Locus architecture and RAG scanning determine antibody diversity.

Diverse mammalian antibody repertoires are produced via distant genomic contacts involving immunoglobulin Igh variable (V), diversity (D), and joining (J) gene segments and result in V(D)J recombination. How such interactions determine V gene usage remains unclear. The recombination-activating gene (RAG) chromatin scanning model posits that RAG recombinase bound to the recombination center (RC) linearly tracks along chromatin by means of cohesin-mediated loop extrusion; a proposition supported by cohesin depletion studies. A mechanistic role for chromatin loop extrusion has also been implicated for Igh locus contraction. In this opinion, we provide perspective on how loop extrusion interfaces with the 3D conformation of the Igh locus and newly identified enhancers that regionally regulate VH gene usage during V(D)J recombination, shaping the preselected repertoire.

Tonsil organoids: peering down the throat of human immunity.

Animal studies and explant cultures of human lymphoid tissues do not reliably model human vaccine responses. A remarkable strategy for reassociation of human tonsillar cells in ex vivo culture leads to organoid formation and provides an exciting new tool to probe human humoral immune responses to infection.

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.

53BP1 Contributes to Igh Locus Chromatin Topology during Class Switch Recombination.

In B lymphocytes, Ig class switch recombination (CSR) is induced by activation-induced cytidine deaminase, which initiates a cascade of events leading to DNA double-strand break formation in switch (S) regions. Resolution of DNA double-strand breaks proceeds through formation of S-S synaptic complexes. S-S synapsis is mediated by a chromatin loop that spans the C region domain of the Igh locus. S-S junctions are joined via a nonhomologous end joining DNA repair process. CSR occurs via an intrachromosomal looping out and deletion mechanism that is 53BP1 dependent. However, the mechanism by which 53BP1 facilitates deletional CSR and inhibits inversional switching events remains unknown. We report a novel architectural role for 53BP1 in Igh chromatin looping in mouse B cells. Long-range interactions between the Eµ and 3'Eα enhancers are significantly diminished in the absence of 53BP1. In contrast, germline transcript promoter:3'Eα looping interactions are unaffected by 53BP1 deficiency. Furthermore, 53BP1 chromatin occupancy at sites in the Igh locus is B cell specific, is correlated with histone H4 lysine 20 marks, and is subject to chromatin spreading. Thus, 53BP1 is required for three-dimensional organization of the Igh locus and provides a plausible explanation for the link with 53BP1 enforcement of deletional CSR.

Computational construction of 3D chromatin ensembles and prediction of functional interactions of alpha-globin locus from 5C data.

Conformation capture technologies measure frequencies of interactions between chromatin regions. However, understanding gene-regulation require knowledge of detailed spatial structures of heterogeneous chromatin in cells. Here we describe the nC-SAC (n-Constrained-Self Avoiding Chromatin) method that transforms experimental interaction frequencies into 3D ensembles of chromatin chains. nC-SAC first distinguishes specific from non-specific interaction frequencies, then generates 3D chromatin ensembles using identified specific interactions as spatial constraints. Application to α-globin locus shows that these constraints (∼20%) drive the formation of ∼99% all experimentally captured interactions, in which ∼30% additional to the imposed constraints is found to be specific. Many novel specific spatial contacts not captured by experiments are also predicted. A subset, of which independent ChIA-PET data are available, is validated to be RNAPII-, CTCF-, and RAD21-mediated. Their positioning in the architectural context of imposed specific interactions from nC-SAC is highly important. Our results also suggest the presence of a many-body structural unit involving α-globin gene, its enhancers, and POL3RK gene for regulating the expression of α-globin in silent cells.

Constraints contributed by chromatin looping limit recombination targeting during Ig class switch recombination.

