Enhancers within the Ig V Gene Region Orchestrate Chromatin Topology and Regulate V Gene Rearrangement Frequency to Shape the B Cell Receptor Repertoire Specificities

Antibody repertoire development is a tightly regulated and complex process. The B-cell receptor is the membrane-bound version of the Ab that the cell will ultimately secrete. At the early stages of B-cell development, the V, D, and J gene segments in the Ig loci of the B lymphocytes undergo a tightly orchestrated series of complex rearrangements to create a functional BCR (1). An Ab molecule is composed of four polypeptide chains, with two identical H chains (Igh) and two identical L chains (Igκ or Igλ) linked together by disulfide bonds (2). The rearrangement of V, D, and J genes at these loci is mediated by the recombination activating genes (Rag1 and Rag2) (3). The RAG complex binds and cleaves the recombination signal sequences (RSSs) flanking each V, D, and J gene segment. Then, through DNA repair mechanisms, the gene segments fuse to ultimately create a V(D)J coding gene (Fig. 1A, 1B). This process occurs sequentially. First, in the Igh locus at the pro–B-cell stage, a D_H-J_H rearrangement takes place, followed by V_H-D_HJ_H rearrangement. Afterward, the L chain V-J rearrangement occurs in pre-B cells. Finally, after the rearranged V(D)J genes from the Igh and Igκ or Igλ loci are successfully transcribed and translated, the newly formed BCR is displayed in the cell surface.

Most of the V(D)J recombination principles apply equally for the H and L chain loci (1–3). However, there are significant differences between the rearrangement in the Igh versus the Igκ loci. First, all the V genes in the Igh locus are located in the same transcriptional orientation as the D-J and C gene segments, and thus all RSSs are in convergent orientation with respect to the D-J rearrangement. In contrast, V genes in the Igκ locus are located in both orientations with respect to the Jκ genes. This specific feature poses a challenge for the currently favored models as to how the RAG complexes find their substrates (V, D, and J gene segments) for recombination (4). Another feature that is different between the Igh versus the Igκ loci is that the V gene rearrangement for the Igh is fixed once it occurs, because all unrearranged D segments are deleted during the first rearrangement. In contrast, the Igκ locus can undergo a series of sequential rearrangements (Fig. 1B). This characteristic allows pre-B cells to test and change the specificity of the B-cell receptor if the newly created L chain gene is nonproductive or results in an autoreactive BCR in a process called “receptor editing” (5, 6).

The quality of the RSS was considered to be the primary factor behind why certain V genes underwent more frequent rearrangement than others for a number of years (3, 7). RSSs have a consensus sequence but very few RSSs have a complete consensus sequence. In vitro studies with recombination substrates have shown that variations from the consensus sequence can diminish recombination by vastly different frequencies (7–12). However, the advent of next-generation sequencing technologies such as chromatin immunoprecipitation sequencing, RNA sequencing, and VDJ sequencing, coupled with bioinformatic analyses, have elucidated several potential reasons why some V genes rearrange more frequently than others, although these are predominantly correlative (13–16). Rearrangement frequency correlates with several factors, including RSS quality, specific transcription factor (TF) binding near a V gene, local epigenetic milieu, nucleosomal accessibility, and three-dimensional (3D) chromatin conformation (13, 15–26). In addition, B cells generated by V(D)J recombination in different physiological locations (bone marrow versus fetal liver versus lamina propria) are known to have different repertoires (27, 28). Although many of these features correlate with the levels of rearrangement, they do not fully account for the differences in V gene rearrangement frequency. In this brief review, we present and summarize the data describing how newly described enhancers located within the large V gene portion of Igκ and Igh loci work as a higher-order mechanism for the regulation of the Ab repertoire formation by regulating chromatin interactions and epigenetic milieu, which in turn alter the 3D chromatin topology of the loci.

