The WTX/AMER1 gene family: evolution, signature and function
© Boutet et al; licensee BioMed Central Ltd. 2010
Received: 4 February 2010
Accepted: 15 September 2010
Published: 15 September 2010
WTX is a novel gene mutated in a proportion of Wilms' tumors and in patients suffering from sclerosing bone dysplasia. On the molecular level WTX has been shown to act as an antagonist of canonical Wnt/β-catenin signaling in fish and mammals thus linking it to an essential pathway involved in normal development and cancer formation. Interestingly, WTX seems to also localize to an intranuclear component called paraspeckles. In spite of the growing interest of molecular biologists in WTX, little is known about its paralogs and its phylogenetic history.
Using the amino-acid sequence of WTX/AMER1 as a tool for the assignment of orthology and paralogy, we here identify two novel proteins, AMER2 and AMER3, as "WTX" related. This Amer gene family is present in all currently available vertebrate genome sequences, but not invertebrate genomes and is characterized by six conserved blocks of sequences. The phylogenetic analysis suggests that the protoAmer gene originated early in the vertebrate lineage and was then duplicated due to whole genome duplications (WGD) giving rise to the three different Amer genes.
Our study represents the first phylogenetic analysis of Amer genes and reveals a new vertebrate specific gene family that is likely to have played an important role in the evolution of this subphylum. Divergent and conserved molecular functions of Wtx/Amer1, Amer2 and Amer3 are discussed.
Early 2007, a search for genes deleted in Wilms' tumors, a pediatric solid tumor of the kidney led to the identification of the X-linked gene WTX (also called AMER1) . Using large-scale interactome mapping a second independent study demonstrated that WTX induces degradation of β-catenin via the proteasome system, thus identifying this gene as an important modulator of this crucial signaling pathway . Wtx/Amer1 also physically interacts with APC , a tumor suppressor gene involved in colorectal cancer . In addition, on the cellular level WTX localizes to subnuclear domains that have been identified as paraspeckles . Recent analysis suggests that WTX may also play an important function during normal development: expression analysis demonstrated a dynamic expression pattern throughout embryogenesis  and mutations have been identified in patients suffering from a range of developmental defects including osteopathia striata congenita with cranial sclerosis (OSCS) and cardiac anomalies . To better characterize the functional and structural properties of the WTX/AMER1 gene it is essential to understand its molecular evolution and its phylogenetic history. Duplications are common events during evolution and are one of the main driving forces for the emergence of new genes that can lead to the appearance of new gene families. The aim of the present study was to identify potential new members of the "WTX/AMER" family, characterize their phylogenetic relationships and analyze their evolutionary history.
Results and Discussion
Wtx/Amer1 is the founding member of a novel vertebrate gene family
To further investigate the evolutionary origin and phylogenetic distribution of the Amer genes, we extended the in silico searches to other available genomic databases. We could identify orthologs of Wtx/Amer1, Amer2 and Amer3 in primates (i.e. Pan troglodyte), and all other studied mammals (i.e. Canis familiaris), including the most basal therian groups, xenarthra (i.e. Dasypus novemcinctus), marsupials (i.e. Monodelphis domestica) and monotremes (i.e. Ornithorhynchus anatinus). The three Amer genes were also present in other tetrapod species such as birds (i.e. Gallus gallus), amphibians (i.e. Xenopus tropicalis) and in actinopterygians (i.e. the teleost fish Danio rerio) (for a complete list of orthologs see additional file 2; Table S1). We were unable to find any ortholog in cyclostome genomes such as the lamprey (Petromyzon marinus), but this is probably due to the fact that this genome has not yet been completely sequenced and assembled.
