Human Molecular Genetics, 2001, Vol. 10, No. 17 1859-1866
© 2001 Oxford University Press
Identification of an endogenous RNA transcribed from the antisense strand of the HFE gene
UMR-CNRS 6061, ‘Génétique et Développement’, 2 avenue du Pr Léon Bernard, CS 34317, 35043 Rennes cedex, France
Received May 15, 2001; Revised and Accepted June 14, 2001.
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ABSTRACT |
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Hereditary haemochromatosis is an autosomal recessive disease which results in iron overload, and it is the most frequently inherited disorder in Caucasian populations. The gene involved (HFE) has recently been identified, and it encodes an MHC class I-like molecule. A 2.7 kb cDNA has been isolated, whereas the HFE gene expression is characterized by an almost ubiquitous mRNA of 4.1 kb in size. The difference between this transcript and the isolated cDNA has not yet been explained. Thus, the 5' end of the HFE gene is still undefined and very little is known about the regulation of its expression. By searching this end, we isolated an antisense transcript originating from the same gene locus. Further investigations (rapid amplification of cDNA ends, RT–PCR experiments and dbEST screening) indicated that this RNA spans exon 1, exon 2, part of intron 1 of the HFE gene and
1 kb upstream of it. This HFE antisense transcript is polyadenylated but displays no open reading frame. A ribonuclease A protection assay definitively demonstrated the biological existence of the HFE antisense RNA, which appears to be expressed in all of the tissues and cell lines tested. Furthermore, in vitro coupled transcription–translation experiments revealed that the HFE expression is decreased by this antisense RNA, indicating that it may play a critical role in the regulation of the HFE gene expression. |
INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Hereditary haemochromatosis is the most frequently inherited disease in the European population. It causes iron overload leading to hepatic cirrhosis, cardiomyopathy and other clinical complications. Although it was localized as early as 1975 in 6p21.3 (1), the HFE gene responsible for the disease has only recently been cloned (2). Mapped at 4.5 Mb telomeric to HLA-A, it encodes a protein related to the non-classical MHC class I molecules. This gene family includes MIC, HLA-E, -F and -G (localized on chromosome 6), MR1 and CD1 (chromosome 1), and FcRn (chromosome 19). Although not anticipated, iron overload observed in both ß2m–/– (3) and HFE–/– mice (4) definitively proved the implication of such a molecule in iron metabolism.
Like the other MHC class I genes, the 12 kb HFE gene is organized in seven exons. Its expression is low and it corresponds to a major ubiquitous 4.1 kb mRNA in addition to other minor mRNAs in most of the tissues tested. The HFE cDNA (U60319), 2.7 kb in size, exhibits an apparently complete open reading frame (ORF). Despite the many experiments performed, the missing 1.4 kb has not yet been identified, and so the HFE gene transcription start site and regulatory regions remain unknown. Some recent studies have reported the existence of HFE alternate transcripts which may correspond to some of the minor mRNAs visualized on northern blots (5–7).
The identification and characterization of the HFE gene promoter is one of the key steps in understanding the regulation of iron metabolism. The HFE protein (43–48 kDa) interacts with the transferrin receptor and reduces its affinity for the ligand (8–10), and so decreases cellular iron uptake. This scheme cannot be entirely applied, however, to the duodenal crypt cells which are believed to regulate intestinal absorption of iron. There is no known biological link between TfR-mediated basolateral iron uptake, which mainly occurs in undifferentiated crypt cells, and DMT1-mediated iron absorption which occurs at the luminal side of the enterocytes. The HFE protein is present in undifferentiated crypt cells of the duodenum, and it may act as a sensor to correctly programme the enterocytes. This programming should result in mature enterocytes with an iron absorption level according to the whole body iron status. The mechanism through which HFE acts is unknown, and it could not be investigated without knowledge of its transcription regulation. Furthermore, identification of the cis-acting sequences involved in HFE transcription is of great interest for gaining an insight into the molecular mechanisms of the development of iron overload in haemochromatosis. Ninety percent of patients in northern Europe are homozygous for the C282Y mutation (review in 11) which prevents the formation of the disulphide bond required for the binding of ß2 microglobulin and subsequent normal processing of the HFE protein (12). A second mutation, H63D, has been observed associated with haemochromatosis. Its seems to play a minor role in the development of the disease, even in the C282Y/H63D compound heterozygous state. Thus, a small percentage of haemochromatosis patients, with a larger group in southern Europe, could not be explained by the C282Y mutation. The genetic heterogeneity of the disease was recently demonstrated by the localization in chromosome 1 of the juvenile haemochromatosis locus, designated HFE 2 (13), and by identification of the HFE 3 gene encoding the transferrin receptor 2, which was mutated in two cases of non-HFE haemochromatosis (14–16). Nevertheless, we could not exclude the possibility that for some of the non-C282Y patients, the disease might result from mutations or anomalies located within the regulatory regions of the HFE gene.
