Characterization of a cluster of sulfatase genes on Xp22.3 suggests gene duplications in an ancestral pseudoautosomal region
Characterization of a cluster of sulfatase genes on Xp22.3 suggests gene duplications in an ancestral pseudoautosomal regionGermana Meroni1,*, Brunella Franco1, Nicoletta Archidiacono2, Silvia Messali1, Grazia Andolfi1, Mariano Rocchi2 and Andrea Ballabio1,3
1Telethon Institute of Genetics and Medicine (TIGEM), San Raffaele Biomedical Science Park, Milano, Italy, 2Institute of Genetics, University of Bari, Bari, Italy and 3Department of Molecular Biology, University of Siena, Siena, Italy
Received November 15, 1995;Revised and Accepted January 5, 1996
An obligatory crossing-over event between the X and Y chromosomes in mammals occurs at each male meiosis within the 2.6 Mb of DNA defining the pseudoautosomal region (PAR). Genes located within or near the human PAR have homologous copies on the X and Y chromosomes, escape X inactivation and appear to be highly divergent throughout evolution. We have characterized the genomic structure of two genes from a recently identified cluster of sulfatase genes (ARSD and ARSE) located in the Xp22.3 region, and of their homologs on the Y chromosome. Our results indicate that the ARSD and ARSE genes from within this cluster have a conserved genomic organization, shared also by another Xp22.3 gene, STS, but completely different from that of all the other sulfatase genes. Sequence analysis of the Y-linked homologs indicate that they represent truncated pseudogenes. Sequence identity values between the X and Y copies of each gene is on average 91%, significantly higher than the values obtained by comparing different members of the family. FISH mapping experiments performed in several primate species revealed an identical localization of the X-linked copies to that in man, but different localizations of the Y homologs. Together, our data indicate that the cluster of sulfatase genes on human Xp22.3 was created through duplication events which probably occurred in an ancestral PAR, and support the view that the PAR has undergone multiple changes during recent mammalian evolution.
Mammalian species have heteromorphic sex chromosomes with very different contents and structures. Proper pairing and segregation of sex chromosomes in the heterogametic sex is ensured by a segment of 100% identity between the X and Y chromosomes, known as the pseudoautosomal region (PAR) (1 -5 ). This region is located at the distal tip of the X and Y chromosome short arms measuring ~2.6 Mb (6 ). It has been hypothesized that the PAR originates from multiple additions and attritions from different autosomes to the sex chromosomes (7 ). The overall frequency of recombination in PAR is exceptionally high, with one obligatory crossing-over event occurring at each male meiosis, which, on average, results in a 10- to 20-fold difference in recombination frequency between this and other regions of the genome (8 ). This high frequency of recombination, together with the presence of repeat sequences in the distal part of the X chromosome short arm (9 ), may predispose to unequal crossing-over events leading to gene duplications and deletions.
An example of a gene family which has arisen through duplications in this region is the MIC2 gene family, composed of two functional genes and one pseudogene. The two genes, MIC2, which encodes a cell surface antigen (10 ), and the Xg blood group gene (XG) (11 ,12 ), are 45% homologous at the protein level and are transcriptionally co-regulated. MIC2 lies within the PAR, while XG has a very peculiar localization, the 5' end of the gene being pseudoautosomal and the 3' X-specific. In addition, an MIC2-related pseudogene (MIC2R), retaining one copy of the first exon and four copies of the fourth and fifth exons of MIC2, is located distal to MIC2 (13 ).
Recently, we identified a second cluster of related genes located in the X-specific region proximal to the MIC2 family, ~150 kb from the pseudoautosomal boundary. This cluster contains three genes, ARSD, ARSE and ARSF, which represent novel members of the sulfatase gene family, encoding enzymes which hydrolyze sulfate ester bonds in a wide variety of substrates (14 ). ARSD, ARSE and ARSF are highly homologous to the STS gene, also located in the Xp22.3 region (15 ).
