Human Molecular Genetics, 2000, Vol. 9, No. 2 203-216
© 2000 Oxford University Press
Identification and characterization of MTR1, a novel gene with homology to melastatin (MLSN1) and the trp gene family located in the BWS-WT2 critical region on chromosome 11p15.5 and showing allele-specific expression
Children’s Hospital, University of Mainz, Langenbeckstrasse 1, D-55101 Mainz, Germany, 1Department of Cancer Genetics, Roswell Park Cancer Institute, Buffalo, NY, USA and 2Department of Biochemistry and McGill Cancer Center, McGill University, Montreal, Canada
Received 17 August 1999; Revised and Accepted 17 November 1999.
DDBJ/EMBL/GenBank accesssion no. AF177473.
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Alterations within human chromosomal region 11p15.5 are associated with the Beckwith–Wiedemann syndrome (BWS) and predisposition to a variety of neoplasias, including Wilms’ tumors (WTs), rhabdoid tumors and rhabdomyosarcomas. To identify candidate genes for 11p15.5-related diseases we compared human genomic sequence with expressed sequence tag and protein databases from different organisms to discover evolutionarily conserved sequences. Herein we describe the identification and characterization of a novel human transcript related to a putative Caenorhabditis elegans protein and the trp (transient receptor potential) gene. The highest homologies are observed with the human TRPC7 and with melastatin 1 (MLSN1), whose transcript is downregulated in metastatic melanomas. Other genes related to and interacting with the trp family include the Grc gene, which codes for a growth factor-regulated channel protein, and PKD1/PKD2, involved in polycystic kidney disease. The novel gene presented here (named MTR1 for MLSN1- and TRP-related gene 1) resides between TSSC4 and KvLQT1. MTR1 is expressed as a 4.5 kb transcript in a variety of fetal and adult tissues. The putative open reading frame is encoded in 24 exons, one of which is alternatively spliced leading to two possible proteins of 872 or 1165 amino acids with several predicted membrane-spanning domains in both versions. MTR1 transcripts are present in a large proportion of WTs and rhabdomyosarcomas. RT–PCR analysis of somatic cell hybrids harboring a single human chromosome 11 demonstrated exclusive expression of MTR1 in cell lines carrying a paternal chromosome 11, indicating allele-specific inactivation of the maternal copy by genomic imprinting.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Cytogenetic and molecular studies have linked several diseases to the human chromosomal 11p15.5 region (reviewed in refs 1,2). The predominant and most complex of these disorders is the Beckwith–Wiedemann syndrome (BWS), which occurs in 1 in 13 700 births (3,4). The main characteristics of this congenital condition are exomphalus, macroglossia and gigantism, combined with an enhanced risk of developing neoplasias. Although the majority of BWS cases are sporadic, rare familial cases segregate in an autosomal dominant fashion with almost exclusive maternal inheritance. Approximately 20% of sporadic BWS cases show paternal uniparental disomy (patUPD) for markers in 11p15.5. Rarely, chromosomal aberrations have been observed in BWS cases involving this region including paternal p15 duplication and maternally derived translocations or inversions with 11p15.3–5 breakpoints. In BWS-associated tumors, loss of heterozygosity (LOH) for 11p15.5 involving the maternal allele is a frequent observation (5). The region associated with tumor suppression in G401 cells and growth suppression in RD cells was mapped to a region between D11S648 and D11S1318 by genetic complementation of subchromosomal transferable fragments (6). This region excludes IGF2 and H19 but encompasses the ‘BWS critical region 1’ [BWSCR1: D11S679–D11S551 (7)], a region defined by chromosomal rearrangement breakpoints in BWS patients. At least eight genes within 11p15.5 show a monoallelic expression pattern including two paternally (KvLQT1-AS [LIT1] and IGF2) and six maternally (IPL, ORCTL2, P57KIP2, KvLQT1, ASCL2 and H19) transcribed genes. This functional inequality of the two alleles in somatic cells is due to an epigenetic modification called genomic imprinting (8). It has been shown that disruption of imprinting leads to developmental dysregulation resulting in malformations and malignancies (9). Loss of imprinting (LOI) of 11p15.5 genes is a common finding in BWS-related tumors (10,11). To explain the phenotypes associated with BWS and the link between tumor initiation and the high frequency of UPD, LOH and LOI observed for 11p15.5, the involvement of a paternally expressed growth-promoting gene (12) and/or a maternally expressed growth/tumor-suppressing gene (13) has been proposed.
