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

Effective Ab-mediated responses depend on a highly diverse Ab repertoire with the ability to bind a wide range of epitopes in disease-causing agents. The generation of this repertoire depends on the somatic recombination of the variable (V), diversity (D), and joining (J) genes in the Ig loci of developing B cells. It has been known for some time that individual V, D, and J gene segments rearrange at different frequencies, but the mechanisms behind this unequal V gene usage have not been well understood. However, recent work has revealed that newly described enhancers scattered throughout the V gene-containing portion of the Ig loci regulate the V gene recombination frequency in a regional manner. Deletion of three of these enhancers revealed that these elements exert many layers of control during V(D)J recombination, including long-range chromatin interactions, epigenetic milieu, chromatin accessibility, and compartmentalization.

An Igh distal enhancer modulates antigen receptor diversity by determining locus conformation.

The mouse Igh locus is organized into a developmentally regulated topologically associated domain (TAD) that is divided into subTADs. Here we identify a series of distal VH enhancers (EVHs) that collaborate to configure the locus. EVHs engage in a network of long-range interactions that interconnect the subTADs and the recombination center at the DHJH gene cluster. Deletion of EVH1 reduces V gene rearrangement in its vicinity and alters discrete chromatin loops and higher order locus conformation. Reduction in the rearrangement of the VH11 gene used in anti-PtC responses is a likely cause of the observed reduced splenic B1 B cell compartment. EVH1 appears to block long-range loop extrusion that in turn contributes to locus contraction and determines the proximity of distant VH genes to the recombination center. EVH1 is a critical architectural and regulatory element that coordinates chromatin conformational states that favor V(D)J rearrangement.

Enhancer-instructed epigenetic landscape and chromatin compartmentalization dictate a primary antibody repertoire protective against specific bacterial pathogens.

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.

Enhancers as regulators of antigen receptor loci three-dimensional chromatin structure.

Enhancers are defined as regulatory elements that control transcription in a cell-type and developmental stage-specific manner. They achieve this by physically interacting with their cognate gene promoters. Significantly, these interactions can occur through long genomic distances since enhancers may not be near their cognate promoters. The optimal coordination of enhancer-regulated transcription is essential for the function and identity of the cell. Although great efforts to fully understand the principles of this type of regulation are ongoing, other potential functions of the long-range chromatin interactions (LRCIs) involving enhancers are largely unexplored. We recently uncovered a new role for enhancer elements in determining the three-dimensional (3D) structure of the immunoglobulin kappa (Igκ) light chain receptor locus suggesting a structural function for these DNA elements. This enhancer-mediated locus configuration shapes the resulting Igκ repertoire. We also propose a role for enhancers as critical components of sub-topologically associating domain (subTAD) formation and nuclear spatial localization.

CHD4 is essential for transcriptional repression and lineage progression in B lymphopoiesis.

Cell lineage specification is a tightly regulated process that is dependent on appropriate expression of lineage and developmental stage-specific transcriptional programs. Here, we show that Chromodomain Helicase DNA-binding protein 4 (CHD4), a major ATPase/helicase subunit of Nucleosome Remodeling and Deacetylase Complexes (NuRD) in lymphocytes, is essential for specification of the early B cell lineage transcriptional program. In the absence of CHD4 in B cell progenitors in vivo, development of these cells is arrested at an early pro-B-like stage that is unresponsive to IL-7 receptor signaling and unable to efficiently complete V(D)J rearrangements at Igh loci. Our studies confirm that chromatin accessibility and transcription of thousands of gene loci are controlled dynamically by CHD4 during early B cell development. Strikingly, CHD4-deficient pro-B cells express transcripts of many non-B cell lineage genes, including genes that are characteristic of other hematopoietic lineages, neuronal cells, and the CNS, lung, pancreas, and other cell types. We conclude that CHD4 inhibits inappropriate transcription in pro-B cells. Together, our data demonstrate the importance of CHD4 in establishing and maintaining an appropriate transcriptome in early B lymphopoiesis via chromatin accessibility.

New insights emerge as antibody repertoire diversification meets chromosome conformation.

