fluorescent protein tagged, epitope tagged, or untagged. Furthermore, v-ErbA is naturally overexpressed in cells infected with AEV, and this strong expression is essential in host cells to mediate transforming activity (5, 9, 13). This phenomenon was previously explained solely by the relatively poor interaction of v-ErbA with TREs. In the presence of T3, increasing amounts of v-ErbA relative to TR are required to increase v-ErbA transcriptional repression (8). This is consistent with our finding that increasing the amount of v-ErbA relative to that of TR increased the degree of mislocalization of the receptor to the cytoplasm, and that this overexpression could partially compensate for the disrupting action of T3 on TR/v-ErbA dimers. It now appears that natural overexpression of the oncoprotein is necessary, in part, to mediate all components of dominant-negative activity, including subcellular mislocalization of TR
and RXR.
In summary, our findings not only increase understanding of the normal cellular response to T3 but also provide important insight into the ontogeny of an oncogene and modulation of gene expression through both compartmentalization and dominant-negative transcription factors. Other dominant-negative variants of TR may be involved in human cancer (70); thus, these findings may have implications for a mechanism for their action as well.
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MATERIALS AND METHODS |
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Transient Transfection Assays
Transient transfection assays and subsequent analysis of fixed NIH/3T3 cells by epifluorescence microscopy were performed as described (14). Sixteen to 24 h after transfection, cells were incubated for 414 h with T3 and 9-cis RA-depleted medium or with medium supplemented with 100 nM T3 and/or 100 nM TSA or 100 nM 9-cis RA (Sigma, St. Louis, MO). The subcellular localization of untagged v-ErbA and HA-v-ErbA was analyzed by indirect immunofluorescence using standard procedures (14). Cells where probed with anti-c-erbA antibodies (1:50, rabbit polyclonal to full-length chicken TR, FL-408; Santa Cruz Biotechnology, Inc., Santa Cruz, CA) or anti-HA (1:200, rabbit polyclonal, S1827, CLONTECH) and labeled secondary antibodies (1:100 and 1:500 respectively, Vector Laboratories, Inc., Burlingame, CA). Confocal images were collected with Laser Sharp 2000 version 5.3 (Bio-Rad, Hercules, CA) using the Radiance 2001 (Bio-Rad) laser scanning system, mounted on an inverted microscope (Nikon Eclipse TE300, Nikon Inc., Melville, NY). The following filter combinations were used: GFP Ex Argon laser (Ar) 514 nm, Dichroic Long Pass (DCLP) 560, high quality filter (HQ) 515/30; DsRed Helium Neon laser 543 nm, DCLP 560, DCLP 650, HQ 600/50; CFP Ar 457 nm, DCLP 500, HQ 485/30; YFP Ar 514 nm, DCLP 500, DCLP 650, HQ 545/40; DsRed He/Ne 570 nm, DCLP 500, DCLP 560, LP 570.
Analysis of v-ErbA Association with Specific Organelles
To analyze the colocalization of v-ErbA with lysosomes or endosomes, cells transfected with GFP or YFP-tagged v-ErbA were stained for 30 min at 37 C with Lysotracker Red DND-99 (100 nM, Molecular Probes, Eugene, OR) or cotransfected with an endosome-targeting vector, pECFP-Endo (CLONTECH). For Golgi and ER colocalization, YFP or GFP-v-ErbA transfected cells were stained for 30 min at 37 C just before the fixation with ER-Tracker Blue-White DPX (500 nM, Molecular Probes), or were cotransfected with a Golgi-specific targeting vector, pECFP-Golgi (CLONTECH). For mitochondrial association, GFP-v-ErbA transfected cells were incubated for 30 min at 37 C with Mitotracker Red (50 nM, Molecular Probes) just before fixation.
Analysis of Nucleocytoplasmic Distribution and Colocalization
Transfection experiments were carried out at least three times for each treatment, with greater than 100 cells analyzed per trial. Scoring of cells was performed blindly, without prior knowledge of treatment. For analysis of nucleocytoplasmic distribution, cells were categorized into four groups based on rigorous criteria for the qualitative assessment of the subcellular distribution of TR: all N, complete nuclear localization; N > C, mainly nuclear; N = C, whole cell distribution; or all C, mainly to entirely cytoplasmic. For analysis of colocalization, cells were categorized into three groups based on qualitative rigorous criteria for the assessment of the relative degree of colocalization of TR
or RXRß with v-ErbA: total, partial, or no colocalization. Log-linear analysis was used to determine the statistical significance of differences in subcellular distribution and colocalization in the presence and absence of v-ErbA and ligand.
