Journal of Heredity Advance Access published August 3, 2007

Journal of Heredity doi:10.1093/jhered/esm056 The American Genetic Association. 2007. All rights reserved. For permissions, please email: journals.permissions@oxfordjournals.org.

Comparative Genomic Structure of Human, Dog, and Cat MHC: HLA, DLA, and FLA
NAOYA YUHKI, THOMAS BECK, ROBERT STEPHENS, BEENA NEELAM,
AND

STEPHEN J. OBRIEN

From the Laboratory of Genomic Diversity, National Cancer Institute at Frederick, Frederick, MD 21702-1201 (Yuhki, OBrien); SAIC-Frederick, National Cancer Institute at Frederick, Frederick, MD 21702-1201 (Beck); the Advanced Biomedical Computing Center, National Cancer Institute at Frederick, Frederick, MD 21702-1201 (Stephens, Neelam). Address correspondence to Dr. Naoya Yuhki at the address above, or e-mail: yuhki@ncifcrf.gov.
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Abstract
Comparisons of the genomic structure of 3 mammalian major histocompatibility complexes (MHCs), human HLA, canine DLA, and feline FLA revealed remarkable structural differences between HLA and the other 2 MHCs. The 4.6-Mb HLA sequence was compared with the 3.9-Mb DLA sequence from 2 supercontigs generated by 7x whole-genome shotgun assembly and 3.3-Mb FLA draft sequence. For FLA, we confirm that 1) feline FLA was split into 2 pieces within the TRIM (member of the tripartite motif) gene family found in human HLA, 2) class II, III, and I regions were placed in the pericentromeric region of the long arm of chromosome B2, and 3) the remaining FLA was located in subtelomeric region of the short arm of chromosome B2. The exact same chromosome break was found in canine DLA structure, where class II, III, and I regions were placed in a pericentromeric region of chromosome 12 whereas the remaining region was located in a subtelomeric region of chromosome 35, suggesting that this chromosome break occurred once before the split of felid and canid more than 55 million years ago. However, significant differences were found in the content of genes in both pericentromeric and subtelomeric regions in DLA and FLA, the gene number, and amplicon structure of class I genes plus 2 other class I genes found on 2 additional chromosomes; canine chromosomes 7 and 18 suggest the dynamic nature in the evolution of MHC class I genes.

The major histocompatibility complex (MHC) plays key roles in controlling both adaptive and innate immune systems (Klein 1986). In the adaptive immune system, both MHC class I and class II antigens recognize, bind, and present peptides to cytotoxic and helper T-cells, respectively (Bjorkman et al. 1987a 1987b; Brown et al. 1988) and initiate cell-to-cell communication between antigen-presenting cells and T-cells by forming immunological synapses and activating T-cells for cellular and humoral immune systems (Grakoui et al. 1999). In addition, a number of gene clusters in this complex encode proteins that play important roles for antigen processing (proteosome subunits, LMP2 and 7, antigen transporters; TAP1 and 2 (Beck et al. 1992; Monaco 1992), antigen loading for the class I antigen, TAPASIN (Ortman et al. 1997), antigen loading for the class II antigens, DM (Fling et al. 1994; Morris et al. 1994) and DO (Alfonso et al. 1999) molecules). In the innate immune system, both classical (HLA-A, -B, -C in humans) and nonclassical (HLA-E, -G) class I (Le Bouteiller et al. 1999; Voles-Gomez et al. 2000) antigens plus class Irelated molecules (MIC-A, -B ) interact with the

natural killer ( NK) receptors KIR ( Parham 2005) and NKG2 antigens and Ly49 and NKG2 ( Takei et al. 2001) antigens in human and mouse, respectively, and inhibit and activate NK-cell functions. In addition to the immunological importance, the MHC provides important tools to study molecular evolution. Extremely polymorphic features of both class I and class II antigens identified in most vertebrates provide large numbers of peptide-binding grooves for both antigens in order to adapt to various pathogens. Natural and balancing selections play pivotal roles to generate and maintain these polymorphisms ( Yuhki and OBrien 1990; Klein et al. 1993). A large-scale sequencing project for the HLA (3.6 Mb) yielded findings of 224 tightly linked genes, including 128 expressed genes and 96 pseudogenes ( MHC Sequencing Consortium 1999). In addition, 2 complete MHC haplotype sequences spanning 4.6 Mb were published (Stewart et al. 2004). In contrast of this large complex structure of the HLA, the chicken MHC B-locus shows a minimal essential MHC

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extending 92 kb and including 19 functional genes, raising questions about the structure of the other MHC systems (Kaufman et al. 1999). We have analyzed 2 additional MHCs, one from a canine generated by the 7x whole-genome shotgun (WGS) assembly (Lindblad-Toh et al. 2005) and the second from a feline generated by Bacterial Artificial chromosome (BAC) sequencing (OBrien and Yuhki 1999; Yuhki et al. 2003; Beck et al. 2005; Pontius J, et al. 2007).