Several 11p15.5 genes with growth-related functions have been characterized but, with the exception of H19, IGF2 and P57KIP2, none has been implicated in BWS and/or BWS-associated neoplasias. IGF2 is a paternally transcribed insulin-related growth factor with a key role in hormone-triggered cell proliferation (14). A significant number of BWS patients and sporadic Wilms’ tumors (WTs) show, respectively, patUPD or LOI for IGF2, and this correlates with increases in protein expression levels (15,16). Transgenic mice with deregulated IGF2 expression show some features of BWS, such as prenatal overgrowth and macroglossia, but neither exomphalus nor a predisposition to tumors (17). H19 is maternally transcribed and codes for an RNA of unknown function but with possible tumor suppressor properties (18–20). The H19 gene locus seems to act as a cis-active element, regulating the transcription of IGF2 and INS. Mice deleted for H19 develop somatic overgrowth similar to BWS, apparently due to the enhanced expression of Igf2 and Ins2, rather than the lack of H19 transcripts (21). The expression pattern of H19 and the fact that no consistent imprinting defects nor mutations in H19 have been described in BWS or WT cases make a direct involvement of H19 in the etiology of these diseases unlikely. The third gene, P57KIP2, is a cyclin-dependent kinase inhibitor with a negative regulatory effect on cell proliferation (22,23) and is preferentially transcribed from the maternal allele (24). Approximately 5% of BWS patients (25–27) show mutations in P57KIP2 predicted to result in a loss of function. To date, this is the only gene within the BWSCR1 that shows functional mutations linked to the overgrowth syndrome. Targeted disruption of p57kip2 in mice results in abdominal muscle defects and kidney dysplasias but also features not common in BWS patients, such as cleft palate, endochondral bone ossification defects, lens cell hyperproliferation and apoptosis (28). Tokino et al. (29) and O’Keefe et al. (27) examined a large number of WTs for genetic alterations in P57KIP2 finding only polymorphisms unlikely to affect protein function. Together, IGF2, H19 and P57KIP2 can account for only some aspects of the BWS phenotype and tumors, suggesting that at least one associated gene remains to be found in the BWSCR1 at 11p15.5.
Clearly a unifying hypothesis for BWS and related malignancies will require an in-depth understanding of the genes in 11p15.5 and characterization of their expression and potential interplay. Accordingly, we report the characterization of a novel evolutionarily conserved sequence within BWSCR1 at 11p15.5. This gene, MTR1 (MLSN1- and TRP-related gene 1), seems to be regulated by genomic imprinting, is transcribed in a variety of tissues (including tumor samples) and shows homology to the trp gene family originally described in Drosophila melanogaster.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Screening for evolutionarily conserved sequences
To identify evolutionarily conserved sequences we compared the previously published genomic sequences of P1-derived artificial chromosome (PAC) clones encompassing the BWSCR1 (for a detailed map see ref. 30, http://gestec.swmed.edu/chromoso.htm ) with the Caenorhabditis elegans database at the Sanger Center. The human DNA sequence was translated in all six possible frames into amino acids and compared with C.elegans proteins (‘Wormpep’ database at http://www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml ). Figure 1 shows the sequence homology at the amino acid level of a segment of the human PAC pDJ915F1 (GenBank accession no. AC003693) to a putative C.elegans protein T01H8.5 (CE13022) which belongs to the group of transient receptor potential (trp) proteins. Homology between the 1863 amino acid C.elegans protein and the putative human protein ranges from 41 to 73% with 26–57% identity and extends in parts over 15.5 kb in the human DNA. The relative order of the protein subfragments with respect to their location in the complete open reading frame (ORF) is also conserved at the human DNA level. BLAST searches of human DNA against the available human expressed sequence tag (EST) and protein databases also revealed a similarity to the human TRP gene family including melastatin (MLSN1) and a single EST hit (AA577486 from a colon tumor). The putative new gene was named MLSN1- and TRP-related gene 1 (MTR1) due to the predicted homologies.
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PCR and hybridization experiments with human MTR1 probes demonstrated that a murine Mtr1 exists as part of a block of synthenic genes on distal mouse chromosome 7 corresponding to human chromosome 11p15.5 (data not shown).