The aim of this work was to identify the HFE transcription start site and promoter. Our experiments unexpectedly led to the discovery of an HFE antisense RNA instead of the 5' end of HFE. This RNA, transcribed from the HFE opposite strand, partially covers the HFE mRNA and may play a significant role in the regulation of the HFE gene expression.
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RESULTS |
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5'-Rapid amplification of cDNA ends (5'-RACE)
In order to isolate the 5' end of the HFE gene, many RACE experiments were performed (oligocapping, Smart 5'-RACE, RACE Marathon), but none of them was successful. Nevertheless, new information was obtained from 5'-RACE carried out on pre-prepared testis cDNA using the Marathon cDNA amplification kit (Clontech). The amplimer obtained corresponds to an
1 kb DNA stretch which includes the 5' end of HFE exon 1, and the upstream 929 bp continuous on the genomic sequence. Surprisingly, this DNA segment also displayed a poly(A) tail resulting from a post-transcriptional event, due to the presence of a consensus polyadenylation signal sequence located at the expected position (–906 bp). These data strongly suggested the existence of an antisense RNA which at least partially overlaps exon 1 of the HFE gene.
Demonstration of the existence of an HFE antisense transcript
To confirm the existence of a transcript covering 1 kb upstream of the HFE gene, RT–PCRs were performed with RNAs from testis and HeLa cells. Reverse transcriptions were carried using an oligo(dT) primer, and the cDNAs were subsequently amplified with primers positioned in either the HFE exon 1, or in the upstream region. As expected, PCR products of appropriate sizes were observed after PCR –881U/+115L or nested PCR –881U/–613L on testis and HeLa cells (data not shown), confirming the transcriptional ability of the region upstream of the HFE gene.
To characterize the orientation of the RNA transcribed from this region, we performed 3'-RACE. Total RNA from HeLa cells was reverse-transcribed using the modified oligo(dT) primer, OL3'-LA. Amplifications were then performed with a forward primer annealing to the OL3'-LA cassette and a reverse primer located upstream of the HFE gene 5' end. Nested RT–PCR consisted of an initial PCR OL32/–25L or OL32/–613L followed by the second PCR OL21/–798L (the positions of each HFE primer are depicted in Fig. 1A). The nested PCR product of 180 bp in size was cloned and sequenced (data not shown). In each clone, we systematically observed a poly(A) tail at the 3' end of the PCR product, located at a previously observed position (–906), in agreement with an RNA transcription in the HFE gene opposite orientation.
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To obtain more detailed information, PCRs were carried out using as the template cDNA obtained from oriented reverse transcription with strand-specific primers: +6982L for the HFE gene transcription strand and –880U for the other strand. PCRs targeting the HFE gene 5' DNA stretch (exons 1 and 2 and the 1 kb HFE upstream segment) were then performed (Fig. 1B). When RNA was reverse transcribed using +6982L primer (HFE sense transcription), no PCR was obtained from the HFE upstream region. In contrast, a PCR product of expected size was systematically observed when the cDNA was primed with –880U (antisense orientation)
In addition, nested oriented RT–PCR B-H primed in the HFE exon 2 produced a fragment of 1.0 and 1.1 kb for testis and HeLa cells, respectively. Sequencing of these two fragments indicated that they resulted from the conservation of part of the HFE intron 1 in the antisense RNA (Fig. 2A).
