Natural Polymorphisms in Tap2 Influence Negative Selection and CD4∶CD8 Lineage Commitment in the Rat

Genetic variation in the major histocompatibility complex (MHC) affects CD4∶CD8 lineage commitment and MHC expression. However, the contribution of specific genes in this gene-dense region has not yet been resolved. Nor has it been established whether the same genes regulate MHC expression and T cell selection. Here, we assessed the impact of natural genetic variation on MHC expression and CD4∶CD8 lineage commitment using two genetic models in the rat. First, we mapped Quantitative Trait Loci (QTLs) associated with variation in MHC class I and II protein expression and the CD4∶CD8 T cell ratio in outbred Heterogeneous Stock rats. We identified 10 QTLs across the genome and found that QTLs for the individual traits colocalized within a region spanning the MHC. To identify the genes underlying these overlapping QTLs, we generated a large panel of MHC-recombinant congenic strains, and refined the QTLs to two adjacent intervals of ∼0.25 Mb in the MHC-I and II regions, respectively. An interaction between these intervals affected MHC class I expression as well as negative selection and lineage commitment of CD8 single-positive (SP) thymocytes. We mapped this effect to the transporter associated with antigen processing 2 (Tap2) in the MHC-II region and the classical MHC class I gene(s) (RT1-A) in the MHC-I region. This interaction was revealed by a recombination between RT1-A and Tap2, which occurred in 0.2% of the rats. Variants of Tap2 have previously been shown to influence the antigenicity of MHC class I molecules by altering the MHC class I ligandome. Our results show that a restricted peptide repertoire on MHC class I molecules leads to reduced negative selection of CD8SP cells. To our knowledge, this is the first study showing how a recombination between natural alleles of genes in the MHC influences lineage commitment of T cells.     

Multiple divergent haplotypes express completely distinct sets of class I MHC genes in zebrafish

The zebrafish is an important animal model for stem cell biology, cancer, and immunology research. Histocompatibility represents a key intersection of these disciplines; however, histocompatibility in zebrafish remains poorly understood. We examined a set of diverse zebrafish class I major histocompatibility complex (MHC) genes that segregate with specific haplotypes at chromosome 19, and for which donor-recipient matching has been shown to improve engraftment after hematopoietic transplantation. Using flanking gene polymorphisms, we identified six distinct chromosome 19 haplotypes. We describe several novel class I U lineage genes and characterize their sequence properties, expression, and haplotype distribution. Altogether, ten full-length zebrafish class I genes were analyzed, mhc1uba through mhc1uka. Expression data and sequence properties indicate that most are candidate classical genes. Several substitutions in putative peptide anchor residues, often shared with deduced MHC molecules from additional teleost species, suggest flexibility in antigen binding. All ten zebrafish class I genes were uniquely assigned among the six haplotypes, with dominant or codominant expression of one to three genes per haplotype. Interestingly, while the divergent MHC haplotypes display variable gene copy number and content, the different genes appear to have ancient origin, with extremely high levels of sequence diversity. Furthermore, haplotype variability extends beyond the MHC genes to include divergent forms of psmb8. The many disparate haplotypes at this locus therefore represent a remarkable form of genomic region configuration polymorphism. Defining the functional MHC genes within these divergent class I haplotypes in zebrafish will provide an important foundation for future studies in immunology and transplantation.     

Disruption and pseudoautosomal localization of the major histocompatibility complex in monotremes

Background

The monotremes, represented by the duck-billed platypus and the echidnas, are the most divergent species within mammals, featuring a flamboyant mix of reptilian, mammalian and specialized characteristics. To understand the evolution of the mammalian major histocompatibility complex (MHC), the analysis of the monotreme genome is vital.

Results

We characterized several MHC containing bacterial artificial chromosome clones from platypus (Ornithorhynchus anatinus) and the short-beaked echidna (Tachyglossus aculeatus) and mapped them onto chromosomes. We discovered that the MHC of monotremes is not contiguous and locates within pseudoautosomal regions of two pairs of their sex chromosomes. The analysis revealed an MHC core region with class I and class II genes on platypus and echidna X3/Y3. Echidna X4/Y4 and platypus Y4/X5 showed synteny to the human distal class III region and beyond. We discovered an intron-containing class I pseudogene on platypus Y4/X5 at a genomic location equivalent to the human HLA-B,C region, suggesting ancestral synteny of the monotreme MHC. Analysis of male meioses from platypus and echidna showed that MHC chromosomes occupy different positions in the meiotic chains of either species.

