Cloning of Fatso (Fto), a novel gene deleted by the Fused toes (Ft)mouse mutation

Thomas Peters,1,2 Katrin Ausmeier, 1 Ulrich Ru ther1,2

1Institut fur Molekularbiologie, Medizinische Hochschule Hannover, 30625 Hannover, Germany2Lehrstuhl fur Entwicklungs- und Molekularbiologie der Tiere (EMT), Heinrich Heine Universtita¨t, Universitatsstr. 1, 40225Dusseldorf, Germany

Received: 5 May 1999 / Accepted: 9 June 1999

Abstract. The Fused toes (Ft) mouse mutation was created byinsertional mutagenesis, resulting in the deletion of several hun-dred kb of genomic sequences of mouse Chromosome (Chr) 8.Mice heterozygous for theFt mutation are characterized by partialsyndactyly of forelimbs and massive thymic hyperplasia indicatingthat programmed cell death is affected. HomozygousFt/Ft em-bryos die at midgestation and show severe malformations of cra-niofacial structures. Furthermore, establishment of left-right asym-metry is random. Here we report on the positional cloning of anovel gene by exon trap analysis of a genomic clone encodingwild-type sequences corresponding to parts of the deletion inFtmutants. RT-PCR experiments demonstrated that the newly iden-tified gene, Fatso (Fto), is expressed throughout embryonic devel-opment. Wide expression was also found in tissues of adult mice.We show that expression ofFto is completely absent in mouseembryonic fibroblasts homozygous for theFt mutation. In addi-tion, we isolated the full-length cDNA which encodes a putative58-kDa protein showing no similarities to known proteins or pro-tein motifs. The expression data ofFto define it as a candidategene involved in processes such as programmed cell death, cra-niofacial development, and establishment of left-right asymmetry.

Introduction

The dominant mouse mutation Fused toes (Ft) was generated by atransgene integration into region D of mouse Chr 8 (van der Ho-even et al. 1994). The insertion of the transgene resulted in thedeletion of several hundred kb of genomic sequences, suggestingthat several genes are affected by the mutation (Ausmeier, Lesche,Peters, and Ru¨ther, unpublished). Mice heterozygous for theFtmutation are characterized by partial syndactyly of forelimbs andthymic hyperplasia. Analysis of cell death in the interdigital web ofthe developing forelimbs and in vitro induction of apoptosis inimmatureFt/+ thymocytes suggested that programmed cell deathis affected in both structures (van der Hoeven et al. 1994). Homo-zygousFt/Ft embryos are growth retarded and die between em-bryonic day (E) 10.5 and E 12.5, exhibiting severe malformationsof craniofacial structures. In addition, these embryos have lost thegenetic control of left-right asymmetry since embryonic turningand heart looping are random (Hemyer et al. 1997). Regarding thevarious phenotypic alterations observed inFt mutant mice, wethink that the molecular and phenotypic understanding of theFtmutation will result in the identification and characterization ofseveral genes involved in limb and craniofacial development aswell as in the control of programmed cell death and left-rightasymmetry.

In previous studies we have described the identification of the

candidate geneFt1, which is located close to the transgene inte-gration site. SinceFt1 has been deleted by the mutation, its ex-pression is completely lost in homozygousFt/Ft embryos (Lescheet al. 1997). However, targeted inactivation ofFt1 was not suffi-cient to result in a phenotype similar toFt, suggesting that addi-tional genes have been affected by theFt mutation (Lesche andRuther unpublished).

To investigate the extension of theFt deletion and to identifyadditional Ft candidate genes, we started genomic walking byscreening of several libraries of the mouse genome to isolateclones encoding the corresponding regions of the wild-type ge-nome. Here we report on the identification of the novel gene Fatso,which has been completely deleted by theFt mutation.

Materials and methods

Exon trapping.Genomic DNA from BAC clone FBAC4432-24I23 (Ge-nome Systems Inc.) was digested withBamHI or BglII respectively andsubcloned into plasmid pSPL3 (Church et al. 1994). Exon amplificationwas performed with the Exon Trapping System (Gibco/BRL) according tothe manufacturer’s instructions. Trapped exons were sequenced on an ABIautomated sequencer.

