Reactivation by exon shuffling of a conserved HLA-DR3-like pseudogene segment in a New World primate species

Abstract

The common marmoset (Callithrix jacchus), a New World monkey species with a limited MHC class II repertoire, is highly susceptible to certain bacterial infections. Genomic analysis of exon 2 sequences documented the existence of only one DRB region configuration harboring three loci. Two of these loci display moderate levels of allelic polymorphism, whereas the -DRB*W12 gene appears to be monomorphic. This study shows that only the Caja-DRB*W16 and -DRB*W12 loci produce functional transcripts. The Caja-DRB1*03 locus is occupied by a pseudogene, given that most of the transcripts, if detected at all, show imperfections and are present at low levels. Moreover, two hybrid transcripts were identified that feature the evolutionarily conserved peptide-binding motif characteristic for the Caja-DRB1*03 gene. Thus, the severely reduced MHC class II repertoire in common marmosets has been expanded by reactivation of a pseudogene segment as a result of exon shuffling.

The common marmoset (Callithrix jacchus) has its natural habitat in the swamps and Atlantic rainforest of northeastern Brazil. This species is used as an animal model to investigate several chronic and infectious diseases (1–5).

The classical Mhc class I and II genes play a key role in controlling various immune functions and generally exhibit a high degree of allelic polymorphism in different vertebrate species. Humans, apes, and Old World monkeys are known to share Mhc class I and II loci, but particular lineages have also been documented to predate speciation events that occurred during primate evolution (6).

Equivalents of HLA class II loci have been detected in common marmosets and display various levels of polymorphism (7–10). For instance, both of the apparently functional Caja-DQA1 and -DQB1 genes are oligomorphic, but the -DP locus seems to be inactivated (7). Despite extensive efforts, only three DRB loci have been identified thus far (7, 11, 12). The Caja-DRB1*03 gene, related to the HLA-DRB1*03 lineage that is characterized by the evolutionarily conserved EYSTS motif, is moderately polymorphic (7, 10, 13). Careful comparison of human and common marmoset nucleotide sequences provided evidence that these class II genes are not truly orthologous and arose as a result of convergent evolution (7, 13). The Caja-DRB*W16 locus exhibits a degree of polymorphism comparable to -DRB1*03, whereas the -DRB*W12 gene appears to be invariant. The monomorphic status of the latter gene, and its capacity to present particular myelin oligodendrocyte glycoprotein peptides, may explain why all common marmosets are susceptible to experimental autoimmune encephalomyelitis induction by immunization with particular autoantigens (14).

As a result of expansion and contraction processes, the primate DRB region shows extensive polymorphism with regard to the number and combination of loci present per haplotype, in species as well as in individuals of a given species. For example, within rhesus macaques >30 different DRB region configurations have been defined, whereas in humans only 5 have been documented (15–17). Common marmosets, mostly born as nonidentical twins, are natural bone-marrow chimeras because of a sharing of blood circulation through placental fusion during gestation. Although haplotype definitions are, therefore, complicated, phenotypic and segregation analyses suggested that every region configuration possesses one Caja-DRB*W12 locus, together with at least one -DRB1*03 and one -DRB*W16 locus (7). Thus, common marmosets apparently lack -DR region configuration polymorphism. Moreover, because the few Caja-DRB loci themselves display only moderate or low degrees of allelic polymorphism, one might expect that these animals are susceptible to contracting particular diseases. Indeed, common marmosets in captivity are especially prone to die from certain viral and bacterial infections (3, 18).

Thus far, only genomic DNA analyses of exon 2 sequences have been performed, and nothing is known about the transcription and expression status of the various loci. To gain insight into the class II repertoire at the transcription level, a thorough cDNA analysis of the different Caja-DRB genes in a well defined population of animals was performed.

Results and Discussion

Caja-DRB Genes and the Presence/Absence of Corresponding Transcripts.