Conclusion

Molecular and cytogenetic analyses reveal new insights into the evolution of the mammalian MHC and the multiple sex chromosome system of monotremes. In addition, our data establish the first homology link between chicken microchromosomes and the smallest chromosomes in the monotreme karyotype. Our results further suggest that segments of the monotreme MHC that now reside on separate chromosomes must once have been syntenic and that the complex sex chromosome system of monotremes is dynamic and still evolving.     

A class I jumping clone places the HLA-G gene approximately 100 kilobases from HLA-H within the HLA-A subregion of the human MHC

By the combination of cosmid cloning, chromosomal jumping, and pulsed-field gel electrophoresis (PFGE), we have fine-mapped the HLA-A subregion of the human major histocompatibility complex (MHC). Through the isolation of a class I jumping clone, the Qa-like HLA-G class I gene has been placed within 100 kb of HLA-H. The tight physical linkage of these class I genes has been further supported by hybridizing PFGE blots with locus-specific probes. It has been found that both of the above class I genes are linked to HLA-A, with HLA-H residing no more than 200 kb from the HLA-A gene. These data support the possible existence of a Qa-like subregion composed of nonclassical HLA class I genes within the human MHC linked telomerically to the HLA-A locus.     

Genomic sequence analysis of the 238-kb swine segment with a cluster of TRIM and olfactory receptor genes located, but with no class I genes, at the distal end of the SLA class I region

Continuous genomic sequence has been previously determined for the swine leukocyte antigen (SLA) class I region from the TNF gene cluster at the border between the major histocompatibility complex (MHC) class III and class I regions to the UBD gene at the telomeric end of the classical class I gene cluster (SLA-1 to SLA-5, SLA-9, SLA-11). To complete the genomic sequence of the entire SLA class I genomic region, we have analyzed the genomic sequences of two BAC clones carrying a continuous 237,633-bp-long segment spanning from the TRIM15 gene to the UBD gene located on the telomeric side of the classical SLA class I gene cluster. Fifteen non-class I genes, including the zinc finger and the tripartite motif (TRIM) ring-finger-related family genes and olfactory receptor genes, were identified in the 238-kilobase (kb) segment, and their location in the segment was similar to their apparent human homologs. In contrast, a human segment (alpha block) spanning about 375 kb from the gene ETF1P1 and from the HLA-J to HLA-F genes was absent from the 238-kb swine segment. We conclude that the gene organization of the MHC non-class I genes located in the telomeric side of the classical SLA class I gene cluster is remarkably similar between the swine and the human segments, although the swine lacks a 375-kb segment corresponding to the human alpha block. The nucleotide sequence data reported in this paper have been submitted to DDBJ, EMBL, and GenBank databases under accession numbers AB158486 and AB158487     

Exchanges of short polymorphic DNA segments predating speciation in feline major histocompatibility complex class I genes

Sequence comparisons of 14 distinct MHC class I cDNA clones isolated from species representing the three major taxonomic lineages of Felidae (domestic cat lineage, ocelot lineage, and pantherine lineage) revealed that feline MHC class I alleles have highly mosaic structures with short polymorphic sequence motifs that are rearranged between alleles of individual MHC loci, between MHC class I genes within cat species, and between homologous MHC loci in different species. The pattern of sequence variation in felids supports the role of the following factors in production and maintenance of MHC variation: (1) gradual spontaneous mutation; (2) selective pressure to conserve certain residues but also to vary in hypervariable regions, notably residues that functionally participate in antigen recognition and presentation; and (3) recombination-mediated gene exchange between alleles and between related genes. The overall amount of genetic variation observed among MHC class I genes in the Felidae family is no greater than the amount of variation within any outbred cat species (i.e., domestic cat, ocelot). The occurrence of equivalent levels of polymorphism plus the simultaneous persistence of the same sequence motifs in divergent feline species suggest that most MHC class I nucleotide site polymorphism predated species divergences. Ancient polymorphisms have been transmitted through the speciation events and modern feline MHC class I alleles were derived by recombinational exchange of polymorphic sequence motifs. Moreover, some of these sequence motifs were found in other mammalian MHC class I genes, such as classical human HLA-B5, nonclassical human HLA-E class I genes, and bovine class I genes. These results raise the prospect of an ancient origin for some motifs, although the possibility of convergence in parallel mammalian radiations cannot be excluded. Correspondence to: N. Yuhki     

Structure and evolution of a member of a new subfamily of GTP-binding proteins mapping to the human MHC class I region

A gene coding for a putative GTP-binding protein, MMR1, has been localized on band C of the murine Chr 17 within or close to the MHC (Denizot et al. 1992). Its human homolog, HSR1, localized to the human MHC class I region, is described in this paper. Its sequence, compared with MMR1, shows that the conceptual proteins encoded by these genes are highly homologous and have thus been subjected to high constraints during evolution. Furthermore, a detailed databank search with HSR1 leads to the characterization of a new subfamily of GTP-binding proteins, of which HSR1 and MMR1 are the only eukaryotic members. The precise localization of HSR1 within the human MHC class I region is also presented.     