RT-PCR analysis and Northern blot hybridization.Total RNA wasisolated from whole mouse embryos, adult mouse tissues, and mouse em-bryonic fibroblasts with the single-step protocol (Chomczynski and Sacchi1987). RT-PCR analysis was performed with Superscript II reverse tran-scriptase (Gibco/BRL) and an oligo (dT) primer for first-strand cDNAsynthesis from total RNA (2mg). Trapped exons 29/10 and 28/10 werelinked by subsequent PCR amplification with primers 29/10-F1 (58-TCTGAGGATGAAAGTGAGGACGAG-38) and 28/10-B1 (58-CCA-CACGGTGAGTGGAACTAAAC-38), resulting in a 221-bp product. Bothprimers were also used to assay for expression of the trapped exons duringembryonic development and in adult tissues. To permit an evaluation ofdifferential RNA loading, expression of the HPRT gene was analyzed withprimers HPRT-5 (58-CACAGGACTAGAACACCTGC-38) and HPRT-3(58-GCTGGTGAAAAGGACCTCT-38). PCR amplifications were carriedout at 94°C for 20 s, 57°C for 60 s, 72°C for 30 s, for 32 cycles, followedby a 7-min extension at 72°C.

Northern blot analysis of total RNA (20mg) was performed by hybrid-ization with cDNA probes and standard Northern blot hybridization pro-tocols.

cDNA isolation.A mouse 9-day embryo cDNA library was constructedby Bernhard Herrmann and provided by the Resource Center/Primary Da-tabase (RZPD) of the German Human Genome Projekt. This library wasscreened by hybridization with the radiolabled 221-bp PCR product (seeRT-PCR analysis). Missing 58 sequences were isolated by rapid amplifi-cation of cDNA ends (RACE; Frohman et al. 1988) with the 58/38 RACEKit (Roche Diagnostics). Of the total RNA isolated from a 12.5-day wholemouse embryo, 2mg was used for reverse transcription with primer Fto-B1350 (58-CCAGGATGGCAGACAGAATCTC-38). Subsequent PCRCorrespondence to:U. Ruther

Mammalian Genome 10, 983–986 (1999).

© Springer-Verlag New York Inc. 1999

Incorporating Mouse Genome

amplification was carried out with nested reverse primers Fto-B750 (58-CAGGTTCTCATCGTGATGCCAG-38) and PAF-B1 (58-TAG-GAAGGTCTGACATGCAGCG-38) and forward primers Oligo d(T)-anchor (58-GACCACGCGTATCGATGTCGACTTTTTTTTTTTTTT-TTV-38) and PCR anchor (58-GACCACGCGTATCGATGTCGAC-38)with the Expand High Fidelity PCR-System (Roche Diagnostics). Ampli-fication conditions were 94°C for 30 s, 58°C for 30 s, 68°C for 2 min, for35 cycles, followed by a 7-min extension at 68°C.

Computer-based sequence analysis.Fto cDNA and protein sequenceswere tested for similarity to known sequences in the National Center forBiotechnology Information database with the BLAST program (Altschul etal. 1997). Motif analysis of Fto was performed with protein sequenceanalysis programs derived from the ISREC (Swiss Institute for Experimen-tal Cancer Research) Profile Scan Server (http://www.isrec.isb-sib.ch/software/PFSCAN-form.html).

Coupled in vitro transcription/translation.35S-labeled Fto was syn-thesized in reticolucyte lysates with the TNT T3/T7 coupled in vitro tran-scription/translation kit (Promega) according to the instructions of themanufacturer.

Accession number.AJ237917

Results

Identification of potential exons deleted in Ft mice.The transgeneintegration that caused theFt phenotype involved deletion of sev-eral hundred kb of genomic sequences of Chr 8. To identify tran-scribed sequences in the genomic deletion, we performed exontrapping of isolated bacterial artificial chromosome (BAC) clones.Exon trap analysis of a BAC clone containing an insert of approxi-mately 170 kb of genomic DNA that was shown to be completelydeleted by theFt mutation (data not shown) resulted in the iden-tification of several putative exons. None of these exons exhibitedany homology to sequences in the GenBank database. To deter-mine whether any of these exons belong to the same gene, wedesigned primers from predicted exons and used these in RT-PCRassays to evaluate whether the sequences could be linked at thetranscript level. We were able to link exon 28/10 (78 bp) and exon29/10 (143 bp) by RT-PCR, resulting in a 221-bp PCR productindicating that the sequences of both exons are located next to eachother on the same transcript (data not shown).

To confirm that the combined sequences of trapped exons areexpressed in tissues affected by theFt mutation, we investigatedthe expression of the transcript by RT-PCR. In adult mice wefound high expression of the transcript in most organs analyzed(Fig. 1A). However, the transcript is not expressed in heart andskin. In addition, expression was detected throughout embryonicdevelopment as early as E 8.5 (Fig. 1B). Northern blot analysis oftotal RNA from mouse embryonic fibroblasts (MEF) revealed ex-pression of the identified transcript (approximately 3.6 kb) in wild-type MEF, but complete absence in MEF homozygous for theFtmutation (Fig. 2). Thus, expression of the identified transcript islost owing to theFt mutation.