In stark contrast to their presence in apparently all vertebrates, no Amer genes could be identified in the genomes of model invertebrates including flies or nematodes and other protostomes such as mollusks (Lottia gigantea) and annelids (Capitella capitata). A similar situation already exists for other Wnt signaling components such as the Dkk family, which was believed to be vertebrate specific . More recently, however, Dkk genes have been described in the diploblastic hydra and sea anemone . It has, therefore, been postulated that gene families are missing in model protostomes due to the fast evolutionary rates and rampant gene loss in Drosophila and Caenorhabditis elegans genomes . To test whether a similar situation may exist for the Amer gene family, we searched for Amer orthologs in a wide range of metazoans, from the most basal (cnidarians and placozoans) to the vertebrates' closest relatives. No hits in any of the analyzed genomes could be detected, not even in non-vertebrate chordates (i.e. Branchiostoma floridae, Ciona intestinalis) and other non-vertebrate deuterostomes such as sea urchin (Strongylocentrotus purpuratus) despite the low occurrence of gene loss in some of these species . Although we cannot rule out the possibility that Amer-related genes are present in some of these species, but have diverged beyond recognition, our data strongly suggest that this gene family is vertebrate specific. While the six conserved blocks of sequences found in the human and mouse proteins are present in all the vertebrate orthologs (Figure 1 and additional file 1, Figure SM1), PROSITE analysis did not identify protein domains of known function (additional file 1, Figure SM2). Taken together, our data support the common origin of the three genes, which seemed to have arisen at the time of the origin of vertebrates.
Phylogenetic relationship of Wtx/Amer1, Amer2 and Amer3
The radial phylogenetic tree (additional file 3, Figure SM6) confirmed the subdivision of the three genes into three different groups.
Divergent and conserved molecular functions of Wtx/Amer1 genes
In this study we reported the first phylogenetic analysis of the Amer gene family and identify Amer3 as a novel family member. The common ancestry of the three Amer genes is supported by the presence of six conserved sequence blocks in all the orthologs, as well as the syntenic conservation with NTMR and ARHGEF paralogs in their three genomic loci. The absence of any sequence similarity to Amer genes in invertebrate genomes and the presence of three Amer paralogs in all studied vertebrate species strongly support the hypothesis that these genes are novel inventions that originated early in vertebrate evolution. Additional functional studies using animal models misexpressing Amer2 and Amer3 or knock out alleles will be required to determine their exact function in embryogenesis. Given the importance of Wtx/Amer1 in normal development and its involvement in human pathologies, it is tempting to speculate that mutations in Amer2 and Amer3 may also underlie developmental disorders.
Database search and sequence queries
Search for Amer homologous proteins in vertebrates was made through the NCBI and Ensembl (http://www.ensembl.org; release 56, september 2009) protein databases (see the complete list in additional file 2; Table S1). In silico proteins were obtained using GenomeScan software (http://genes.mit.edu/genomescan.html). In order to find potential orthologs in invertebrate genomes, mouse Wtx/Amer1, Amer2 and Amer3 were blasted against the amphioxus (http://genome.jgi-psf.org/Brafl1/Brafl1.home.html), the sea urchin (http://www.spbase.org/SpBase/) the sea anemone (Nematostella vectensis, http://genome.jgi-psf.org/Nemve1/Nemve1.home.html), annelid (Lottia gigantea, http://genome.jgi-psf.org/Lotgi1/Lotgi1.home.html), mollusk (Capitella capitata, http://genome.jgi-psf.org/Capca1/Capca1.home.html) and placozoan (Trichoplax adhaerens, http://genome.jgi-psf.org/Triad1/Triad1.home.html) genomes using tblastn under unrestrictive conditions (e-value = 100). No hits were found. The absence of Wtx/Amer1 from the Drosophila genome has been previously reported .
Alignment and phylogenetic analysis
Amino acid sequences of Wtx/Amer1, Amer2, Amer3 were aligned using Clustal W  and manually corrected with BioEdit [14, 15]. Bayesian Inference trees were performed using MrBayes 3.1.2 [16, 17] with the model recommended by ProTest 1.4  under the Akaike information and the Bayesian information criterions. We used the JTT + I + G model for both trees. Convergence was reached when the value for the standard deviation of split frequencies stayed below 0.01. Burn-in was determined by plotting parameters across all runs for both analysis: all trees prior to convergence were discarded and consensus trees were calculated for the remaining trees. We used one MrBayes run of 1,000,000 generations and 453,000 generation burn-in for the tree presented in Figure 3A and one MrBayes run of 2,000,000 generations and 1,893,000 generation burn-in for the tree presented in Figure SM6 (additional file 3). The trees were viewed and edited with the TreeExplorer programme in MEGA 4.0 . Maximum likelihood analysis was conducted using RAxML version 7.0.3  using a JTT model of evolution, 1000 bootstrap replicates and the rapid bootstrapping algorithm. The phylogenetic tree obtained using ML is consistent with the one obtained by Bayesian inference (data not shown). Alignment files used to calculate the phylogenetic trees are presented in Figure SM7 and SM8 (additional file 4 and 5 respectively).
