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The Igh locus contains ~100 VH gene segments over an almost 3Mb genomic interval. (A) (Upper panel) Diagram of the Igh locus indicating VH, D, JH, and CH exons and regulatory elements (not to scale). The intronic Eμ and 3′Eα super-enhancers and intergenic control region 1 (IGCR1), composed of two divergent CBEs, are critical regulatory elements. CBE orientation is indicated by (purple) triangle direction. The 3′ regulatory region (3′RR) is a composite of nine CBEs located at the 3′ boundary of the Igh locus adjacent to 3′Eα super-enhancer. Sites I, II, and III (purple circles) anchor the sub-topologically associating domain (Sub-TADs) A, B, and C. The VHS107 family along with nine smaller VH families comprise the intermediate VH segments. The interspersed distal VH gene segments are composed of the VHJ558 and VH3609 families and are located at the 5’ end of the locus. (Lower panel) The VH7183 (blue bars) and VHQ52 families (red bars) are located at the DHJH-proximal end of the locus. Each DHJH -proximal VH exon is paired with a recombination signal sequence (not shown) and a CBE (purple triangle). The CBE associated with VH5-1 exon is non-functional (gray triangle). VH81X (VH5-2) is the second VH exon gene relative to IGCR1. (B) Schematic of the stepwise process of V(D)J recombination. DH-JH rearrangement precedes VH-DHJH recombination.
Source publication
Vast repertoires of unique antigen receptors are created in developing B and T lymphocytes. The antigen receptor loci contain many variable (V), diversity (D) and joining (J) gene segments that are arrayed across very large genomic expanses and are joined to form variable-region exons of expressed immunoglobulins and T cell receptors. This process...
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Citations
... However, individual single-cell transcriptomes have so far not been paired with germline assemblies from the same donor. Haplotyped structural variants and heterogeneity of immunoglobulin V, D, J, and C gene segments may affect the antibody repertoire of a given individual, representing the potential of future precision medicine based on individual variation (Kidd et al. 2016;Kenter et al. 2021;Peres et al. 2023). ...
Immunoglobulin (IGH, IGK, IGL) loci in the human genome are highly polymorphic regions that encode the building blocks of the light and heavy chain IG proteins that dimerize to form antibodies. The processes of V(D)J recombination and somatic hypermutation in B cells are responsible for creating an enormous reservoir of highly specific antibodies capable of binding a vast array of possible antigens. However, the antibody repertoire is fundamentally limited by the set of variable (V), diversity (D), and joining (J) alleles present in the germline IG loci. To better understand how the germline IG haplotypes contribute to the expressed antibody repertoire, we combined genome sequencing of the germline IG loci with single-cell transcriptome sequencing of B cells from the same donor. Sequencing and assembly of the germline IG loci captured the IGH locus in a single fully-phased contig where the maternal and paternal contributions to the germline V, D, and J repertoire can be fully resolved. The B cells were collected following a measles, mumps, and rubella (MMR) vaccination, resulting in a population of cells that were activated in response to this specific immune challenge. Single-cell, full-length transcriptome sequencing of these B cells resulted in whole transcriptome characterization of each cell, as well as highly-accurate consensus sequences for the somatically rearranged and hypermutated light and heavy chain IG transcripts. A subset of antibodies synthesized based on their consensus heavy and light chain transcript sequences demonstrated binding to measles antigens and neutralization of measles live virus.
... IGH V(D)J gene rearrangement is mediated by the RAG complex (RAG1/2), which binds to recombination signal sequences (RSS) that flank IGHV, IGHD, and IGHJ genes to form the recombination center (RC). Most of our knowledge regarding specific factors involved in Ab repertoire regulation, including the process of V(D)J recombination, is derived from studies of inbred mice [14][15][16][17][18][19][20][21][22][23][24]. For example, the chromatin landscape of the C57BL/6 Igh locus is characterized by three loops anchored at boundaries of topologically associated domains (TADs) [20,25] associated with convergent CTCF binding motifs [23,26]. ...
... For example, the chromatin landscape of the C57BL/6 Igh locus is characterized by three loops anchored at boundaries of topologically associated domains (TADs) [20,25] associated with convergent CTCF binding motifs [23,26]. The probability of a gene being incorporated into the RC can be described as a function with multiple inputs, including 1) ability of RAG to bind the gene RSS 2) gene location within a TAD 3) spatial relationship of the gene to convergent CTCF motifs 4) gene-proximal DNA accessibility, and 5) binding of specific transcription factors that influence enhancer activity and enhancer-promoter contacts [14,15,19,20,25]. In addition, there are several well-described cis-elements in the C57BL/6 haplotype that regulate initial assembly of the RC: the intronic enhancer, Eμ, and the intergenic control region 1 (IGCR1) [22][23][24]26,27]. ...