In our panel of 15 selected marmosets, a total of 22 full-length cDNAs could be defined (Fig. 1). Nearly all of the Caja-DRB*W16 cDNAs defined earlier by exon 2 analyses are present and characterized by abundant transcription levels. The Caja-DRB*W16 transcripts have all of the characteristics necessary to produce bona fide gene products (Fig. 2). In some animals, three distinct transcripts were detected. This finding is probably due to the chimeric status of the animals, although we cannot exclude the possibility that some region configurations harbor duplicated loci.

Fig. 1.

Caja-DRB alleles detected in a panel of 15 common marmosets. Shading is used to indicate whether the respective allele is detected only on the genomic (exon 2) level (black shading), or on both the genomic and cDNA levels (gray shading). Animals m02040 and m02041 were tested only on cDNA. Because it was impossible to develop -DRB lineage-specific primers that did not crossreact with other DRB cDNAs, real-time PCR results were not reliable. Therefore, transcription levels were determined by counting the number of clones. For every individual, at least 60 clones were analyzed. All transcripts of DRB*W16 and of the DRB1*0307/DRB1*0312 alleles were detected in at least 50% of the clones, and DRB*W12 alleles were detected in ≈25% of the clones. The transcripts of DRB1*03L alleles, however, were found at a much lower extent, namely in <5% of the clones tested.

Fig. 2.

Alignment of deduced Caja-DRB amino acid sequences, given in conventional one-letter code. A dash or dot indicates identity with the consensus or deletion of an amino acid, respectively, whereas ∗ indicates that this part of the sequence is unknown. Amino acids or deletions, which are typical for the -DRB1*03 and -DRB*W16 lineages, are shown in yellow or blue, respectively. Consequently, the names of the hybrid genes, -DRB1*0307 and -DRB1*0312, are shown in both blue and yellow. L, leader peptide and first four amino acids of the coding sequence; β₁, β₁ domain; β₂, β₂ domain; TM, transmembrane region; CYT, cytoplasmic tail; 3′UT, 3′ untranslated region.

Thus far, the Caja-DRB*W12 locus has been considered to be monomorphic. During this study, a second allele differing by only two codons producing amino acid replacements was observed and designated Caja-DRB*W1202 (Fig. 2). A specially designed set of primers allowed us to detect at least one transcript in all animals tested (Fig. 1). These results, and relevant pedigree data, support the observation that a functional Caja-DRB*W12 gene is present on every haplotype.

In contrast to the two other DRB loci, most of the transcripts corresponding to the various Caja-DRB1*03 alleles are not detectable and possibly represent null alleles. Despite the use of various sets of primer, in our panel of 15 animals only eight different -DRB1*03 cDNAs were defined (Figs. 1 and 2). Moreover, six of eight of these transcripts show inconsistencies, such as the presence of several initiation codons (ATG) in exon 1 that may impair the signal peptide function, as well as an eight-codon-long deletion of the cytoplasmic domain (Fig. 2). The cytoplasmic domain of the DR β chain is considered to play a vital role in signal transduction (19) and/or to affect antigen presentation by influencing the level of class II-associated invariant chain peptides/class II complexes and an efficient endoplasmatic reticulum egress (20, 21). In addition, truncation of either the α or β cytoplasmic tails is known to virtually eliminate internalization of DR molecules and subsequent presentation of certain viral antigens (22). The combination of these two errors will likely result in a gene product that is functionally affected. Furthermore, in comparison with the other two loci, most of the Caja-DRB1*03 transcripts, if detected at all, display very low transcription levels (Fig. 1). This finding again suggests that most Caja-DRB1*03 genes are dysfunctional. For that reason, these alleles have been named Caja-DRB1*0301L, and so on (see Fig. 2), where the suffix L defines the low expression status of the allele, analogous to the HLA nomenclature system (23).

Reactivation of a Pseudogene Segment by Exon Shuffling.