Transcripts, alternative splicing and genomic structure of MTR1
Analysis of the genomic MTR1 subregion with GRAIL and Genescan predicted 24 exons encompassing the putative ORF (~3.9 kb) and spanning 18.5 kb. The location of MTR1 is proximal to TSSC4 and distal to the first 5' exon of the KvLQT1 gene, thus placing this new gene in the BWSCR1. To directly demonstrate that predicted exons were transcribed into mRNA, we carried out exon-connection experiments in order to amplify overlapping portions of the gene. Since the BWSCR1 is implicated in both BWS and WTs, we generated cDNAs from human fetal and adult kidneys. Since the amplification products spanned introns, a distinction between PCR products arising from transcribed sequences or untranscribed genomic DNA could be made. The minimal set of primers essential for covering the MTR1 ORF were MTR1E1F/MTR1E16R and MTR1E15F/MTR1E24R. Reverse transcription–polymerase chain reaction (RT–PCR) analyses from adult and fetal kidney RNA preparations yielded products of the expected size. PCR products were subcloned and sequenced on both strands. Interestingly, one Genescan-predicted exon (exon 19) could not be found in the clone obtained by RT–PCR. However, an additional exon, not predicted by the informatics approach described above, was reproducibly included in the RT–PCR product (exon 23A). Furthermore, the primer pair MTR1E15F/MTR1E24R generated two PCR products from the input cDNA, differing in one exon (exon 18). We tentatively conclude that this exon is alternatively spliced. Comparison of the genomic sequence with that of the MTR1 transcript enabled us to determine the precise exon–intron boundaries of the MTR1 gene (Fig. 2).
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Within the KvLQT1 gene an antisense transcript (LIT1 or KvLQT1-AS) could be located, which is transcribed in the same direction as MTR1; furthermore, it is also paternally expressed and maps nearby (31,32). As the 3' end of KvLQT1-AS has not been reported yet, we carried out exon-connection experiments on reverse transcribed RNA expressing both transcripts. We did not detect any RT–PCR products derived from a spliced LIT1/MTR1 transcript (data not shown). Thus, it seems to be unlikely that both transcripts are part of one single gene.
Expression of MTR1
The amplification product of the primer pair MTR1E15F/MTRE24R was gel purified and used to determine the expression profile and transcript size of the MTR1 gene on multiple tissue northern (MTN) blots. To exclude cross-hybridization to genes from the TRP gene family, Southern blots of restricted genomic DNA and genomic pDJ915F1 were hybridized under identical conditions. In the Southern blots identical signals were obtained in genomic DNA fragments as well as in the PAC clone fragments, thus demonstrating a specific hybridization, without indications for cross-hybridization. A 4.5 kb transcript could be detected in a variety of fetal and adult tissues (Fig. 3A). Since the confirmed exons comprise only 3913 bp, these results suggest that ~500 bp of untranslated mRNA sequence remain to be identified. On northern blots, MTR1 shows significant expression in fetal liver, kidney and spleen, as well as in adult kidney, liver, small intestine, lung, pancreas, colon, prostate, pancreas and the gland tissues (Fig. 3B). The MTN result shows that MTR1 is transcribed in a large number of tissues, although only one EST of this gene was present in the EST database. Since the two putative spliceforms of MTR1 differ in exon 18 only (175 bp) resolution of the two transcripts would not be expected on northern blots.
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Protein structure predictions
The predicted structure of the RT-generated MTR1 cDNA indicated several interesting features. Due to the alternatively spliced exon (exon 18), at least two isoforms are expected. Exon 1 contains an almost ideal Kozak consensus sequence, suggesting that initiation of translation may occur at this position. The longest putative ORF with this initiation codon and including exon 18 encompasses 3495 bp, coding for a predicted polypeptide of 1165 amino acids (Fig. 4). The expected molecular weight is 131.45 kDa. Secondary structure prediction algorithms suggest a protein with several transmembrane domains (six predicted with SOSUI and one additional seventh domain in the N-terminal region predicted with PSORT II). The second splice version encodes a smaller ORF of 872 amino acids (Fig. 4). This putative protein shares the first 869 amino acids with the longer MTR1 protein, consisting of 4–5 transmembrane domains. The expected molecular weight is 97.74 kDa. All prediction programs locate both MTR1 proteins most likely in the plasma membrane with the C-terminus inside the cell.
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Paternal expression of MTR1 mRNA
As previously described, the preferential monoallelic expression of imprinted genes is maintained in somatic cell hybrids carrying a single human chromosome 11 (31,33). This enables the use of cell hybrids containing either a maternally or a paternally derived human 11p15 chromosomal region as a model system to identify imprinted genes from the BWSCR1 area.
We tested MTR1 gene expression by RT–PCR in a subset of the somatic-cell hybrids which were previously described by Gabriel et al. (33). These included two cell lines with a maternally derived human chromosome 11 (GM7300, GM13400) and three somatic cell hybrids with a paternal copy of 11p15 (GM10927B, GM11944, GM11941). To prove the maintainance of the epigenetic chromatin states, we tested the hybrids for differential methylation at a previously described maternally methylated NotI restriction site within the KvLQT1 gene region (31–33). All cell hybrids tested showed the methylation according to their parental origin (Fig. 5A, maternal hybridization signal at 4.2 kb, paternal at 2.7 kb), thus confirming the expected epigenetic chromatin states. To verify the integrity of the 11p15.5 transcripts, we then tested the cell hybrids for the presence of a coding fragment of NAP1L4 as a biallelic transcript. Figure 5B shows the RT–PCR products obtained for the abovementioned gene fragment, demonstrating the presence of 11p15.5 in the hybrids under study.