... IRF4, PAX5) [15,19,20,26,[28][29][30]. However, to date, these models have not systematically considered the impact of genetic variation that can occur between IG haplotypes [14]. In humans, haplotype diversity is a hallmark of the IG loci. ...
The expressed antibody repertoire is a critical determinant of immune-related phenotypes. Antibody-encoding transcripts are distinct from other expressed genes because they are transcribed from somatically rearranged gene segments. Human antibodies are composed of two identical heavy and light chain polypeptides derived from genes in the immunoglobulin heavy chain (IGH) locus and one of two light chain loci. The combinatorial diversity that results from antibody gene rearrangement and the pairing of different heavy and light chains contributes to the immense diversity of the baseline antibody repertoire. During rearrangement, antibody gene selection is mediated by factors that influence chromatin architecture, promoter/enhancer activity, and V(D)J recombination. Interindividual variation in the composition of the antibody repertoire associates with germline variation in IGH, implicating polymorphism in antibody gene regulation. Determining how IGH variants directly mediate gene regulation will require integration of these variants with other functional genomic datasets. Here, we argue that standard approaches using short reads have limited utility for characterizing regulatory regions in IGH at haplotype-resolution. Using simulated and ChIP-seq reads, we define features of IGH that limit use of short reads and a single reference genome, namely 1) the highly duplicated nature of DNA sequence in IGH and 2) structural polymorphisms that are frequent in the population. We demonstrate that personalized diploid references enhance performance of short-read data for characterizing mappable portions of the locus, while also showing that long-read profiling tools will ultimately be needed to fully resolve functional impacts of IGH germline variation on expressed antibody repertoires.
... We next investigated whether Kidins220 plays a role in the usage of specific V-and J-genes. The murine Igh locus consists of more than 100 Igh-V-, 8-12 Igh-D-and 4 Igh-J-genes (Kenter et al., 2021). We compared the use of each gene segment between CTRL and B-KO cells by performing a Pearson correlation test. ...
The ratio between κ and λ light chain (LC)-expressing B cells varies considerably between species. We recently identified Kinase D-interacting substrate of 220 kDa (Kidins220) as an interaction partner of the BCR. In vivo ablation of Kidins220 in B cells resulted in a marked reduction of λLC-expressing B cells. Kidins220 knockout B cells fail to open and recombine the genes of the Igl locus, even in genetic scenarios where the Igk genes cannot be rearranged or where the κLC confers autoreactivity. Igk gene recombination and expression in Kidins220-deficient B cells is normal. Kidins220 regulates the development of λLC B cells by enhancing the survival of developing B cells and thereby extending the time-window in which the Igl locus opens and the genes are rearranged and transcribed. Further, our data suggest that Kidins220 guarantees optimal pre-BCR and BCR signaling to induce Igl locus opening and gene recombination during B cell development and receptor editing.
... We currently understand little about the genetic factors, and thus the associated molecular mechanisms, that dictate the regulation of the human Ab response. In fact, much of what we understand about the specific genomic factors involved in Ab repertoire development and variability comes from inbred animal models [32][33][34][35] , even though such questions would have greater relevance to health if addressed in outbred human populations 22 . These limitations continue to impede our understanding of the contribution of IGH polymorphism to disease risk, infection and response to vaccines and therapeutics 22,31,36,37 . ...
... For example, the mouse IG loci partition into distinct regions, marked by specific regulatory marks, including TFBS and histone modification signatures, many of which, alongside RSS variation, have been associated with intra-gene V(D)J recombination frequency differences 32,84,85 . The mouse IG loci are also characterized by 3-dimensional structure, TADs and sub-TADs that are associated with complex interactions between gene promoters and enhancers that coordinate V(D)J recombination in pre-B cells 35,53,[86][87][88] . In contrast to mouse, functional genomic elements dictating V(D)J recombination in the human IGH locus have not been characterized in depth; nonetheless, our intersection of guQTLs with publicly available annotation sets revealed enrichments in cis-regulatory elements and TFBS involved in V(D)J recombination in animal models. ...
Variation in the antibody response has been linked to differential outcomes in disease, and suboptimal vaccine and therapeutic responsiveness, the determinants of which have not been fully elucidated. Countering models that presume antibodies are generated largely by stochastic processes, we demonstrate that polymorphisms within the immunoglobulin heavy chain locus (IGH) impact the naive and antigen-experienced antibody repertoire, indicating that genetics predisposes individuals to mount qualitatively and quantitatively different antibody responses. We pair recently developed long-read genomic sequencing methods with antibody repertoire profiling to comprehensively resolve IGH genetic variation, including novel structural variants, single nucleotide variants, and genes and alleles. We show that IGH germline variants determine the presence and frequency of antibody genes in the expressed repertoire, including those enriched in functional elements linked to V(D)J recombination, and overlapping disease-associated variants. These results illuminate the power of leveraging IGH genetics to better understand the regulation, function, and dynamics of the antibody response in disease.