Vast repertoires of unique antigen receptors are created in developing 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. This process creates the potential for an organism to respond to large numbers of different pathogens. Here, we consider the underlying molecular mechanisms that favor some V genes for recombination prior to selection of the final antigen receptor repertoire. We discuss chromatin structures that form in antigen receptor loci to permit spatial proximity among the V, D, and J gene segments and how these relate to the generation of antigen receptor diversity.

Reprogramming the antigen specificity of B cells using genome-editing technologies.

We have developed a method to introduce novel paratopes into the human antibody repertoire by modifying the immunoglobulin (Ig) genes of mature B cells directly using genome editing technologies. We used CRISPR-Cas9 in a homology directed repair strategy, to replace the heavy chain (HC) variable region in B cell lines with that from an HIV broadly neutralizing antibody (bnAb), PG9. Our strategy is designed to function in cells that have undergone VDJ recombination using any combination of variable (V), diversity (D) and joining (J) genes. The modified locus expresses PG9 HC which pairs with native light chains (LCs) resulting in the cell surface expression of HIV specific B cell receptors (BCRs). Endogenous activation-induced cytidine deaminase (AID) in engineered cells allowed for Ig class switching and generated BCR variants with improved HIV neutralizing activity. Thus, BCRs engineered in this way retain the genetic flexibility normally required for affinity maturation during adaptive immune responses. Peripheral blood derived primary B cells from three different donors were edited using this strategy. Engineered cells could bind the PG9 epitope and sequenced mRNA showed PG9 HC transcribed as several different isotypes after culture with CD40 ligand and IL-4.

A B-Cell-Specific Enhancer Orchestrates Nuclear Architecture to Generate a Diverse Antigen Receptor Repertoire.

The genome is organized into topologically associated domains (TADs) that enclose smaller subTADs. Here, we identify and characterize an enhancer that is located in the middle of the V gene region of the immunoglobulin kappa light chain (Igκ) locus that becomes active preceding the stage at which this locus undergoes V(D)J recombination. This enhancer is a hub of long-range chromatin interactions connecting subTADs in the V gene region with the recombination center at the J genes. Deletion of this element results in a highly altered long-range chromatin interaction pattern across the locus and, importantly, affects individual V gene utilization locus-wide. These results indicate the existence of an enhancer-dependent framework in the Igκ locus and further suggest that the composition of the diverse antibody repertoire is regulated in a subTAD-specific manner. This enhancer thus plays a structural role in orchestrating the proper folding of the Igκ locus in preparation for V(D)J recombination.

Epigenetic Enhancer Marks and Transcription Factor Binding Influence Vκ Gene Rearrangement in Pre-B Cells and Pro-B Cells.

To date there has not been a study directly comparing relative Igκ rearrangement frequencies obtained from genomic DNA (gDNA) and cDNA and since each approach has potential biases, this is an important issue to clarify. Here we used deep sequencing to compare the unbiased gDNA and RNA Igκ repertoire from the same pre-B cell pool. We find that ~20% of Vκ genes have rearrangement frequencies ≥2-fold up or down in RNA vs. DNA libraries, including many members of the Vκ3, Vκ4, and Vκ6 families. Regression analysis indicates Ikaros and E2A binding are associated with strong promoters. Within the pre-B cell repertoire, we observed that individual Vκ genes rearranged at very different frequencies, and also displayed very different Jκ usage. Regression analysis revealed that the greatly unequal Vκ gene rearrangement frequencies are best predicted by epigenetic marks of enhancers. In particular, the levels of newly arising H3K4me1 peaks associated with many Vκ genes in pre-B cells are most predictive of rearrangement levels. Since H3K4me1 is associated with long range chromatin interactions which are created during locus contraction, our data provides mechanistic insight into unequal rearrangement levels. Comparison of Igκ rearrangements occurring in pro-B cells and pre-B cells from the same mice reveal a pro-B cell bias toward usage of Jκ-distal Vκ genes, particularly Vκ10-96 and Vκ1-135. Regression analysis indicates that PU.1 binding is the highest predictor of Vκ gene rearrangement frequency in pro-B cells. Lastly, the repertoires of iEκ-/- pre-B cells reveal that iEκ actively influences Vκ gene usage, particularly Vκ3 family genes, overlapping with a zone of iEκ-regulated germline transcription. These represent new roles for iEκ in addition to its critical function in promoting overall Igκ rearrangement. Together, this study provides insight into many aspects of Igκ repertoire formation.