Structure of a classical MHC class I molecule that binds "non-classical" ligands

Chicken YF1 genes share a close sequence relationship with classical MHC class I loci but map outside of the core MHC region. To obtain insights into their function, we determined the structure of the YF1*7.1/β₂-microgloblin complex by X-ray crystallography at 1.3 Å resolution. It exhibits the architecture typical of classical MHC class I molecules but possesses a hydrophobic binding groove that contains a non-peptidic ligand. This finding prompted us to reconstitute YF1*7.1 also with various self-lipids. Seven additional YF1*7.1 structures were solved, but only polyethyleneglycol molecules could be modeled into the electron density within the binding groove. However, an assessment of YF1*7.1 by native isoelectric focusing indicated that the molecules were also able to bind nonself-lipids. The ability of YF1*7.1 to interact with hydrophobic ligands is unprecedented among classical MHC class I proteins and might aid the chicken immune system to recognize a diverse ligand repertoire with a minimal number of MHC class I molecules.     

A class I jumping clone places the HLA-G gene approximately 100 kilobases from HLA-H within the HLA-A subregion of the human MHC

By the combination of cosmid cloning, chromosomal jumping, and pulsed-field gel electrophoresis (PFGE), we have fine-mapped the HLA-A subregion of the human major histocompatibility complex (MHC). Through the isolation of a class I jumping clone, the Q alpha-like HLA-G class I gene has been placed within 100 kb of HLA-H. The tight physical linkage of these class I genes has been further supported by hybridizing PFGE blots with locus-specific probes. It has been found that both of the above class I genes are linked to HLA-A, with HLA-H residing no more than 200 kb from the HLA-A gene. These data support the possible existence of a Q alpha-like subregion composed of nonclassical HLA class I genes within the human MHC linked telomerically to the HLA-A locus.     

Molecular characterization,balancing selection,and genomic organization of the tree shrew (Tupaia belangeri) MHC class I gene

The major histocompatibility complex (MHC) class I genes play a pivotal role in the adaptive immune response among vertebrates. Accordingly, in numerous mammals the genomic structure and molecular characterization of MHC class I genes have been thoroughly investigated. To date, however, little is known about these genes in tree shrews, despite the increasingly popularity of its usage as an animal model. To address this deficiency, we analyzed the structure and characteristic of the tree shrew MHC class I genes (Tube-MHC I) and performed a comparative gene analysis of the tree shrew and other mammal species. We found that the full-length cDNA sequence of the tree shrew MHC class I is 1074 bp in length. The deduced peptide is composed of 357 amino acids containing a leader peptide, an α1 and α2 domain, an α3 domain, a transmembrane domain and a cytoplasmic domain. Among these peptides, the cysteines, CD8⁺ interaction and N-glycosylation sites are all well conserved. Furthermore, the genomic sequence of the tree shrew MHC class I gene was identified to be 3180 bp in length, containing 8 exons and 7 introns. In 21 MHC class I sequences, we conducted an extensive study of nucleotide substitutions. The results indicated that in the peptide binding region (PBR) the rate of non-synonymous substitutions (dN) to synonymous substitutions (dS) was greater than 1, suggesting balancing selection at the PBR. These findings provide valuable contributions in furthering our understanding of the structure, molecular polymorphism, and function of the MHC class I genes in tree shrews, further improving their utility as an animal model in biomedical research.     

Gene duplication and fragmentation in the zebra finch major histocompatibility complex

Background

Due to its high polymorphism and importance for disease resistance, the major histocompatibility complex (MHC) has been an important focus of many vertebrate genome projects. Avian MHC organization is of particular interest because the chicken Gallus gallus, the avian species with the best characterized MHC, possesses a highly streamlined minimal essential MHC, which is linked to resistance against specific pathogens. It remains unclear the extent to which this organization describes the situation in other birds and whether it represents a derived or ancestral condition. The sequencing of the zebra finch Taeniopygia guttata genome, in combination with targeted bacterial artificial chromosome (BAC) sequencing, has allowed us to characterize an MHC from a highly divergent and diverse avian lineage, the passerines.