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The three somatic cell hybrids containing the paternally derived chromosome 11 showed an MTR1 RT–PCR product (GM11941 only following Southern blot hybridization), whereas no product was observed from hybrids containing a maternal copy of chromosome 11. The PCR products obtained in this experiment were confirmed to be derived from MTR1 by hybridization of the blotted gel with a radioactively labeled MTR1 probe and by direct sequencing of one of the PCR products (data not shown). The data indicate that the MTR1 gene shows (preferential) paternal expression.
Expression of MTR1 in tumor RNAs
Approximately 5–15% of WTs are due to mutations within the Wilms’ tumor gene 1 (WT1), located on chromosomal region 11p13 and encoding a zinc finger-containing transcription factor. WTs are heterogenous with variable contents of blastemic, stromal and epithelial tissue. We therefore selected a representative number of 47 WTs, including two cases with LOH at chromosome 11p (WT128cl, WT188) and two WT cell lines (WT128cl, WCCS). Each tumor was histologically characterized and analyzed for WT1 mutations. WT128 and WT127 were the only WTs that had a mutation in WT1 [WT128 premature stop codon published by Löbbert et al. (34), WT127 premature stop codon in zinc finger 2 (exon 8) (data not shown)]. Equal amounts of total RNA (2 µg) from the 47 tumors, five unaffected control tissues (fetal and adult kidney), and four DNA probes of different concentrations (1–25 ng) were used to prepare an RNA dot blot. After hybridization with a radioactively labeled MTR1 cDNA fragment, signals could be detected in varying amounts in almost all WT samples. On average these signals were higher than in the normal tissues (Fig. 6). Hybridization signals were randomly confirmed by RT–PCR on the tumor samples with the primer pairs MTR1E15F/MTR1E16R and MTR1E17F/MTR1E21R. The latter primer pair encompasses the alternatively spliced exon 18. All RT–PCRs showing amplification with these primers displayed both splice versions of MTR1 (data not shown). Both WT cell lines showed marginal MTR1 signals on hybridization. We interpret these signals as blot background, which is in agreement with the negative RT–PCR results. In addition, three BWS patient tumor samples (without WT1 mutation) were analyzed by RT–PCR, showing moderate amounts of MTR1 transcript.
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As alterations in 11p15.5, especially in BWSCR1, show correlation not only with the development of WTs but also with rhabdomyosarcoma (35), tissue samples of three rhabdomyosarcomas were included in this study. RT–PCR with the abovementioned MTR1 primer pairs showed amplification, indicating that MTR1 is also expressed in this tumor type (data not shown).
Translocation of the MTR1 gene in a rhabdoid tumor (TM87-16)
Malignant rhabdoid tumors (MRTs) belong to the tumor group of soft tissue sarcomas and are extremely aggressive, with a high metastatic potential. They were first thought to be variants of WTs but, as they have been observed in a variety of extrarenal sites, they are now regarded as a distinct tumor type. Karnes et al. (36) established the MRT cell line TM87-16 from a pleural effusion of a retroperitoneal mass diagnosed in a 21-month-old male Caucasian. Chromosomal analysis showed a reciprocal translocation t(11;22)(p15.5;q11.23), the breakpoint of which is located in the genomic region spanned by PAC clone pDJ915F1 (30), the same clone that contains MTR1. Since the exact breakpoint has not been determined, we amplified genomic fragments for MTR1 and used them in a fluorescence in situ hybridization (FISH) analysis of TM87-16 metaphase spreads. The FISH experiment showed that MTR1 has been completely translocated onto the derivative chromosome 22, without being disrupted (Fig. 7). Therefore, the translocation breakpoint in TM87-16 is proximal to MTR1. RT–PCR analysis of total RNA from TM87-16 with primers MTR1E15F/MTR1E16R showed moderate MTR1 expression (data not shown).