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Characterization of the antisense transcript
Northern blot hybridizations. Two probes were hybridized on in-house or commercial northern blots (multiple tissues northern blots I and II). These probes were localized at the antisense RNA 3' end (–800/–632) and in the insertion of HFE intron 1 (i1 622/i1 869). Despite a long exposure (up to 15 days), no signals, or almost none, were obtained for these two probes. Only the intronic probe (i1 622/i1 869) seemed to hybridize to an RNA 6 kb in size. The signal was very weak and only observed on our blot [8–10 µg poly(A)+ RNA per lane] in HeLa and Caco2 cells.
Ribonuclease A protection. In order to enhance the sensitivity of our experiment, we performed a ribonuclease protection assay using a probe covering the antisense intronic insertion. An aliquot of 4 µg of poly(A)+ RNA from HeLa cells was hybridized to probes corresponding to the sense or to the antisense of HFE gene transcription in order to prove the antisense orientation of the transcript. The sense probe was completely protected by HeLa cells RNA, whereas the antisense probe was degraded (Fig. 3). These data confirm that the intronic portion used as the probe is completely included in an RNA transcribed from the complementary strand of the HFE gene.
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EST. dbEST screening at regular intervals with the 1 kb DNA segment upstream of the HFE gene led to three ESTs. These 3' ESTs, AI671175, AI916326 and AI955100, are derived from three different HFE antisense oligo(dT)-primed cDNA clones [I.M.A.G.E. Consortium CloneID 2305638, 2293728 and 2475717, respectively (19)] which come from pancreas (2293728) or pooled germ cell tumours (2305638 and 2475717). These three identical and overlapping ESTs displayed the same poly(A) tail and the polyadenylation signal at the previously observed position (–906). Structures and alignments of these clones are depicted in Figure 4.
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The two clones 2305638 and 2293728, available from the UK Human Genome Mapping Project Resource Centre, were sequenced. Only the insert of clone 2293728 was long enough to provide further information about the structure of the antisense transcript: its insert covers the 1 kb upstream HFE region, the whole of HFE exon 1 and the first 1337 bases of intron 1 (Fig. 4).
RT–PCR specific for the HFE antisense RNA. Conservation of part of HFE gene intron 1 was visualized in clone 2293728 and in both HeLa cells and testis by RT–PCR. According to these antisense structural data, an RT–PCR targeting intron 1 was performed on RNA from small intestine, liver, testis, colon, pancreas, HeLa cells, HepG2 cells and I407 cells. After oligo(dT) reverse transcription, the amplification i1 622U/i1 869L was performed (location indicated in Fig. 2A). A positive signal corresponding to the 250 bp amplification was observed in all of the tissues and cell lines tested (Fig. 2B). Thus, the antisense RNA expression is not restricted to testis and HeLa cells.
ORF. ORFs were investigated by the MapDraw program (20). This program identifies an ORF recognized by the occurrence of an in-frame stop codon upstream of a putative start codon with a minimum of 80 amino acids of coding sequence. All of the antisense structures identified were tested and insufficient ORF was retrieved.
Interference of the HFE antisense transcript with the HFE gene expression
To study the potential negative regulation of the HFE gene expression by the antisense RNA, coupled transcription–translation of the HFE protein was performed in the presence and absence of this transcript. The cDNA clone 2293728, covering 1 kb upstream the HFE gene, the first exon and part of the first intron, was transcribed and the resulting RNA was used to study its effect on the HFE protein expression. As depicted in Figure 5, the presence of 300 ng of antisense RNA significantly decreased the amount of expressed HFE protein. This decrease was specific to the presence of the antisense molecule, since addition of an external control RNA had no effect on the level of HFE expression. Thus, at least in vitro, expression of the HFE protein is negatively regulated by the antisense transcript.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The initial goal of this work was to identify the HFE transcription start site and promoter. Therefore, we used different RT–PCR based approaches: oligocapping and 5'-RACE. For the reverse transcription step, various conditions were tested: different enzymes (AMV or MMLV; see Materials and Methods), temperatures and times for RNA denaturation (70–90°C for 5–10 min) and different temperatures and times for reverse transcription (42–50°C for 45–90 min). None of these conditions allowed us to isolate the 5' end of the HFE gene. Of these experiments, the Smart 5'-RACE gave us the best result: this RACE stopped at position +7.
