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

Abstract

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.

Fig. 1

Comprehensive analysis of Class I gene relationships in zebrafish. Following alignment of predicted amino acid sequences from zebrafish Class I genes, neighbor-joining trees were constructed and bootstrap values over 70% are shown. Our analysis revealed 12 genes of the U lineage, 10 genes of the Z lineage, and 17 genes of the L lineage. Sequences assigned as allelic variants are marked with asterisks. Chromosomal location is given in parentheses for genes that map to the current reference genome (Zv9)

Fig. 2

Genomic organization of zebrafish Class I genes on chromosome 19. The top panel displays gene organization for haplotype A including PAC clones AL675121, AL672185 and AL672164 as assembled on an AB chromosome containing sequence variants from the zebrafish AB strain. The bottom panel is haplotype B consisting of BAC clones FP102097, FP085393, and FP074889 from the zebrafish Tuebingen strain assembled on chromosome 19 in the most recent reference genome version, Zv9. For both haplotypes the genes that are oriented in the forward sequence direction (>) are placed above the lines labeled with the genomic sequence identifiers, while genes located in the reverse direction (<) are found below. The Class I genes are located in a highly divergent center region highlighted in pink shading, while other genes are present in an adjacent conserved framework. Haplotype A contains three Class I major histocompatibility genes, uda, uea and ufa that are separated by abcb3/Tap2 genes. Haplotype B contains two Class I major histocompatibility genes, uba and uca separated by a tapbp gene. Some of the conserved flanking genes encode proteins that interact directly with the Class I molecule, including tapbp on the 5′ side, along with a distal abcb3/Tap2 gene on the 3′ side. Others are involved in peptide processing, such as the 3′ flanking psmb8-11 genes

Fig. 3

Expression patterns of zebrafish Class I genes from six haplotypes. Tissues (gill, kidney, liver) were dissected from adult male zebrafish homozygous for a single haplotype (A through F). Following RNA extraction and cDNA synthesis, qPCR data were generated using gene specific primers and normalized to β₂-microglobulin. Data represent the average of three fish for each haplotype. Similar expression levels and patterns of Class I genes for each haplotype were also observed in the spleen, heart, intestine, and testis from all fish tested (data not shown)

Fig. 4

Linkage analysis for haplotypes C through F. A fish heterozygous for haplotypes C and F was crossed with a fish heterozygous for haplotypes D and E. Tail clips from offspring were analyzed both by genotyping for haplotype flanking allele sequences and also by examining which MHC genes were expressed by qPCR. Genotyping results for these offspring are given in the top panel. The expression levels of the different MHC genes (normalized to β₂-microglobulin) are provided for one representative fish heterozygous for each of the four haplotype combinations in the bottom panel

Fig. 5

Genomic Southern blot analysis of chromosome 19 haplotypes A through F. Lane M contains DNA Molecular Weight Marker II (Roche). Lane A has genomic DNA obtained from fish homozygous for haplotype A with predicted bands for uda, uea and ufa at 10,283, 1349, and 6145 bp, respectively. Lane B (Haplotype B) displays two bands consistent with predicted sizes for uba and uca at 18,913 and 7509 bp, respectively. For the remaining haplotypes no genomic sequence information is available, thus restriction digest fragment sizes could not be predicted. For haplotype C (Lane C), bands were observed at 4 and 7 kb, corresponding to the two genes expressed by this haplotype, uja and uka. We note that an additional band at approximately 2 kb was detected in samples from haplotypes C, D, and E, likely representing hybridization with an uncharacterized U lineage gene ula found on chromosome 22, as the predicted restriction fragment of this gene is 2041 bp. Haplotype D (Lane D) had only one specific band at approximately 2.5 kb, consistent with our expression data indicating only the presence of uga. Haplotype E (Lane E) had bands at approximately 5.5 and 1.5 kb. Thus, in addition to uia, another gene with unknown sequence is likely to be found in Haplotype E. Haplotype F (Lane F) had only one specific band at approximately 4 kb, consistent with the expression of only uha

Fig. 6

Multiple sequence alignment for zebrafish Class I U lineage genes. Coding sequences were aligned and translated using the MEGA 4 program. This analysis emphasizes the high levels of diversity present throughout the sequences. Conserved structural residues are highlighted in blue, predicted peptide anchor residues are highlighted in red, and predicted sites of protein-protein interactions are highlighted in green. Within the alignment, dots indicate amino acids that are identical and dashes indicate gaps in the sequence. Conserved features are also indicated with symbols above the alignment: predicted salt bridges (s), peptide anchor residues (*), phosphorylation sites (/), acidic residues for binding to CD8 (a), β₂-microglobulin contacts (b), cysteine residues involved in disulfide bonds (c), and a glycosylation site (g)

Fig. 7

Model of zebrafish Class I gene distribution among six haplotypes. The specific order of the genes for haplotypes C through F is presently unknown, but MHC genes associated with each haplotype are presented in the context of known linked gene polymorphisms in psmb8 and zbtb22b that distinguish these haplotypes from one another and from haplotypes A and B. Specific polymorphisms are illustrated within each allele relative to the haplotype B reference sequence. For haplotype D the divergent psmb8f allele is depicted with cross-hatches. An unknown Class I gene associated with haplotype E is also indicated