Human Molecular Genetics Advance Access originally published online on October 11, 2005
Human Molecular Genetics 2005 14(23):3557-3564; doi:10.1093/hmg/ddi383
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Deletions in the polyAlanine-containing transcription factor FOXL2 lead to intranuclear aggregation
INSERM U709 Génomique et Epigénétique des Pathologies Placentaires and Universités Paris V & VII, Paris 75014, France
* To whom correspondence should be addressed at: Hôpital Cochin, Pavillon Baudelocque, 123 Bd de Port Royal, Paris 75014, France. Tel: +33 143262826; Fax: +33 143264408; Email: veitia{at}cochin.inserm.fr
Received August 9, 2005; Accepted October 5, 2005
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALAKNOWLEDGEMENTSREFERENCES |
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Mutations of FOXL2, a gene encoding a forkhead transcription factor, have been shown to cause the blepharophimosis–ptosis–epicanthus inversus syndrome. This genetic disorder is characterized by eyelid and craniofacial abnormalities associated or not with premature ovarian failure. We have previously shown that mutant FOXL2 with an expanded polyAlanine (polyAla) tract forms large aggregates both in the nucleus and in the cytoplasm of transfected cells, whereas the wild-type protein localizes in the nucleus in a rather diffuse manner. Premature stop codons in FOXL2 have been considered so far as null alleles. However, we demonstrate here that such nonsense mutations may lead to the production of N-terminally truncated proteins by re-initiation of translation downstream of the stop codon. Surprisingly, the truncated proteins strongly aggregate in the nucleus, partially localize in the cytoplasm and retain a fraction of the wild-type protein. We also show that a complete deletion of the polyAla tract of FOXL2 induces a significant intranuclear aggregation. Our results enlarge the spectrum of mutations inducing FOXL2 aggregation.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALAKNOWLEDGEMENTSREFERENCES |
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FOXL2 is a single-exon gene encoding a protein of 376 amino acids. It contains a characteristic domain with DNA binding activity: the forkhead. FOXL2 also presents a polyAlanine tract (polyAla) of 14 residues conserved in mammals (1
). FOXL2 is expressed in the peri-ocular tissues as well as in the somatic compartment of the fetal and adult ovaries (1,2). Mutations of FOXL2 have been shown to cause the blepharophimosis–ptosis–epicanthus inversus syndrome, a rare autosomal dominant disorder (BPES, MIM110100) (2). In BPES Type I, eyelid and slight craniofacial abnormalities are associated with premature ovarian failure (POF), whereas in BPES Type II, only the eyelid malformations are observed (3). A large spectrum of FOXL2 mutations has been found in the coding sequence of the gene (details in the human FOXL2 mutation database at http://medgen.ugent.be/foxl2). Mutations expected to lead to a truncated protein are essentially responsible for BPES Type I. This is the case of nonsense and frameshift mutations located in the 5' end of the open reading frame (ORF), upstream of the region encoding the forkhead domain or within it. In contrast, mutations leading to elongated proteins often lead to BPES Type II. For instance, 30% of the mutations detected in the ORF results in an expansion of the polyAla, from 14 to 24 residues, and is mainly responsible for BPES Type II (4).
We have previously shown in transfection experiments that mutant FOXL2 with a tract of 24 Alas (i.e. FOXL2-Ala24) is localized both in the nucleus and in the cytoplasm of COS-7 cells (Supplementary Material, Fig. S1). The mutant protein forms large cytoplasmic aggregates, whereas FOXL2-Ala14, the wild-type version, localizes exclusively in the nucleus in a rather diffuse manner (5). There are eight other known genes in which polyAla expansions have been shown to cause human disease (6). Notably, aggregation has been reported for other disease-causing polyAla-expanded proteins such as PABPN1 (7), Hoxd13, SOX3, RUNX2, HOXA13 (8), Arx (9) and PHOX2B (10).
