Human pseudoautosomal boundary-like sequences: expression and involvement in evolutionary formation of the present-day pseudoautosomal boundary of human sex chromosomes
Human pseudoautosomal boundary-like sequences: expression and involvement in evolutionary formation of the present-day pseudoautosomal boundary of human sex chromosomesTatsuo Fukagawa, Yasukazu Nakamura, Katsuzumi Okumura1, Masahiro Nogami1, Asako Ando2, Hidetoshi Inoko2, Naruya Saitou and Toshimichi Ikemura*
Department of Evolutionary Genetics, National Institute of Genetics, and The Graduate University for Advanced Studies, Mishima, Shizuoka-ken 411, Japan, 1Faculty of Bioresources, Mie University, Tsu, Mie-ken 514, Japan and 2School of Medicine, Tokai University, Bohseidai, Isehara, Kanagawa-ken 259-11, Japan
Received August 23, 1995;Revised and Accepted October 10, 1995
The human genome is composed of long-range mosaic structures of G+C% (GC%), which are thought to be related to chromosome bands. We previously identified a boundary of Mb-level domains of GC% mosaic structures in the human major histocompatibility complex (MHC) and found in the domain boundary a sequence very similar to pseudoautosomal boundary (PAB) sequences of human sex chromosomes. We designated it `PABL' and found many PABLs in the human genome. By analysis of six genomic and six transcribed PABLs, a core and consensus sequence of about 650 nt was defined; the 3'- and 5'-edges of the PABLs were strictly conserved. Northern blot analysis showed sizes of PABL transcripts to be 5-10 kb in length. Divergence time of PABLs was estimated to be 60-120 million years ago by analysis of human PABLs and PABXY1 of seven primates, and the evolutionary rates deduced showed PABLs to have been under selective constraints. A model for evolutionary formation of the present pseudoautosomal boundary was proposed by postulation of illegitimate recombination between two PABLs.
The human genome, like the genomes of warm-blooded vertebrates in general, has long-range G+C% (GC%) mosaic structures related to chromosome bands: Giemsa-dark G bands are composed mainly of AT-rich sequences, T bands (a subgroup of Giemsa-pale R bands) are of GC-rich sequences, and ordinary R bands are heterogeneous and appear to be intermediate (1 -6 ). Bernardi et al. called the GC% mosaic domains `isochores' (1 ). DNA replication timing, gene density, CpG island density, codon usage, chromosome condensation, repeat sequence density, and chromosome behavior such as recombination are related to chromosome bands and to long-range GC% mosaic domains (5 -12 ). Because chromosome bands are structures observed by microscopes, precise location of their boundaries may seem meaningless. However, considering the various genome behaviors connected with chromosome bands, we may precisely locate their boundaries by using informative landmarks on genomic DNA. Boundaries may be structurally assigned where there are clear GC% transition points, and characteristic signals may be found that punctuate and/or differentiate distinct functions, such as a signal that switches from early to late DNA replication. We previously reported a boundary of long-range GC% mosaic domains in the human major histocompatibility complex (MHC) disclosing a sharp GC% transition, and found in the boundary region high sequence similarity with the pseudoautosomal boundary (PAB) sequence in the short arms of human sex chromosomes (13 ).
Human sex chromosomes have two functionally distinct regions, sex-specific sequences and pseudoautosomal regions (PARs). X and Y chromosomes exchange DNA sequences through homologous recombination in PARs during each male meiosis, and PAR sequences in the two chromosomes are practically identical because of the obligatory recombination (14 -17 ). There are two PARs for human sex chromosomes: PAR1 is at the distal ends of the short arms of the X and Y chromosomes and PAR2 is at the ends of the long arms (17 ,18 ). The interface between the PAR1 of 2.6 Mb and the sex-specific region is the pseudoautosomal boundary, PAB1, and therefore PAB1 is the centromeric limit to recombination in PAR1. Ellis and Goodfellow (16 ) and Ellis et al. (19 ,20 ) reported sequences around the interface, PABX1 and PABY1 sequences (abbreviated PABXY1). A sequence found in the boundary of long-range GC% mosaic domains in the MHC is very similar to PABXY1 being the functional interface in sex chromosomes. We designated the sequence found in the MHC `pseudoautosomal boundary-like sequence 1', abbreviated PABL1 (13 ). With the PABL1 segment as a probe, many copies of pseudoautosomal boundary-like sequences (PABLs) were detected by Southern blot hybridization against genomic DNAs. In the present study we characterized both genomic and transcribed PABLs.