... We describe almost double the numbers of expressed TCRV alleles previously identified and a 30% increase in the total number of TRAJ alleles, suggesting that allelic variation in TCR genes is comparable to that in IGH genes. 10,12 In contrast to the IGH locus, 11,51 we found that genomic deletions are relatively infrequent in the TCR loci. One previously described common deletion encompassing TRBV4-3 was identifiable here through loss of expression of this gene in multiple individuals. ...
Human T cell receptors (TCRs) are critical for mediating immune responses to pathogens and tumors and regulating self-antigen recognition. Yet, variations in the genes encoding TCRs remain insufficiently defined. Detailed analysis of expressed TCR alpha, beta, gamma, and delta genes in 45 donors from four human populations-African, East Asian, South Asian, and European-revealed 175 additional TCR variable and junctional alleles. Most of these contained coding changes and were present at widely differing frequencies in the populations, a finding confirmed using DNA samples from the 1000 Genomes Project. Importantly, we identified three Neanderthal-derived, introgressed TCR regions including a highly divergent TRGV4 variant, which mediated altered butyrophilin-like molecule 3 (BTNL3) ligand reactivity and was frequent in all modern Eurasian population groups. Our results demonstrate remarkable variation in TCR genes in both individuals and populations, providing a strong incentive for including allelic variation in studies of TCR function in human biology.
... Furthermore, disruption of enhancer function has been increasingly associated with disease, specifically involving genetic variants in transcription factor binding sites 87 . Polymorphisms also can affect the recombination frequencies of specific V genes and are correlated with disease [88][89][90] . It seems likely that polymorphisms associated with antigen receptor loci enhancers affect V-gene rearrangement frequencies in human B cells, with implications for human disease and vaccine development. ...
Antigen receptor loci are organized into variable (V), diversity (D) and joining (J) gene segments that rearrange to generate antigen receptor repertoires. Here, we identified an enhancer (E34) in the murine immunoglobulin kappa (Igk) locus that instructed rearrangement of Vκ genes located in a sub-topologically associating domain, including a Vκ gene encoding for antibodies targeting bacterial phosphorylcholine. We show that E34 instructs the nuclear repositioning of the E34 sub-topologically associating domain from a recombination-repressive compartment to a recombination-permissive compartment that is marked by equivalent activating histone modifications. Finally, we found that E34-instructed Vκ-Jκ rearrangement was essential to combat Streptococcus pneumoniae but not methicillin-resistant Staphylococcus aureus or influenza infections. We propose that the merging of Vκ genes with Jκ elements is instructed by one-dimensional epigenetic information imposed by enhancers across Vκ and Jκ genomic regions. The data also reveal how enhancers generate distinct antibody repertoires that provide protection against lethal bacterial infection.
... Non-coding transcription and chromatin remodeling are crucial (21). More recently, it has been shown that the mouse IGH locus is a topologically associated domain (TAD) organized into three chromatin loops or sub-TAD's required for adequate spatial approximation of IGHV genes to the recombination center by chromatin loop extrusion (33,44). Chromatin extrusion is mediated by the cohesin ring protein complex by extruding chromatin between convergently oriented CTCF-binding elements (CBE's). ...
Background
The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model vertebrates.
Methods
The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other vertebrates. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human, Xenopus tropicalis, Danio rerio (zebrafish), and eight tetrapod reference genomes were used for comparison.
Results
Canonical A. mexicanum heavy chain (IGH), lambda (IGL), sigma (IGS), and the putative surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes and there is no clan I IGHV genes. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality.
Conclusion
The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of Ig gene loss which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.
... Non-coding transcription and chromatin remodeling are crucial (Schatz and Ji 2011). More recently, it has been shown that the mouse IGH locus is a topologically associated domain (TAD) organized into three chromatin loops or sub-TAD's required for adequate spatial approximation of IGHV genes to the recombination center by chromatin loop extrusion (Kenter, Watson, and Spille 2021;Zhang et al. 2022). Chromatin extrusion is mediated by the cohesin ring protein complex by extruding chromatin between convergently oriented CTCF-binding elements (CBE's). ...