We have also shown that FOXL2-Ala24 retains a fraction of the normal FOXL2-Ala14 protein in nuclear aggregates, suggesting that the expanded protein may play a dominant negative effect (5). A similar phenomenon has concomitantly been described for Hoxd13, SOX3, RUNX2, HOXA13 (8) and PHOX2B (10). Furthermore, it has been shown that ubiquitin and the proteasome co-localize with the aggregates of polyAla-expanded proteins and that the aggregation process is countered or relieved by the over-expression of various chaperones (8,11,12).
Premature stop codons are commonly thought to result in truncated proteins devoid of the normal C-terminus (C-term) and expected to be inactive. In multi-exon genes, premature stop codons occurring upstream of the last exon–exon junction lead to the activation of the nonsense mediated decay (NMD) (13,14). This results in a specific degradation of the mutant mRNA. In single-exon genes, as FOXL2, NMD has not been described. For single-exon genes, the presence of a premature stop codon may lead to three main outcomes: (i) the production of a truncated peptide lacking the C-term, (ii) translational decoding of the stop codon as a sense codon (read-through) or (iii) translation re-initiation at an internal methionine codon downstream of the premature stop codon. Read-through would lead to a low-level production of the full-length protein while translation re-start may yield an oligopeptide corresponding to the expected N-terminal (N-term) region (standard initiation) as well as an N-term truncated product (resulting from internal re-initiation). This mechanism has been proposed to explain why premature stop codons can result in mild forms of several diseases and has been shown to occur even in multi-exon genes (15,16).
Premature nonsense codons in FOXL2 have been considered so far as null alleles. As a single-exon gene, FOXL2 is not expected, according to our current knowledge, to be targeted by the NMD. Therefore, we have examined the possibility that either translation re-initiation or read-through can take place in the case of FOXL2 nonsense mutations. We demonstrate in transfection experiments that premature nonsense mutations result in the production of N-term truncated proteins by re-initiation of translation downstream of the stop codon. Surprisingly, the truncated proteins strongly aggregate in the nucleus and partially localize in the cytoplasm. Furthermore, we show that the N-term truncated proteins retain a fraction of the wild-type protein in the nuclear aggregates.
Recently, a deletion of the polyAla tract of FOXL2 removing 10 Ala residues was reported to be associated with POF in absence of BPES (17). We also show that a complete deletion of the polyAla tract also induces a significant intranuclear aggregation. We discuss the potential implications of our findings.
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RESULTS AND DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALAKNOWLEDGEMENTSREFERENCES |
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FOXL2 mRNA carrying a premature stop codon is translated and results in N-term truncated products that aggregate massively in the nucleus
To explore a possible re-initiation of translation downstream of a premature stop codon or read-through in the context of FOXL2, we have expressed its ORF containing a nonsense codon, as a fusion product with the green fluorescent protein (GFP) at the C-term of FOXL2 (i.e. FOXL2-E19X-GFP construct) (Fig. 1). Direct observation by fluorescence microscopy shows that a fusion protein is indeed produced. This demonstrates that the region downstream of the nonsense codon can be translated. Interestingly, the fluorescence observed was not diffuse, as one might have expected. In fact, intranuclear aggregates were detected in all transfected cells (Fig. 2B). Moreover,
70% of transfected cells displayed a characteristic cytoplasmic decoration not observed in the transfection with FOXL2-Ala14-GFP (wild-type) (Fig. 2A). This decoration reminds the distribution of mitochondria in COS-7 cells. To test this, we used Mitofluor Red to stain mitochondria and we found a co-localization of both green and red fluorescences (Supplementary Material, Fig. S1). The significance of the interaction between a derivative of FOXL2 and mitochondria remains unexplained. As the characteristic intranuclear aggregation and cytoplasmic decoration have never been observed for the wild-type protein, we supposed that the fusion protein(s) did not contain the full-length FOXL2. The alternative was the production of N-term truncated products. To explore this possibility, we performed a western blot analysis on extracts of transfected cells using a monoclonal anti-GFP antibody. We detected a GFP fusion protein that was smaller than the one detected for the fusion with the wild-type ORF (65 versus 70 kDa) (Fig. 3). Examination of the sequence of FOXL2 revealed the existence of eight in-frame AUG codons downstream of the nonsense mutation. The first AUG codon downstream of the stop mutation lies within a perfect Kozak consensus sequence (i.e. GCC AUG G) located in the region encoding the N-term portion of the forkhead domain. Thus, translation re-initiation at the 65th codon of the ORF (i.e. AUG65) would lead to a product having a molecular weight of 63 kDa, in agreement with our western blot results. We detected another band that corresponds to a product resulting from the initiation of translation at either codon AUG137 or AUG149 of the FOXL2 ORF. For reasons explained subsequently, we propose that AUG137 is the most likely translation initiation site. This band appeared also, but very faintly, in single transfections with the wild-type construct.
