GENOMICS

45, 168–174 (1997) GE974915

ARTICLE NO.

The Genomic Organization and the Full Coding Region of the Human PAX7 Gene Eugene Vorobyov,* Ilja Mertsalov,* Barbara Dockhorn-Dworniczak,† Bernd Dworniczak,* and Ju¨rgen Horst*,1 *Institut fu¨r Humangenetik and †Gerhard-Domagk Institut, Westfa¨lische Wilhelms-Universita¨t, 48149 Mu¨nster, Germany Received March 10, 1997; accepted July 3, 1997

The PAX7 gene encodes a transcription factor that is a member of the PAX family of developmental control genes. In addition to playing a role in embryogenesis, PAX genes appear to be of importance in a number of diverse human diseases and cancers. The PAX7 gene maps to human chromosomal region 1p36 and therefore is a potential candidate for human disorders linked to this region. In particular, a rearrangement of the PAX7 gene by chromosomal translocation is frequently found in alveolar rhabdomyosarcoma tumors. Here, we cloned a cDNA containing the full coding region of the human PAX7 gene and determined its genomic organization. The gene encodes a predicted protein of 520 amino acids that is 47 amino acids longer at the carboxy end than the highly related PAX3 protein. The coding region of the gene is interrupted by seven introns, the positions and lengths of which are similar to those of the corresponding introns of the PAX3 gene. Sequence data for exon/intron boundaries of PAX7 exons 1, 5, 6, 7, and 8 were determined and, together with previously published data for exons 2, 3, and 4, provide the complete sequence information for mutation analysis of the human PAX7 gene. q 1997 Academic Press

INTRODUCTION

To date nine paired box (PAX) genes from mouse and human have been isolated on the basis of their homology to the paired box sequence of Drosophila. PAX genes encode transcription factors that play a crucial role in vertebrate embryogenesis. Recently, it was shown that certain congenital malformations in mice and humans are associated with Pax mutations (for review see Stuart and Gruss, 1995). One group within Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under Accession Nos. X96743– X96748. 1 To whom correspondence should be addressed at Institut fu¨r Humangenetik, Vesaliusweg 12-14, 48149 Mu¨nster, Germany. Telephone: (0251) 835-5401. Fax: (0251) 835-6995. E-mail: horst@ uni-muenster.de.

0888-7543/97 $25.00 Copyright q 1997 by Academic Press All rights of reproduction in any form reserved.

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the PAX gene family consists of two paralogous genes, PAX3 and PAX7. These genes show a high degree of homology and a similar expression pattern. Pax3 mutations have been linked to the splotch phenotype in mice (Epstein et al., 1991; Goulding et al., 1993; Vogan et al., 1993) and with the equivalent human genetic disease—Waardenburg syndrome (Tassabehji et al., 1992; Baldwin et al., 1992; Hoth et al., 1993). Furthermore, PAX3 or PAX7 are rearranged by chromosome translocations t(2;13)(q35;q14) and t(1;13)(p36;q14), respectively, in the pediatric cancer alveolar rhabdomyosarcoma (Barr et al., 1993; Shapiro et al., 1993a; Davis et al., 1994). Because of these translocations, the aminoterminal DNA-binding domains of PAX3 and PAX7 are combined with the transcriptional regulation domain of FKHR (a member of the fork head domain family of transcriptional factors), and the respective fusion genes are abundantly transcribed in the tumor cells. The human PAX7 gene is located on chromosome 1p36.2 (Scha¨fer and Mattei, 1993; Shapiro et al., 1993b; Stapleton et al., 1993). This region is frequently deleted or rearranged in a variety of tumors derived from cells of neuroectodermal or mesodermal origin, including neuroblastoma, melanoma, and pheochromocytoma, as well as breast cancer and leiomyoma (for reference see Report of the Second International Workshop on Human Chromosome 1 Mapping, 1996). During mouse embryogenesis, the Pax7 gene is expressed in the developing brain, the neural tube, and the dermamyotome (Jostes et al., 1991), suggesting a role for the gene in the specification of neural and muscle cells, two types of cells that are affected in some of the above tumors. As such, one could suggest that the PAX7 gene is a potential candidate for a tumor suppressor gene. To facilitate further identification of human diseases associated with a dysfunction of the PAX7 gene it was important to define the complete coding sequence and genomic structure of the gene. During analysis of rhabdomyosarcoma tumors we found both forms of the above-mentioned chromosomal translocations, t(2;13) and t(1;13). In the tumor containing a PAX3–FKHR translocation, the intact PAX7 gene was expressed at a detectable level. From this tumor, using the RACE