Cutting Edge: Proper Orientation of CTCF Sites in Cer Is Required for Normal Jκ-Distal and Jκ-Proximal Vκ Gene Usage.

Igκ locus contraction and Vκ gene usage are controlled by Cer, a cis-acting sequence in the Vκ-Jκ intervening region. This effect is attributed to two CTCF-binding sites within Cer that are oriented toward the Vκ gene region. However, the importance of Cer CTCF orientation in regulating VκJκ rearrangement is unknown. We used CRISPR/Cas9 editing to delete and invert Cer in murine Abl pro-B cell lines. This revealed that Cer orientation is critical because clones with either an inverted or deleted Cer element show skewing toward Jκ-proximal Vκ gene usage. However, only Cer deletion increased Jκ-proximal Vκ germline transcription, suggesting an insulating function of Cer. Lastly, circularized chromosome conformation capture interaction data show that Cer CTCF orientation regulates long-range interactions with inversion clones displaying fewer interactions with regions in the middle and distal parts of the Vκ locus and more interactions to downstream regions compared with wild-type or deletion clones.

Variable Extent of Lineage-Specificity and Developmental Stage-Specificity of Cohesin and CCCTC-Binding Factor Binding Within the Immunoglobulin and T Cell Receptor Loci.

CCCTC-binding factor (CTCF) is largely responsible for the 3D architecture of the genome, in concert with the action of cohesin, through the creation of long-range chromatin loops. Cohesin is hypothesized to be the main driver of these long-range chromatin interactions by the process of loop extrusion. Here, we performed ChIP-seq for CTCF and cohesin in two stages each of T and B cell differentiation and examined the binding pattern in all six antigen receptor (AgR) loci in these lymphocyte progenitors and in mature T and B cells, ES cells, and fibroblasts. The four large AgR loci have many bound CTCF sites, most of which are only occupied in lymphocytes, while only the CTCF sites at the end of each locus near the enhancers or J genes tend to be bound in non-lymphoid cells also. However, despite the generalized lymphocyte restriction of CTCF binding in AgR loci, the Igκ locus is the only locus that also shows significant lineage-specificity (T vs. B cells) and developmental stage-specificity (pre-B vs. pro-B) in CTCF binding. We show that cohesin binding shows greater lineage- and stage-specificity than CTCF at most AgR loci, providing more specificity to the loops. We also show that the culture of pro-B cells in IL7, a common practice to expand the number of cells before ChIP-seq, results in a CTCF-binding pattern resembling pre-B cells, as well as other epigenetic and transcriptional characteristics of pre-B cells. Analysis of the orientation of the CTCF sites show that all sites within the large V portions of the Igh and TCRß loci have the same orientation. This suggests either a lack of requirement for convergent CTCF sites creating loops, or indicates an absence of any loops between CTCF sites within the V region portion of those loci but only loops to the convergent sites at the D-J-enhancer end of each locus. The V region portions of the Igκ and TCRα/δ loci, by contrast, have CTCF sites in both orientations, providing many options for creating CTCF-mediated convergent loops throughout the loci. CTCF/cohesin loops, along with transcription factors, drives contraction of AgR loci to facilitate the creation of a diverse repertoire of antibodies and T cell receptors.

YY1 plays an essential role at all stages of B-cell differentiation.

Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor shown to be essential for pro-B-cell development. However, the role of YY1 in other B-cell populations has never been investigated. Recent bioinformatics analysis data have implicated YY1 in the germinal center (GC) B-cell transcriptional program. In accord with this prediction, we demonstrated that deletion of YY1 by Cγ1-Cre completely prevented differentiation of GC B cells and plasma cells. To determine if YY1 was also required for the differentiation of other B-cell populations, we deleted YY1 with CD19-Cre and found that all peripheral B-cell subsets, including B1 B cells, require YY1 for their differentiation. Transitional 1 (T1) B cells were the most dependent upon YY1, being sensitive to even a half-dosage of YY1 and also to short-term YY1 deletion by tamoxifen-induced Cre. We show that YY1 exerts its effects, in part, by promoting B-cell survival and proliferation. ChIP-sequencing shows that YY1 predominantly binds to promoters, and pathway analysis of the genes that bind YY1 show enrichment in ribosomal functions, mitochondrial functions such as bioenergetics, and functions related to transcription such as mRNA splicing. By RNA-sequencing analysis of differentially expressed genes, we demonstrated that YY1 normally activates genes involved in mitochondrial bioenergetics, whereas it normally down-regulates genes involved in transcription, mRNA splicing, NF-κB signaling pathways, the AP-1 transcription factor network, chromatin remodeling, cytokine signaling pathways, cell adhesion, and cell proliferation. Our results show the crucial role that YY1 plays in regulating broad general processes throughout all stages of B-cell differentiation.

A structural hierarchy mediated by multiple nuclear factors establishes IgH locus conformation.

Conformation of antigen receptor gene loci spatially juxtaposes rearranging gene segments in the appropriate cell lineage and developmental stage. We describe a three-step pathway that establishes the structure of the 2.8-Mb immunoglobulin heavy chain gene (IgH) locus in pro-B cells. Each step uses a different transcription factor and leads to increasing levels of structural organization. CTCF mediates one level of compaction that folds the locus into several 250- to 400-kb subdomains, and Pax5 further compacts the 2-Mb region that encodes variable (VH) gene segments. The 5' and 3' domains are brought together by the transcription factor YY1 to establish the configuration within which gene recombination initiates. Such stepwise mechanisms may apply more generally to establish regulatory fine structure within megabase-sized topologically associated domains.

CTCF and ncRNA Regulate the Three-Dimensional Structure of Antigen Receptor Loci to Facilitate V(D)J Recombination.

At both the immunoglobulin heavy and kappa light chain loci, there are >100 functional variable (V) genes spread over >2 Mb that must move into close proximity in 3D space to the (D)J genes to create a diverse repertoire of antibodies. Similar events take place at the T cell receptor (TCR) loci to create a wide repertoire of TCRs. In this review, we will discuss the role of CTCF in forming rosette-like structures at the antigen receptor (AgR) loci, and the varied roles it plays in alternately facilitating and repressing V(D)J rearrangements. In addition, non-coding RNAs, also known as germline transcription, can shape the 3D configuration of the Igh locus, and presumably that of the other AgR loci. At the Igh locus, this could occur by gathering the regions being transcribed in the VH locus into the same transcription factory where Iµ is being transcribed. Since the Iµ promoter, Eµ, is adjacent to the DJH rearrangement to which one V gene will ultimately rearrange, the process of germline transcription itself, prominent in the distal half of the VH locus, may play an important and direct role in locus compaction. Finally, we will discuss the impact of the transcriptional and epigenetic landscape of the Igh locus on VH gene rearrangement frequencies.

Targeted chromatin profiling reveals novel enhancers in Ig H and Ig L chain Loci.

The assembly and expression of mouse Ag receptor genes are controlled by a collection of cis-acting regulatory elements, including transcriptional promoters and enhancers. Although many powerful enhancers have been identified for Ig (Ig) and TCR (Tcr) loci, it remained unclear whether additional regulatory elements remain undiscovered. In this study, we use chromatin profiling of pro-B cells to define 38 epigenetic states in mouse Ag receptor loci, each of which reflects a distinct regulatory potential. One of these chromatin states corresponds to known transcriptional enhancers and identifies a new set of candidate elements in all three Ig loci. Four of the candidates were subjected to functional assays, and all four exhibit enhancer activity in B but not in T lineage cells. The new regulatory elements identified by focused chromatin profiling most likely have important functions in the creation, refinement, and expression of Ig repertoires.

Flexible ordering of antibody class switch and V(D)J joining during B-cell ontogeny.

V(D)J joining is mediated by RAG recombinase during early B-lymphocyte development in the bone marrow (BM). Activation-induced deaminase initiates isotype switching in mature B cells of secondary lymphoid structures. Previous studies questioned the strict ontological partitioning of these processes. We show that pro-B cells undergo robust switching to a subset of immunoglobulin H (IgH) isotypes. Chromatin studies reveal that in pro-B cells, the spatial organization of the Igh locus may restrict switching to this subset of isotypes. We demonstrate that in the BM, V(D)J joining and switching are interchangeably inducible, providing an explanation for the hyper-IgE phenotype of Omenn syndrome.