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-satellite and green signals to the genomic MTR1 region including the putative promoter region 5' of MTR1. The cell line shows a reciprocal t(11;22)(p15.5;q11.23) translocation, with location of the chromosome 11 breakpoint in the genomic region spanned by PAC clone pDJ915F1 (30), the same clone that contains MTR1. Hybridization signals were only obtained on the intact chromosome 11 and the derivative chromosome 22, thus demonstrating complete translocation of MTR1 onto the derivative chromosome 22, without disruption of the gene. |
DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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The intensive study of chromosomal aberrations in neoplasias (37–39) and the characterization of rare breakpoints in the complex overgrowth syndrome BWS (30,40) helped to link these diseases to the BWSCR1 in 11p15.5 between the markers D11S648 and D11S551 (41). Molecular data, including the experiments involving the transfer of subchromosomal fragments into tumor cell lines, showed that 11p15.5 harbors at least one gene with oncogenic potential and/or one tumor suppressor gene (6,42). The mechanisms involved in disease development could include disruption of developmental genes or impairment of their regulation. Breakpoints associated with sporadic cases of BWS cluster in the area within or near the KvLQT1 gene, an ion channel gene associated with cardiovascular diseases when mutated. Due to an absence of phenotypic overlap between BWS and long-QT syndrome (LQT), the KvLQT1 gene itself is unlikely to be involved in the initiation of neoplasias or the dysmorphology of BWS. Therefore, other genes located within the BWSCR1 must be responsible for these disorders.
Most 11p15.5 genes, such as IPL, IMPT1/ORCTL2 and KvLQT1 were isolated using positional cloning strategies. With increasing sequence data available from the Human Genome Project, other gene isolation strategies will prove to be more effective. The recently isolated TSSC4 gene (43) was initially detected by the use of an exon-trapping strategy which identified a number of exons mapping to an area between KvLQT1 and IGF2. They were used to isolate, in silico, EST contigs, which then helped to determine the gene structure by comparison with the genomic sequence of the region. Clearly, a limiting component of this approach is that rare transcripts are under-represented in the EST databases. Another approach to identify genes and important regulatory sequences takes advantage of the fact that genomic regions with essential functions are evolutionarily conserved. This approach can be achieved by cross-hybridization of genomic fragments against cDNA/genomic libraries of other species (zoo-blots, e.g. ref. 44) although this can produce hybridization artifacts resulting in false positive signals.
Since whole genome sequencing has made substantial progress in such organisms as Saccharomyces cerevisiae and C.elegans, it is now also possible to perform in silico analysis of cross-species relatedness. In this paper, we describe the use of comparative sequence analysis of the human PAC clone pDJ915F1 (GenBank accession no. AC003693) with the C.elegans database, that led to the isolation of the human MTR1 gene. The homology of MTR1 to the C.elegans trp protein-related gene CE13022 extends over large parts of the ORF, with average homologies of 50% at the amino acid level. Genescan and GRAIL analyses enabled a more detailed exon–intron structure prediction of the human MTR1 on pDJ915F1. The homology in the coding region between C.elegans and human MTR1 extends from exon 2 to exon 18 of the predicted 24 exons.
It is surprising that, although MTR1 seems to be expressed at the level of a northern blot, only one EST clone (GenBank accession no. AA577486) representing this locus could be detected in the databases. This suggests either a rare transcript, a restriction site within the 3' end of the cDNA that was used for directed subcloning of the cDNA libraries (e.g. NotI), or a ‘difficult-to-clone’ transcript (due to instability of the mRNA, toxicity of the cDNA or secondary structure impedence of the RT). RT–PCR on RNA isolated from normal kidney tissue was performed to demonstrate that MTR1 is indeed transcribed, and not an unprocessed pseudogene. The predicted exons were amplified within overlapping cDNA fragments, with a minimal set of two products being necessary to cover the complete 3913 bp-spanning ORF. During this process of characterization it became evident that two alternatively spliced isoforms of MTR1 exist. These arise as a result of alternative splicing of exon 18. Both splice versions could be detected in RNA from all tested tissues expressing MTR1 (data not shown).
The resulting protein encoded by the larger of the two ORFs shows several predicted transmembrane domains, numbering either six or seven depending on the prediction algorithm used. Transmembrane prediction is based on physicochemical properties of a protein, leading to transmembrane helical structures. PSORT II predicts transmembrane data with an accuracy of 66% (45), whereas SOSUI detects the transmembrane helices with an 85–96% accuracy (46, reviewed at http://www.tuat.ac.jp/~mitaku/adv-sosui/about.html ). Based on this information, we suggest that the most likely structure for the MTR1 protein is one that contains six transmembrane domains, predicted by the SOSUI software, as shown in Figures 4 and 8.