In contrast to the Caja-DRB1*03L alleles, the -DRB1*0307 and -DRB1*0312 transcripts are characterized by high expression levels and were subjected to further analysis. The Caja-DRB1*0307 and -DRB1*0312 allotypes posses the EYSTS motif, encoded by exon 2 (Fig. 2), which is characteristic of the DRB1*03 lineage to which HLA-DRB1*03 also belongs (6). This motif is apparently very important because it is present in many species, playing a key role in immune response against mycobacteria. Sequence comparisons illustrated that, for these two particular alleles, only exon 2 matches all of the features of the Caja-DRB1*03 locus, whereas the other exons align perfectly with the -DRB*W16 gene. The alignment with the -DRB*W16 gene is manifested most prominently for exons 1 and 4, which encode the leader peptide and the cytoplasmatic domain, respectively (Fig. 2). The data suggest that an apparently intact exon 2 originating from a Caja-DRB1*03 pseudogene has been embedded into the context of a -DRB*W16 gene as a result of an exon-shuffling event. The resulting recombinant is transcribed at normal levels and has all of the features that allow translation. As could be expected on the basis of their hybrid character, Caja-DRB1*0307 and -DRB1*0312 form a unique branch in the phylogenetic tree (Fig. 3).

Fig. 3.

Phylogenetic tree constructed according to the neighbor-joining method, illustrating the evolutionary relationships between alleles of various primate species. Bootstrap values are indicated. The alleles depicted in this tree were selected from this study or from the ImMunoGeneTics/Non-Human Primates database (23). Dog leukocyte antigen (DLA) -DRB is chosen as outgroup. Caja, Callithrix jacchus; Saoe, Saguinus oedipus; Gogo, Gorilla gorilla; Mamu, Macaca mulatta; Mane, Macaca nemestrina.

The MHC class II repertoire of common marmosets is even more condensed than previously thought. Prior work documented the absence of Caja-DPB and the presence of only a few -DQ alleles, and only three -DRB loci displaying low or moderate levels of polymorphism (7). The present study makes clear that Caja-DRB*W16 represents the major locus with regard to expression levels, whereas the oligomorphic -DRB*W12 gene products are present to only a moderate degree, and -DRB1*03, if transcribed at all, is present at low levels. On the basis of this low expression level and the presence of several genetic errors, Caja-DRB1*03 is considered to be a pseudogene. The existence of this pseudogene further reduces the limited MHC class II repertoire in common marmosets. However, the seriously condensed repertoire in marmosets seems to have been expanded by the recruitment of the Caja-DRB1*0307 and -DRB1*0312 genes, both of which are also characterized by high expression levels. The origin of these two hybrid genes can be explained by an exon-shuffling event that resulted from the exchange of exon 2 between highly related homologous loci (Fig. 4). The exon-shuffling phenomenon is considered to be one of the main mechanisms propelling the origin of novel genes, and consequently protein evolution, especially in multigenic families (24, 25). The second possible product of this exon-shuffling event, a hybrid Caja-DRB transcript with an exon 2 originating from DRB*W16, was not observed in our panel. One must realize that such a gene would remain undetected during genomic exon 2 sequencing, and also during cDNA analysis, because there is probably no or low transcription. In the case of animal 9523, in which the hybrid Caja-DRB1*0307 allele is observed, the presence of -DRB*W1601 exon 2 can be shown by genomic DNA sequencing, but the corresponding transcript is not detected (Fig. 1). Therefore, it is plausible that this exon is part of a hybrid Caja-DRB1*16N gene that is not transcribed.

Fig. 4.

Schematic representation of the exon-shuffling event producing a novel Caja-DRB gene.

Functional Implications.

MHC class II molecules are heterodimers composed of an α chain and a β chain. Most of the polymorphic codons of the Mhc-DRB gene map to exon 2 encoding the β₁ domain, which plays a crucial role in the formation of the peptide-binding site (26). The β₁ domain of the Caja-DRB1*0307 and -DRB1*0312 gene products possesses the EYSTS motif (Fig. 2), which is characteristic for the Mhc-DRB1*03 lineage and is present in many non-human primate species (6, 27). Peptide-binding and T cell recognition studies demonstrated that this motif might have been conserved throughout primate evolution as a result of functional constraints and selective advantages (28). The importance of this motif is further illustrated by the fact that it has been generated several times de novo during evolution in New World primate and other species through convergent evolution (7, 29, 30). Consequently, the limited peptide-binding repertoire of the common marmoset has been expanded significantly by the reactivation of a segment from a pseudogene, as the result of an exon-shuffling event. The MHC region is scattered with pseudogenes and pseudogene segments (31–35). This study clearly documents that an intact, but old, polymorphic gene segment may be recruited to again play an active role in immune response.