Materials and Methods

Sequence The human HLA sequence was downloaded from the Sanger Web site. The canine DLA, 2 supercontigs, were obtained from Broad Institute (supercontigs 38195 and 40588). Feline MHC draft sequence was generated by shotgun sequencing with an ABI3730XL analyzer and Phred base calling Phrap (Ewing and Green 1998; Ewing et al. 1998) assembly and Consed (Gordon et al. 1998, 2001) from selected BAC clones based on feline MHC BAC maps (Beck et al. 2005). Sequence Comparisons Sequence comparisons were performed by the PIPMAKER program (Schwartz et al. 2000). Gene identification and annotation was done by GENSCAN (Burge and Karlin 1997) and BLAST program against the human Refseq database (Altschul et al. 1990). Fluorescent In Situ Hybridization Fluorescent in situ hybridization (FISH) was done using a standard condition with fluorescein isothiocyanate (FITC-) and digoxigenin-labeled BAC clone DNA (Modi et al. 1987).

BAT1 gene through OCT3 gene. Thirty class I genes/gene fragments were found in this subregion. Of these genes, 17 class I genes were confirmed by the GENSCAN, BLASTP, and BLASTN programs. In addition, 4 MHC class Irelated (MIC ) genes were found. This region spans approximately 600 kbp adjacent to the class III region. 2. Central class I subregion: The central class I subregion was defined as a region from OCT3 gene through the pericentromeric region of the long arm of feline chromosome B2, spanning approximately 800 kbp (Figure 1C). Twentyseven framework human genes, which have no class I or class IIrelated function and conserved in human HLA and murine H2 homologues, were found and scattered in this region. Two class I gene/gene fragments were located near the end of this region. One class I gene (I-Rp) appears to be a gene fragment based on dotplot analysis using a full-length MHC class I cDNA sequence (data not shown), whereas another class I gene (I-S ) appears to be a full-length MHC class I gene. This I-S class I gene was confirmed by the GENSCAN program as well. Those 2 class I gene/gene fragments were located on the centromeric side from the TRIM39 gene. 3. Distal class I subregion: Distal class I subregion was defined as a region between the subtelomeric region and a chromosomal break point, near the TRIM26 gene through the third olfactory receptor gene OLFR homologue. This region spans approximately 300 kbp and encodes 10 framework genes; however, no class I genes or gene fragments were identified. Alpha Satellite and Telomere Repeats Two dotter dotplot analyses of the BAC sequence were performed in order to obtain data that can show evidence of the pericentromeric and subtelomeric localizations of the central and distal class I subregions (Figure 2A,B). One BAC clone 329i22, which encodes TRIM39, and 2 class I gene/gene fragments were analyzed by self-to-self dotplot program in order to detect repeat sequence structures. This analysis detected 3 clusters of alpha satellite repeats, which were found near the centromeric and telomeric regions in the domestic cat chromosomes (Modi et al. 1988), suggesting that this 329i22 BAC clone has subheterochromatin-like features (Figure 2A). Similar dotplot analysis indicated that BAC clone 46j10 has both alpha satellite and 47 units of telomeric repeats, suggesting that the 46j10 sequence has characteristics of the subtelomeric region (Figure 2B). Canine MHC Two supercontigs, which encode the entire canine MHC sequences defined as sequence-encoding genes from HKET through UBD, plus 3 olfactory receptor genes were obtained and analyzed. One supercontig, 38195, which is 9 472 360 bp in size includes two-thirds of the MHC genes (SYNGAP1 to TRIM39) in the 2.9-Mbp region. The other supercontig, 40588, which is 2 661 921 bp in size includes the rest of the MHC genes (TRIM26 to olfactory receptor genes).

