Extensive analysis of D-J-C arrangements allows the identification of different mechanisms enhancing the diversity in sheep T cell receptor β-chain repertoire

<p>Abstract</p> <p>Background</p> <p>In most species of mammals, the <it>TRB </it>locus has the common feature of a library of <it>TRBV </it>genes positioned at the 5'- end of two in tandem aligned D-J-C gene clusters, each composed of a single <it>TRBD </it>gene, 6-7 <it>TRBJ </it>genes and one <it>TRBC </it>gene. An enhancer located at the 3'end of the last <it>TRBC </it>and a well-defined promoter situated at the 5'end of the <it>TRBD </it>gene and/or a undefined promoter situated at the 5'end of the <it>TRBD2 </it>are sufficient to generate the full recombinase accessibility at the locus. In ruminant species, the 3'end of the <it>TRB </it>locus is characterized by the presence of three D-J-C clusters, each constituted by a single <it>TRBD</it>, 5-7 <it>TRBJ </it>and one <it>TRBC </it>genes with the center cluster showing a structure combined with the clusters upstream and downstream, suggesting that a unequal crossover occurred in the duplication. An enhancer downstream the last <it>TRBC</it>, and a promoter at the 5'-end of each <it>TRBD </it>gene are also present.</p> <p>Results</p> <p>In this paper we focused our attention on the analysis of a large number of sheep TR β-chain transcripts derived from four different lymphoid tissues of three diverse sheep breed animals to certify the use and frequency of the three gene clusters in the β-chain repertoire. As the sheep <it>TRB </it>locus genomic organization is known, the exact interpretation of the V-D-J rearrangements was fully determined. Our results clearly demonstrate that sheep β-chain constitutes a level of variability that is substantially larger than that described in other mammalian species. This is due not only to the increase of the number of D and J genes available to the somatic recombination, but also to the presence of the trans-rearrangement process. Moreover, the functional complexity of β-chain repertoire is resolved by other mechanisms such as alternative cis- and trans-splicing and recombinational diversification that seems to affect the variety of the constant region.</p> <p>Conclusion</p> <p>All together our data demonstrate that a disparate set of molecular mechanisms operate to perform a diversified repertoire in the sheep β-chain and this could confer some special biological properties to the corresponding αβ T cells in the ruminant lineage.</p

Genomic organization and recombinational unit duplication-driven evolution of ovine and bovine T cell receptor gamma loci

<p>Abstract</p> <p>Background</p> <p>In humans and mice ("γδ low species") less than 5% of the peripheral blood T lymphocytes are gamma/delta T cells, whereas in chicken and artiodactyls ("γδ high species") gamma/delta T cells represent about half of the T cells in peripheral blood. In cattle and sheep (Bovidae) two paralogous T cell receptor gamma loci (TRG1 and TRG2) have been found. TRG1 is located on 4q3.1, within a region of homology with the human TRG locus on chromosome 7, while TRG2 localizes on 4q2.2 and appears to be unique to ruminants. The purpose of this study was the sequencing of the genomic regions encompassing both loci in a "γδ high" organism and the analysis of their evolutionary history.</p> <p>Results</p> <p>We obtained the contiguous genomic sequences of the complete sheep TRG1 and TRG2 loci gene repertoire and we performed cattle/sheep sequence analysis comparison using data available through public databases. Dot plot similarity matrix comparing the two sheep loci with each other has shown that variable (V), joining (J) and constant (C) genes have evolved through a series of duplication events involving either entire cassettes, each containing the basic V-J-J-C recombinational unit, or single V genes. The phylogenetic behaviour of the eight enhancer-like elements found in the sheep, compared with the single copy present in the human TRG locus, and evidence from concordant insertions of repetitive elements in all analyzed TRGJ blocks allowed us to infer an evolutionary scenario which highlights the genetic "flexibility" of this region and the duplication-driven evolution of gene cassettes. The strong similarity of the human and Bovidae intergenic J-J-C regions, which display an enhancer-like element at their 3' ends, further supports their key role in duplications.</p> <p>Conclusion</p> <p>We propose that only duplications of entire J-J-C regions that possessed an enhancer-like element at their 3' end, and acquired at least one V segment at their 5' end, were selected and fixed as functional recombinational units.</p

New insight into the genomic structure of dog T cell receptor beta (TRB) locus inferred from expression analysis