Using the PABL1 segment found in the human MHC as a probe, we isolated about 150 independent cosmid clones having PABLs. Figure 1 shows alignment of genomic PABLs sequenced thus far and PABXY1 plus their flanking sequences; the sequence orientation for PABXY1 is from telomere to centromere (i.e., from PAR1 to the sex-specific region). Similarity for all pairs of PABLs and PABXY1 were high (~80% nucleotide identity), and the 3'-terminus of the similar region among all PABLs corresponds closely to the PABXY1 homology terminus reported by Ellis et al. (19 ,20 ) as where the X and Y chromosome sequences diverge completely (sex-specific in Fig. 2 ).
Although multiple copies of PABLs were found in the human genome, genomic sequences in the recent GenBank (Release 87 including update data; 1995) exhibiting significant similarity with the PABLs were confined to PABXY1. In the case of expressed sequence tag (EST) sequences, however, eleven ESTs were similar (~80% nucleotide identity) to separate portions of PABLs (Fig. 2 ), suggesting that some, if not all, PABLs are transcribable and presumably have functions. For identification of the characteristics of the predicted PABL transcripts, six human cDNA libraries of different tissues and cells were screened with the PABL1 segment as a probe. Clones were obtained from all six libraries, and inserts of all 20 cDNA clones analyzed were longer than the PABL size. We then sequenced the following six cDNA clones: Mo1 and Mo2 from the monocyte library; Bc4 from the B-cell library; Sk13 from the skin library; and Sp2 and Sp3 from the spleen library. Alignment around the 5'-terminal portion of all available PABLs found in the cDNA clones including ESTs (T92306 and R12279) is shown in Figure 3 A. The similarity terminus of the 5'-portion of these transcribed PABLs corresponds closely to that defined for genomic PABLs. It should first be noted that a portion of the Mo2 cDNA sequence was practically identical to one of the ESTs mentioned above, HSAAAAWAH. The identity portion spanned not only the PABL sequence but also the entire flanking region of 266 nt HSAAAAWAH, predicting the EST to correspond to a portion of the Mo2 transcript and the transcript to be fairly abundant. Sequences of three recently reported ESTs (R12279, R07404, and T99392, derived from at least two independent cDNA libraries) were almost identical with each other, indicating the transcripts are also abundant.
The six cDNAs of PABLs analyzed were longer than the PABL size. For estimation of the intact sizes of PABL transcripts, northern blot analysis of human total or polyA+ RNA fraction was done with the PABL1 probe. Figure 4 A shows the results for the total RNA extracted from B-cell line GM01416D. Broad bands mainly with mobility slower than that of 28S rRNA and estimated to be 5-10 kb in length were detected. The results for the polyA+ RNA fractions were practically the same for total RNA (Fig. 4 B). All samples shown in Figure 4 gave signals, although expression levels seemed to differ. The broad bands shown are likely to correspond to many transcripts hybridized with PABL sequences, and their length being long was consistent with the finding that the PABL cDNAs were longer than the PABL size. For detection of a single transcript corresponding to a unique cDNA, a Sp2 cDNA fragment deprived of its PABL core was used as the hybridization probe, and in this case a single sharp band of 7.5 kb was detected (Fig. 4 C).
