Location, location, location: the evolutionary history of CD1 genes and the NKR-P1/ligand systems.

CD1 genes encode cell surface molecules that present lipid antigens to various kinds of T lymphocytes of the immune system. The structures of CD1 genes and molecules are like the major histocompatibility complex (MHC) class I system, the loading of antigen and the tissue distribution for CD1 molecules are like those in the class II system, and phylogenetic analyses place CD1 between class I and class II sequences, altogether leading to the notion that CD1 is a third ancient system of antigen presentation molecules. However, thus far, CD1 genes have only been described in mammals, birds and reptiles, leaving major questions as to their origin and evolution. In this review, we recount a little history of the field so far and then consider what has been learned about the structure and functional attributes of CD1 genes and molecules in marsupials, birds and reptiles. We describe the central conundrum of CD1 evolution, the genomic location of CD1 genes in the MHC and/or MHC paralogous regions in different animals, considering the three models of evolutionary history that have been proposed. We describe the natural killer (NK) receptors NKR-P1 and ligands, also found in different genomic locations for different animals. We discuss the consequence of these three models, one of which includes the repudiation of a guiding principle for the last 20 years, that two rounds of genome-wide duplication at the base of the vertebrates provided the extra MHC genes necessary for the emergence of adaptive immune system of jawed vertebrates.

Sequence of a complete chicken BG haplotype shows dynamic expansion and contraction of two gene lineages with particular expression patterns.

Many genes important in immunity are found as multigene families. The butyrophilin genes are members of the B7 family, playing diverse roles in co-regulation and perhaps in antigen presentation. In humans, a fixed number of butyrophilin genes are found in and around the major histocompatibility complex (MHC), and show striking association with particular autoimmune diseases. In chickens, BG genes encode homologues with somewhat different domain organisation. Only a few BG genes have been characterised, one involved in actin-myosin interaction in the intestinal brush border, and another implicated in resistance to viral diseases. We characterise all BG genes in B12 chickens, finding a multigene family organised as tandem repeats in the BG region outside the MHC, a single gene in the MHC (the BF-BL region), and another single gene on a different chromosome. There is a precise cell and tissue expression for each gene, but overall there are two kinds, those expressed by haemopoietic cells and those expressed in tissues (presumably non-haemopoietic cells), correlating with two different kinds of promoters and 5' untranslated regions (5'UTR). However, the multigene family in the BG region contains many hybrid genes, suggesting recombination and/or deletion as major evolutionary forces. We identify BG genes in the chicken whole genome shotgun sequence, as well as by comparison to other haplotypes by fibre fluorescence in situ hybridisation, confirming dynamic expansion and contraction within the BG region. Thus, the BG genes in chickens are undergoing much more rapid evolution compared to their homologues in mammals, for reasons yet to be understood.

Expression of the leukemic prognostic marker CD7 is linked to epigenetic modifications in chronic myeloid leukemia.

BACKGROUND: Expression levels of the cell surface glycoprotein, CD7, and the serine protease, elastase 2 (ELA2), in the leukemic cells of patients with chronic myeloid leukemia (CML) have been associated with clinical outcome. However, little is known about the mechanisms that underlie the variable expression of these genes in the leukemic cells. RESULTS: To address this question, we compared the level of their expression with the DNA methylation and histone acetylation status of 5' sequences of both genes in leukemic cell lines and primitive (lin-CD34+) leukemic cells from chronic phase CML patients. DNA methylation of the ELA2 gene promoter did not correlate with its expression pattern in lin-CD34+ cells from chronic phase CML patient samples even though there was clear differential DNA methylation of this locus in ELA2-expressing and non-expressing cell lines. In contrast, we found a strong relation between CD7 expression and transcription-permissive chromatin modifications, both at the level of DNA methylation and histone acetylation with evidence of hypomethylation of the CD7 promoter region in the lin-CD34+ cells from CML patients with high CD7 expression. CONCLUSION: These findings indicate a link between epigenetic modifications and CD7 expression in primitive CML cells.