Enhancers are DNA regulatory elements that play a crucial role in gene expression (29). Enhancers can contain a diverse combination of consensus binding sequences for universal and lineage- and developmental stage–specific TFs (30, 31). Once bound, TFs can recruit additional proteins, such as coactivators, to the enhancer or the target gene’s promoter region to augment transcriptional activity (29, 32). Recruitment of these TFs facilitates the upregulation or activation of specific genes or clusters of genes. Although enhancers are often adjacent to the genes they regulate, they can also be located far from the regulated genes, sometimes even hundreds of thousands of base pairs away. Enhancers also function regardless of their orientation relative to the target gene or regulatory circuit. Overall, enhancers play a critical role in controlling gene expression in a precise and context-specific manner, allowing cells to maintain their identity and respond to environmental cues.

Enhancers also shape the chromatin topology via enhancer–enhancer and enhancer–promoter interactions (33–35). Enhancers induce promoter activity by moving closer together in space to establish a physical interaction. Current models for enhancer–enhancer and enhancer–promoter interactions propose that local phase separation, mediated by weak multivalent interactions among enhancer-associated molecules, drives spatial proximity between these regulatory elements with analogous states of activity and/or biophysical properties (29, 36–42). TFs and coactivators bound to the enhancers containing intrinsically disordered regions (IDRs) drive the formation of these phase-separated condensates through a process called liquid-liquid phase separation (LLPS) (Fig. 1C) (37–41, 43–46). The formation of biomolecular condensates, also known as membrane-less condensates or droplets, by LLPS is emerging as a fundamental principle in regulating cellular processes, including the initiation and maintenance of gene transcriptional programs (36, 38, 43, 47).

Previously identified and well-characterized enhancers in the Ag receptor loci are all located near the J and C region genes. These enhancers regulate chromatin accessibility, histone post-translational modifications, recruitment of RAG complexes, noncoding transcription, rearrangement, and transcription of the rearranged gene (1). For the Igκ locus, there are three enhancers near the Jκ and Cκ genes: iEκ, 3′Eκ, and Ed. Deletion of iEκ or 3′Eκ causes a moderate to severe decrease in total Igκ rearrangement, whereas the simultaneous deletion of both eliminates all rearrangement (21, 48, 49). A reduction in B cell numbers often accompanies these deletions. In contrast, deletion of the Ed does not cause any rearrangement defect, but it affects the transcription of the rearranged Vκ-Jκ gene segment (50).

Much more recently, enhancers have been identified in the large V gene–containing regions of the TCR and BCR loci (26, 51, 52). These enhancers have been identified by traditional enhancer characteristics: enrichment of H3K4me1 and H3K27ac and binding of key TFs and often Brg1 and p300. These characteristics are commonalities between these enhancers and the enhancers near the J, D, and C genes. However, unlike enhancers near the J and C genes, deleting these V region–located enhancers did not affect overall levels of rearrangement or total B-cell numbers. Instead, their deletion severely affects the rearrangement of nearby V genes, and the levels of V gene rearrangement in a 0.8–1.5 Mb region surrounding the enhancer are also modestly reduced. We describe the findings about three of these enhancers that have been well characterized recently.