Here is an updated report on the genomic organization of T cell receptor beta (TRB) locus in the domestic dog (Canis lupus familiaris) as inferred from comparative genomics and expression analysis. The most interesting results we found were a second TRBD–J–C cluster, which is absent from the reference genome sequence, and the annotation of two additional TRBV genes. In dogs, TRB locus consists of a library of 37 TRBV genes positioned at the 50 end of two in tandem aligned D–J–C gene clusters, each composed of a single TRBD, 6 TRBJ and one TRBC genes, followed by a single TRBV gene with an inverted transcriptional orientation. The TRB genes are distributed in less than 300 kb, making the canine locus, one of the smaller mammalian TRB locus studied so far. The small size may be ascribed to reduced gene duplication occurrences and a lower density of total interspersed repeats compared to humans and mice. Despite the low TRBV gene content, a large and diversified beta chain repertoire is displayed in the dog peripheral blood. A full usage of TRBV and TRBJ genes, including pseudogenes, and a high level of allelic polymorphism contribute to generate diversity. Finally, this study suggests that the overall TRB locus organization is evolutionarily conserved supporting the dog as a highly suited model system for immune development and diseases

The Genomic Organisation of the TRA/TRD Locus Validates the Peculiar Characteristics of Dromedary δ-Chain Expression

The role of gamma/delta T cells in vertebrate immunity is still an unsolved puzzle. Species such as humans and mice display a low percentage of these T lymphocytes (i.e., “ gamma/delta low species”) with a restricted diversity of gamma/delta T cell receptors (TR). Conversely, artiodactyl species (i.e., “ gamma/delta high species”) account for a high proportion of gamma/delta T cells with large gamma and delta chain repertoires. The genomic organisation of the gamma TR (TRG) and delta (TRD) loci has been determined in sheep and cattle, noting that a wide number of germline genes that encode for gamma and delta chains characterise their genomes. Taking advantage of the current improved version of the genome assembly, we have investigated the genomic structure and gene content of the dromedary TRD locus, which, as in the other mammalian species, is nested within the TR alpha (TRA) genes. The most remarkable finding was the identification of a very limited number of variable germline genes (TRDV) compared to sheep and cattle, which supports our previous expression analyses for which the somatic hypermutation mechanism is able to enlarge and diversify the primary repertoire of dromedary delta chains. Furthermore, the comparison between genomic and expressed sequences reveals that D genes, up to four incorporated in a transcript, greatly contribute to the increased diversity of the dromedary delta chain antigen binding-site

Overview of the Germline and Expressed Repertoires of the TRB Genes in Sus scrofa

The α/β T cell receptor (TR) is a complex heterodimer that recognizes antigenic peptides and binds to major histocompatibility complex (MH) molecules. Both α and β chains are encoded by different genes localized on two distinct chromosomal loci: TRA and TRB. The present study employed the recent release of the swine genome assembly to define the genomic organization of the TRB locus. According to the sequencing data, the pig TRB locus spans approximately 400 kb of genomic DNA and consists of 38 TRBV genes belonging to 24 subgroups located upstream of three in tandem TRBD-J-C clusters, which are followed by a TRBV gene in an inverted transcriptional orientation. Comparative analysis confirms that the general organization of the TRB locus is similar among mammalian species, but the number of germline TRBV genes varies greatly even between species belonging to the same order, determining the diversity and specificity of the immune response. However, sequence analysis of the TRB locus also suggests the presence of blocks of conserved homology in the genomic region across mammals. Furthermore, by analysing a public cDNA collection, we identified the usage pattern of the TRBV, TRBD, and TRBJ genes in the adult pig TRB repertoire, and we noted that the expressed TRBV repertoire seems to be broader and more diverse than the germline repertoire, in line with the presence of a high level of TRBV gene polymorphisms. Because the nucleotide differences seems to be principally concentrated in the CDR2 region, it is reasonable to presume that most T cell β-chain diversity can be related to polymorphisms in pig MH molecules. Domestic pigs represent a valuable animal model as they are even more anatomically, genetically and physiologically similar to humans than are mice. Therefore, present knowledge on the genomic organization of the pig TRB locus allows the collection of increased information on the basic aspects of the porcine immune system and contributes to filling the gaps left by rodent models