GST Pull-Down and Coimmunoprecipitation Assays
Direct interaction between TR and v-ErbA was examined by GST pull-down assays. The GST-TR
fusion protein was expressed in Escherichia coli BL21-Codon Plus (DE3)-RIL cells (Stratagene, La Jolla, CA). After induction with 0.5 M isopropyl-ß-D-thiogalactopyranoside at 30 C, bacterial cells were harvested and sonicated in B-PER Bacterial Protein Extraction Reagent (Pierce, Rockford, IL) supplemented with 500 µg/ml lysozyme. GST-TR
was purified using Glutathione Sepharose 4B resin (Amersham Pharmacia Biotech, Arlington Heights, IL) and eluted with reduced glutathione, followed by dialysis against PBS. Purified protein was bound to Immobilized Glutathione gel in Mini-Spin Columns (Pierce), according to the manufacturers instructions. Radiolabeled TR
, v-ErbA, and ribosomal protein L5 were translated in vitro using the TNT-coupled transcription/translation system (Promega) in the presence of 35S-methionine (Amersham) and SP6 or T3 RNA polymerase. Pull-down assays were carried out using the Profound Pull-Down GST Assay Kit (Pierce), according to the manufacturers instructions. Samples were analyzed by 10% SDS-PAGE and fluorography was performed as described (14).
For coimmunoprecipitation assays, COS-1 cells were cotransfected with GFP-TR and DsRed-v-ErbA expression plasmids in 100-mm plates. Twenty hours after transfection, transfection medium was replaced with DMEM containing 10% fetal bovine serum minus methionine (Invitrogen Life Technologies, Carlsbad, CA), supplemented with 50 µCi/ml 35S-methionine. Cells were lysed 48 h after transfection in M-PER Reagent (Pierce) and incubated with anti-GFP antibody (CLONTECH Living Colors full-length A.v. polyclonal) bound to AminoLink Plus Coupling Gel in Mini-Spin Columns (Pierce), according to the manufacturers instructions. After elution, immunoprecipitated antigen samples were concentrated using PAGEprep Protein Clean-Up and Enrichment Kit (Pierce) and analyzed by 10% SDS-PAGE and fluorography. The identity of radiolabeled protein bands was confirmed by comparing with known size standards (Bio-Rad Kaleidoscope prestained protein molecular weight standards) and by Western blot analysis with anti-GFP and anti-DsRed-specific antibodies (CLONTECH Living Colors A.v. monoclonal #JL-8; CLONTECH Living Colors DsRed monoclonal).
FRET Analysis
NIH/3T3 cells were transfected with expression vectors for unfused CFP, unfused YFP or CFP-YFP fusion protein alone, or cotransfected with equal quantities of expression plasmids for both unfused CFP and YFP, or either CFP-TR or CFP-RXRß and YFP-v-ErbA. Transfected cells were fixed as described above. To measure the efficiency of FRET analysis via sensitized emission of YFP, all images were collected with 2p excitation. In monolayer cells, 2p excitation is reported to yield a FRET signal that is less affected by donor concentration than in one photon confocal FRET microscopy (45). However, the photobleaching rate under 2p excitation is higher at the focal plane than in 1p microscopy (73, 74, 75, 76). The use of fixed cells and identical settings, allowed scanning of each sample only once for each wavelength. This helped reduced 2p high-order photobleaching and associated drawbacks. Nevertheless, it is important when designing an experiment using this technique on samples other than fixed monolayer cells to keep in mind the higher photobleaching properties at the focal plane of 2p excitation. The 2p-excitation was achieved with a Ti/Si laser line (MaiTai, Spectra-Physics, Mountain View, CA), connected to a Radiance 2100 (Bio-Rad) confocal microscope, using the same settings for each image collected (gain, offset, zoom, laser power). The channels used to collect the pictures were as follows: donor (CFP), 2p-820 nm, HQ 485/30; FRET 2p-820 nm, DCLP 500, HQ 528/50; acceptor (YFP) 2p-920 nm, DCLP 500, HQ 528/50. To reduce potential photobleaching CFP and FRET channels were collected simultaneously. Using the setcol display feature, the background (Bkg) was set as pixels with an intensity of five or less. To quantify the data, a correction algorithm was used that allows a more precise and more quantitative approach to FRET (44, 45). The correction algorithm was applied using scripts written with scientific imaging software (Scanalytics, Fairfax, VA) according to the following formula:
where cF corresponds to the corrected FRET signal; D, F, and A represent the images collected under the donor, FRET, and acceptor channels respectively, in samples transfected by: d, donor; a, acceptor; or a,b, both donor and acceptor, respectively. The index i represents the given range of intensity, and n was set to 5 bit (=32), delimiting a 3-bit (=8) range of intensity (for example, A3a,b represents all the pixels that have an intensity between 24 and 31, from an image of cells coexpressing the donor and acceptor that was collected under the acceptor channel). The background (Bkg) or saturated pixels (Sat) of these images were set to zero using masks before the analysis. The cross-talk coefficients rd and ra were calculated for each range of intensity, i, using 12 different sets of images each from cells expressing donor or acceptor alone from three different slides. These cells were chosen with various intensity levels so that the entire dynamic range could be covered for the correction. In this case, Fa and Fd represent the sets of pixels with the same coordinates as in Aia and Did, respectively.