An enhancer in the Igκ locus, E88, was the first of these V gene region regulatory elements to be functionally characterized in detail (26). We named these by their proximity to the closest functional Vκ gene, in this case Igkv1-88. E88 deletion in mice shows a profound defect in the rearrangement of the Vκ genes nearby, with a milder effect in the rearrangement of other genes in a wider area (∼1.5 Mb) surrounding E88 (Fig. 1D). Noncoding (germline) transcription of V genes nearby E88, but not at a distance, was also decreased. High-throughput chromosome conformation capture (HiC) analysis revealed that the Igκ locus is located inside a discrete topologically associating domain (TAD) and also demonstrated the presence of subTADs in Vκ gene region. TADs and compartment domains are the primary modes of a high order of chromatin folding (53–56). TADs display a high frequency of interactions between regions within their chromatin domain and a lower frequency between regions in adjacent chromatin domains, and they can be observed in HiC studies as dominant triangles off the diagonal (54, 55). SubTADs are smaller domains inside TADs with similar interaction patterns that tend to form in a cell type–specific manner (57). The HiC data reveal that the E88 enhancer likely regulates Vκ rearrangement in a subTAD-specific manner, with the 1.5-Mb region that showed reduced rearrangement levels correlating with two subTADs (26). Moreover, we demonstrated that E88 was a hub of local and long-range chromatin interactions across the Igκ locus. These interactions included interactions with several enhancers and promoters, including the traditional enhancers near the Jκ and Cκ genes (iEκ, 3′Eκ, and Ed). Deletion of E88 also showed that interactions across the Igκ locus were drastically affected. Furthermore, changes in the interaction patterns from iEκ (∼1.8 to 2.3 kb away from the Jκ genes) with the rest of the V gene region of the Igκ locus in E88^−/− mice correlated with increased or reduced rearrangement frequency. Moreover, these data showed that interactions in wild-type mice correlated with the deposition of H3K4me1, and loss of interactions correlated with the reduction of H3K4me1 (26, 52). Together, these data suggest that the interaction frequency between regulatory elements with enhancer characteristics facilitates Vκ-Jκ rearrangement. Overall, this work revealed that this novel enhancer in the V gene region shapes the chromatin topology through long-range interactions and that it directly affects the pattern of rearrangement of Vκ genes over a 1.5-Mb region, with the most profound effect for Vκ genes near E88.

Building on these findings, we tested the hypothesis that enhancers in the Vκ gene region control the rearrangement of specific Vκ genes with relevant physiological functions. Specifically, we identified and characterized an enhancer, E34, near a Vκ gene, Igkv7-33, that encodes the L chain of the natural Ab EO6/T15 (58, 59). This Ab recognizes the molecule phosphorylcholine (PC) present in the cell wall of many pathogenic bacteria, including Streptococcus pneumoniae (60, 61). EO6/T15 also binds PC in oxidized low-density lipoproteins, which can prevent the progression of atherosclerosis (62, 63). EO6/T15 Abs are present in naive mice without any previous foreign Ag exposure, presumably due to natural exposure to oxidized low-density lipoprotein (61). Basal levels of EO6/T15 are sufficient to protect mice after S. pneumoniae infection (61). EO6/T15⁺ B cells are the dominant cell producing Abs after exposure to PC-containing Ags. By deleting E34 in mice, we demonstrated that the E34 enhancer controls Igkv7-33 gene rearrangement frequency (59). Furthermore, due to this low level of Igkv7-33 rearrangement, EO6/T15 Ab and EO6/T15⁺ B-cell steady-state levels were both reduced. As expected, EO6/T15⁺ B-cell responses were negatively impacted after stimulation with a relevant Ag due to a low level of EO6/T15⁺ B cells in the E34^−/− mice. The repertoire changes caused by E34 deletion increased the susceptibility to S. pneumoniae infection but not to other bacterial or viral pathogens examined.

HiC studies showed that the deletion of E34 changed the chromatin interaction landscape in the Igκ locus (59). Chromatin interactions involving the areas around E34 were substantially depleted in E34^−/− mice. As with E88, interaction pattern alterations from the iEκ to the rest of the Vκ gene region correlated with rearrangement frequency. The loss of specific chromatin interactions was also correlated with loss or reduction of CCCTC-binding factor (CTCF) and H3K4me1 binding at specific sites. Furthermore, comprehensive analysis of these HiC data revealed that the E34 deletion affected subTAD boundaries. Combining the HiC and the Vκ rearrangement data, we showed that Vκ gene reduced rearrangement was contained in a specific subTAD, similar to the results with E88 mice. A study by Karki et al. arrives at a similar conclusion (64). They showed that noncoding transcription from Vκ gene promoters is activated in a subTAD-specific manner. Using microscopy, they also showed that the actively transcribing subTAD in a specific allele had been translocated to a region with high transcription and enriched RNA polymerase close to the highly transcribed Jκ genes (64). This repositioning facilitated the rearrangement of the Vκ genes in a specific subTAD.