The genes for several human sulfatases have been cloned and mutations in these genes have been identified in patients with specific metabolic disorders (16 -18 ). All sulfatases share a significant degree of sequence homology along their entire length and they are highly conserved throughout evolution, even in very distantly related species, such as sea urchin and bacteria (19 ,20 ). Full-length cDNAs of ARSD and ARSE were isolated and fully characterized, while a single exon of the ARSF gene was identified. Mutation analysis implicated ARSE as the gene involved in X-linked recessive chondrodysplasia punctata (CDPX; MIM302950), a rare form of bone dysplasia (14 ). Albeit X-specific, these genes show several features that are typical of pseudoautosomal genes. They escape from X-inactivation, they have a homolog on the Y chromosome, and they are not conserved in the mouse. These features are also shared by the STS (15 ) and KAL genes (21 -24 ), which are located 4 and 5 Mb, respectively, proximal to the newly identified cluster of sulfatase genes (ARSD, ARSE and ARSF).
Here, we report the characterization of the genomic structures of the ARSD and ARSE genes on Xp22.3, and the identification, mapping assignment, and partial sequence analysis of homologous loci on the Y chromosome. These results, together with mapping data derived from the analysis of other primate species, shed new light on the evolution of mammalian sex chromosomes and of the sulfatase gene family.
Figure 1 shows a schematic representation of the genomic region containing the ARSD and ARSE genes (14 ,25 ). Three previously identified cosmids (clones 48, 75 and 76) were used to characterize the genomic structures of these two genes. Southern blot analysis performed on these cosmid DNAs enabled us to assign each exon to a specific EcoRI fragment (Fig. 1 ). Together, the two genes span a region of ~60 kb and the orientation of transcription is in both cases from the centromere towards the telomere. We performed long-range PCR experiments to establish the distance between the two genes using cosmid 75 and YAC 455B10 as DNA templates. PCR amplification using a forward primer designed from the sequence of the first ARSD exon and a reverse primer designed from the last ARSE exon allowed us to demonstrate that the two genes are separated by only 6 kb (data not shown).
Previous experiments suggest that both the ARSD and ARSE cDNAs cross-hybridize to fragments derived from the Y chromosome. This finding was not surprising, as other non-pseudoautosomal genes from the distal short arm of the X chromosome, including STS and KAL, have a non-functional homolog located on the Y chromosome (23 ,24 ,31 -34 ). The cDNAs of the ARSD and ARSE genes were used as probes to screen a human Y-chromosome YAC library which was purchased from Research Genetics. Two YAC clones, yOX197 and yOX103, were identified in this library; however, subsequent experiments revealed a discrepancy between our mapping data and the position of these YAC clones within the previously published map by Foote et al. (35 ). PCR experiments using STSs DYS235 and DYS236 (35 ) demonstrated that these YACs do not correspond to the original clones yOX197 and yOX103. This is likely to be due to sample mix up which occurred at the company distributing the YAC clones, as previously observed by other groups (D. Vollrath, personal communication). Figure 3 shows the result of the Southern blot analysis carried out on EcoRI-digested plugs from the two overlapping Y chromosome YAC clones. Hybridization using ARSD and ARSE cDNAs as probes showed that each YAC contains both the ARSD and ARSE homologs, suggesting that these two genes are organized in a tandem array also on the Y chromosome. The two cDNAs detected several Y-specific fragments which were also detected in a 4Y human cell line but were absent in the X-derived YAC clones, NB6F12 and 455B10, containing the two genes. All Y-specific bands detected on genomic DNA were also present in the two Y-derived YACs, which thus contain the entire ARSD and ARSE homologs. To characterize the ARSD and ARSE-Y homologs in more detail, we hybridized each individual PCR-amplified exon from these two genes to the same Southern blot. The results are schematically summarized in Figure 1 . All ARSD exons appeared to be conserved on the Y, with the exception of exon 5 for which no Y-specific hybridization fragment was observed. The situation was completely different for ARSE, in which only four out of 11 exons (exons 1A, 8, 9 and 10) were shared by the Y homolog.