Obligatory pairing and crossover in the PAR ensure accurate segregation of the sex chromosomes during male meiosis. An unusually high rate of homologous recombination in PAR1 (20-fold the genome average) seems to arise from a mechanism that promotes obligatory physical association and the successive crossover between the sex chromosomes. The strict limit to terminate the high frequency recombination in PAR1 is the pseudoautosomal boundary 1 (PAB1). An Alu element is situated in PAB1 of the human Y chromosome, PABY1, but not in PABX1 (19 ). Ellis et al. defined first the Alu element as the strict boundary of PAR1. A later study of the boundaries of PAR1 in Old World monkeys, however, found no Alu repeats on the PABY1 (20 ). In spite of the lack of the Alu element, they proposed the Alu-insertion site itself as the strict boundary of PAR1 for the following reasons. In pair-wise comparisons of the X and Y boundary sequences within each species of several hominoids and Old World monkeys, they found sequences about 220 nt long downstream of the Alu insertion site to be more divergent between sex chromosomes than the sequences of the Alu-upstream region (~78% vs. ~97% nucleotide identity; see Fig. 2 for human PABXY1), whether or not the Alu element was present. By extensive analysis of the nucleotide substitution patterns, they concluded that the Alu-insertion site, which corresponds to the abrupt transition between the high- and reduced homology regions, is the strict limit for high-frequency recombination in PAR1 and thus the exact boundary of PAR1 (20 ). The position of the boundary in Old World monkeys and hominoids was practically the same. This shows that the limit of PAR1 recombination was at the Alu-insertion site before divergence of lineages of Old World monkeys and great apes, and that an Alu element was inserted into the preexisting boundary of the Y chromosome in the great ape lineage. As did Ellis et al., this site is called the `Alu-insertion site' in this paper whether or not the Alu element is present; the high- and reduced-homology regions are thus called the Alu-upstream and Alu-downstream regions, respectively. Downstream of the Alu-downstream region, there is no similarity between X and Y sequences, which are called the sex-specific regions in Figure 2 .
Sequences around the Alu-insertion site being the exact interface that divides sex chromosomes into two functionally distinct regions are known to be strictly conserved in species of hominoids and Old World monkeys (20 ). At the insertion site there is a 10 nt sequence strictly conserved even among PABLs, although the Alu element is absent (see Fig. 1 ). Strict sequence conservation around this Alu-insertion site, as well as strict conservation of the 5'- and 3'-termini, during evolution may be related to possible functions of PABXY1 and PABLs. In our previous paper, we reported a rather unexpected finding that the sequence similarity of the Alu-downstream portion of PABX1 or PABY1 with the PABL2 (82.2 or 80.5% nucleotide identity) is higher than that between PABX1 and PABY1 (77.8% identity) and the similarity among PABX1, PABY1, and PABL1 is equivalent (13 ). For clarifying evolutionary processes that formed the present PABXY1 and PABLs and investigating functional constraints on the sequences, phylogenetic relationships and evolutionary rates were examined for all available PABLs. The reported PABXY1 sequences of great apes and Old World monkeys (20 ) were included for estimation of the evolutionary rates. Divergence between great apes and Old World monkeys is postulated to have occurred 25 million years ago. Because of the PAR characteristic, PABX1 and PABY1 sequences upstream of the Alu-insertion site are practically identical within a single species. For avoidance of complications arising from this peculiar characteristic, individual PABXY1 and PABLs were divided into two regions and unrooted phylogenetic trees were separately constructed by the neighbor-joining method (Fig. 5 ). Trees obtained from evolutionary distances on the basis of one- and two-parameter methods were identical in their branching patterns or topologies, and branch lengths were nearly the same.