Creation of the two isoforms of rodent NKG2D was driven by a B1 retrotransposon insertion.

The mouse gene for the natural killer (NK) cell-activating receptor Nkg2d produces two protein isoforms, NKG2D-S and NKG2D-L, which differ by 13 amino acids at the N-terminus and have different signalling capabilities. These two isoforms are produced through differential splicing, but their regulation has not been investigated. In this study, we show that rat Nkg2d has the same splicing pattern as that of the mouse, and we mapped transcriptional start sites in both species. We found that the splice forms arise from alternative promoters and that the NKG2D-L promoter is derived from a rodent B1 retrotransposon that inserted before mouse-rat divergence. This B1 insertion is associated with loss of a nearby splice acceptor site that subsequently allowed creation of the short NKG2D isoform found in mouse but not human. Transient reporter assays indicate that the B1 element is a strong promoter with no inherent lymphoid tissue-specificity. We have also identified different binding sites for the ETS family member GABP within both the mouse and rat B1 elements that are necessary for high-promoter activity and for full Nkg2d-L expression. These findings demonstrate that a retroelement insertion has led to gene-regulatory change and functional diversification of rodent NKG2D.

Avian NK activities, cells and receptors.

Natural killer (NK) activity has been examined in birds for over 30 years, but evidence that avian NK activity plays crucial roles in disease is only suggestive. In chickens, NK activity is mediated by TCR0 cells in the intestinal epithelium, but elsewhere subsets of alphabeta and gammadelta T cells (NKT cells) may be more important. There are few lectin-like NK receptor genes, located in the genomic region syntenic with the natural killer complex (NKC) as well as the major histocompatibility complex (MHC). In contrast, a huge number of Ig-like receptor genes are located in a region syntenic with the leukocyte receptor complex (LRC).

High allelic polymorphism, moderate sequence diversity and diversifying selection for B-NK but not B-lec, the pair of lectin-like receptor genes in the chicken MHC.

We previously characterised the C-type lectin-like receptor genes B-NK and B-lec, located next to each other in opposite orientations in the chicken major histocompatibility complex (MHC). We showed that B-NK is an inhibitory receptor expressed on natural killer cells, whereas B-lec is an activation-induced receptor with a broader expression pattern. It is interesting to note that the chicken MHC has been linked with resistance or susceptibility to Marek's disease virus (MDV), an oncogenic herpes virus. Recent reports show that the C-type lectin-like receptors in mouse and rat (Ly49H, NKR-P1 and Clr) are associated with resistance to another herpesvirus, cytomegalovirus (CMV). Therefore, B-NK and B-lec are potential candidate genes for the MHC-mediated resistance to MDV. In this paper, we report that both genes encode glycosylated type II membrane proteins that form disulphide-linked homodimers. The gene sequences from nine lines of domestic chicken representing seven haplotypes show that B-lec is well conserved between the different haplotypes, apparently under purifying selection. In contrast, B-NK has high allelic polymorphism and moderate sequence diversity, with 21 nucleotide changes in the complementary deoxyribonucleic acids (cDNAs) resulting in 20 amino acid substitutions. The allelic variations include substitutions, an indel and loss/gain of three predicted N-linked glycosylation sites. Strikingly, there is as much as 7% divergence between protein sequences of B-NK from different haplotypes, greater than the difference observed between the highly polymorphic human KIR NK receptors. Analysis of ds and dn reveal evidence of strong positive selection for B-NK to be polymorphic at the protein level, and modelling demonstrates significant variation between haplotypes in the predicted ligand binding face of B-NK.

A role for DNA hypomethylation and histone acetylation in maintaining allele-specific expression of mouse NKG2A in developing and mature NK cells.