The plasmid expressing human β-catenin was kindly provided by Jurgen Behrens (University of Erlangen, Germany). The TOP-FLASH and FOP-FLASH luciferase reporter constructs have been described previously . Briefly, the TOP-FLASH construct is designed to measure transcriptional activation mediated by β-catenin and FOP-FLASH is the mutated counterpart of the TOP-FLASH plasmid. Mouse Wtx/Amer1, Amer2 and Amer3 expression plasmids were obtained by PCR amplification of the coding sequence from BAC constructs (bMQ-277D5 for Wtx/Amer1, bMQ-344G22 for Amer2 and bMQ-123E19 for Amer3) (Geneservice, Cambridge, UK)) and insertion into the pAcGFP1-C1 plasmid (Clontech). All constructs were verified by DNA sequencing.
Detection of Wtx/Amer1 and Amer2 variants by RT-PCR
Total RNA from E9.5 to E14.5 dpc mouse embryos or Wtx/Amer1, Amer2 or Amer3 transfected HEK293T cells was extracted using TRIzol reagent (Invitrogen) and RNA purification was performed using the RNeasy Mini kit (Qiagen) and Rnase-free DNAse digestion (Qiagen). cDNA were synthesized from 1 μg of RNA using the MMLV reverse transcriptase system and random hexamers (Invitrogen). All PCR assays were performed using the GoTaq® Green Mix for PCR (Promega). Amplification of the N-terminus of Wtx/Amer1, Amer2 and Amer3 was performed using three different pairs of primers for each gene. Primer sequences used in Figure 2 are as follows: Wtx/Amer1, foward: tgaggcaacagaaggacca, reverse: tggagagtcaacaggatgaagctgttcaa; Amer2, foward: atggactcgcattgtgagtgcg, reverse: cgagctcccatctgcaaa; Amer3, foward: gaggagaggaaagaccttcatc, reverse: tcccagaacttgttgaagtctg. The other primer pairs used in this assay are available on demand. Briefly, all forward primers were located in the non-coding exon 1 or at the 5'end of exon 2 and all reverse primers just downstream of the internal splicing acceptor. PCR products were sequenced (SeqLAB, Sequence Laboratories, Göttingen, Germany) to check the specificity of Amer variants.
HEK293T cells were cultured in DMEM supplemented with 10% (v/v) fetal calf serum, in a 5% CO2 humidified atmosphere. HEK293T cells were transiently transfected with the TOP-FLASH reporter or its mutated counterpart (FOP-FLASH) and β-catenin with either Wtx/Amer1, Amer2 and Amer3 expression plasmids (200 ng each). Cells were also co-transfected with 50 ng of CMV (cytomegalovirus)-β-galactosidase as an internal control. Transient transfection of expression plasmids was performed with Fugene according to manufacturers instructions (Invitrogen). After 36 h, luciferase activity was measured according to the Luciferase Assay System (Promega) and data were normalized to β-galactosidase activity and plotted. Luciferase activities obtained after transfection with the FOP-FLASH construct are not shown as they were close to zero for each condition.
APC membrane recruitment
adenomatous polyposis coli
whole duplication genome
nuclear localization signal
osteopathia striata congenital with cranial sclerosis
We wish to acknowledge Ignacio Maeso for help with phylogenetic analysis and valuable comments concerning the manuscript. We also thank Michael Clarkson for continuous discussion in the lab. We are grateful to Jurgen Behrens (University of Erlangen, Germany) for the β-catenin plasmid and Laurent Ruel and Miguel Caetano Monteiro (CNRS UMR6543, Nice University, France) for providing the TOP-FLASH and FOP-FLASH plasmids. A.B. and G.C. are supported by ARC (French national council for cancer research) and a Marie-Curie fellowship as part of the InterDeC PhD program. The research in the laboratory is supported by grants from ARC (grant 1130) and the Association for International Cancer Research (AICR grant 09-0752).
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