At a standard 850 band level, the human MHC harboring PABL1 is on a wide R band, 6p21.3, which has been assigned to a T-type R band (T bands); T bands are an evidently heat-stable subgroup of R bands and mainly composed of GC-rich sequences. By high-resolution banding, a thin G positive subband, 6p21.32, was found within the MHC (23 ). On the basis of detailed base composition analysis, Fukagawa et al. (13 ) predicted that the genome portion harboring the evidently AT-rich 200 kb around the junction between classes II and III constitutes a portion of the thin G positive subband. PABL1 was within the AT-rich domain but close to the domain edge, suggesting PABL1 is located near a chromosome band boundary. Considering the wide range of functional behaviors of chromosome bands and GC% mosaic domains, their boundaries are most probably composed of multiple signals and structures related with multiple functions. Near the GC% mosaic boundary in the MHC the following characteristic structures have been found: on the GC-rich side a dense 20 kb Alu cluster and on the GC-poor side a dense 30 kb LINE-l cluster and PABL1 (13 ). If these characteristics are found for other boundaries, it will add to our comprehensive understanding of both DNA sequences and chromosome structures. We focus here on GC% distribution and band structures around PABXY1. PAR1 has been assigned to an R band (Xp22.33 and Yp11.32), and judging from the high density of CpG islands a major portion of 2.6 Mb of PAR1 is thought GC-rich (17 ). The boundaries with the neighboring G-positive subbands, Xp22.32 and Yp11.31, appear rather close to PABXY1 (17 ,24 ) (Fig. 6 ). PABXY1 and their neighboring sex-specific sequences, including SRY, RPS4Y, and ZFY located downstream of PABY1, are AT-rich (20 ). We therefore previously predicted that PABXY1 is near a boundary of the long-range GC% mosaic domains and of chromosome bands in the sex chromosomes (13 ). Very recently, analyzing a contiguous 41 kb sequence harboring PABY1 (22.5 kb of PARY1 and 18.5 kb of Y-specific sequence), Whitfield et al. (25 ) showed that the PARY1 and Y-specific portions differ dramatically in GC%. It thus becomes clear that not only PABL1 in the MHC but also PABY1 are located in the GC% mosaic boundaries and therefore genome characteristics in regions harboring these elements are similar.
Functionally important molecules evolve more slowly than less important molecules (26 ). PABLs probably have biological functions because they are transcribable and their evolutionary rates were slower than nonfunctional regions. Concerning the functions of boundaries of chromosome bands and GC%-mosaic domains, the switching of DNA replication timing is an important candidate (11 ,13 ). Multi-color FISH of interphase nuclei (27 ), in which contiguous [lambda] phage and cosmid clones in the MHC (13 ,28 ) were used as probes, showed that PABL1 is located in a switching region of DNA replication timing (Okumura et al., in preparation). Major portions of the methylated and transcriptionally inactive X chromosome replicate very late in S phase, but PAR1 escaping from the X-inactivation is thought to replicate much earlier (29 ,30 ). Therefore, a switch in DNA-replication timing, at least in the inactivated X chromosome, presumably occurs near PAB1. PABLs including PABXY1 may be related to possible signals for DNA replication timing such as a pause signal for the replication. Considering that high frequency-recombination in PAR1 between sex chromosomes terminates abruptly at PAB1, PABLs are expected to be involved in a certain process of recombination. A model for evolutionary formation of the present pseudoautosomal boundaries is proposed below by postulation of illegitimate recombination between two PABLs.
PABX1 was found within an intron of PBDX (pseudoautosomal boundary divided on the X chromosome), which encodes the Xga antigen and was recently renamed XG (31 ,32 ). When RT-PCR analyses were done on human total RNA from a B-cell line, primers designed for the present cDNAs reproducibly gave PCR products with the expected sizes under standard conditions (data not shown). However, primers designed for the PABX1 region showed only occasionally a faint band of the expected size under the same or modified conditions. RNA molecules corresponding to the present cDNAs may be more abundant or have longer life-span than the RNA of the XG intron.