Materials and Methods

Animals.

For this study, common marmosets were selected from two breeding colonies: 4 animals from the Institute of Technology, Zürich-Schwerzenbach, and 11 animals from the Biomedical Primate Research Centre’s pedigreed breeding colony. Most of these animals were analyzed for Caja-DRB polymorphism by exon 2 sequence analyses, as well as full-length cDNA sequencing of Caja-DRB transcripts.

DNA/RNA Isolation and Exon 2 Amplification of Genomic DNA.

Genomic DNA was extracted from immortalized B lymphocytes by a standard salting-out procedure, and RNA was isolated by using the RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s recommendations. Amplification of exon 2 was performed as described recently for rhesus macaques by using generic DRB primers (36), or with primers specific for common marmosets (7, 10).

cDNA Synthesis, Amplification, and RT-PCR.

cDNA was synthesized from freshly extracted mRNA by using the Universal RiboClone cDNA synthesis system (Promega) according to the manufacturer’s recommendations. Full-length sequences were amplified by PCR from 10 μl of cDNA by using primers developed for human DRB5′ and 3′ untranslated sequences (37). PCR mixtures and conditions were as described in ref. 38, employing a commercially available Taq polymerase (Invitrogen). RT-PCR was performed directly on RNA by using the Qiagen OneStep RT-PCR kit according to the manufacturer’s guidelines. The primers used for RT-PCR are the same as those indicated above (37). In addition, the following internal primers specific for Caja-cDRB exon 1 and exon 4, respectively, were used: 5′Caja-cDRBex1, 5′-TGC RTG GCA GYG CTG ACA GTG-3′; and 3′Caja-cDRBex4, 5′-GCC CTG CCC CAA GGA AGA GC-3′. Twenty-five amplification cycles then followed, with 45 s at 94°C, 45 s at 60°C, and 1 min at 72°C. A final extension step for 30 min at 72°C was added.

RACE.

5′ RACE was performed directly on RNA by using the 5′ RACE system for rapid amplification of cDNA ends, Version 2.0 (Invitrogen) according to the manufacturer’s guidelines. The 3′ primer used for first-strand cDNA synthesis was a gene-specific primer for Caja-DRB1*03 and -DRB*W16: Caja GSP1 5′RACE W16-DRB1 5′-GGA AGG TCC AGT CTC CAT TC-3′. For the PCR of dC-tailed cDNA, a nested gene-specific primer was used: Caja GSP2 5′RACE all, 5′-ACC TGA CTT CAA TGC TGC CTG G-3′. The cycling parameters were 2 min at 94°C, followed by 32 cycles of 45 s at 94°C, 45 s at 60°C, and 90 s at 72°C. A final extension step was added for 30 min at 72°C.

We performed a 3′ RACE directly on the RNA by using the 3′ RACE system for rapid amplification of cDNA ends, Version D (Invitrogen) according to the manufacturer’s guidelines. Use of 3′ RACE takes advantage of the natural poly(A) tail found in mRNA as a generic priming site for PCR. The primers used for amplification of the target cDNA were Caja-DRB*W12, -DRB*W16, and -DRB1*03 exon-2-specific primers, as published (10). The cycling parameters were 3 min at 94°C, followed by 25 cycles of 45 s at 94°C, 45 s at 60°C, and 1 min at 72°C. A final extension step was performed for 30 min at 72°C.

Cloning and Sequencing.

Genomic exon 2 amplicons and/or full-length Caja-DRB PCR products were cloned and sequenced by using the InsT/Aclone PCR product cloning kit (Fermentas, St. Leon-Roth, Germany) as described (36). Sequencing reactions were performed with the BigDye Terminator cycle sequencing kit (Applied Biosystems), and the samples were run on the ABI 3100 genetic analyzer (Applied Biosystems). Analysis of the sequences was performed with sequence navigator (Applied Biosystems).