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(rapid amplification of the cDNA ends) procedure on the basis of the previously published partial sequence of PAX7 cDNA (Scha¨fer et al., 1994), we cloned a cDNA encompassing the full coding region of the PAX7 gene. In addition, we isolated approximately 80 kb of the human PAX7 genomic sequence that allowed us to establish the exon/intron organization of the gene. In this report, we present the genomic structure and the nucleotide sequence data required for further analysis of the human PAX7 gene. MATERIALS AND METHODS Materials. Enzymes were obtained from Boehringer Mannheim, except for those used in sequencing reactions, reverse transcription, and RACE. Radioactively labeled nucleotides were purchased from Amersham. The alveolar rhabdomyosarcomas (No. 282 and No. 438) were diagnosed and classified according to the recommendations of the Intergroup Rhabdomyosarcoma Study pathology committee (Asmar et al., 1994). Analysis of tumor RNA. Total RNA was extracted from snapfrozen tumor samples according to standard procedures. Five micrograms of total RNA from tumor tissue was reverse transcribed using MoMLV (GIBCO BRL) and 100 ng of oligo(dT) primer (Pharmacia) in a 30-ml reaction. One-twentieth of this reaction was used as template for PCR. The PAX3-, PAX7-, and FKHR-specific primers were as described (Davis et al., 1994; Galili et al., 1993). PCR was performed for 40 cycles at 947C for 45 s, 607C for 45 s, and 737C for 2 min. PCR products were subcloned into vector pGEM-T (Promega) and sequenced. cDNA cloning. To isolate cDNAs of the human PAX7 gene we used the RACE procedure. Based on the previously published partial sequence of the human PAX7 cDNA (Scha¨fer et al., 1994), we designed two nested pairs of oligonucleotides: P1 and P2 for 5*RACE and P3 and P4 for 3*RACE. RACE experiments were performed with the 5*-AmpliFINDER RACE kit (Clontech) and the 3*RACE system (GIBCO BRL) following the manufacturers’ protocols. Finally, we designed primers P5 and P6 for the 5*- and 3*-ends of cDNAs resulting from RACE, to amplify full-length PAX7 cDNA. An oligo(dT)primed cDNA from the 3*RACE reverse transcription reaction was used as template for the PCR. The amplification conditions were 947C for 45 s, 627C for 1 min, and 727C for 3 min, repeated for 40 cycles. PCR products were subcloned into the pGEM-T vector and sequenced. Oligonucleotide sequences. The oligonucleotide sequences used were P1, 5*-GCTCCCTTCACCCCTGCTGCGA-3*; P2, 5*-GTCGGCAAAAAATCGCTTCCCGT-3*; P3, 5*-CAGCACCACCGGCTACAGCG-3*; P4, 5*-GCTATCAGTACGGCCAGTACGGCCA-3*; P5, 5*GAAAGCTGGTGTGGAGGGAGA-3*; and P6, 5*-GCAGAGGCTTAGCCAGGAGAT-3*. Isolation and mapping of genomic clones. A human genomic library in the Lambda FIX II vector (Stratagene) was screened with six different restriction fragments from the human PAX7 cDNA using standard methods (Sambrook et al., 1989). Genomic clones were restriction mapped and probed with oligonucleotides spaced throughout the cDNA sequence. Oligonucleotide probes were labeled with T4 polynucleotide kinase. Exon-containing restriction fragments were subcloned into pBluescript KS (/) (Stratagene) and used as sequencing templates with the same oligonucleotides as those used for hybridization to cross the exon/intron junctions. DNA sequencing. Double-stranded DNA sequencing was performed by dideoxy-chain termination (Sanger et al., 1977) using T7 DNA polymerase (Pharmacia). G/C-rich regions were resolved by substituting 7-deaza-dGTP in all reactions. Either internal primers designed from the cDNA sequence or external universal primers for pBluescript were employed. Sequences were determined on both strands in most cases. The HUSAR/UWGCG computer program