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To confirm the translation re-initiation at AUG65, we transfected a construct corresponding to a deletion of the first 64 codons of FOXL2 (FOXL2-
1-64-GFP) (Fig. 1). As expected, the expression of FOXL2-1-64-GFP resulted in strong aggregation in the nucleus and staining of the cytoplasm (data not shown). Western blot analysis showed that the construct led to the expression of the same two proteins as mentioned earlier (i.e. resulting from FOXL2-E19X-GFP) (Fig. 3).
To quantify the relative amount of the N-term truncated product resulting from translation re-initiation and the wild-type product, we performed a co-transfection experiment using the same amount of plasmid encoding FOXL2-E19X-GFP and FOXL2-Ala14-GFP. Comparison of the signal strengths indicated that FOXL2 N-term truncated products were expressed at 25% of the wild-type level. It is worth noting that, even when the expression of the N-term truncated product was weaker than that of the WT protein (i.e. western blot results), aggregation was observed in all transfected cells, whereas no aggregation was detected for the normal protein. This suggests that the aggregation observed in transfected cells expressing the truncated product is not an artifact of protein over-expression. To rule out a possible artifact of aggregation induced by the GFP in the fusion protein, we performed similar transfection experiments with a construct driving the expression of the FOXL2-E19X not fused to the GFP. Subsequent detection of FOXL2 was carried out with an antibody directed against the C-term of the protein that we have described elsewhere (1). According to our expectations, the immunofluorescence results recapitulated what has been described earlier for the fusion protein (Fig. 2C and D).
Only one premature nonsense mutation located upstream AUG65 has been identified in a BPES (Type I) patient: Q53X (18). Thus, we have tested whether re-initiation of translation could also occur in this context using the construct FOXL2-Q53X-GFP (Fig. 1). Fluorescence microscopy showed that a fusion protein was indeed produced and nuclear aggregates were found, but weaker than for FOXL2-E19X-GFP. Surprisingly, the western blot failed to detect the band corresponding to translation initiation at AUG65. The only product detected was the one corresponding to initiation at AUG137. It is worth noting that Met137 is localized at the C-term end of the forkhead, just before a polypeptide segment rich in basic amino acids, which functions as a nuclear localization signal (i.e. MFEKGNYRRRRRMK). This explains why the fusion protein is localized in the nucleus and justifies our previous assumption that AUG137 is preferred to AUG149 for translation initiation. These results show that translation re-initiation at AUG65 occurs only when the nonsense mutation is closer to the natural initiation site. To further explore this, we tested two other constructs containing nonsense mutations at two intermediate positions: P31X and G41X (FOXL2-P31X-GFP and FOXL2-G41X-GFP constructs) (Fig. 1). A western blot analysis showed that for the nonsense mutation located at codon 31, re-initiation of translation took place and resulted in the production of the same two proteins as for FOXL2-E19X-GFP (Fig. 3). Moreover, the efficiency of translation (re-initiation) was similar for both constructs. In contrast, when the nonsense mutation was located at codon 41, only AUG137 was used as start codon. These latter results reinforce our supposition that re-initiation of translation at AUG65 is dependent on the location of the nonsense mutation and suggests that there is a ‘critical’ position located between 31 and 41 that modulates in a sharp fashion (on or off) translation resumption. Our results with the constructs FOXL2-E19X-GFP and FOXL2-P31X-GFP show that translation can indeed be re-initiated downstream of a premature stop codon and lead to N-term truncated products that strongly aggregate in the nucleus. However, absence of substantial translation initiation at AUG65 from the constructs FOXL2-G41X-GFP and FOXL2-Q53X-GFP suggests two possibilities: (i) the longer the region that has been translated (i.e. FOXL2:1-40 and 1-52 versus FOXL2:1-18 and 1-30), the weaker the chances of restarting translation or (ii) there must be a minimum distance between the premature stop and the next AUG in a good Kozac environment for successful re-initiation. In the case of the ATRX gene, the mutation Q37X leads to translation restart at AUG40 (15), which suggests that there is no absolute need of a minimum distance. The first hypothesis deserves further studies.
