Pseudoautosomal genes in man

Genes and sequences that exchange between the eutherian sex chromosomes during male meiosis fail to show complete sex linkage (Koller & Darlington, 1934; Haldane, 1936). The mode of inheritance of such genes and sequences has been termed pseudoautosomal (Burgoyne, 1982). For many years, evidence supporting the existence of the pseudoautosomal region was indirect and based on assumptions about chromsosomal behaviour during male meiosis. More recently, the existence of the pseudoautosomal region in man and mouse has been confirmed by a combination of molecular and classical genetic analysis.

In mouse, the Sxr mutation was shown to be caused by the pseudoautosomal inheritance of the male sexdetermining gene (Tdy) (Evans, Burtenshaw & Cattanach, 1982; Singh & Jones, 1982). Sts, the gene encoding the microsomal enzyme steroid sulphatase, is present on both the X and Y chromosomes in mouse and its inheritance is almost certainly pseudoautosomal (Keitges, Rivest, Siniscalco & Gartler, 1985; Weissenbach et al., this symposium; Bishop et al., this symposium). A third indication of the pseudoautosomal region in the mouse is the inheritance of a retroviral sequence, MOV-15. newly integrated into the Y chromosome. Unlike Sts or Sxr. which show no sex linkage, MOV-15 exchanges between the sex chromosomes with a frequency of about 10%, implying the existence of a recombination gradient in the mouse pseudoautosomal region (Harbers, Soriano, Muller & Jaenisch, 1986).

In man, the first suggestion of the existence of a human pseudoautosomal region came from the chromosomal localization of the MIC2 gene. A monoclonal antibody, 12E7, which defines a human cell surface antigen encoded by the MIC2 gene was used to map the gene to both the X and Y chromosomes and the sublocalization on the X chromosome was well within the X-Y pairing region. Consistent with the X and Y localization, the MIC2 gene was found to escape inactivation on the inactive X chromosome in female cells. Formal proof of the existence of the pseudoautosomal region, however, was obtained from studies of the inheritance of several DNA sequences isolated at random from the human Y chromosome (Cooke, Brown & Rappold, 1985; Simmler et al. 1985; Rouyer et al. 1986). These studies demonstrated that in male meiosis a single obligate recombination event occurs in the distal portion of the short arms of the X and Y chromosomes. The position of crossing over is variable and a gradient of recombination with respect to Y-specific sequences is observed. Cloning the MIC2 gene provided DNA probes which were used to demonstrate that MIC2 also exchanged during male meiosis, but X/Y recombination events which extend to include the MIC2 gene occur with a much lower frequency than the other pseudoautosomal markers (Goodfellow, Darling, Thomas & Goodfellow, 1986).

The steroid sulphatase gene, STS, in man differs from its homologue in mouse and is not pseudoautosomal (Craig & Tolley, 1986; Fraser et al., this symposium). MIC2 is the only known human pseudoautosomal gene. We will consider here the biology and genetics of MIC2 and the use of MIC2 probes for studying X-inactivation. Finally, we will describe the relationship between MIC2 and the X-linked locus XG which has led us to postulate the existence of a second pseudoautosomal gene in man.

The monoclonal antibody 12E7 was produced by Levy, Dilley, Fox & Warnke (1979). The antibody reacts with a cell surface antigen found on all human cells tested, with the exception of spermatozoa (Goodfellow, 1983). The 12E7 antigen is also found on the surface of gorilla and chimpanzee cells but is absent from the cells of orang-utan and other primates as well as all other animal species tested (Goodfellow, 1983). Table 1 describes additional monoclonal antibodies, all of which react with the 12E7 antigen. Although several of these antibodies are of different immunoglobulin class and recognize different antigenic epitopes, they share the unusual property of failing to immunoprecipitate the 12E7 antigen despite recognizing it by immunoblotting (Banting, Pym & Goodfellow, 1985).