Unifying model for molecular determinants of the preselection Vß repertoire.

The primary antigen receptor repertoire is sculpted by the process of V(D)J recombination, which must strike a balance between diversification and favoring gene segments with specialized functions. The precise determinants of how often gene segments are chosen to complete variable region coding exons remain elusive. We quantified Vß use in the preselection Tcrb repertoire and report relative contributions of 13 distinct features that may shape their recombination efficiencies, including transcription, chromatin environment, spatial proximity to their DßJß targets, and predicted quality of recombination signal sequences (RSSs). We show that, in contrast to functional Vß gene segments, all pseudo-Vß segments are sequestered in transcriptionally silent chromatin, which effectively suppresses wasteful recombination. Importantly, computational analyses provide a unifying model, revealing a minimum set of five parameters that are predictive of Vß use, dominated by chromatin modifications associated with transcription, but largely independent of precise spatial proximity to DßJß clusters. This learned model-building strategy may be useful in predicting the relative contributions of epigenetic, spatial, and RSS features in shaping preselection V repertoires at other antigen receptor loci. Ultimately, such models may also predict how designed or naturally occurring alterations of these loci perturb the preselection use of variable gene segments.

Deep sequencing of the murine IgH repertoire reveals complex regulation of nonrandom V gene rearrangement frequencies.

A diverse Ab repertoire is formed through the rearrangement of V, D, and J segments at the IgH (Igh) loci. The C57BL/6 murine Igh locus has >100 functional VH gene segments that can recombine to a rearranged DJH. Although the nonrandom usage of VH genes is well documented, it is not clear what elements determine recombination frequency. To answer this question, we conducted deep sequencing of 5'-RACE products of the Igh repertoire in pro-B cells, amplified in an unbiased manner. Chromatin immunoprecipitation-sequencing results for several histone modifications and RNA polymerase II binding, RNA-sequencing for sense and antisense noncoding germline transcripts, and proximity to CCCTC-binding factor (CTCF) and Rad21 sites were compared with the usage of individual V genes. Computational analyses assessed the relative importance of these various accessibility elements. These elements divide the Igh locus into four epigenetically and transcriptionally distinct domains, and our computational analyses reveal different regulatory mechanisms for each region. Proximal V genes are relatively devoid of active histone marks and noncoding RNA in general, but having a CTCF site near their recombination signal sequence is critical, suggesting that being positioned near the base of the chromatin loops is important for rearrangement. In contrast, distal V genes have higher levels of histone marks and noncoding RNA, which may compensate for their poorer recombination signal sequences and for being distant from CTCF sites. Thus, the Igh locus has evolved a complex system for the regulation of V(D)J rearrangement that is different for each of the four domains that comprise this locus.

Tcra gene recombination is supported by a Tcra enhancer- and CTCF-dependent chromatin hub.

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4(-)CD8(-) double-negative thymocytes and Tcra gene segments in CD4(+)CD8(+) double-positive thymocytes. Initial V(α)-to-J(α) recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E(α)) interacts directly with V(α) and J(α) gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E(α) promotes interactions between these V(α) and J(α) segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E(α) and to many locus promoters, biases E(α) to interact with these promoters, and is required for efficient V(α)-J(α) recombination. Our data indicate that E(α) and CTCF cooperate to create a developmentally regulated chromatin hub that supports V(α)-J(α) synapsis and recombination.

Noncoding transcription within the Igh distal V(H) region at PAIR elements affects the 3D structure of the Igh locus in pro-B cells.

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eµ, the start site of Iµ germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eµ-independent. YY1(-/-) pro-B cells are greatly impaired in distal V(H) gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eµ interactions. ChIP-seq shows high level YY1 binding only at Eµ, but low levels near some antisense promoters. PAIR-Eµ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V(H) genes close to the DJ(H) rearrangement which is adjacent to Eµ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V(H) genes to undergo efficient rearrangement.