A novel gene deleted in Ft mice.To identify the complete cDNA,we screened a cDNA library derived from mouse embryos at E 9.0with the 221-bp PCR product. Three cDNA clones were identified(MPMGc559N2210Q3, MPMGc559B1598Q3, and MPMGc-559J0125Q3), abbreviated hereafter as N2210, B1598, and J0125.To obtain the full-length cDNA sequence, we performed a RACEanalysis extending the obtained cDNA sequence of the longestcDNA clone J0125 by 10 nucleotides. The total size of the isolatedcDNA sequence (Fig. 3) is 3.6 kb and corresponds well to thefull-length transcript, given the observed size of the transcriptdetected by Northern blot hybridization (Fig. 2). In addition, twoclassical polyadenylation sites are present at the 38 end of thecDNA. Therefore, we conclude that we isolated the full-lengthcDNA of the identified transcript. Alignment of the cDNA se-quence to DNA databases did not show any significant homologiesto known sequences (see Discussion for details). Thus, the newlyidentified sequence must be the product of a novel gene. Southernanalysis of genomic DNA with probes derived from the isolatedcDNA revealed a complete deletion of these sequences inFt/Ftmice (data not shown). Moreover, this analysis suggested this geneto have a size of at least 250 kb (data not shown), which promptedus to name the newly identified gene Fatso (Fto).

Analysis of the cDNA sequence revealed an open readingframe (ORF) encoding a putative protein of 502 amino acid resi-dues (Fig. 3). The putative translation start site is located at nucleo-tide 38. However, the sequence surrounding this potential startcodon is not consistent with a consensus Kozak sequence (Kozak1996). The assumed translational initiation codon is only includedin cDNA clone J0125 (Fig. 3), and we used this cDNA clone in acoupled in vitro transcription/translation assay. We could detectthe in vitro translation of a protein product of approximately 58kDa, whereas the other two identified cDNA clones B1598 andN2210 failed to produce any protein in this experiment (Fig. 4).Deletion of nucleotides 1582 to 3541 of cDNA clone J0125 (DJ)

Fig. 1. Semi-quantitative RT-PCR analysis ofRNA isolated from adult mice (a) and embryos (b)with a forward primer from trapped exon 29/10and a reverse primer from trapped exon (28/10(top lane). Bottom lanes show expression of theHPRT gene as control for RNA loading. Lanesare: (a) 1, water control; 2–12, RNA from adultmice isolated from spleen (2), liver (3), thymus(4), heart (5), lung (6), testis (7), kidney (8), brain(9), muscle (10), skin (11), and salivary glands(12); (b) 1, water control; 2–7, RNA fromembryos at E8.5, E9.5, E10.5, E11.5, E12.5, andE13.5, respectively.

Fig. 2. Northern-blot analysis of mouse embryonic fibroblasts isolatedfrom wild-type (+/+), heterozygous (Ft/+) and homozygous (Ft/Ft) em-bryos. Total RNA was hybridized with a probe containing trapped exons28/10 and 29/10. The arrow head marks the position of the 3.6-kb tran-script. The lower panel provides loading controls by hybridization with aGAPDH probe.

T. Peters et al.: Cloning of Fatso984

also resulted in the detection of the 58-kDa protein product, veri-fying that the ORF deduced from the cDNA sequence is indeedencoding the in vitro translated protein. The size of the proteinproduct corresponds well to the theoretical mass calculated fromthe sequence (57,98 kDa) (Fig. 4).

Computer-based motif searching failed to identify any evi-dence for significant functional homologies to known proteins.However, there is a potential bipartite nuclear localization signal atthe N-terminal end of the putative protein (Fig. 3).

Discussion

In this study we have presented the identification of a novel gene,Fto, which has been deleted by theFt mutation on mouse Chr 8.

The Fto cDNA encodes a putative protein of 502 amino acidresidues. Although the translational initiation codon fails to meet aconsensus Kozak sequence, it is sufficient for the correct transla-tion of the putative Fto protein as demonstrated by in vitro tran-scription/translation of cDNA clone J0125 andDJ. Since we werenot able to identify any previously described protein motifs ordomains, the function of the protein encoded byFto remains to beassessed. However, the presence of a bipartite nuclear localizationsignal at the N-terminus of the protein suggests a role in thenucleus.