The clone pSGl was isolated by screening a bacteriophage λgtll cDNA expression library with the 12E7 antibody (Darling et al. 1986a). Three lines of evidence support the conclusion that this is a cDNA clone corresponding to the MIC2 gene. First, the chromosomal localization of the genomic sequences recognized by pSGl is the same as the MIC2 gene (see below). Second, 12E7 antigen-positive mouse cells have been constructed by DNA-mediated gene transfer using human DNA. Antigen-positive primary and secondary transfectants contain human sequences which react with pSGl (B. Pym et al. unpublished observations). Furthermore, transfectants expressing increased amounts of 12E7 antigen have amplified the sequences reacting with pSGl. Third, a monoclonal antibody, MSGB1, has been produced by immunizing a mouse with a peptide, the sequence of which was derived from a conceptual translation of the nucleotide sequence of pSGl (Darling et al. 1986b). The MSGB1 antibody reacts with the 12E7 antigen. Although sequence analysis suggests that pSGl contains all of the coding sequence of the MIC2 antigen, comparison with the message suggests that pSGl does not contain the 5’ untranslated region of the M1C2 transcript. A putative full-length clone, pNT23, has been isolated by screening a new cDNA library constructed with mRNA from the human teratocarcinoma cell line NTERA2 (M. V. Wiles and S. Darling, unpublished results).

Cosmid and bacteriophage  genomic libraries have been screened with the pSGl clone and subclones derived from it. Over 50 kb of human genomic DNA has been isolated. This DNA contains the predicted 5’ end of the gene, but still lacks sequences at the 3’ end. Only the CpG-rich region (HTF island, see Bird, 1986) at the 5’ end of the gene has been analysed in detail.

The MIC2 gene was originally mapped to both the X and Y chromosome by exploiting the species specificity of the 12E7 antibody in somatic cell genetics (Goodfellow, 1983). Further localization of the Xlocated M1C2 gene was achieved by studying hybrids containing different fragments of the X chromosome. Two hybrids are of particular interest. The hybrid UCLAB2 contains an X chromosome which has a short-arm terminal deletion of about 5x10⁶bp; this deletion removes the entire pseudoautosomal region as well as the genes MIC2 and STS and several DNA sequences known to be restricted to the X chromosome. This result places an upper limit on the size of the pseudoautosomal region (Curry el al. 1984). The hybrid 817×175 contains an X-Y translocation chromosome which has resulted in the loss of MIC2 but not STS. This makes M1C2 the most distal of the known genes on the X chromosome (Mondello et al. 1987).

A similar conclusion has been reached by Geller, Shapiro & Mohandas (1986). The regional localization of the MIC2 gene on the Y chromosome was achieved by in situ hybridization using the pSGl probe (Buckle et al. 1985). In the human genome, only two sites of hybridization to pSGJ were found: the end of the X chromosome short arm and the end of the Y chromosome short arm. This localization is within the pairing region and is consistent with MIC2 being part of the pseudoautosomal region.

DNA marker studies were undertaken to test for pseudoautosomal inheritance of MIC2. Numerous restriction fragment length polymorphisms were found to be detected with the probe pSGl. Unfortunately, the cDNA probe proved particularly difficult to use, as patterns of hybridization to DNA digested with most restriction enzymes tested produced multiple bands, many of which showed variation in the population. This complexity in the patterns of hybridization observed made it difficult to identify alleles. Simplification of the Southern blot patterns was obtained by using genomic probes. Like other pseudoautosomal DNA sequences, isolated at random from the Y chromosome, the genomic MIC2 probes also recognized a large number of polymorphic sites. The MIC2 polymorphisms appear to be due to simple base pair changes rather than more complex deletions or duplications.

In families ascertained at random, we have found a single X/Y recombination event including MIC2 in 46 informative male meiosis. Testing the same families with other pseudoautosomal markers allowed us to construct a male meiotic map of the region. MIC2 is the most proximal of the pseudoautosomal loci and shows no detectable linkage with the most distal pseudoautosomal marker DXYS14 which is known to be close to the telomere (Cooke et al. 1985). These results are important for two reasons. First, the pseudoautosomal region is maximally 5×10⁶bp long and may be considerably smaller. 50 % recombination in 5×10⁶bp is some ten times higher than the expected average for that amount of DNA sequence in man (Renwick, 1969). It is also a considerably greater rate of recombination than is found over the same region in females (Fig. 1). Interestingly we, and others, have found no evidence for double recombinants in the pseudoautosomal region. Second, it has been argued on theoretical grounds (Bengtsson & Goodfellow, 1987) that the male sex-determining gene, TDF, will lie close to the pseudoautosomal region of man. If this is the case M1C2 is a useful flanking marker for TDF.