One of the more significant findings in the E34 study was that the deletion of E34 changed the chromatin compartmentalization of the E34 subTAD (59). Compartment domains are defined by a high-order association of distinct genomic loci from HiC data using principal component analysis. Each compartment is associated with patterns of active (A) compartment and inactive or repressed (B) compartment histone marks (56, 65, 66). Active transcription is characteristic of A compartments, whereas repressed transcription is characteristic of B compartments. Compartmental domains are smaller than TADs and are present inside, between, or overlapping CTCF loops in mammals (66–68). They are also classified as active A and inactive B domains. We show that the E34-containing subTAD switches from a compartment that is shared by the rest of the Igκ locus to a compartment that is enriched with repressive epigenetic marks, mainly H3K9me3.

Using multiomics, loss-of-function, and in vivo pathogenesis approaches, we connected functional layers to decipher the mechanisms of the selection of the V gene usage during V(D)J recombination. We merged the chromatin conformation data from HiC with epigenetic profiling and transcriptomic data to reveal the depth of the changes caused by E34 deletion. Enrichment for H3K4me1, H3K27ac, CTCF, and H3K27me3 was reduced in a subTAD that contained the E34 enhancer. Chromatin accessibility was also severely affected. Chromatin accessibility and H3K4me1 enrichment have been associated with rearrangement fitness, whereas H3K4me1, H3K27ac, and CTCF have been associated with chromatin interactions (13, 15, 16, 69, 70). Furthermore, the translocation of chromatin domains between A and B compartments is correlated with changes in epigenetic signatures, transcription, chromatin accessibility, and intra- and interchromatin interaction frequency (71–74). All these features are intimately interconnected and play critical roles in cell development, identity, and function. Our findings that E34 controls all of these features illustrate how enhancers in the Igκ locus have coopted these mechanisms to regulate specific Vκ gene rearrangement. To our knowledge, these findings are the first demonstration that proper chromatin architecture and ensuing recombination within a locus are essential for an effective immune response.

Four enhancers spread across the V_H gene portion of the Igh locus were recently characterized by one of us (A.J.F.), together with colleagues from the Kenter laboratory, based on the same characteristics that we used to identify the Igκ enhancers described above (75). We deleted one of these enhancers (E_VH1) in mice. Similar to E34 and E88 deletion, E_VH1^−/− mice show defects in rearrangement of a large area of V_H genes, especially nearby E_VH1 (Fig. 1D). Interestingly, B1a B cells specific for phosphatidylcholine (PtC) were reduced in E_VH1^−/− mice. PtC is exposed in senescent cells and is also present in some pathogenic bacteria, including Brucella melitensis and Pseudomonas aeruginosa (76, 77). B1a B cells specific for PtC frequently use the IGHV11-2, which was reduced ∼2-fold in E_VH1^−/− mice, which may explain the reduction in splenic B1a B cells (78, 79).

Virtual 4C studies reveal the presence of an enhancer network coordinated by E_VH1 (75). A viewpoint at E_VH1 shows interactions with E_VH2, E_VH3, and E_VH4 as well as with Eu, and deletion of E_VH1 eliminates interactions with all of these other enhancers. With a viewpoint at Eu, wild-type mice show strong interactions at E_VH1. Deletion of E_VH1 eliminates the interaction of Eu with E_VH1, but now a new interaction can be seen at E_VH2, and the weaker interaction at E_VH3 is maintained. These observations were confirmed using fluorescence in situ hybridization analyses. This enhancer network likely instructs the chromatin topology of the Igh locus.