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Results
Two-Color FISH Feline BAC clones 329i22 and 46j10 encode TRIM39, MHCI-Rp, and MHCI-S genes and TRIM26, TRIM15, and TRIM10 genes, respectively. These BAC clones were labeled with digoxigenin (329i22) and FITC (46j10) (329i22: pink color, 46j10: green color) and hybridized with domestic cat chromosomal DNA. The results indicated that digoxigenin and FITC showed signals on a centromere of the long arm and the telomere of the short arm of feline chromosome B2, respectively (Figure 1A, 1B). Annotation of MHC Class I Region in the Domestic Cat Analysis of an approximately 1.8-Mbp sequence of the feline MHC class I region (2006 August freeze) revealed 72 coding genes (FLAI-A through OLFR3) (Table 1). Based on this analysis, we assigned 3 subregions as follows: 1. Proximal class I subregion (Figure 1C): Proximal class I subregion was defined as a region adjacent to the

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Figure 1. (A) Locations of TRIM39, 26, 15, 10 genes in HLA, H2, compared with gene contents and order in FLA. BAC clone 329i22 and 46j10 encode TRIM39, class I-Rp, and -S genes and TRIM26, TRIM15, and TRIM10 genes, respectively. BAC clone 329i22 and 46j10 were labeled with digoxigenin and FITC, respectively, and hybridized on feline chromosome (fcaB2). BAC sequence coordinates are shown. Location of BAC clone 329i22 and 46j10, indicating each location on telomere of the short arm and centromere of the long arm of fcaB2. (B) G-banding of feline chromosome B2 and schematic views of fcaB2 and its synthenic cfa chromosomes, cfa35, 12, 1. (C) Schematic view of gene contents and order in FLA. The FLA class I region spans approximately 1700 kbp not including (peri)centromeric and (sub)telomeric repeats. A break point was indicated by double slashes. Left and right sides of the double slashes are pericentromeric and subtelomeric regions, respectively, and rearranged by a chromosomal inversion.

Detailed gene contents and gene order were presented in Figure 3A,B, where canine MHCs split into 2 DNA fragments at the point where the feline MHC split occurred between TRIM39 and TRIM26. Two-thirds of canine MHCs (SYNGAP1 to TRIM39) are located on or near the centromeric border of canine chromosome 12 and acquired 4 genes

(ARG1, CRSP3, ATP5L, and ENPP3) from the HSA 6p13 region. The remaining canine MHCs (500 kbp from TRIM26 to olfactory receptor genes) were found on the telomeric region of canine chromosome 35 and have acquired 4 processed pseudogenes (DSG3, EEF1A2, MGC35023, MGC35023)

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and placed on the field. This process was continued in both x and y axes to fill out the entire sequence comparison filed. As results, contiguously similar sequences of x and y axes were recognized as positive 45-degree solid lines and sequence inversion events were recognized as negative 45degree solid lines. Sequence insertion was recognized as discontinuity of one axis but not the other. Discontinuity of solid lines suggests rapid sequence divergence (i.e., speciesspecific sequence amplification in each species) in those regions. Results indicated a mosaic structure of highly conserved and species-specific unique regions throughout the MHC. Figure 4 shows a combination of 45-degree straight lines (sharp boxes) and disrupted lines (fuzzy boxes) in the dotplot between 4.6-Mbp HLA (x axis) and 2.9-Mb (cfa12) and 1.0-Mb (cfa35) DLA ( y axis). From right to left, blue boxes represent extended class II region, class III region, and central class I framework regions in comparison between HLA and cfa12 DLA. Three relatively short conserved regions in comparison between HLA and cfa35 DLA represent 2 class I framework gene regions adjacent to the missing HLA-A corresponding region shown as a straight red line, plus an additional conserved region beyond olfactory receptor gene clusters. Three types of disrupted patterns were observed. First, the class II region has recognizable short straight lines with gaps and shifts, suggesting gene amplifications and deletions. Second, unrecognizable conserved patterns, suggesting species-unique evolution, were found in HLA-B and -C class I gene regions, near centromeric and telomeric regions in DLA and olfactory receptor gene clusters. Third, 2 conserved lines (2 sharp boxes) were located without a gap in y axis but with a gap in x axis, indicating a large deletion of HLA-A class I corresponding region in DLA.