Protein aggregation has been reported for several kinds of mutations including polyGln and polyAla tract expansions, missense mutations and mutations leading to over-expression of wild-type proteins (7,19). Our results provide the first demonstration of protein aggregation induced by nonsense mutations due to expression of an ‘unexpected’ N-term truncated protein.
The first 18 amino acids of the forkhead domain play a major role in the solubility of FOXL2
To map the amino acid segment whose deletion leads to FOXL2 aggregation, we made a series of constructs driving the expression of GFP fusion proteins containing several deletions in the N-term region (FOXL2-1-9-GFP, 1-30, 1-46, 1-64) (Fig. 1). We noticed a proportionality between the length of the deletion and the weak aggregation resulting from the first three constructs. As expected, 100% of cells transfected with the FOXL2-1-64-GFP construct displayed a strong nuclear aggregation (Fig. 4). Thus, the polypeptide segment between positions 47 and 64, which corresponds to the first 18 amino acids of the forkhead domain, seems to play a major role in FOXL2 solubility. In addition, 70% of cells transfected with the FOXL2-1-64-GFP construct showed the cytoplasmic decoration, not observed in transfections with the other constructs. To further confirm the implication of the protein segment between amino acids 47 and 64 in FOXL2 solubility, we performed a transfection experiment with a construct driving the expression of an internally deleted fusion protein (FOXL2-47-64-GFP) (Fig. 1). We observed a strong nuclear aggregation (Fig. 4) of the fusion protein. Taken together, these results suggest that the N-term portion of the forkhead domain of FOXL2 is required to keep the protein in a soluble state (i.e. the wild-type intranuclear distribution pattern). The forkhead domain is composed of three alpha helices and two characteristic large loops or ‘wings’. The structure of this domain in a complex with a DNA target has been resolved for the forkhead factor HNF3
/FOXA3 (20). Comparisons of the amino acid sequences of FOXA3 and FOXL2 reveal that six out of the 18 amino acids involved in solubility belong to the first helix of the forkhead. Aggregation of the protein lacking the first 18 amino acids of the forkhead might arise from misfolding of the protein as a result of the misfolding of the forkhead itself.
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To assess the aggreability of the forkhead domain lacking the first 18 amino acids, we performed transfection experiments with two novel constructs: one to express the whole forkhead domain alone fused to the GFP (FOXL2
1-46153-376-GFP) and another to express a truncated forkhead domain lacking the first 18 amino acids fused to the GFP (FOXL21-64153-376-GFP). The whole forkhead fused to the GFP was found essentially in the nucleus in a diffuse manner (Fig. 2E). The truncated forkhead fused to the GFP did not aggregate in the nucleus but preferentially localized in some regions of the nucleus reminding the nucleoli (Fig. 2F). These observations suggest that if aggregation of the FOXL2-47-64-GFP protein is due to a deletion in the forkhead, it takes place only in the context of a longer protein. This also points to the existence of other regions that promote FOXL2 aggregation.