The long-range restriction map constructed using pulsed-field gel electrophoresis techniques is consistent with the meiotic map of the pseudoautosomal region. Preliminary results suggest that M1C2 lies less than 5×10⁵bp away from sequences which are Y specific (C. Pritchard, personal communication).

Biochemical studies have been severely hampered because the available antibodies (see Table 1) fail to immunoprecipitatc the 12E7 antigen reproducibly. The alternative approach of immunoblotting. though of limited value for immunological and biosynthetic analysis, demonstrated that the MIC2 product is a cell surface molecule of 32·5 × 10³M_r with a pl of about 5·0 (Banting el al. 1985).

Sequence analysis of the M1C2 cDNA defined a long open reading frame. To check that the correct open reading frame had been identified, mice were immunized with a synthetic peptide corresponding to part of this sequence (Fig. 2). Monoclonal antibodies were produced that recognize both the immunizing peptide and the 32·5 × 10³M_rMIC2 cell surface product. MSGB1 is typical of these antibodies (Table 1). Whilst none of these antibodies immunoprecipitate specific polypeptides their ability to recognize the 32·5×10³M_r cell surface molecule in immunoblot analysis demonstrates that the synthetic peptide sequence does correspond to part of the MIC2 gene product, therefore, the correct open reading frame has been chosen for conceptual translation.

Recent epitope mapping studies have shown that all the antibodies listed in Table 1 recognize short, adjacent and/or overlapping continuous epitopes in the conceptual translation (G. S. Banting unpublished results). Since these antibodies bind to the surface of cells, this part of the sequence must be extracellular; definition of the transmembrane domain would allow predictions to be made regarding orientation of the molecule in the plasma membrane. Hydropathy profiles show two long hydrophobic stretches within the conceptual amino acid sequence, one at the extreme amino terminus. Such sequences could serve as transmembrane domains or signal sequences (Kyte & Doolittle. 1982).

Computer-assisted searches of available protein databases have provided no clue as to function since, with few exceptions, they have revealed no significant homology between the MIC2 product and any previously published sequence. All the exceptions are matches found between the amino terminal region of the MIC2 conceptual translation and the signal sequences of several precursor proteins. It is therefore likely that the amino terminus acts as a signal sequence directing the molecule to the cell surface.

The lack of homology between the MIC2 product and previously described sequences suggests that the molecule may have a novel function. We have chosen to raise antibodies against synthetic peptides corresponding to potentially interesting regions of the conceptual translation in an attempt to investigate this function.

It would be predicted on gene-dosage grounds that MIC2 would escape X-inactivation. Using somatic cell genetics it was found that MIC2 does escape X-inactivation and cloning MIC2 has provided a tool for studying the molecular basis of X-inactivation.

Control of gene expression in mammalian cells is still poorly understood. Sequences rich in the CpG dinucleotide pair at the 5’ ends of genes have been implicated as playing an important role in their expression (Bird, 1986). Because of the large number of Hpall sites present in the G + C-rich regions, the descriptive term ‘HTF-island’ (HpaW tiny fragment island) has been applied to such regions. A general feature of HTF-islands is a lack of 5-methylcytosine residues and hypomethylation within HTF-islands is positively correlated with gene activity. Analysis of the HTF-island of two X-linked genes (PGK and HPRT) has shown methylation of the HTF-islands on the transcriptionally silent inactive X chromosome, whereas the same islands on the active X are unmethylated (Yen et al. 1984; Keith, Singer-Sam & Riggs. 1986).