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BLAST analysis revealed homology between the longest MTR1 isoform and the human TRPC gene family (best homology with TRPC7). The four known human TRPC genes (TRPC1, -3, -6, -7) encode proteins with sequence similarity to Drosophila TRP. Considering the initial homology to the C.elegans trp gene that was used to detect MTR1, this finding is not unexpected. Among the human TRP family two subgroups can be defined based on sequence similarity consisting of TRPC1, -3 and -6 on the one hand and TRPC7 on the other hand. Like TRPC7, MLSN1 is another distant member of the human TRP gene family. It contains 27 exons and codes for a 1533 amino acid protein. Due to the loss of MLSN1 expression in highly metastatic malignant melanoma cells, a role for the gene product in suppression of metastasis has been suggested (47). Both MLSN1 and MTR1 have the greatest degree of amino acid homology to TRPC7. The similarity of MTR1 to TRPC7 and the remaining TRP family is not limited to sequence homology. The predicted transmembrane domains in MTR1 cluster in a corresponding number and with a very similar spacing as the transmembrane domains of TRP gene family members. The TRP gene family generally has six transmembrane domains although TRPC3 and TRPC7 contain seven predicted membrane-spanning domains (as predicted by the SOSUI program). A highly conserved EWKFAR motif shortly after the last transmembrane domain is present in the proteins coded by >90% of all TRP genes (48). In cTRP (C.elegans trp) the motif is EWKFHR, in MLSN1 it is VWKFQR and in MTR1 it is FWKFQR (differences underlined). The alternatively spliced version of MTR1 leads to an interruption of the TRP-related protein before the last two proposed transmembrane domains, thus reducing the degree of similarity to the TRP gene products. Figure 8 shows the similarity of MTR1 to C.elegans CE13022, MLSN1 and TRPC7 and to the more distant human TRPC1 gene product illustrating the points discussed above. Additional evidence supporting the relatedness of MTR1 and the TRP gene family is the predicted cytoplasmic localization of the C-terminus of the (predicted) protein.
Aside from the transmembrane segments and domains mentioned above, the TRPC genes differ in their amino acid sequence. TRPC7 is only ~20% identical to TRPC1 and TRPC3. They also differ regarding their expression profiles: TRPC7 is expressed only in brain (49), whereas TRPC3 expression is almost ubiquitous (50). The main difference between MTR1 and the basic TRP gene family is the lack of ankyrin repeats within MTR1, a feature that MTR1 shares with MLSN1 (47). These adaptor epitopes link plasma membrane proteins to cytoplasmic proteins through a 33 amino acid repeated motif. They also may function in protein–protein interactions among plasma membrane proteins, mediating the activation of the channels (51). MTR1, MLSN1 and TRPC7 thus seem to be more distant relatives of the TRP calcium channel family. The TRP gene family seems to be crucial for agonist-activated capacitive Ca2+ entry in cells (50). This ion influx replenishes Ca2+ stores depleted by a variety of stimuli and is essential for appropriate cellular responses to hormones and growth factors (50).
Recently, another gene, VRL-1, with similar domains has been characterized (52). It encodes a vanilloid receptor (VR1)-related protein with three ankyrin repeats, six transmembrane domains, a cytoplasmic C-terminus and a putative pore-loop region. VRL-1 is activated by heat, resulting in cation-mediated currents (e.g. Ca2+) in VRL-1-expressing cells. A murine homolog of VR1, named Grc (growth factor-regulated channel) has been shown to mediate Ca2+ entry after activation by IGF-1 (53). IGF-1, like the previously mentioned IGF-2, is a growth factor that promotes progression of the cell cycle in several cell types and may also play an important role in tumorigenesis (54). As the inhibition of calcium entry blocks the growth-promoting effect of IGF-1, stimulation of calcium entry should be essential for growth induction. As in Grc, VR1/VRL-1 and MLSN1, the region of highest homology between MTR1 and the Trp genes is centered around the putative pore region and the last transmembrane domain, suggesting a similar function of MTR1 in the Ca2+ regulation in cells.
It has been shown (55) that in Drosophila trp proteins interact with related trpl proteins [40% identical over the N-terminal ~700 amino acids (56)], forming characteristically different channels compared with homomultimer proteins. Another heteromultimeric association of a TRP gene family member is the association of TRPC1 with PKD2 (57). Mutations in PKD2 and PKD1—each one coding for a membrane protein—are responsible for the majority of cases of autosomal dominant polycystic kidney disease (ADPKD). Patients develop kidney and liver cysts which result in renal failure in 50% of these cases (58). PKD2 can form a complex with TRPC1, where the C-terminal domains are sufficient to mediate the association (57). A possible effect of PKD2 in modulating the function of voltage-activated Ca2+ channels of the Trp family is a current hypothesis (57). It has been shown that PKD1 also interacts with multiple members of the TRPC family independently of PKD2, and overexpression of all three proteins does not disturb pairwise interaction. Thus, it is likely that mutations in PKD2 or PKD1 may result in aberrant responsiveness to Ca2+ store depletion (57). Such a heteromultimeric association between two proteins may also be the case with MTR1 (if MTR1 behaves according to the structural predictions) and other members of the membrane proteins including the TRPC gene family. Alternatively, the two MTR1 isoforms may interact with each other.