To study the evolution of the sulfatase cluster in Xp22.3, we have determined the localization of the ARSD and ARSE genes on the sex chromosomes of several primate species. We used an X-derived cosmid containing ARSD (cosmid 76) and a Y-derived YAC containing both ARSD-Y and ARSE-Y as probes in fluorescence in situ hybridization (FISH) experiments performed on metaphase spreads from man (HSA), common chimpanzee (PTR), gorilla (GGO) and macaca (MFA) (Fig. 6 ).
We have characterized a cluster of sulfatase genes located on Xp22.3. This cluster includes the ARSD and ARSE genes, for which the full-length cDNAs were isolated and completely characterized, and the ARSF gene, for which only one exon was identified by genomic sequencing (14 ). ARSD and ARSE span together a region of 60 kb located 150 kb proximal to the pseudoautosomal boundary (PABX) and are both transcribed from the centromere towards the telomere. ARSD is divided into 10 small exons, while ARSE has an additional 5' untranslated exon. The two genes are separated by only 6 kb. The alignment of the genomic structures revealed a perfect conservation of the intron-exon junctions of these two new sulfatase genes, with the splicing occurring at exactly the same position in the two genes. Interestingly, another sulfatase gene, STS, which is located on Xp22.3 ~4 Mb from ARSD and ARSE, has an identical genomic structure which is completely different from that reported for the other members of the sulfatase gene family. All members of this gene family share a significant degree of sequence homology along their entire length and they are highly conserved throughout evolution. The high degree of sequence conservation suggests that this family of proteins has been generated by gene duplication from a common ancestral gene. Major differences among the present day mammalian sulfatases, such as substrate specificity and tissue pattern of expression, are most likely the result of mutation and selection that occurred during evolution. Our findings strongly suggest that the ARSD, ARSE, ARSF and STS genes originated from a common ancestral gene through a series of duplication events which occurred recently during evolution.
Like other genes isolated from the same region of the X chromosome, the two new sulfatases have a pseudogene on the Y chromosome. All but one of the exons of the ARSD gene are present on the Y, while ARSE-Y shows only exons 1A, 8, 9 and 10. Several deletions, insertions and substitutions leading to stop codons and frameshifts were identified, indicating that ARSD-Y and ARSE-Y are non-functional, truncated pseudogenes. Moreover, RT-PCR experiments performed on hybrids containing only the human Y chromosome indicated that these pseudogenes are not transcribed (data not shown). The isolation of Y-specific overlapping YAC clones containing both ARSD-Y and ARSE-Y allowed us to demonstrate the presence of a tandem array also on the Y chromosome. In situ hybridization and deletion mapping assigned the two homologs to the long arm of the Y chromosome, near the centromere and proximal to the KALP homolog, with the order on the Y chromosome being CEN-(XGPY, ARSD-Y, ARSE-Y)-KALP-STSP.
Both the presence of sequences homologous to the ARSD and ARSE genes on the Y chromosome and the evidence that these genes escape X inactivation (14 ), suggest that they were part of an ancestral PAR, as previously suggested for both the STS and KAL genes (23 ,31 ,32 ). In agreement with this hypothesis, no murine ARSD or ARSE homolog could be identified, as in most human pseudoautosomal genes (5 ). An ancestral pseudoautosomal location of ARSD and ARSE is consistent with their origin through duplication events. These may have been caused by the high frequency of recombination present in this region, with one obligatory crossing-over occurring at each male meiosis. Additional factors predisposing to gene duplications in this region are the presence of several repeat sequences, such as the STIR family of repeats (9 ,36 ), and the presence, within the pairing region, of the pseudoautosomal boundary resulting in the juxtaposition of segments with 100% X-Y homology with segments having very little or no homology.