In addition, unlike the probes corresponding to the partial or complete HFE ORF, no signal could be visualized on northern blot using a probe located upstream of the translation start site (positions 80–180, data not shown). In parallel, we used the Repeat Masker program provided by the sequence annotation tool NIX (UK HGMP Resource Centre server) to identify within the 80 kb HFE upstream genomic DNA all of the sequences which do not correspond to a human repeat. Systematically probing these sequences on northern blots did not reveal the HFE 4.1 kb mRNA (data not shown). The 5' end of HFE still remains an enigma: although an article has been published about a putative HFE promoter (21), no formal proof has been provided to indicate if it is continuous on the genome, or located more distantly due to a splicing event occurring in the hitherto described HFE exon 1.
Surprisingly, a 5'-RACE experiment using the Marathon kit allowed us to identify the 3' end of an HFE antisense mRNA instead of isolating the 5' end of HFE. This transcript has a 3' end located at position –929. This position correlates well with the presence of a consensus polyadenylation signal AATAAA in the genomic sequence at position –906. Initially isolated from testis, it was also observed by RT–PCR in other tissues and cellular types. Three cDNA clones from the EST database present the same orientation. Sequencing of RT–PCR products and cDNA clones revealed that it is continuous on the genomic sequence at 929 bp upstream of the HFE gene, and covers the first exon 1 and part of intron 1 of HFE. The expression level of this HFE antisense transcript in the cell seems to be very low. RNase protection assays on mRNA from HeLa cells definitively demonstrated that the transcribed segment derived from HFE intron 1 is specifically included in the antisense RNA. Moreover, in vitro studies of the HFE protein expression revealed that it is negatively regulated in the presence of this antisense transcript.
Regulation of gene expression by antisense transcripts is well established in prokaryotic systems (22), and there are an increasing number of examples of endogenous antisense RNAs in eukaryotic organisms (23). The sense–antisense transcription units identified to date in eukaryotes present a variety of different structures. Regions of overlapping transcription have been described at both the 5' (24–28) and 3' ends (29–32), and in some instances the sense and antisense transcripts share exonic regions (33,34). A striking example is the recent observation within a single 60 kb fragment in the HLA class I region of the presence of all of these structures (35). Nevertheless, the exact effects of antisense RNA on its sense counterpart have yet to be established, and they may be as diverse as their structure. It is apparent that mammalian cells have devoted genetic information and systems to processing RNA:RNA hybrids, and it is becoming clear that there may be many more genes than previously suspected for which natural antisense RNAs exist. There is some experimental evidence for their regulatory functions in eukaryotes; they have been shown to be expressed in competition with sense transcripts (36), or to pair with the sense transcript and affect post-transcriptional events such as splicing (31), cytoplasmic stability (37,38) and translation (39). If antisense RNAs naturally have regulatory effects on their complementary mRNAs, this is likely to be the result of (i) modification of the sense mRNA through RNA editing by double-stranded RNA adenosine deaminases, (ii) degradation by ribonucleases specific for RNA:RNA hybrids or (iii) inactivation by blocking the sense mRNA translation. Many of these antisense transcripts do not fulfil conventional criteria for defining mRNAs: polyadenylation, splicing and presence of ORFs. There is evidence for an absence of effective translation of such transcripts and for their implication in the regulation of sense RNA expression. The HFE antisense RNA described in this study presents a poly(A) tail but displays no ORF, so it could play a significant role in the regulation of the HFE gene expression.