Unlike the data from the E34 and E88 studies, relative chromatin interaction frequency from the Eu enhancer, which is located near the D_H-J_H genes, does not correlate with relative rearrangement frequency (E_VH1^−/− over wild-type V gene rearrangement) (75). In contrast, this correlation is very clear in the E88 and E34 studies (26, 59). Other studies also show a correlation between TF binding and enrichment of H3K4me1, an enhancer-associated epigenetic modification near the Vκ genes, with their rearrangement frequency (13, 16). There is also a correlation between active histone marks and rearrangement frequency for many V_H genes. However, in contrast, studies in the Igh locus show a correlation between rearrangement frequency and CTCF binding near proximal V_H genes, and deletion of some of those CTCF sites shows their requirement for proximal V_H gene rearrangement (14, 15, 80, 81). In sum, this suggests that the function of the enhancer network on the Igh locus differs in some ways from that in the Igκ locus.

A defining characteristic of some enhancers is their cell type specific activity (30, 82). Enhancers in the Ig loci can ensure the proper timing for rearrangement during development by recruiting lineage-specific TFs. Pioneer TFs, such as EBF, have an important role because they can bind heterochromatin and drive the recruitment of histone acetyltransferases, such as p300, and methyltransferases that eventually allow recruitment chromatin remodeler complexes such as BRG1 (30, 45, 82, 83). EBF, p300, and Brg1 bind to E34, E88, and E_VH1. E2A is another key TF bound to these three enhancers. In contrast, Pax5 enrichment is much higher in E34 than in E88 or E_VH1, which might explain some developmental differences in activation between these enhancers. E88 and E_VH1 are more active in pro-B cells compared with E34, which is more active in pre-B cells. All of these TFs are associated with B cell–specific enhancer function, recruitment of chromatin remodeling complexes, chromatin interactions, and the establishment and maintenance of the 3D genome structure (82, 84–88). Collectively, this allows the binding of coactivators, such as Mediator. Finally, these processes, coupled with the recruitment of RNA polymerase II, eventually activate transcription and chromatin accessibility.

Enhancers located in the V gene region of the Ig loci serve a variety of functions. First, they help to determine the proper chromatin compartmentalization and topology of the V genes in a large region surrounding them, which is likely delimited by loop domains or subTADs. Deletion of any of these three enhancers leads to significant changes in the subTAD structure and a decrease in long-range interactions emanating from the region of the deleted enhancer. Second, they influence the epigenetic landscape of the surrounding area, including the enrichment of activation-associated epigenetic marks and the binding of other factors. Third, they open the chromatin over the V gene RSS, making it available for RAG-mediated cleavage. Therefore, the deletion of any of these enhancers results in a similar pattern of severe defects in the rearrangement of nearby genes and a more modest decrease in a larger area, often demarcated by subTAD boundaries (Fig. 1E, 1F). These multiple layers of control ensure proper V gene rearrangement, guaranteeing the appropriate representation of specific V genes in the BCR and Ab repertoires.

Loop extrusion and compartmentalization are the two major mechanisms of chromosome organization (66, 68, 89–95). TADs and subTADs are formed by cohesin-dependent loop extrusion and are typically, but not always, demarcated by CTCF bound sites at the boundaries (68, 89, 91, 94) (Fig. 2A). Cohesin complexes are loaded onto the DNA fiber by NIPBL to initiate loop extrusion (89, 96, 97). NIPBL is usually present at enhancer and promoter regions throughout the genome (32, 98). Once loaded into the chromatin fiber, cohesin complexes extrude DNA until they reach a boundary element, typically CTCF-enriched sites that are usually in a convergent orientation. Compartment and compartmental domains are created by a process called LLPS, which depends on the multivalent interactions of factors associated with chromatin and histone modifications specific to each type of compartment (66, 99, 100) (Fig. 2B). On one hand, chromatin with repressive epigenetic signatures tends to cluster, driven by the HP1 proteins and the Polycomb complex (72, 95, 100–102). On the other hand, the clustering of active chromatin is partially mediated by IDRs in proteins such as BRD4, Mediator, RNA polymerase II, and a variety of TFs (37–41, 44–46, 103, 104). Active transcription by RNA polymerase II and the produced RNA can also contribute to this process (39, 41, 46, 105–108).