Another cluster of homologous genes, MIC2, MIC2R and XG (11 ), which is located within the present day PAR, only 150 kb apart from the ARSD gene, may have been generated through the same mechanism. We postulate that the movement of the pseudoautosomal boundary to a more distal location on Xp, with a reduction in size of the PAR, changed the status of these genes from pseudoautosomal to X-linked. A lack of recombination between the X and Y copies of these genes then caused divergence between the X and Y copies with subsequent loss of function of the Y homologs. Average sequence identities between the X and Y copies of the genes were 91.2% for ARSD and 90.9% for ARSE. Sequence identity reported between STS-X and STSP was 88% (31 ). Together, these data indicate that each gene is more similar to its Y-homolog than to other members of the cluster, strongly supporting the hypothesis that duplication events occurred before the X and Y copies of these genes started to diverge, i.e. while they were still pseudoautosomal. Sequence data also suggest that the two Y-linked homologs lost their function as soon as they started to diverge from the ARSD and ARSE genes. In fact no significant differences were found in X-Y sequence identities between exonic and intronic regions, nor in the mutation rate among different codon positions, suggesting that ARSD-Y and ARSE-Y have not been subject to selective pressure to maintain their function.
The Y-chromosome YAC library was purchased from Research Genetics (catalog #95030). The 214 clones were arrayed in one filter which was used for the subsequent screening. For the YAC characterization, total yeast DNA was prepared in 100 µl of Seaplaque (FMC, Rockland, ME) agarose plugs and used for all subsequent procedures. YAC insert size was determined by PFGE analysis using the CEPH configuration according to previously reported procedures (40 ).
Restriction digestions, Southern blot analysis, amplification and subcloning into Bluescript vector were carried out according to standard procedures.
All nucleotide sequences were determined using a Sequenase sequencing kit (USB) and the alignment program Bestfit (Genetics Computer Group, Madison, WI) was used to analyze sequence identity of X/Y homologous loci.
Metaphase spreads were obtained from PHA-stimulated peripheral blood lymphocytes (HSA), and from lymphoblastoid cell lines of common chimpanzee (Pan troglodytes), gorilla (Gorilla gorilla) and macaca (Macaca fascicularis). Chromosome preparations were hybridized in situ with probes labeled with biotin or digoxigenin by nick translation, essentially as described by Lichter et al. (41 ), with minor modifications. Briefly, 600 ng of labeled probe was used for each experiment; hybridization was performed at 37oC in 2* SSC, 50% (v/v) formamide, 10% (w/v) dextran sulfate, 5 µg COT1 DNA (BRL) and 3 µg sonicated salmon sperm DNA, in a volume of 10 µl. Post-hybridization washing: hybridization experiments on human metaphases were washed at 42oC in 2* SSC-50% formamide (*3), followed by three washes in 0.1* SSC at 60oC. Probes from YACs were produced by Alu-PCR amplification as described (42 ). Biotin-labeled DNA was detected with Cy3-conjugated avidin (BDS, PA), while digoxigenin-labeled DNA was detected using FITC-conjugated anti-digoxigenin antibody (Boehringer Mannheim).
Chromosome identification was obtained by simultaneous DAPI staining that produced a Q-banding pattern. Digital images were obtained using a Zeiss Axioplan epifluorescence microscope equipped with a cooled CCD camera (Princeton Instruments, NJ). Cy3, FITC and DAPI fluorescence, detected using specific filter set combinations (Chroma Technology, VT), were recorded separately as gray scale images. The filter set used allows capturing of Cy3, FITC and DAPI signals without any image shifting. Pseudocoloring and image merging were performed using the Adobe Photoshop software.
We thank Giovanna Camerino, Peter Goodfellow and Elena Rugarli for helpful suggestions. This work was supported by the Italian Telethon Foundation and by Associazione Italiana per la Ricerca sul Cancro.
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