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Discussion
Figure 2. (A) Self-dotplot analysis of BAC 329i22 sequence. A105 270-bp sequence was compared with self-using dotter program. Arrows indicate 3 clusters of feline alpha satellite repeats (FASAT). (B) Self-dotplot analysis of BAC 46j10 sequence. A54 599-bp sequence was compared with self-using dotter program, detecting both FASAT and 47 units of telomeric repeats ( TTAGGG).

near the telomere region. No class I gene or gene fragments were identified in this region. Dotplot Comparison of HLA and DLA In order to reveal the difference of genomic structure, a direct sequence comparison of 2 MHCs, HLA and DLA, dotplot analysis (Erik et al. 1995) was attempted. Intersperse repeat sequences were first masked, and every 100-bp sequence segments were compared against the other sequence. The sequence similarity was color graded (white, gray, and black)

Here we report 2 unique MHC genomic structures of the feline and canine MHC regions. Two-color FISH clearly indicated that the MHC break occurred within the TRIM gene family region, specifically between TRIM39 and TRIM26 (Figure 1A). The same MHC break was also found in the canine MHC from 2 supercontigs of 7x WGS assembly (Figure 3A,B). These results can also be visualized as FLA, DLA, and HLA class I gene contents in Figure 6. Because the split of felids and canids are reported to be most ancient in Carnivora species, 55 million years ago (MYA) (Murphy et al. 2001), this split MHC structure may be a common form in Carnivora species. We have attempted to compare G-banding patterns of the feline chromosome (fcaB2) and canine chromosomes (cfa35, 12, 1). The result showed recognizable similar G-banding pattern in the location of the MHC (Figure 1B). Similar conclusions are made by examining the G-banding patterns of Carnivora chromosomes (Nash et al. 2001; OBrien et al. 2006). Genomic structure of 9 mammalian MHCs was presented in Figure 5. Though this is a rough sketch of the MHC structure, it is clear that chromosome breaks/inversion/centro-

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Figure 3. (A) The 3000-kb DLA region from supercontig 38195 and its gene contents and gene order. This 3-Mbp DLA spans from SYNGAP1 through TRIM39 plus gene contents transduced from HSA 6q13 near centromere of cfa12. Coordinates from 7x canine whole-genome shotgun sequence are shown in (A) and (B). (B) The 500-kb DLA region from supercontig 40588 and its gene contents and gene order. This 500-kbp DLA spans from RAN to TRIM26 plus 4 pseudogenes near telomere of cfa35.

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Figure 4. Dotplot analysis of the HLA 4.6 Mbp and 2 DLA fragments, 2.9 Mbp and 1.0 Mbp. Conserved and nonconserved regions are sharp and fuzzy boxed, respectively. Approximate borders of class II, III, and I regions are shown above and to the right of the graph.

mere invasion occurred in the MHC region during evolution of each species. For example, nearly all mammalian MHC class II regions retain DP, DQ, and DR subregions; however, DP regions tends to become nonfunctional as observed in dog DLA, cat FLA, and equine MHC. In FLA, the entire DQ region was deleted after the split of canids and felids. Moreover, in bovine BoLA, DNA break in the class II region followed by the DNA inversion caused the loss and gain of DP and DY/DI class II regions, respectively (Band et al. 1998). Other notable examples are shown in this paper; the feline MHC split occurred once more than 55 MYA and was located near the centromere and telomere regions by a chromosomal inversion (fcaB2). In canids, the same segments found in the FLA are located near centromere and telomere of 2 separate chromosomes (cfa12 and 35). In swine MHC, centromere invasion was found in the border of the class II and III regions (Renard et al. 2006). Recent mouse genome assembly also shows that a segmental break in its MHC occurred in the distal class I region, where approximately 2.8-Mbp segments from TRIM27 (RFP) to SCGN genes are located on chromosome mmu13, apart from mmu17 where the rest of the MHCs are located

(Mouse Genome Sequencing Consortium 2002). Thus, an integrity of the MHC defined by HLA (SYNGAP1 through SCGN) is shown in a few exceptional cases in mammalian MHC. The direct sequence comparison between HLA and DLA performed by the PIPMAKER program shows the mosaic structure of several hundred conserved and nonconserved kilobase segments. In nonconserved regions, the class II region was clearly reorganized by gene amplification and deletion as described elsewhere (Debenham et al. 2005). The class I region was probably rearranged by gene amplification of a small amplicon (i.e., class I gene plus MIC in HLA (Shiina et al. 1999) and class I gene plus BAT1 in FLA, unknown in DLA) in species-specific fashion. A similar pattern was observed in the olfactory receptor gene cluster, suggesting that the gene amplification seen in class I genes may have taken place in this cluster. It is noteworthy to describe the gene organization near heterochromatin regions, where ancient chromosome break points have not remained the same clear-cut sequences as the original ones, but rather this region functions as a vacuum of new genes/ rearrangements.