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package (DKFZ, Heidelberg, Germany) was used for contig assembly and sequence comparisons.

RESULTS

Cloning and Characterization of the PAX7 cDNA Sequencing of the PCR products obtained by RT-PCR analysis of the alveolar rhabdomyosarcomas revealed a PAX7/FKHR fusion transcript in tumor 438 and a PAX3/FKHR transcript in tumor 282 (data not shown). Furthermore, RNA from tumor 282 containing intact PAX7 transcripts was used as template to clone PAX7 cDNAs. To isolate the 3*- and 5*-ends of the human PAX7 gene we performed RACE. These experiments yielded a 547-bp product in 5*RACE and a 330-bp product in 3*RACE. Based on the sequences obtained from RACE we designed primers P5 for the 5*-end and P6 for the 3*-end to amplify the full-length PAX7 sequence. As a result, a 2213-bp RT-PCR product was obtained, cloned, and sequenced. This cDNA contains the complete coding region of the human PAX7 gene, including 599 bp in the 5*- and 100 bp in the 3*-untranslated regions. The human PAX7 cDNA sequence was verified by comparing with the sequences of genomic clones. At the 5*-end there is an in-frame stop codon 36 amino acids upstream of the first methionine codon. At the 3*-end there is a consensus AATAAA polyadenylation signal 18 nucleotides 5* of the polyadenylation site in the cDNA cloned by 3*RACE. A sequence identical to the 3*-end of the cDNA was found in exon 8 of the gene. From these data we conclude that the human PAX7 gene encodes a predicted protein of 520 amino acids. Genomic Organization of the PAX7 Gene A total of 39 genomic clones were isolated. All of the PAX7 exons were contained within three groups of nonoverlapping phage clones. Analysis of the genomic clones resulted in the identification of eight exons. The exon/intron organization of the human PAX7 gene is depicted graphically in Fig. 1. Intron lengths were determined by restriction endonuclease mapping and sizing gel analysis. The overall length of the gene was estimated to be more than 80 kb. The exon/intron boundaries of the gene were determined by sequencing each exon and both flanking intron sequences. The nucleotide and deduced amino acid sequences of all exons were identical with the cDNA-derived sequences. All introns are flanked by the consensus sequences of splice donor GT and acceptor AG sites. The sequence data for exons 1, 5, 6, 7, and 8 are shown in Fig. 2. The DNA sequences and exon/intron structure of exons 2, 3, and 4 have been reported previously by Burri et al. (1989). The human PAX3 and PAX7 coding sequences have been shown to share a high degree of homology (Burri et al., 1989; Davis et al., 1994; Scha¨fer et al., 1994). Recently, the genomic organization of the human PAX3

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FIG. 1. The genomic organization of the human PAX7 gene. (A) Schematic representation of the PAX7 locus. Exons are indicated by numbered black boxes. The positions of genomic phage clones are shown below by open bars. (B) Three nonoverlapping genomic regions covering the PAX7 gene are represented. The locations of the eight exons and some restriction sites are indicated. Translation start codon (ATG) and termination codon (TAA) are shown. R, EcoRI; S, SalI; X, XbaI; N, NotI.