There is an HTF-island at the 5’ end of the MIC2 gene. Preliminary sequence analysis of 1·2 kb around the presumed first exon indicates that it is very GC rich (67%) with abundant CpG dinucleotide pairs. Southern blot analysis of genomic DNAs doubly digested with HindIII and rare cutting methylationsensitive restriction enzymes reveal a cluster of unmethylated sites including BssHII. SocII. NarI and NaeI (Fig. 3). Comparison of hybridization patterns to DNAs prepared from peripheral blood leukocytes of males (XY). females (XX) and Turners syndrome female (XO) proved that there were no differences in methylation of sites within the HTF-island on the active X, inactive X or Y chromosome. DNAs from a number of lymphoblastoid B cell lines show a similar lack of methylation. The HTF-islands associated with PGK or HPRT are methylated on the inactive X chromosome in XX individuals (Yen et al. 1984; Keith et al. 1986). Lack of methylation of the MIC2 HTF-island on the inactive X chromosome correlates with escape from inactivation. It is similarly unmethylated on the Y chromosome carrying an active gene. Methylation within the body of MIC2 is, however, variable as measured by comparison of hybridization to Mspl- and Wpall-digested DNAs. In DNA prepared from blood cells from Turners syndrome females (X^active O) two Hpail sites we have examined are always cleaved whereas DNAs prepared from blood of normal females (X^active x^active) show that those sites are either not cleaved or are only partially cleaved on the inactive X chromosome (C. Mondello, unpublished observation).

One plausible interpretation of these results is that escape from methylation and perhaps X-inactivation generally is an intrinsic property of the HTF-island of M1C2. Alternatively, the HTF-islands of genes which are X-inactivated may be differentially susceptible to methylation on the inactive X chromosome.

The expression of the 12E7 antigen on red blood cells is polymorphic. Two phenotypes can be recognized: high level 12E7-antigen expression and low level 12E7-antigen expression. There is a complex interaction between the XG blood group locus and the 12E7-antigen polymorphism. All individuals that are XG^a-antigen positive are high level expressors of the 12E7 antigen. XG^a-antigen-negative males can be either high or low level expressors of 12E7 antigen. These results suggested the existence of a Y-located gene, YG, which was polymorphic and regulated the level of 12E7-antigen expression on red blood cells (Goodfellow & Tippett, 1981). Formal proof of a Y-located genetic element was obtained from sib-analysis. It was predicted that XG^a-antigen-negative brothers would all share the same 12E7-antigen phenotype. Of about 200 brothers tested, 194 shared the same 12E7-antigen phenotype. This result can only be explained by the influence of a Y-located gene (Tippett, Shaw, Green & Daniels, 1986). The six exceptional cases were originally thought to be artefactual due to technical problems, however, at least in one case, the exception is real. When we investigated the one exceptional brother and his family by DNA analysis it was discovered that he was recombinant at MIC2. This result has led us to postulate a new model in which a c/x-acting regulator controls both XG^a-antigen expression and 12E7-antigen expression on red blood cells (Fig. 4). The regulator. XGR. is polymorphic with two alleles. The A allele induces the structural XG locus to produce the XG^a antigen and induces the MIC2 locus to produce high levels of the 12E7 antigen in red blood cells. The B allele induces only low levels of the 12E7 antigen and fails to induce the expression of the XG^a antigen. The regulator is postulated to be pseudoautosomal in order to explain the exceptional case described above. This theory is explained in more detail in Goodfellow. Pritchard, Tippett & Goodfellow (1987).

Molecular and genetic analysis of the MIC2 gene of man has revealed important clues as to the structure of the pseudoautosomal region of man. We anticipate that further analysis of this system will provide insights into the mechanism of X-inactivation and the evolution of the pseudoautosomal region. MIC2 is a flanking marker for the pseudoautosomal and X- and Y-chromosome-specific regions. That location makes M1C2 a convenient starting point for experiments designed to clone TDF.

We would like to thank our many collaborators who have contributed to the unravelling of the complexities of the MIC2 system. This manuscript was prepared with editorial assistance from Mrs C. Middlemiss.