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.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Germline deletion of Igh 3' regulatory region elements hs 5, 6, 7 (hs5-7) affects B cell-specific regulation, rearrangement, and insulation of the Igh locus.

Regulatory elements located within an ∼28-kb region 3' of the Igh gene cluster (3' regulatory region) are required for class switch recombination and for high levels of IgH expression in plasma cells. We previously defined novel DNase I hypersensitive sites (hs) 5, 6, 7 immediately downstream of this region. The hs 5-7 region (hs5-7) contains a high density of binding sites for CCCTC-binding factor (CTCF), a zinc finger protein associated with mammalian insulator activity, and is an anchor for interactions with CTCF sites flanking the D(H) region. To test the function of hs5-7, we generated mice with an 8-kb deletion encompassing all three hs elements. B cells from hs5-7 knockout (KO) (hs5-7KO) mice showed a modest increase in expression of the nearest downstream gene. In addition, Igh alleles in hs5-7KO mice were in a less contracted configuration compared with wild-type Igh alleles and showed a 2-fold increase in the usage of proximal V(H)7183 gene families. Hs5-7KO mice were essentially indistinguishable from wild-type mice in B cell development, allelic regulation, class switch recombination, and chromosomal looping. We conclude that hs5-7, a high-density CTCF-binding region at the 3' end of the Igh locus, impacts usage of V(H) regions as far as 500 kb away.

Epigenetic and 3-dimensional regulation of V(D)J rearrangement of immunoglobulin genes.

V(D)J recombination is a crucial component of the adaptive immune response, allowing for the production of a diverse antigen receptor repertoire (Ig and TCR). This review will focus on how epigenetic regulation and 3-dimensional (3D) interactions may control V(D)J recombination at Ig loci. The interplay between transcription factors and post-translational modifications at the Igh, Igκ, and Igλ loci will be highlighted. Furthermore, we propose that the spatial organization and epigenetic boundaries of each Ig loci before and during V(D)J recombination may be influenced in part by the CTCF/cohesin complex. Taken together, the many epigenetic and 3D layers of control ensure that Ig loci are only rearranged at appropriate stages of B cell development.

CCCTC-binding factor (CTCF) and cohesin influence the genomic architecture of the Igh locus and antisense transcription in pro-B cells.

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V(H) genes to be brought in proximity with D(H)-J(H) segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the V(H) region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5' of DFL16 and the 3' regulatory region, and also the intronic enhancer (Eµ), creating a D(H)-J(H)-Eµ-C(H) domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D(H) region and parts of the V(H) locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.

Compound haploinsufficiencies of Ebf1 and Runx1 genes impede B cell lineage progression.

Early B cell factor (EBF)1 is essential for B lineage specification. Previously, we demonstrated the synergistic activation of Cd79a (mb-1) genes by EBF1 and its functional partner, RUNX1. Here, we identified consequences of Ebf1 haploinsufficiency together with haploinsufficiency of Runx1 genes in mice. Although numbers of "committed" pro-B cells were maintained in Ebf1(+/-)Runx1(+/-) (ER(het)) mice, activation of B cell-specific gene transcription was depressed in these cells. Expression of genes encoding Aiolos, kappa0 sterile transcripts, CD2 and CD25 were reduced and delayed in ER(het) pro-B cells, whereas surface expression of BP-1 was increased on late pro-B cells in ER(het) mice. Late pre-B and immature and mature B cells were decreased in the bone marrow of Ebf1(+/-) (E(het)) mice and were nearly absent in ER(het) mice. Although we did not observe significant effects of haploinsuficiencies on IgH or Igkappa rearrangements, a relative lack of Iglambda rearrangements was detected in E(het) and ER(het) pre-B cells. Together, these observations suggest that B cell lineage progression is impaired at multiple stages in the bone marrow of E(het) and ER(het) mice. Furthermore, enforced expression of EBF1 and RUNX1 in terminally differentiated plasmacytoma cells activated multiple early B cell-specific genes synergistically. Collectively, these studies illuminate the effects of reduced Ebf1 dosage and the compounding effects of reduced Runx1 dosage. Our data confirm and extend the importance of EBF1 in regulating target genes and Ig gene rearrangements necessary for B cell lineage specification, developmental progression, and homeostasis.

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.

The epigenetic profile of Ig genes is dynamically regulated during B cell differentiation and is modulated by pre-B cell receptor signaling.

Ag receptor loci poised for V(D)J rearrangement undergo germline transcription (GT) of unrearranged genes, and the accessible gene segments are associated with posttranslational modifications (PTM) on histones. In this study, we performed a comprehensive analysis of the dynamic changes of four PTM throughout B and T cell differentiation in freshly isolated ex vivo cells. Methylation of lysines 4 and 79 of histone H3, and acetylation of H3, demonstrated stage and lineage specificity, and were most pronounced at the J segments of loci poised for, or undergoing, rearrangement, except for dimethylation of H3K4, which was more equally distributed on V, D, and J genes. Focusing on the IgL loci, we demonstrated there are no active PTM in the absence of pre-BCR signaling. The kappa locus GT and PTM on Jkappa genes are rapidly induced following pre-BCR signaling in large pre-B cells. In contrast, the lambda locus shows greatly delayed onset of GT and PTM, which do not reach high levels until the immature B cell compartment, the stage at which receptor editing is initiated. Analysis of MiEkappa(-/-) mice shows that this enhancer plays a key role in inducing not only GT, but PTM. Using an inducible pre-B cell line, we demonstrate that active PTM on Jkappa genes occur after GT is initiated, indicating that histone PTM do not make the Jkappa region accessible, but conversely, GT may play a role in adding PTM. Our data indicate that the epigenetic profile of IgL genes is dramatically modulated by pre-BCR signaling and B cell differentiation status.

Cutting edge: developmental stage-specific recruitment of cohesin to CTCF sites throughout immunoglobulin loci during B lymphocyte development.

Contraction of the large Igh and Igkappa loci brings all V genes, spanning >2.5 Mb in each locus, in proximity to DJ(H) or J(kappa) genes. CCCTC-binding factor (CTCF) is a transcription factor that regulates gene expression by long-range chromosomal looping. We therefore hypothesized that CTCF may be crucial for the contraction of the Ig loci, but no CTCF sites have been described in any V loci. Using ChIP-chip, we demonstrated many CTCF sites in the V(H) and V(kappa) regions. However, CTCF enrichment in the Igh locus, but not the Igkappa locus, was largely unchanged throughout differentiation, suggesting that CTCF binding alone cannot be responsible for stage-specific looping. Because cohesin can colocalize with CTCF, we performed chromatin immunoprecipitation for the cohesin subunit Rad21 and found lineage and stage-specific Rad21 recruitment to CTCF in all Ig loci. The differential binding of cohesin to CTCF sites may promote multiple loop formation and thus effective V(D)J recombination.