Comparative Analysis of the TRB Locus in the Camelus Genus

T cells can be separated into two major subsets based on the heterodimer that forms their T cell receptors. αβ T cells have receptors consisting of α and β chains, while γδ T cells are composed of γ and δ chains. αβ T cells play an essential role within the adaptive immune responses against pathogens. The recent genomic characterization of the Camelus dromedarius T cell receptor β (TRB) locus has allowed us to infer the structure of this locus from the draft genome sequences of its wild and domestic Bactrian congeners, Camelus ferus and Camelus bactrianus. The general structural organization of the wild and domestic Bactrian TRB locus is similar to that of the dromedary, with a pool of TRBV genes positioned at the 5′ end of D-J-C clusters, followed by a single TRBV gene located at the 3′ end with an inverted transcriptional orientation. Despite the fragmented nature of the assemblies, comparative genomics reveals the existence of a perfect co-linearity between the three Old World camel TRB genomic sequences, which enables the transfer of information from one sequence to another and the filling of gaps in the genomic sequences. A virtual camelid TRB locus is hypothesized with the presence of 33 TRBV genes distributed in 26 subgroups. Likewise, in the artiodactyl species, three in-tandem D-J-C clusters, each composed of one TRBD gene, six or seven TRBJ genes, and one TRBC gene, are placed at the 3′ end of the locus. As reported in the ruminant species, a group of four functional TRY genes at the 5′ end and only one gene at the 3′ end, complete the camelid TRB locus. Although the gene content is similar, differences are observed in the TRBV functional repertoire, and genes that are functional in one species are pseudogenes in the other species. Hence, variations in the functional repertoire between dromedary, wild and domestic Bactrian camels, rather than differences in the gene content, may represent the molecular basis explaining the disparity in the TRB repertoire between the Camelus species. Finally, our data contribute to the knowledge about the evolutionary history of Old World camelids

Sheep (Ovis aries) T cell receptor alpha (TRA) and delta (TRD) genes and genomic organization of the TRA/TRD locus

In mammals, T cells develop along two discrete pathways characterized by expression of either the αβ or the γδ T cell receptors. Human and mouse display a low peripheral blood γδ T cell percentage ("γδ low species") while sheep, bovine and pig accounts for a high proportion of γδ T lymphocytes ("γδ high species"). While the T cell receptor alpha (TRA) and delta (TRD) genes and the genomic organization of the TRA/TRD locus has been determined in human and mouse, this information is still poorly known in artiodactyl species, such as sheep

Extensive analysis of D-J-C arrangements allows the identification of different mechanisms enhancing the diversity in sheep T cell receptor β-chain repertoire

Abstract Backgroun

The expansion of the TRB and TRG genes in domestic goats (Capra hircus) is characteristic of the ruminant species

Goats (Capra hircus), one of the first domesticated species, are economically important for milk and meat production, and their broad geographical distribution reflects their successful adaptation to diverse environmental conditions. Despite the relevance of this species, the genetic research on the goat traits is limited compared to other domestic species. Thanks to the latest goat reference genomic sequence (ARS1), which is considered to be one of the most continuous assemblies in livestock, we deduced the genomic structure of the T cell receptor beta (TRB) and gamma (TRG) loci in this ruminant species

Genomic and comparative analysis of the T cell receptor gamma locus in two Equus species

The genus Equus is the only extant genus of the Equidae family, which belongs to Perissodactyla, an order of mammals characterized by an odd number of toes (odd-toes ungulates). Taking advantage of the latest release of the genome assembly, we studied, for the first time in two organisms belonging to the Equus genus, the horse (Equus caballus) and the donkey (Equus asinus), the T cell receptor gamma (TRG) locus encoding the gamma chain of the γδ T cell receptor. Forty-five Variable (TRGV) genes belonging to the seven IMGT-NC validated mammalian TRGV subgroups, 25 Joining (TRGJ) and 17 Constant (TRGC) genes organized in 17 V-J-(J)-C cassettes, in tandem on about 1100 Kb, characterize the horse TRG locus, making the horse TRG locus the one with the greatest extension and with a significantly higher number of genes than the orthologous loci of the other mammalian species. A clonotype analysis of an RNA-seq transcriptomic dataset derived from spleen of an adult healthy horse, using the complete set of the horse TRGJ germline gene sequences as a probe, revealed that, in addition to the most prominent V-J rearrangements within each cassette, there is a relevant proportion of trans-cassette V-J recombination, whereby the same TRGV genes can recombine with different TRGJ genes spliced to the corresponding TRGC genes. This recombinant event strongly contributes to the diversity of the γ chain repertoire. In the donkey TRG locus, 34 TRGV, 21 TRGJ and 14 TRGC genes distributed in 14 V-J-(J)-C cassettes were found in a region of approximately 860 kb. Although the donkey’s TRG is smaller than that of the horse, in Equus genus, this is still the second largest locus so far found in any mammalian species. Finally, the comparative analysis highlighted differences in size and gene content between the horse and donkey TRG loci, despite belonging to the same genus, indicating a good level of diversification within Equus. These data is in agreement with the evolutionary idea of the existence of a Equus recent common ancestor in rapid evolution, for which a mutation rate between horses and donkeys is more comparable to that between species belonging to different genera rather than to species of the same genus