Background
The axolotl, Ambystoma mexicanum is a unique biological model for complete tissue regeneration. Is a neotenic endangered species and is highly susceptible to environmental stress, including infectious disease. In contrast to other amphibians, the axolotl is particularly vulnerable to certain viral infections. Like other salamanders, the axolotl genome is one of the largest (32 Gb) and the impact of genome size on Ig loci architecture is unknown. To better understand the immune response in axolotl, we aimed to characterize the immunoglobulin loci of A. mexicanum and compare it with other model tetrapods.
Methods
The most recently published genome sequence of A. mexicanum (V6) was used for alignment-based annotation and manual curation using previously described axolotl Ig sequences or reference sequences from other tetrapods. Gene models were further curated using A. mexicanum spleen RNA-seq data. Human reference genomes, Xenopus tropicalis , and Danio rerio (zebrafish) were used for comparison.
Results
Canonical A. mexicanum Heavy chain (IGH), lambda (IGL), sigma (IGS) and Surrogate light chain (SLC) loci were identified. No kappa locus was found. More than half of the IGHV genes and the IGHF gene are pseudogenes, there are no clan I IGHV genes and CDRH3 diversity is restricted. Although the IGH locus size is proportional to genome size, we found local size restriction in the IGHM gene and in the V gene intergenic distances. In addition, there were V genes with abnormally large V-intron sizes, which correlated with loss of gene functionality.
Conclusion
The A. mexicanum immunoglobulin loci share the same general genome architecture as most studied tetrapods. Consistent with its large genome, Ig loci are larger; however, local size restrictions indicate evolutionary constraints likely to be imposed by high transcriptional demand of certain Ig genes, as well as the V(D)J recombination over very long genomic distance ranges. The A. mexicanum has undergone an extensive process of pseudogenization which partially explains a reduced potential repertoire diversity that may contribute to its impaired antibody response.
... Our approach (which we call desYgnator) relies on a hierarchy of experimental controls as well as information and statistical theories and provides new recommendations for unbiased comparison of RGM between individuals that were previously thought to be identical (Fig. 2). Our results indicate that inter-individual differences in VDJ recombination are not only influenced by genetic differences in germline gene repertoires, such as germline gene polymorphisms or structural variation (Kenter et al. 2021), but also influenced by nongenetic differences (e.g., epigenetics). Indeed, it has been previously shown that epigenetic mechanisms intervene in the regulation of VDJ recombination (Pulivarthy et al. 2016). ...
The process of recombination between variable (V), diversity (D), and joining (J) immunoglobulin (Ig) gene segments determines an individual's naive Ig repertoire and, consequently, (auto)antigen recognition. VDJ recombination follows prob-abilistic rules that can be modeled statistically. So far, it remains unknown whether VDJ recombination rules differ between individuals. If these rules differed, identical (auto)antigen-specific Ig sequences would be generated with individual-specific probabilities, signifying that the available Ig sequence space is individual specific. We devised a sensitivity-tested distance measure that enables inter-individual comparison of VDJ recombination models. We discovered, accounting for several sources of noise as well as allelic variation in Ig sequencing data, that not only unrelated individuals but also human mono-zygotic twins and even inbred mice possess statistically distinguishable immunoglobulin recombination models. This suggests that, in addition to genetic, there is also nongenetic modulation of VDJ recombination. We demonstrate that population-wide individualized VDJ recombination can result in orders of magnitude of difference in the probability to generate (auto)antigen-specific Ig sequences. Our findings have implications for immune receptor-based individualized medicine approaches relevant to vaccination, infection, and autoimmunity.
... Different expression levels of Ig V alleles in heterozygous individuals have been reported in multiple studies (Fig. 4B) [23,37,38], but the reason for such uneven expression remains unclear. It is likely that non-coding regions might play a role in the regulation of Ig expression, but this has been very little explored [105]. Although the non-coding regions have received slightly more attention in the past years [37,106], polymorphisms in these regions remain poorly characterised. ...
Immunoglobulins (Ig) play an important role in the immune system both when expressed as antigen receptors on the cell surface of B cells and as antibodies secreted into extracellular fluids. The advent of high-throughput sequencing methods has enabled the investigation of human Ig repertoires at unprecedented depth. This has led to the discovery of many previously unreported germline Ig alleles. Moreover, it is becoming clear that convergent and stereotypic antibody responses are common where different individuals recognise defined antigenic epitopes with the use of the same Ig V genes. Thus, germline V gene variation is increasingly being linked to the differential capacity of generating an effective immune response, which might lead to varying disease susceptibility. Here, we review recent evidence of how germline variation in Ig genes impacts the Ig repertoire and its subsequent effects on the adaptive immune response in vaccination, infection, and autoimmunity.