The efficiency of FRET was then calculated for each sample using the following formula:
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where E(%) is the percent efficiency of FRET. corresponds to the ratio of
/
in Hoppe et al. (44) or to (
dd/
dd)x Qd in Elanglovan et al. (45). The efficiency of FRET for the construct expressing a CFP-YFP fusion protein was determined to be approximately 30% at pH 8.5 in vitro (pH of the mounting media used) (44).
Was thus calculated by averaging 12 different sets of images collected from three different slides of cells transfected by the CFP-YFP fusion construct, according to the following equation:
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To ensure that the signal measured was due to FRET, 12 images from cells transfected solely with unfused CFP and YFP were collected from three different slides. Finally, for cells expressing YFP-TR or YFP-RXRß cotransfected with CFP-v-ErbA in the presence or absence of T3 or RA, respectively, the images were collected blind from a total of 12 slides (three slides per treatment) without knowledge of treatment. The cells chosen for analysis were solely cells with obvious colocalization of YFP-TR
or YFP-RXRß with CFP-v-ErbA. Distance was calculated according to the following formula:
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For pbFRET, the signal was determined using the following formula:
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Heterokaryon Assays
Heterokaryon assays were performed as described (14). In brief, NIH/3T3 cells were transfected with YFP-TR and/or CFP-v-ErbA. Twenty to 24 h after transfection, HeLa cells were trypsinized and plated at high density with transfected NIH/3T3 cells. Three to 4 h after seeding, HeLa cells were fused to the transfected NIH/3T3 cells with polyethylene glycol at 50% wt/vol in 75 mM HEPES (Roche, Indianapolis, IN). LMB and/or cycloheximide (Sigma) were added after transfection at a final concentration of 5 ng/ml for the LMB, and 50 µg/ml for the first 30 min then 100 µg/ml thereafter for the cycloheximide. The heterokaryons were left to shuttle 68 h. The integrity of the heterokaryons was confirmed using transmission microscopy, and shuttling was indicated by the presence of YFP-TR
in both the transfected NIH/3T3 and untransfected HeLa cell nuclei. For differential staining of nuclei, the coverslips were incubated in a 100-µl drop of Dulbeccos PBS containing 0.5 µmol of To-Pro3 and/or 10 µg/ml of Hoechst for 15 min in the dark, then rinsed three times with Dulbeccos PBS for 5 min before being mounted in VectaShield.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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First Published Online January 13, 2005
Abbreviations: AEV, Avian erythroblastosis virus; Ar, Ex argon laser; CFP, cyan fluorescent protein; CRM1, chromosome region maintenance 1; DCLP, Dichroic Long Pass; DsRed, red fluorescent protein; ER, endoplasmic reticulum; FRET, fluorescence resonance energy transfer; GFP, green fluorescent protein; GST, glutathione-S-transferase; HA, hemagglutinin; LMB, leptomycin B; 1p and 2p, one- and two-photon excitation; pbFRET, photobleaching of the acceptor; RA, retinoic acid; RXR, retinoid X receptor; TR, thyroid hormone receptor
; TRE, thyroid hormone-responsive element; TSA, trichostatin A; YFP, yellow fluorescent protein.
Received for publication May 18, 2004. Accepted for publication January 7, 2005.
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REFERENCES |
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