The fact that MTR1 seems to be imprinted showing paternal expression makes it an interesting candidate gene, which may be involved in the etiology of BWS and/or some of the neoplasias associated with 11p15.5 alterations. As mentioned above, a paternally transcribed growth-promoting gene would fit into the picture of a candidate gene for these diseases. It remains to be seen whether MTR1 meets the requirements to function as a channel gene or as a channel modulator and whether it plays a role in hormone and growth factor responses, as suggested by the structural homology. Nevertheless, MTR1 is transcribed in tissues involved in BWS malformations and tumors [i.e. kidney and liver as well as WTs and rhabdomyosarcomas (Figs 5 and 6)]. In the analyzed rhabdoid tumor cell line TM87-16 MTR1 is translocated completely to the derivative chromosome 22 (Fig. 7). This translocation does not result in enhanced gene activation. Further studies involving functional and mutational analyses are necessary to define the role that MTR1 may play in the pathogenesis of BWS and/or neoplasias associated with the BWSCR1.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
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Sequence analysis
DNA and EST database searches were performed using the BLAST programs on the Sanger Center server (http://www.sanger.ac.uk/Projects/C_elegans/blast_server.shtml ) for searches on C.elegans databases and on the NCBI server (http://www.ncbi.nlm.nih.gov/ ) for all other comparisons. Genomic sequences were analyzed for putative exon sequences using the Genescan software package (http://www.hgmp.mrc.ac.uk/ GenomeWeb/nuc-geneid.html ). Comparison analyses between proteins were done using the Clustal program package at the Genebee server (http://www.genebee.msu.su/ ). PCR primers which flank the predicted ORF of MTR1 were synthesized and used to amplify cDNA fragments that were directly sequenced after removal of unincorporated primers on an ABI377 sequencer (Applied Biosystems, Weiterstadt, Germany). Larger cDNA fragments were subcloned in pBluescript and sequenced with internal primers as well as with the flanking T3 and T7 primers.
RT–PCR analysis
Total RNA (2 µg) was reverse transcribed after DNase I digestion, using 250 pmol oligo(dT) primer and 300 U of MMLV reverse transcriptase (Gibco BRL, Eggenstein, Germany) in a total volume of 40 µl. Two microliters of this RT were analyzed in a 20 µl PCR, using the following gene-specific primers:
MTR1E15F, 5'-gtgctgtcttcctgctcac-3',
MTR1E16R, 5'-tgacacccacgatgaacagg-3',
MTR1E17F, 5'-ggacttcatggtgttcacgc-3',
MTR1E21R, 5'-cgtggtactccacaatcagg-3',
MTR1E1F, 5'-ccatgcaggatgtccaaggc-3',
MTR1E24R, 5'-tcaggcaacacaagtcagg-3'.
Primer pairs MTR1E15F/MTR1E16R and MTR1E17F/MTR1E21R were used to demonstrate transcription of the MTR1 gene in RTs with the following PCR conditions: initial denaturation for 3 min at 94°C, 35 cycles (denaturation for 1 min at 94°C, annealing for 1 min at 58°C, extension for 1 min at 72°C) and a final polishing step of 7 min at 72°C, using 1.5 mM MgCl2, 25 pmol of each primer and 5 U Taq polymerase. Primer pairs MTR1E1F/MTR1E16R and MTR1E15F/MTR1E24R were used to amplify the complete ORF in two overlapping fragments, using the Expand PCR system with conditions according to the manufacturer’s protocol (Boehringer Mannheim, Mannheim, Germany).