The most obvious candidate region to promote aggregation is the polyAla tract. Indeed, we have previously shown that the expansion of the polyAla tract of FOXL2 induces aggregation and mislocalization of the mutant protein. To test a possible effect of the normal polyAla tract on the aggregation process of the N-term truncated FOXL2, we transfected a construct driving the expression of FOXL2--1-64-GFP without the polyAla (i.e. FOXL2-1-64Ala0-GFP). The fusion protein strongly aggregated in the nucleus, in the same manner as FOXL2-1-64-GFP, showing that regions outside the polyAla are involved in the aggregation of truncated FOXL2 (data not shown).
To test whether the C-term region, downstream of the polyAla, was responsible for the aggregation of the N-term truncated proteins, we transfected a construct driving the expression of an N-term truncated FOXL2-GFP fusion protein lacking the C-term region. The product aggregated in the nucleus in the same manner as FOXL2-1-64-GFP (data not shown). Thus, the segment of 68 amino acids between the fkh and the polyAla appears to contain a key determinant of aggregation of FOXL2-1-64 as its presence is sufficient to induce strong nuclear aggregation. These results suggest that either (i) a misfolded fkh domain requires the 68 amino acid segment to aggregate (as it may provide, e.g. a multimerization domain) or (ii) the 68 amino acids located between the fkh and the polyAla are prone to aggregation even in absence of most of the forkhead domain. Indeed, the fact that the protein starting at Methionine 137, basically lacking the forkhead, strongly aggregates in the nucleus favors the second idea.
On evolutionary grounds, it is worth noting that the ORF of FOXL2 in all mammalian sequences available starts with two Met codons. It is tempting to suggest that this is a way to ensure that FOXL2 translation is always started at either AUG1 or AUG2 and not at AUG65 or AUG137, which would lead to the accumulation of aggregates in the nucleus over time.
The N-term truncated FOXL2 partially retains the normal protein in the intranuclear aggregates
The production of a truncated protein may have a dominant negative effect. First, this truncated product may antagonize the normal activity of FOXL2. For example, a partially truncated forkhead could still bind the DNA target or some co-factors of FOXL2 and prevent their interaction with the normal protein. Secondly, the truncated product could retain the normal protein in the intranuclear aggregates. This phenomenon has been noticed in our previous study where we have shown that polyAla-expanded FOXL2 retains the normal protein in the aggregates (5). Thus, we have explored this possibility for FOXL2 containing a nonsense mutation. Specifically, we have co-transfected FOXL2-Ala14-GFP acting as a fluorescent reporter with the same DNA amount of either FOXL2-E19X non-GFP or FOXL2-Ala14 non-GFP (as a control). The fluorescent reporter was detected in a significantly higher proportion of nuclear aggregates in the co-transfection with FOXL2-E19X non-GFP (10±0.6% of cells with nuclear aggregates) than in the co-transfection with FOXL2-Ala14 non-GFP (2.7±1.2%, P=0.03). This suggests that in addition to inducing intranuclear aggregation in all transfected cells, FOXL2-E19X is able to retain a fraction of the wild-type protein in the aggregates. The aggregation of the N-term truncated proteins resulting from nonsense mutations located upstream of codon 31 might lead to a more severe phenotype, due to a potential dominant negative effect, than the phenotype observed in cases of null mutations.