Significant ORFs were not found for the present cDNA sequences, for either PABLs or their flanks. Computer searches could assign no protein-coding capacity. It should also be noted here that intact size of PABL transcripts ranges from 5 to 10 kb but the length of cDNA sequences obtained in this study is 2 kb or less. Therefore we can not exclude the possibility that protein-coding regions are present in the unanalyzed portions. Alternatively, these results may suggest that the functional form of the transcripts is RNA molecules. PABL1 and PABXY1 were both found in experiments that searched for molecular structures and signals differentiating global characteristics in the human genome. Large PABL transcripts may be necessary for recognition or differentiation of global genome characteristics. XIST RNA which is 17 kb long has been thought important in such determination and differentiation in the inactivated X chromosome (33 ).
Recently Ellis et al. (31 ) proposed a model for the evolutionary formation of the present-day pseudoautosomal boundary of the short arms, PABXY1. They hypothesized a pericentric inversion of the Y chromosome with one break point in ancestral XG and the other break point 5 kb distal to the ancestral SRY believed to be on the earlier long arm (see legend of Fig. 7 ). Here we propose a model in which the hypothesized inversion occurred by illegitimate recombination between the two PABL elements, one PABL in the ancestral XG and the other near the ancestral SRY; before recombination, the two elements on the earlier Y chromosome were ordinary PABLs (Fig. 7 A). After recombination, the characteristics of a pseudoautosomal boundary was acquired and the recombinant PABL became PABY1 (Fig. 7 C).
A cosmid library constructed from the total human DNA of an HLA homogeneous B cell line on vector pWE15 by Inoko et al. (34 ) and a [lambda]-EMBL3 library constructed from the human DNA of peripheral blood cells (35 ) were used. Six human cDNA libraries cloned on vector [lambda]gt10 or [lambda]gt11 were obtained from Clontech (Palo Alto, CA): placenta 5'-stretch plus ([lambda]gt11), placenta ([lambda]gt11), monocyte ([lambda]gt11), B cell ([lambda]gt10), spleen ([lambda]gt10), and skin fibroblast ([lambda]gt10). Cloning of PABLs, their subcloning into pUC118, sequencing, and database searches of GenBank/EMBL/DDBJ, as well as of PIR and Swiss-Prot protein databases, were done as described previously (13 ,28 ).
Alignments and calculations of sequence identity were done with the MALIGN program available on DDBJ. For identification of the core sequence of PABLs, all pairs of sequences with PABLs or PABXY1 were first aligned, and multiple alignments of the similar portions thus found were done as described by Hein (36 ). Phylogenetic trees were constructed by using the neighbor-joining method (37 ). Evolutionary rates were estimated as described by Nei (38 ).
The authors are very grateful to Dr N. Ellis for kindly providing us with his unpublished sequence and valuable comments; to Drs K. Sugaya, T. Tenzen and K. Matsumoto for discussion; to Mrs Y. Miyauchi for technical assistance. The computers at the DDBJ and the Human Genome Center of Japan were used. This work was supported by a JSPS Fellowship for Japanese Junior Scientists to T.F., Grant-in-Aid for Creative Basic Research (Human Genome Project) to T.I. and for Scientific Research on Priority Areas (Genome Informatics) to T.I., and by Grants-in-Aid of Scientific Research from the Ministry of Education, Science and Culture of Japan to T.F. and T.I.
MHC, major histocompatibility complex; PAB, pseudoautosomal boundary; PABL, pseudoautosomal boundary-like sequence; PABX1, pseudoautosomal boundary sequence of short arm of X chromosome; PABY1, pseudoautosomal boundary sequence of short arm of Y chromosome; PABXY1, PABX1 and PABY1 sequences; PAR1, pseudoautosomal region of short arm of sex chromosomes; PAR2, pseudoautosomal region of long arm of sex chromosomes; EST, expressed sequence tag.
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*To whom correspondence should be addressed
Sequence data from this article have been deposited with the GenBank/EMBL/DDBJ Data Libraries under Accession Nos. D55638 (Bc4), D55639 (Mo1), D55640 (Mo2), D55641 (Sk13), D55642 (PABLSp2), D55643 (Sp2), D55644 (Sp3), D63517 (PABL consensus sequence)
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