Strikingly, a BLAST database search with the isolated full-length cDNA ofFto revealed that a fragment spanning nucleotides161–779 is almost identical (98%) to the reverse sequence of exon1 and 58 flanking region of the murine somatostatin receptor 2gene (Sst2) (AJ005518, nucleotides 995–1610; Kraus et al. 1998).SinceSst2 was previously mapped to mouse Chr 11 (Brinkmeierand Camper 1997), we were interested to know whether theremight be a possibility that both genes share the same sequences.However, Northern blot analysis of total RNA ofAtT20cells thatexpress both genes,Fto andSst2, showed that the identical cDNAfragment specifically hybridizes only to the 3.6-kbFto transcript.We could never detect hybridization of this probe to the 2.2-kbtranscript ofSst2. In addition, RT-PCR analysis ofFto and Sst2cDNA confirmed that these sequences can exclusively be found aspart of the cDNA sequence of theFto gene. Therefore, the previ-ously described cDNA clone encoding the B isoform of theSst2gene (Vanetti et al. 1992) is most likely a hybrid clone.

Since theFt mutation resulted in the deletion of several hun-dred kb of genomic sequences, we think that several genes wereaffected. The occurrence of dominant and recessive phenotypicalterations observed inFt mice also supports this multigene defecthypothesis because several molecular mechanisms appear to bedisturbed by theFt mutation (for example, programmed cell death,limb and craniofacial development, control of left-right asymme-try).

So far we have described two different genes,Ft1 andFto,which have been deleted by the mutation. However, we have evi-dence from at least three more genes that are affected by theFtmutation (Peters, Ausmeier, and Ru¨ther, unpublished).

The Ft1 gene encodes an evolutionarily conserved protein

Fig. 3. cDNA sequence and deduced protein sequence ofFto. The cDNAsequence (AJ237917) was assembled from three identified cDNA clonesand several 58RACE clones. 58 ends of the identified cDNA clones J0125,B1598, and N2210 are located at positions 11, 43 and 800, respectively(bold). The open reading frame extends from nucleotides 38 to 1544 andis translated into a putative protein of 502 amino acid residues. The po-tential bipartite nuclear localization signal at the N-terminal end of theputative Fto protein is underlined. TheEcoRV restriction site at position1582 used for the generation of cDNA subcloneDJ as well as two classicalpolyadenylation signals (AATAAA) at starting at positions 3229 and 3521,respectively, are underlined.

Fig. 4. In vitro translation of Fto protein. cDNA clones J0125, B1598, andN2210 as well as the cDNA subcloneDJ generated byEcoRV digestionindicated as J, B, N andDJ, respectively, were used in a coupled in vitrotranscription/translation assay. The arrow head marks the position of the58-kDa protein.

T. Peters et al.: Cloning of Fatso 985

showing weak similarities to ubiquitin conjugating enzymes(Lesche et al., 1997). However, targeted inactivation ofFt1 alonewas not sufficient to result in a phenotype similar to Fused toes(Lesche and Ru¨ther unpublished). Nevertheless, sinceFt1 ix ex-pressed in structures affected by theFt mutation (for example,limb buds, neural tube), we cannot exclude that the loss ofFt1might contribute to theFt phenotype.

The newly identified gene,Fto, so far is the largest gene de-leted by theFt mutation covering more than 250 kb of genomicsequences. It is widely expressed in adult tissues of wild-typemice. In addition, we detected expression by RT-PCR from E 8.5onwards throughout embryonic development. We also assume thatFto is expressed at even earlier stages. To assess the spatial patternof Fto expression, we hybridized a variety of different antisenseprobes to whole-mount embryos isolated from several develop-mental stages. We could never detect a specific expression, butrather a wide distribution ofFto transcripts (data not shown). This,together with the presented RT-PCR data, indicates thatFto is acandidate gene for being involved in mechanisms like pro-grammed cell death, limb development, craniofacial development,and the control of left-right asymmetry, since it is expressed at astage during embryonic development when the phenotypic alter-ations of homozygousFt/Ft embryos start to manifest. Inactivationof Fto will allow elucidation of its function in embryonic devel-opment and its contribution to the Ft phenotype. However, only acombined inactivation ofFto and otherFt candidate genes mightfinally lead to the manifestation of the completeFt phenotype.

Acknowledgments.We are grateful to the Resource Center of the GermanHuman Genome Project for providing the mouse cDNA library and clones.We thank Lars Grotewold and Jens Bo¨se for critical reading of the manu-script. This work was supported by the Deutsche ForschungsgemeinschaftSFB 271, TP B6.

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