Results

The zebra finch MHC exhibits a complex structure and history involving gene duplication and fragmentation. The zebra finch MHC includes multiple Class I and Class II genes, some of which appear to be pseudogenes, and spans a much more extensive genomic region than the chicken MHC, as evidenced by the presence of MHC genes on each of seven BACs spanning 739 kb. Cytogenetic (FISH) evidence and the genome assembly itself place core MHC genes on as many as four chromosomes with TAP and Class I genes mapping to different chromosomes. MHC Class II regions are further characterized by high endogenous retroviral content. Lastly, we find strong evidence of selection acting on sites within passerine MHC Class I and Class II genes.

Conclusion

The zebra finch MHC differs markedly from that of the chicken, the only other bird species with a complete genome sequence. The apparent lack of synteny between TAP and the expressed MHC Class I locus is in fact reminiscent of a pattern seen in some mammalian lineages and may represent convergent evolution. Our analyses of the zebra finch MHC suggest a complex history involving chromosomal fission, gene duplication and translocation in the history of the MHC in birds, and highlight striking differences in MHC structure and organization among avian lineages.     

Unprecedented intraspecific diversity of the MHC class I region of a teleost medaka, Oryzias latipes

The major histocompatibility complex (MHC) is present at a single chromosomal locus of all jawed vertebrate analyzed so far, from sharks to mammals, except for teleosts whose orthologs of the mammalian MHC-encoded genes are dispersed at several chromosomal loci. Even in teleosts, several class IA genes and those genes directly involved in class I antigen presentation preserve their linkage, defining the teleost MHC class I region. We determined the complete nucleotide sequence of the MHC class I region of the inbred HNI strain of medaka, Oryzias latipes (northern Japan population-derived), from four overlapping bacterial artificial chromosome (BAC) clones spanning 540,982 bp, and compared it with the published sequence of the corresponding region of the inbred Hd-rR strain of medaka (425,935 bp, southern Japan population-derived) as the first extensive study of intraspecies polymorphisms of the ectotherm MHC regions. A segment of about 100 kb in the middle of the compared sequences encompassing two class Ia genes and two immunoproteasome subunit genes, PSMB8 and PSMB10, was so divergent between these two inbred strains that a reliable sequence alignment could not be made. The rest of the compared region (about 320 kb) showed a fair correspondence, and an approximately 96% nucleotide identity was observed upon gap-free segmental alignment. These results indicate that the medaka MHC class I region contains an ∼100-kb polymorphic core, which is most probably evolving adaptively by accumulation of point mutations and extensive genetic rearrangements such as insertions, deletions and duplications. The nucleotide sequence data of HNI MHC class I region reported in this paper have been submitted to the DDBJ/EMBL/GenBank and were assigned the accession number AB183488.     

The chimpanzee major histocompatibility complex class II DR subregion contains an unexpectedly high number of beta-chain genes

The major histocompatibility complex (MHC) class II DR subregion of the chimpanzee was studied by restriction fragment length polymorphism (RFLP) analysis. Genomic DNA obtained from a panel of 94 chimpanzees was digested with the restriction enzyme Taq I and hybridized with an HLA-DR probe specific for the 3' untranslated (UT) region. Such a screening revealed the existence of 14 distinct DRB/Taq I gene-associated fragments allowing the definition of 11 haplotypes. Segregation studies proved that the number of chimpanzee class II DRB/Taq I fragments is not constant and varies from three to six depending on the haplotype. Comparison of these data with a human reference panel manifested that some MHC DRB/Taq I fragments are shared by man and chimpanzee. Moreover, the number of HLA-DRB/Taq I gene-associated fragments detected in a panel of homozygous typing cells varies from one to three and corresponds with the number of HLA-DRB genes present for most haplotypes. However, a discrepancy is observed for the HLA-DR4,-DR7, and -DR9 haplotypes since a fourth HLA-DRB pseudogene present within these haplotypes lacks its 3' UT region and thus is not detected with the probe used. These results suggest that chimpanzees have a higher maximum number of DRB genes per haplotype than man. As a consequence, some chimpanzee haplotypes must show a dissimilar organization of the MHC DR subregion compared to their human equivalents. The implications of these findings are discussed in the context of the trans-species theory of MHC polymorphism. Address correspondence and offprint requests to: R. E. Bontrop.     