In the Igh locus, proximity between the V_H and D_HJ_H regions is thought to be achieved through loop extrusion, likely aided by the enhancer network (4, 18, 75, 80, 81, 109–112). Loop extrusion drives V_H to D_HJ_H rearrangement by a process that involves RAG-mediated chromatin scanning for compatible convergent RSSs between the D_HJ_H segment and the V_H genes (Fig. 2C). As discussed by Zhang et al. (4), the loop extrusion mechanism that assists RAG scanning does not necessarily need to begin at the recombination center over the D_H-J_H genes, but it can initiate in the V_H gene region. Perhaps, the enhancers in this area, such as E_VH1, serve as cohesin-loading elements, through NIPBL, for loop extrusion initiation favoring rearrangement of the V_H genes nearby. The distinct configuration of the Igh locus, where all the V_H gene RSSs are in convergent orientation to the D_HJ_H RSSs, facilitates RAG scanning–mediated rearrangement involving deletion of the intervening DNA (18, 80, 81, 109, 112). Furthermore, the inversion of a part or all of the Igh locus was shown to inhibit the rearrangement of the V_H genes present in these inverted DNA segments, indicating that correct RSS directionality is a requirement for a RAG scanning–mediated rearrangement (111, 112). In contrast, Vκ-Jκ rearrangement involves either deletion or inversion of the intervening DNA sequences due to the fact that Vκ genes are positioned in either forward or reverse orientations with respect to the Jκ genes. This renders loop extrusion–dependent, RAG-mediated chromatin scanning insufficient to drive Vκ to Jκ rearrangement, raising the question whether Igκ locus rearrangement fundamentally differs from that of the Igh locus.

Our current and previous data suggest that enhancers instruct Vκ-Jκ rearrangement by bringing the Vκ genes into close physical proximity to the Jκ genes (26, 52, 59). These data also indicate that the superenhancer (formed by the iEκ, 3′Eκ, and Ed enhancers) located near the Jκ genes is in direct physical contact with other enhancers, including E34 and E88, and with enhancer-associated epigenetic marks across the Vκ gene region (26, 52, 59, 113, 114). These enhancer-associated epigenetic marks are frequently found on the RSSs, gene body, and promoters of the Vκ genes, likely facilitating the linking of the Jκ gene RSS with the Vκ gene RSS. Furthermore, enhancers in the Vκ gene region of the Igκ locus regulate the compartmentalization of the chromatin domain by controlling the epigenetic status of the domain (59). This agrees with the observations that compartmental domains correlate directly with chromatin features (66–68, 115). A number of studies suggest that enhancer–enhancer and enhancer–promoter interactions are mediated by LLPS (29, 36–42). Chromatin domains with similar epigenetic signatures tend to cluster in 3D space (66–68, 115). Moreover, recent studies revealed that nuclear condensates act as mechano-active chromatin to draw paired genomic regions that are separated by vast genomic distances together while mechanically opposing nontargeted regions located in the vicinity to merge into the condensate (103). In a similar fashion, we propose that the Igκ superenhancer pulls the Jκ region and the E34 or E88 enhancers and loop domains into close physical proximity in a shared nuclear condensate (Fig. 2D).