Deletion of the polyala of FOXL2 induces protein aggregation sensitive to chaperone activity
On the basis of our previous findings, we hypothesized that other alterations in the FOXL2 protein might also lead to aggregation. Notably, it is known that a partial deletion of the polyAla tract of FOXL2 (Ala 4) is associated with POF (17). Thus, to test a potential aggregation of a similar mutant, we transfected a construct expressing a polyAla-less FOXL2 fused to the GFP (i.e. FOXL2-Ala0-GFP) (Fig. 1). We observed a significantly higher proportion of cells carrying intranuclear aggregates when transfected with FOXL2-Ala0-GFP (22.3±4.2%) than in cells transfected with FOXL2-Ala14-GFP constructs (3.7±1.5%, P=0.03). The aggregates were clearly different from those observed with FOXL2-1-64 or FOXL2-Ala24 (Figure 2G). To have an additional proof that they contained abnormally folded FOXL2-Ala0-GFP, we tested the effect of the over-expression of a chaperone, whose construct was available in the laboratory, on the aggregation process. We found that over-expression of Hsp40 significantly reduced the proportion of cells carrying nuclear aggregates. Indeed, only 7.8±1.7% of cells co-transfected with FOXL2-Ala0-GFP and pCDNA3.1-Hsp40 carried intranuclear aggregates, when compared with 21.3±1.5% of cells co-transfected with FOXL2-Ala0-GFP and empty pCDNA3.1 (control assay, P=0.03). Thus, it appears that strong structural or physico-chemical constraints are imposed to the length of the polyAla tract: expansion induces cytoplasmic retention as well as cytoplasmic and nuclear aggregation. On the other hand, contraction would also be deleterious in the context of the ovarian proteome, as the protein might adopt an incorrect conformation leading to intranuclear aggregation. This may explain the high conservation of the length of the polyAla tract in mammals (21).
All in all, we show that premature nonsense mutations in the FOXL2 mRNA result in the production of N-term truncated proteins by re-initiation of translation downstream of the stop codon. Moreover, the aggregates of the truncated proteins are able to retain the normal FOXL2. We also show that a complete deletion of the polyAla tract induces a significant intranuclear aggregation. These results suggest that a perturbation of structural or physico-chemical determinants involved in the solubility of a protein, may lead to aggregation and disease.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALAKNOWLEDGEMENTSREFERENCES |
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Plasmid constructs
The constructs expressing FOXL2-Ala14-GFP and FOXL2-Ala14 non-GFP are those described by Caburet et al. (5
). FOXL2-P31X-GFP, FOXL2-G41X-GFP, FOXL2-Q53X-GFP and FOXL2--47-64-GFP were obtained by junction-PCR (22). Detailed information available upon request. All other deletion constructs were obtained by PCR using appropriate primers (the plasmid pCDNA3.1-FOXL2-Ala14 was used as template). FOXL2-E19X-GFP and FOXL2-E19X non-GFP were obtained by PCR amplification using a forward primer to introduce a stop mutation at position 55 of the ORF and two reverse primers: one to express the protein fused to the GFP and another to express FOXL2-E19X alone. All these PCR products were cloned into pCDNA3.1-CT-GFP-topoTA cloning vector (Invitrogen). FOXL2-ALA0-GFP construct was obtained by ligating two separated PCR products using primers containing a HinfI restriction site. The PCR products (5' and 3' arms) were digested by HinfI and ligated. After a second PCR amplification, the whole fragment was cloned into pCDNA3.1-CT-GFP-topoTA cloning vector. The FOXL2-1-64ALA0-GFP construct was obtained by PCR amplification using appropriate primers and pCDNA3.1-FOXL2-ALA0-GFP as template. All constructs were sequenced to exclude the presence of PCR-induced mutations. All DNA concentrations were determined spectrophotometrically and corroborated by agarose gel electrophoresis.
Cell culture and transfections
COS-7 cells were seeded 24 h before transfection in Dulbecco's modified Eagle's medium (DMEM; Gibco-Invitrogen) containing 10% fetal calf serum (FCS; Gibco-Invitrogen) and 1% penicillin/streptomycin at a concentration of 35 000 cells per well in 24 well plates containing sterile cover slips. The cells were transfected using the calcium phosphate method and rinsed 24 h after transfection (23). All single transfections were performed using 500 ng of DNA (per well). Co-transfections with either pCDNA-Hsp40 or pCDNA-Hsp27 (available in the laboratory) and empty pCDNA3.1 vector control were performed using 200 ng of FOXL2-ALA0-GFP DNA construct and 800 ng of Hsp40 construct or empty vector. Co-transfections of FOXL2-Ala14-GFP and FOXL2-E19X were performed using 500 ng of each. Forty-eight hours after transfection, cells were washed with phosphate buffered saline (PBS) and fixed for 15 min with 4% paraformaldehyde (PFA). Mitochondria were stained with Mitofluor Red 589 according to the instructions of the manufacturer (Molecular probes, 1 µM). Nuclei were stained with Hoescht reagent (1/500) and cover slips were mounted on slides using fluorescence mounting medium (DAKO; CA). Transfected cells were visualized using epifluorescence (Nikon E600) or confocal (Leica TCS SP2) fluorescence microscopy. The Hsp40 construct is a generous gift from H.L. Paulson.