gene was determined completely (Burri et al., 1989; Hoth et al., 1993; Macina et al., 1995). The coding region of the gene is divided into eight exons by short introns 1, 2, 3, and 6 and long introns 4, 5, and 7, analogously to the structure of the PAX7 gene shown in Fig. 1. To compare the intron locations of the human PAX7 and PAX3 genes we compiled the complete sequence of the PAX3 cDNA using partial sequences published previously (Burri et al., 1989; Hoth et al., 1993; Macina et al., 1995). The data in Fig. 3 demonstrate that the intron locations of the human PAX7 gene are precisely the same as those of the human PAX3 gene. There is an alternative splice site for PAX7 exon 4, located six nucleotides upstream of the splice site that is conservative for this exon in both PAX3 and PAX7 genes. This alternative splice junction was described by Scha¨fer et al. (1994), and we have also isolated both splice forms of the PAX7 cDNA by RT-PCR using human fetal brain RNA as template (data not shown). DISCUSSION

Here we report a further characterization of the human PAX7 cDNA as well as the genomic organization of the PAX7 gene. The previously published cDNA sequence (Scha¨fer et al., 1994) includes 102 bp of 5*-untranslated region (UTR) and a sequence coding for 467 amino acids of the PAX7 gene. However, in this sequence a putative start ATG codon could not be con-

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firmed due to the lack of an in-frame stop codon in the 5*UTR. Our sequence data show an in-frame stop codon 108 bp upstream of the apparent initiator ATG. There are no other in-frame ATGs in this 108-bp sequence. Therefore, we conclude that the predicted ATG is the actual start codon of the PAX7 gene. We have not yet determined the initiation site of transcription and hence we do not know the exact length of the 5*UTR. In regard to the 3*-end of the PAX7 gene, the cDNA cloned by 3*RACE exhibits a consensus polyadenylation signal 18 nucleotides upstream of the polyadenylation site. A sequence identical to the 3*-end of the cDNA was identified in the genomic sequence of exon 8. Therefore, exon 8 (consisting of 508 nucleotides: 406 coding and 102 noncoding) is the most downstream exon in the PAX7 gene. In summary, these data suggest that the human PAX7 gene encodes a predicted protein of 520 amino acids which is 47 amino acids longer at the carboxy end than the highly related PAX3 protein (Fig. 3). A search of the DNA and protein databases failed to identify significant homologies between the sequence coding the last 47 amino acids of PAX7 protein and any other sequences. To define the genomic structure of the PAX7 gene, human genomic clones were isolated. We cloned approximately 80 kb of genomic DNA containing the full coding region of the human PAX7 gene. We determined that the coding region of the gene consists of eight exons. The exon/intron boundaries of exons 1, 5, 6, 7,

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FIG. 2. Partial genomic nucleotide sequence of the human PAX7 gene. (A) Exon 1. The first nucleotide of the adenine residue of the ATG initiation codon is designated /1. (B) Exons 5, 6, 7, and 8. The exonic sequences are shown in uppercase letters. Translated amino acids are shown below the corresponding DNA sequence. The upstream in-frame stop codon and the polyadenylation signal are shown in boldface.