Southern and northern analyses
Genomic DNAs and total RNA were prepared by standard methods [proteinase K/phenol treatment and RNeasy columns (Qiagen, Hilden, Germany), respectively]. For Southern blotting 10 µg of genomic DNA were completely digested with appropriate amounts of the restriction endonucleases. After electrophoresis on a 0.8% agarose gel, the DNA was depurinated for 15 min with 0.25 M HCl and denatured for 30 min with 0.5 N NaOH/1.5 M NaCl. The transfer of DNA and RNA fragments onto Hybond N+ membrane (Amersham, Freiburg, Germany) was performed with 10x SSC for 48 h, followed by ultraviolet crosslinking. Northern dot-blots were obtained by dotting 2 µg of total RNA in a symmetric array on a nylon membrane (Hybond N+; Amersham), followed by ultraviolet crosslinking. In addition, multiple human fetal tissue northern blots (Clontech, Palo Alto, CA) were used for more detailed analysis of MTR1 expression. Complementary DNA probes of the MTR1 gene were amplified by PCR and gel purified with the Qiaquick gel extraction kit (Qiagen) according to the manufacturer’s protocol and 32P-radiolabeled using a nick-translation standard protocol. Hybridization of all blots was at 68°C in Expresshyb solution (Clontech) for 1–2 h, using 2 x 106 c.p.m./ml of probe. Post-hybridization washes were performed following the suggestions of the manufacturer of Expresshyb solution (Clontech). Autoradiography was performed for 16–120 h with intensifying screens at –80°C.
Cell lines
Somatic cell hybrids containing a paternal (GM10927B, GM11944, GM11941) or maternal (GM07300, GM13400) chromosome 11 in a rodent background were maintained as described previously (33). Rhabdoid cell line TM87-16 (36) was maintained in Dulbecco’s modified Eagle’s medium, supplemented with 10% heat-inactivated fetal calf serum, PEN/Strep (Sigma-Aldrich, Deisenhofen, Germany) and glutamine.
Assessment of allelic expression
cDNAs from the somatic cell hybrids containing either the paternal or maternal chromosome 11 were amplified using the primers and amplification protocols described above. PCR products were separated on a 2% agarose gel and transferred onto a membrane as described above. Products were verified by hybridization with a 32P-labeled cDNA probe and direct sequencing of one PCR product. Controls for DNA contamination were performed for all sets of primers with identical RT–PCR conditions but without RT of the prepared RNA.
FISH analysis
FISH on metaphase chromosomes was performed as described previously (59,60). Metaphase spreads from TM87-16 cells were prepared as described previously (34,59,60). Genomic fragments encompassing the MTR1 gene were amplified with flanking primers, using the Expand PCR system (Boehringer Mannheim) according to the manufacturer’s protocol. These fragments were nick-translated with DIG-11-dUTP according to Boehringer Mannheim’s protocols and hybridized to the denatured chromosomes overnight at 37°C in a humidified chamber. Post-hybridization washes were performed at 42°C in 50% formamide, 1x SSC three times for 5 min and in 0.3x SSC three times for 5 min. Immunodetection of the hybridized sequences was performed using a mouse anti-DIG-antibody (Boehringer Mannheim), diluted 1:1000 in 4x SSC, 0.1% Tween-20, and a TRITC-labeled rabbit anti-mouse IgG (Boehringer Mannheim) diluted 1:100, followed by a signal amplification with TRITC-labeled anti-rabbit IgG (Boehringer Mannheim) diluted 1:64. Chromosomes were counterstained with 4',6-diamidino-2-phenyl-indole, and covered with Vectashield (Vector Laboratories, Burlingame, CA). FISH was recorded using a High-Performance-CCD camera (Applied Imaging, Linz, Germany) and analyzed with the Cytovision 2.21 software (Applied Imaging).
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ACKNOWLEDGEMENTS |
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The authors thank A. Langer, J. Busch, M. Engel, O. Steinbeck and N. Krieger for technical assistance and J. Bukur for valuable discussions. Rhabdomyosarcoma tissue samples were provided by U. Kontny (Children’s Hospital, University of Freiburg, Germany). The work was funded by a grant of the Deutsche Forschungsgemeinschaft to B.Z. and A.W. J.P. is an MRC scientist supported by an NCIC grant. M.H. was supported by NIH grant CA63333.
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FOOTNOTES |
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+ To whom correspondance should be addressed. Tel: +49 6131 17 6826; Fax: +49 6131 17 5528; Email: prawitt@wserv.kinder.klinik.uni-mainz.de
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REFERENCES |
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M. Paulsen, O. El-Maarri, S. Engemann, M. Strodicke, O. Franck, K. Davies, R. Reinhardt, W. Reik, and J. Walter Sequence conservation and variability of imprinting in the Beckwith-Wiedemann syndrome gene cluster in human and mouse Hum. Mol. Genet., July 22, 2000; 9(12): 1829 - 1841. [Abstract] [Full Text] [PDF]
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X.-Z. S. Xu, F. Moebius, D. L. Gill, and C. Montell Regulation of melastatin, a TRP-related protein, through interaction with a cytoplasmic isoform PNAS, September 11, 2001; 98(19): 10692 - 10697. [Abstract] [Full Text] [PDF]
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