Immunofluorescence
COS-7 cells were transfected as described earlier using 500 ng of the FOXL2-E19X construct. Forty-eight hours after transfection, cells were washed with PBS, fixed during 15 min in 4% PFA, permeabilized for 15 min in PBS/10% FCS/0.4% Triton X-100 and blocked with 5% skimmed milk in PBS/0.05% Tween-20. Cover slips were incubated overnight with the anti-FOXL2 primary antibody (diluted 1/500). This antibody is directed against the C-term of FOXL2. Cells were washed three times in PBS/0.05% Tween-20 and incubated for 1 h with a secondary RITC-conjugated anti-rabbit IgG antibody (diluted 1/500). Nuclei were stained with the Hoescht reagent (1/500) and cover slips were mounted onto the slides using a fluorescence mounting medium (DAKO; CA) and visualized using standard epifluorescence microscopy (Nikon E600).
Protein analysis
COS-7 cells were seeded 24 h prior to transfection on flasks of 25 cm2 in DMEM 10% FCS 1% penicillin/streptomycin at 375 000 cells per flask. The cells were transfected using the calcium phosphate method and rinsed 24 h after transfection. Single transfections were performed using 5 µg DNA/flask and co-transfections were performed using 5 µg of each DNA. Forty-eight hours after transfection, cells were scrapped directly in Laemmli buffer, sonicated for 20 s (three times), diluted in Laemmli buffer and then denatured for 10 min at 90°C. Protein extracts were then electrophoresed and electrotransfered onto nitrocellulose membranes (Amersham). Membrane were blocked with 5% non-fat milk in PBS/0.05% Tween-20 for 1 h, incubated overnight with anti-GFP primary antibody at 4°C (diluted 1/1000; Roche) and 45 min with secondary antibody anti-mouse IgG-peroxydase (Amersham Life Science) diluted 1/5000. Enhanced chemiluminescence detection was carried out as described by the manufacturer (Amersham Biosciences). Quantification of relative amount of wild-type FOXL2 and truncated FOXL2 resulting from translation re-initiation was performed using NIH-image (three independent experiments).
Statistical analysis of FOXL2 variants nuclear aggregation
Nuclear aggregation was scored visually using standard fluorescence microscopy. For each transfection condition, 300–400 GFP–positive COS-7 cells were analysed from two different experiments, and the proportion of cells with intranuclear aggregates was counted. To estimate the significance of the differences between the mean percentages of cells carrying aggregates, two statistical tests were used: the parametric Student's t-test and a randomization test (5).
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SUPPLEMENTARY MATERIAL |
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Supplementary Material is available at HMG Online.
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AKNOWLEDGEMENTS |
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TOPABSTRACTINTRODUCTIONRESULTS AND DISCUSSIONMATERIALS AND METHODSSUPPLEMENTARY MATERIALAKNOWLEDGEMENTSREFERENCES |
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The authors wish to thank Sandrine Caburet and Julie Cocquet for their helpful comments and discussions. We thank H.L. Paulson for the Hsp40 expression vector. We thank two anonymous referees for their constructive comments and suggestions. This work was supported by the French Ministry of Research, the INSERM. M.F. and R.A.V. are supported by the University of Paris VII.
Conflicts of Interest statement. None declared.
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