Sequences, annotation and single nucleotide polymorphism of the major histocompatibility complex in the domestic cat

Two sequences of major histocompatibility complex (MHC) regions in the domestic cat, 2.976 and 0.362 Mbps, which were separated by an ancient chromosome break (55-80 MYA) and followed by a chromosomal inversion were annotated in detail. Gene annotation of this MHC was completed and identified 183 possible coding regions, 147 human homologues, possible functional genes and 36 pseudo/unidentified genes) by GENSCAN and BLASTN, BLASTP RepeatMasker programs. The first region spans 2.976 Mbp sequence, which encodes six classical class II antigens (three DRA and three DRB antigens) lacking the functional DP, DQ regions, nine antigen processing molecules (DOA/DOB, DMA/DMB, TAPASIN, and LMP2/LMP7,TAP1/TAP2), 52 class III genes, nineteen class I genes/gene fragments (FLAI-A to FLAI-S). Three class I genes (FLAI-H, I-K, I-E) may encode functional classical class I antigens based on deduced amino acid sequence and promoter structure. The second region spans 0.362 Mbp sequence encoding no class I genes and 18 cross-species conserved genes, excluding class I, II and their functionally related/associated genes, namely framework genes, including three olfactory receptor genes. One previously identified feline endogenous retrovirus, a baboon retrovirus derived sequence (ECE1) and two new endogenous retrovirus sequences, similar to brown bat endogenous retrovirus (FERVmlu1, FERVmlu2) were found within a 140 Kbp interval in the middle of class I region. MHC SNPs were examined based on comparisons of this BAC sequence and MHC homozygous 1.9x WGS sequences and found that 11,654 SNPs in 2.84 Mbp (0.00411 SNP per bp), which is 2.4 times higher rate than average heterozygous region in the WGS (0.0017 SNP per bp genome), and slightly higher than the SNP rate observed in human MHC (0.00337 SNP per bp).     

Polymorphic Alu insertions within the Major Histocompatibility Complex class I genomic region: a brief review

Most polymorphic Alu insertions (POALINs) belong to a subgroup of the Alu multicopy retrotransposon family of short interspersed nucleotide elements (SINEs) that are categorized as AluYb8 and AluYa5. The number of AluYb8/AluYa5 members (approximately 4,492 copies) is significantly less than the approximately one million fixed Alu copies per human genome. We have studied the presence of POALINs within the Major Histocompatibility Complex (MHC) class I region on the short arm of chromosome 6 (6p21.3) because this region has a high gene density, many genes with immune system functions, large sequence variations and diversity, duplications and redundancy, and a strong association with more than 100 different diseases. Since little is known about POALINs within the MHC genomic region, we undertook to identify some of the members of the AluYb8/AluYa5 subfamily and to study their frequency of distribution and genetic characteristics in different populations. As a result of our comparative genomic analyses, we identified the insertion sites for five POALINs distributed within the MHC class I region. This brief review outlines the locations of the insertions and sequence features of the five MHC POALINs, their single site and haplotype frequencies in different geographic populations, and their association with different HLA class I genes and disease. We show that the MHC POALINs have a potential value as lineage and linkage markers for the study of human population genetics, disease associations, genomic diversity and evolution.     

Fine-Mapping the Genetic Association of the Major Histocompatibility Complex in Multiple Sclerosis: HLA and Non-HLA Effects

The major histocompatibility complex (MHC) region is strongly associated with multiple sclerosis (MS) susceptibility. HLA-DRB1*15:01 has the strongest effect, and several other alleles have been reported at different levels of validation. Using SNP data from genome-wide studies, we imputed and tested classical alleles and amino acid polymorphisms in 8 classical human leukocyte antigen (HLA) genes in 5,091 cases and 9,595 controls. We identified 11 statistically independent effects overall: 6 HLA-DRB1 and one DPB1 alleles in class II, one HLA-A and two B alleles in class I, and one signal in a region spanning from MICB to LST1. This genomic segment does not contain any HLA class I or II genes and provides robust evidence for the involvement of a non-HLA risk allele within the MHC. Interestingly, this region contains the TNF gene, the cognate ligand of the well-validated TNFRSF1A MS susceptibility gene. The classical HLA effects can be explained to some extent by polymorphic amino acid positions in the peptide-binding grooves. This study dissects the independent effects in the MHC, a critical region for MS susceptibility that harbors multiple risk alleles.