Yuhki et al.  Comparative Genomic Structure of Human, Dog, and Cat MHCs

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Figure 5. A schematic view of 9 mammalian MHCs. Two major mammalian groups, Euarchontoglires including primates (Human [MHC Sequencing Consortium 1999], chimpanzee [Anzai et al. 2003], rhesus macaque [Daza-Vamenta et al. 2004], and rodents [mouse {Kumanovics et al. 2003}, rat {Hurt et al. 2004}]) and Laurasiatheria including carnivores (cat and dog), Perissodactyla (horse [Gustafson et al. 2003]), and Certartiodactyla (pig [Renard et al. 2006], cattle [Lewin et al. 1999]) are shown. Cat and dog separated 55 MYA represent the most ancient split in carnivores. Carnivores and Perissodactyla separated 80 MYA. In class II region, DQ/DR are highly conserved, except FLA (DQ deletion), whereas in class I region, HLA-B, -C are highly conserved.

In the HLA class I region, HLA-A is the largest class I region compared with the HLA-B, -C, -E regions, spanning 550 kbp from HTEX4 to MOG, including at least 11 class I genes (HLA-A, -G, -F as functional genes). In FLA, HTEX4 to MOG spans only 55 kbp, and no class I gene/gene segments were found. The HLA-E gene is also missing in FLA. In DLA, the HLA-A corresponding region and the HLA-E-92 genes are also deleted. The HLA-B, -C regions occupy in a short segment of human HLA; however both FLA and DLA have a multiple number of class I genes (5 DLA [Wagner et al. 2005] and 17 FLA class I genes). It is clear by sequence comparisons that each FLA and DLA class I genes were amplified in species-specific manners and are not orthologous to HLA class I genes. Remarkably, FLA has at least one full-length class I gene at the break point near TRIM39, where mouse H2, M1M10 class I genes, which may function as an escort molecule for pheromone receptors, are located (Loconto et al. 2003). In DLA, 2 other class I genes (Wagner 2003), ctpg26 and DLA-79, are located

on 2 additional chromosomes, cfa7 and cfa18. Though cptg26 appeared to be a processed pseudogene, DLA-79 has a moderate level of polymorphism, suggesting this may encode a functional class I antigen. In addition, a number of olfactory receptor-like genes plus an OCT3-like gene were linked with DLA-79 on cfa18 (Figure 6). Of the19 class I genes in FLA, at least 3 genes have conserved amino acid residues in the peptide-binding region (data not shown). This may suggest that FLA has 3 peptidepresenting class I genes, like human and mouse MHCs. At least 4 MHC class Irelated (MIC) gene homologues were found in FLA HLA-B/C region; however, no MIC-like sequences were found in DLA HLA-B/Ccorresponding region. This may suggest distinct NK control mechanisms in dog and cat.

Funding
National Cancer Institute, National Institutes of Health (N01-CO-12400).

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Journal of Heredity

Figure 6. Comparison of gene content and order in class I region of FLA, DLA, and HLA. Orthologous genes in FLA, DLA, and HLA were connected with lines. FLA has 17 class I genes in HLA-B, -C regions, plus 2 class I genes in HLA-92 region, whereas HLA-E, -A regions are deleted. DLA has 5 class I genes in HLA-B, -C regions and no class I genes are found in HLA-E, -92, -A regions. Two class I genes, one in cfa7 and another in cfa18 (cptg26, DLA-79) are found. Triangle indicates deletion of corresponding HLA class I subregions (HLA-E, -92, -A).

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Acknowledgments
The authors thank Lisa Maslan for her technical support. Neither does the content of this publication necessarily reflect the views or policies of the Department of Health and Human Services nor does mention of trade names, commercial products, or organizations imply endorsement by the US Government.

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This work was presented in poster form at the Third International Conference on Advances in Canine and Feline Genomics and Inherited Diseases, Davis, CA, August 2006. Corresponding Editor: Urs Giger

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