The repertoire of receptors that is expressed by NK cells is critical for their ability to kill virally infected or transformed cells. However, the molecular mechanisms that determine whether and when NK receptor genes are transcribed during hemopoiesis remain unclear. In this study, we show that hypomethylation of a CpG-rich region in the mouse NKG2A gene is associated with transcription of NKG2A in ex vivo NK cells and NK cell lines. This observation was extended to various developmental stages of NK cells sorted from bone marrow, in which we demonstrate that the CpGs are methylated in the NKG2A-negative stages (hemopoietic stem cells, NK progenitors, and NKG2A-negative NK cells), and hypomethylated specifically in the NKG2A-positive NK cells. Furthermore, we provide evidence that DNA methylation is important in maintaining the allele-specific expression of NKG2A. Finally, we show that acetylated histones are associated with the CpG-rich region in NKG2A positive, but not negative, cell lines, and that treatment with the histone deacetylase inhibitor trichostatin A alone is sufficient to induce NKG2A expression. Treatment with the methyltransferase inhibitor 5-azacytidine only is insufficient to induce transcription, but cotreatment with both drugs resulted in a significantly greater induction, suggesting a cooperative role for DNA methylation and histone acetylation status in regulating gene expression. These results enhance our understanding of the formation and maintenance of NK receptor repertoires in developing and mature NK cells.

Two CD1 genes map to the chicken MHC, indicating that CD1 genes are ancient and likely to have been present in the primordial MHC.

CD1 molecules play an important role in the immune system, presenting lipid-containing antigens to T and NKT cells. CD1 genes have long been thought to be as ancient as MHC class I and II genes, based on various arguments, but thus far they have been described only in mammals. Here we describe two CD1 genes in chickens, demonstrating that the CD1 system was present in the last common ancestor of mammals and birds at least 300 million years ago. In phylogenetic analysis, these sequences cluster with CD1 sequences from other species but are not obviously like any particular CD1 isotype. Sequence analysis suggests that the expressed proteins bind hydrophobic molecules and are recycled through intracellular vesicles. RNA expression is strong in lymphoid tissues but weaker to undetectable in some nonlymphoid tissues. Flow cytometry confirms expression from one gene on B cells. Based on Southern blotting and cloning, only two such CD1 genes are detected, located approximately 800 nucleotides apart and in the same transcriptional orientation. The sequence of one gene is nearly identical in six chicken lines. By mapping with a backcross family, this gene could not be separated from the chicken MHC on chromosome 16. Mining the draft chicken genome sequence shows that chicken has only these two CD1 genes located approximately 50 kb from the classical class I genes. The unexpected location of these genes in the chicken MHC suggests the CD1 system was present in the primordial MHC and is thus approximately 600 million years old.

Characterization of the chicken C-type lectin-like receptors B-NK and B-lec suggests that the NK complex and the MHC share a common ancestral region.

The sequencing of the chicken MHC led to the identification of two open reading frames, designated B-NK and B-lec, that were predicted to encode C-type lectin domains. C-type lectin domains are not encoded in the MHC of any animal described to date; therefore, this observation was completely unexpected, particularly given that the chicken has a "minimal essential MHC." In this study, we describe the initial characterization of the B-NK and B-lec genes, and show that they share greatest homology with C-type lectin-like receptors encoded in the human NK complex (NKC), in particular NKR-P1 and lectin-like transcript 1 (LLT1), respectively. In common with NKR-P1 and LLT1, B-NK and B-lec are located next to each other and transcribed in opposite orientation. Like human NKR-P1, B-NK has a functional inhibitory signaling motif in the cytoplasmic tail and is expressed in NK cells. In contrast, B-lec contains an endocytosis motif in the cytoplasmic tail, and like LLT1, is an early activation Ag. Further analysis leads us to propose that there are four subgroups of C-type lectin-like receptors in the NKC, which arose as a result of duplication events. Moreover, this analysis suggests that the NKC may be considered a fifth paralogous region, and therefore shares an ancient common origin with the MHC. This provides evidence that C-type lectin-like receptors were present in the preduplication, primordial MHC region, and suggests that an original function of MHC molecules was for recognition by NK cell receptors encoded nearby.