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and 8 were established with nucleotide sequencing and, together with the previously published sequence data for exons 2, 3, and 4 (Burri et al., 1989), demonstrate the complete genomic sequence organization for the coding region of the human PAX7 gene. A comparison of the genomic structures of PAX7 and PAX3 revealed a very similar organization of both genes. As shown in Fig. 3, the positions of all exon boundaries are conserved between PAX7 and PAX3. It is known that the PAX3–FKHR translocation in alveolar rhabdomyosarcomas involves sequences within the seventh intron of the PAX3 gene, resulting in fusion of exon 7 of PAX3 to exon 2 of FKHR (Macina et al., 1995; Davis et al., 1995). From the sequences of PAX7– FKHR and PAX3–FKHR cDNAs, it was possible to deduce that the fusion points in the resulting chimeric proteins are at homologous positions in the PAX7 and PAX3 moieties (Davis et al., 1994). Our results now show that the position of fusion in the PAX7–FKHR chimeric protein corresponds to the position of intron 7 of PAX7, indicating that sequences within intron 7 are involved in the recombination process, as in the PAX3–FKHR translocation. Further analysis will be required to determine the precise position(s) of recombination and if possibly repetitive sequences are involved in the recombination process, as in the PAX3 gene (Macina et al., 1995). To conclude, the extensive homologies found between PAX3 and PAX7 indicate that they are derived from a common ancestral gene. The underlying evolutionary development of those genes that are located on different chromosomes will be a topic of further investigation. The human PAX7 was mapped to chromosome 1p36, which is known to be a multiple tumor associated region. Therefore, it seems to be important to analyze this gene for mutations in the human diseases associated with alterations in the locus 1p36.2. For example, deletion of the region 1p36 is the most common cytogenetic abnormality observed in neuroblastomas. Loss of heterozygosity for 1p is generally found in 30–40% of the respective patients. Recent findings indicate the presence of two tumor suppressor loci on chromosomal bands 1p35–p36 involved in neuroblastoma development. Deletion of a more proximal suppressor gene is associated with N-myc amplification while a distal, probably imprinted, suppressor is deleted in N-myc single copy cases (Schleiermacher et al., 1994; Caron et al., 1995; Cheng et al., 1995). Six genes were mapped to the 1p36.2 region: the dominant-negative helix–loop– helix gene ID3, a transcription factor gene DAN, a cell cycle-regulated kinase gene CDC2L1, tumor necrosis factor receptor gene TNFR2, the transcriptional regulator E2F-2, and the PAX7 gene. Four of those proposed candidate genes (ID3, DAN, CDC2L1, and TNFR2)

were demonstrated to be located outside of the distal consensus deletion region (DCDR) (White et al., 1995). Recently, from mapping analyses it became evident that ID3 and E2F-2 are separated by a 25-kb interval (Ellmeier et al., 1996). These data suggest that E2F-2 is likely to be located outside of the DCDR as well. Therefore, so far only PAX7 remains as a probable candidate for a tumor suppressor gene in the respective chromosomal region. However, PAX7 was localized to a large consensus deletion region in these tumors. Hence, to identify the connection between this gene and the genesis of any of these malignancies, it is necessary to find more subtle alterations in the PAX7 genomic structure. In addition, it would be of interest to investigate the PAX7 gene for mutations in those Waardenburg syndrome patients in whom PAX3 gene mutations could not be identified. So far, PAX3 mutations have been detected in 78% of families with definite Type 1 or Type 3 Waardenburg syndrome (WS) (Tassabehji et al., 1995). Linkage analysis indicates that WS is genetically heterogeneous and that defects in PAX3 may not be the cause of WS in many families (Farrer et al., 1992). The high degree of similarity of PAX3 and PAX7 genes within the coding region and in the genomic organization and expression pattern leads us to question whether the WS cases showing no PAX3 mutations are due to alterations in the PAX7 gene structure. The sequence data presented here are a prerequisite for detailed studies also in this regard. ACKNOWLEDGMENTS We thank Dr. Klaus Wilke for reading and editing the manuscript. This study was initiated by Dr. D. Plachov and supported by the Volkswagen Stiftung, the Deutsche Forschungsgemeinschaft, the Mildred Scheel Stiftung, and the European Community.

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FIG. 3. Comparison of the intron locations of PAX7 and PAX3 genes. The nucleotides of both sequences are numbered with respect to the translation initiation codon. The intron locations are shown with solid points. Vertical lines indicate identity between the nucleotide sequences. The amino acid sequences of both genes are shown above and below the corresponding DNA sequences. Boldface amino acids indicate the paired domain (PD), octapeptide (OP), and paired type homeodomain (HD).

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