Furthermore, recent studies in cell lines by the Alt laboratory (112), or in mice by the Busslinger laboratory (116), support the model that the Igh and Igκ loci use different chromatin folding principles to achieve V gene recombination. Their findings demonstrate that the structure of the Igκ locus and Vκ-Jκ recombination patterns remain mostly unaffected by changes in Wapl levels. Wapl is responsible for unloading the cohesin complex from the chromatin, which in turn impacts its resident time. Thus, changes in Wapl dynamics directly affect loop extrusion. In contrast with the lack of effect of Wapl levels on Igk rearrangement, the Igh locus chromatin folding and V_H to D_H-J_H recombination were highly susceptible to changes in Wapl expression (111, 112).

Studies using acute protein depletion and high-resolution chromatin conformation capture approaches reveal that most short-range or midrange enhancer–promoter interactions are largely cohesin independent, with the caveat that cohesin-dependent loops created before the acute depletion may be maintained after the short-term depletion (117–119). However, some enhancer–promoter interactions that rely on cohesin were also found in these studies, primarily long-range contacts (120, 121). Furthermore, depletion of cohesin or NIPBL caused superenhancers to colocalize, generating many de novo interactions (68, 89). This observation suggests that the disruption of loop extrusion may facilitate enhancer–enhancer interactions located in different TAD, subTADs, or CTCF loops. The lack of significant changes in Vκ-Jκ rearrangement in cell lines with low levels of Wapl and pre-B cells with high levels of Wapl suggest that enhancer interactions and the enhancer network in the Igκ locus are predominantly cohesin independent, reinforcing the idea that the Igκ and Igh loci use different chromatin folding principles to bring the V genes to the recombination centers to facilitate V(D)J recombination (111, 112).

Although the loop extrusion–mediated RAG-scanning mechanism elegantly explains the rearrangement process in the Igh locus, the orientation of the Vκ genes in the Igκ locus makes this mechanism insufficient to facilitate Vκ-to-Jκ rearrangement (4, 112, 116). Mechanistic principles of enhancer-mediated interactions and chromatin compartmentalization could explain how Vκ gene segments’ inversional rearrangement is facilitated. Unlike loop extrusion–mediated V_H to D_HJ_H proximity, the juxtaposition between the Vκ and Jκ gene segments created by the enhancers’ physicochemical properties is not bound by directionality. This would allow the chromatin fiber in the Vκ gene region to engage the RAG complexes over the Jκ genes in both orientations (Fig. 2E). This orientation-independent interaction would facilitate the rearrangement of the Vκ genes in forward or reverse orientations.

Enhancers in the V gene region of the Ig loci add a critical layer of control to the formation of the BCR repertoire by V(D)J recombination and likely the TCR repertoire as well. To date, germline deletion of these elements, two in the Igκ locus and one in the Igh locus, show a remarkably similar phenotype in that V genes in the vicinity of the deleted enhancer are significantly impaired in rearrangement. These deletions also revealed that these enhancers are involved in dominant long-range chromatin interactions with a few other enhancers in the V gene–containing part of the locus, along with the traditionally defined enhancers at the C region end of the loci. In this way, they shape the 3D structure of their cognate Ig locus. It remains to be determined whether other enhancers within the V gene–containing part of the loci regulate the 3D chromatin structure and/or other specific V gene rearrangement to generate proper and specific Ab responses to additional evolutionarily conserved microbial epitopes and autoantigens.

A diverse repertoire is critical for fighting harmful pathogens and eliminating cancerous cells, among others. However, in the arms race between the host and pathogens, the immune system must be prepared for an overwhelming and forceful attack from pathogens in order for the host to survive those critical early stages of infection. Effectively including V gene segments in Abs that target specific conserved epitopes in pathogens confers an advantage to the host. There are several examples of V gene germline-encoded specificities for pathogen or virulent factor-associated epitopes (122, 123). However, without a functional level of these V gene segments in the repertoire, this protective characteristic would be ineffective. Enhancers provide appropriate functionality to ensure that these V genes are represented in the repertoire at optimal levels that enable early responses to pathogens.

The authors have no financial conflicts of interest.