The study reported here demonstrates that the HFE locus is subject to bidirectional transcription leading to the expression of an HFE antisense transcript. In vitro studies strongly suggested that this RNA represses the HFE protein expression. Nevertheless, further studies are needed to investigate its involvement at the transcription and/or translation steps and its subsequent effect in the iron metabolism regulation.
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MATERIALS AND METHODS |
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All of the primers and sequences described in this study are designated in relation to the +1 position of the published HFE gene (Z92910); primers are listed in Table 1.
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Cell lines and RNA preparation
Total or poly(A)+ RNAs were extracted from different human cell lines: Daudi (Burkitt’s lymphoma), HeLa (cervical adenocarcinoma), Caco2 (colon adenocarcinoma), K562 (erythroleukaemia) and MDA (breast cancer). Extraction was performed with the RNAzolTM B system (Quantum Bioprobe, Montreuil, France) and poly(A)+ RNAs were obtained using oligo(dT) columns (Amersham Pharmacia Biotech, Uppsala, Sweden), in accordance with the manufacturer’s recommendations. Human liver and small intestine poly(A)+ RNAs were purchased from Clontech (Palo Alto, CA).
Marathon cDNA library screening
A library of double-stranded cDNA ligated to anchors at both of their ends was used to search the 5' end of the HFE gene (Marathon cDNA library; Clontech). This library was prepared from testicular RNA. The screening consisted of nested PCR using the upper primers in the Marathon cassette and the lower primers located at the known 5' end of the HFE gene (+e2 3732L and +e1 201L). We performed amplifications by a touchdown procedure as described previously (17) with an annealing temperature cut-down of 0.5°C per cycle. Amplifications were performed in 50 µl with Taq polymerase (2.5 U; Promega, Madison, WI) and 0.2 µM of each primer for 20 cycles of 30 s at 94°C, 30 s of annealing (temperature of the first cycle: 65°C) and 4 min at 72°C followed by 15 identical cycles with a constant annealing temperature of 50°C. The resulting band was cloned in pGEM-T vector (Promega) and sequenced by the automated fluorescent method using dye-terminators on sequencer ABI 377 (Perkin Elmer Applied Biosystems, Foster City, CA), in accordance with the manufacturer’s recommendations.
RT–PCR
For all of the experiments, 1 µg of total RNA was reverse-transcribed for 1 h at 42°C in a final volume of 20 µl using 200 U MMLV reverse transcriptase (Superscript II; Life Technologies Gibco BRL, Rockville, MD) or an AMV reverse transcriptase (AMV-X; Takara, Shuzo, Shiga, Japan). An aliquot of 2 µl was used for nested PCR in the region upstream of the known 5' end of HFE with the following primers: –881U/+115L followed by –881U/–613L. All RT–PCRs described in this article were performed by the previously described touchdown procedure with an annealing time of 1 min for 1 kb.
Gene-specific primers +6982L and –880U were used to prime the reverse transcription according to the HFE sense or antisense orientation respectively. RT–PCRs were then performed targeting the known 5' end of HFE and then the 1 kb HFE upstream sequence by using the following primers. First RT–PCR, A (–881U/+e2 3702L) or B (–548U/+e2 3702L); nested RT–PCR, C [–881U/–618L (262 bp)], D [–595U/–464L (131 bp)], E [–148U/–40L (108 bp)], F [–40U/+e1 206L (244 bp)], G [–79U/+e1 206L (126 bp)] and H [–53U/+e2 3639L (280 bp)].
Amplification was also performed on oligo(dT)-primed single-stranded cDNA with the primers +i1 622U and +i1 869L in order to confirm the conservation of an HFE intron 1 portion in the antisense transcript. RT–PCR conditions were 30 cycles of 30 s at 95°C, 30 s at 55°C and 1 min at 72°C.
3'-RACE
Reverse transcription was performed with the modified oligo(dT) primer OL3'-LA. Amplifications were carried out with the forward primers complementary to the anchor (OL32 and OL21) and the reverse primers located upstream of the 5' end of the HFE gene (–25L, –613L and –798L).
Northern blot hybridization
Probes were hybridized to a northern blot containing 8 µg of mRNA from each cell line described above and to the human-adult multiple tissue northern blots I (heart, brain, placenta, lung, liver, skeletal muscle, kidney and pancreas) and II (spleen, thymus, prostate, testis, ovary, small intestine, colon, peripheral blood leukocyte) (Clontech). An aliquot of 50 ng of probe corresponding to antisense 3' end (–800/–632) or a portion of HFE intron 1 (i1 622/i1 869) was labelled with 5 µl of [
-32P]dCTP (500 µCi) using the Rediprime II kit (Amersham Pharmacia Biotech). Hybridizations were performed overnight under conventional conditions at 42°C with 50% formamide buffer. The final washes consisted of (i) 0.5x SSC/0.1% SDS at 65°C for the commercial blots and (ii) 0.2x SSC/0.1% SDS at 42°C for the laboratory blot. Membranes were exposed to X-ray film (Kodak BIOMAX) at –80°C.
Ribonuclease protection assay
A 720 bp fragment from HFE intron 1 (i1 622/i1 1341) covering the segment included in the antisense RNA was cloned in the two orientations into the pGEM-T vector (Promega) and digested by the NotI restriction enzyme (New England Biolabs, Beverly, MA). In vitro transcription was performed with T7 RNA polymerase (Promega). Samples of 4 µg of polyadenylated RNA were denatured with the [-32P]UTP-labelled riboprobe (5–10 x 104 c.p.m.) for 5 min at 90°C and hybridized overnight at 45°C. RNA:RNA hybrids were subjected to digestion with RNase A (10 µg/ml). After RNase inactivation with proteinase K (0.3 mg/ml) and SDS (0.6%), samples were extracted, precipitated with ethanol and then analysed by electrophoresis on a 5.25% polyacrylamide–6 M urea denaturing gel. To visualize the controls, autoradiographs were performed at –80°C on X-OMAT films (Eastman Kodak, Rochester, NY). For the signal enhancement assays, the gels were exposed to a storage phosphor screen and the phosphorimager Storm was used (Molecular Dynamics, Sunnyvale, CA).
In vitro coupled transcription–translation
An aliquot of 1 µg of the circular plasmid pGEM-T containing the complete HFE coding sequence obtained by RT–PCR from human placenta poly(A)+ RNA (positions 179–1209) was transcribed and translated using the TNT® T7-coupled reticulocyte lysate system (Promega) according to the manufacturer’s recommendations. This experiment was performed in the presence and in the absence of 300 ng of an antisense RNA corresponding to the clone 2293728 (structure depicted in Fig. 4) or of 300 ng of a control RNA (positions –300 to +600 of the talin mRNA, GenBank accession no. AF177198). Antisense or control RNA were transcribed by Sp6 or T3 RNA polymerase, respectively, from 1 µg of linear plasmid according to the manufacturer’s recommendations (Roche Molecular Biochemicals, Meylan, France). RNA was then denaturated for 10 min at 65°C before being added to the HFE transcription–translation reaction mix. The reaction was performed at 37°C for 1.5 h in the presence of [35S]methionine. SDS–PAGE was carried out under reducing conditions in a Mini-Protean electrophoresis unit (Bio-Rad) according to Laemmli (18): 1/25 to 1/5 of the different reactions were loaded. The gel was then fixed for 20 min in a solution of 25% ethanol and 10% acetic acid, amplified with the Amplify solution (Amersham Pharmacia), dried under vaccum and exposed for 24 h at –80°C.
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ACKNOWLEDGEMENTS |
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We thank Phillip Jordan for revising this manuscript. This work was supported by the Conseil Regional de Bretagne, the CNRS and the Association pour la Recherche contre le Cancer.
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FOOTNOTES |
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+ To whom correspondence should be addressed. Tel: +33 2 99 33 62 21; Fax: +33 2 99 33 62 00; Email: jean.mosser@univ-rennes1.fr Present address: Agnès C. Thénié, LEDAC,UMR 5538, Institut Albert Bonniot, Rond point de la Chantourne, 38706 La Tronche cedex, France
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