Engagement of promoters with distal elements in long-range looping interactions has been implicated in regulation of Ig class switch recombination (CSR). The principles determining the spatial and regulatory relationships among Igh transcriptional elements remain poorly defined. We examined the chromosome conformation of C region (CH) loci that are targeted for CSR in a cytokine-dependent fashion in mature B lymphocytes. Germline transcription (GLT) of the γ1 and ε CH loci is controlled by two transcription factors, IL-4-inducible STAT6 and LPS-activated NF-κB. We showed that although STAT6 deficiency triggered loss of GLT, deletion of NF-κB p50 abolished both GLT and γ1 locus:enhancer looping. Thus, chromatin looping between CH loci and Igh enhancers is independent of GLT production and STAT6, whereas the establishment and maintenance of these chromatin contacts requires NF-κB p50. Comparative analysis of the endogenous γ1 locus and a knock-in heterologous promoter in mice identified the promoter per se as the interactive looping element and showed that transcription elongation is dispensable for promoter/enhancer interactions. Interposition of the LPS-responsive heterologous promoter between the LPS-inducible γ3 and γ2b loci altered GLT expression and essentially abolished direct IgG2b switching while maintaining a sequential µâ†’γ3→γ2b format. Our study provides evidence that promoter/enhancer looping interactions can introduce negative constraints on distal promoters and affect their ability to engage in germline transcription and determine CSR targeting.

AID targeting is dependent on RNA polymerase II pausing.

Activation induced deaminase (AID) is globally targeted to immunoglobulin loci, preferentially focused to switch (S) regions and variable (V) regions, and prone to attack hotspot motifs. Nevertheless, AID deamination is not exclusive to Ig loci and the rules regulating AID targeting remain unclear. Transcription is critically required for class switch recombination and somatic hypermutation. Here, I consider the unique features associated with S region transcription leading to RNA polymerase II pausing, that in turn promote the introduction of activating chromatin remodeling, histone modifications and recruitment of AID to targeted S regions. These findings allow for a better understanding of the interplay between transcription, AID targeting and mistargeting to Ig and non-Ig loci.

Spatial confinement is a major determinant of the folding landscape of human chromosomes.

The global architecture of the cell nucleus and the spatial organization of chromatin play important roles in gene expression and nuclear function. Single-cell imaging and chromosome conformation capture-based techniques provide a wealth of information on the spatial organization of chromosomes. However, a mechanistic model that can account for all observed scaling behaviors governing long-range chromatin interactions is missing. Here we describe a model called constrained self-avoiding chromatin (C-SAC) for studying spatial structures of chromosomes, as the available space is a key determinant of chromosome folding. We studied large ensembles of model chromatin chains with appropriate fiber diameter, persistence length and excluded volume under spatial confinement. We show that the equilibrium ensemble of randomly folded chromosomes in the confined nuclear volume gives rise to the experimentally observed higher-order architecture of human chromosomes, including average scaling properties of mean-square spatial distance, end-to-end distance, contact probability and their chromosome-to-chromosome variabilities. Our results indicate that the overall structure of a human chromosome is dictated by the spatial confinement of the nuclear space, which may undergo significant tissue- and developmental stage-specific size changes.

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.

AID-dependent histone acetylation is detected in immunoglobulin S regions.

Class switch recombination (CSR) is regulated by the expression of activation-induced deaminase (AID) and germline transcripts (GLTs). AID-dependent double-strand breaks (DSBs) are introduced into switch (S) regions and stimulate CSR. Although histone acetylation (Ac) has been well documented in transcription regulation, its role in DNA damage repair remains largely unexplored. The 1B4.B6 B cell line and normal splenic B cells were activated to undergo CSR and analyzed for histone Ac by chromatin immunoprecipitation (ChIP). A detailed study of the Igamma3-Sgamma3-Cgamma3 locus demonstrated that acetylated histones are focused to the Igamma3 exon and the Sgamma3 region but not to the intergenic areas. Histone H3 Ac is strongly correlated with GLT expression at four S regions, whereas H4 Ac was better associated with B cell activation and AID expression. To more directly examine the relationship between H4 Ac and AID, LPS-activated AID KO and WT B cells were analyzed and express comparable levels of GLTs. In AID-deficient B cells, both histones H3 and H4 are reduced where H4 is more severely affected as compared with WT cells. Our findings raise the intriguing possibility that histone H4 Ac at S regions is a marker for chromatin modifications associated with DSB repair during CSR.

Switch region identity plays an important role in Ig class switch recombination.

Ig class switch recombination (CSR) is regulated through long-range intrachromosomal interactions between germline transcript promoters and enhancers to initiate transcription and create chromatin accessible to activation-induced deaminase attack. CSR occurs between switch (S) regions that flank Cmu and downstream C(H) regions and functions via an intrachromosomal deletional event between the donor Smicro region and a downstream S region. It is unclear to what extent S region primary sequence influences differential targeting of CSR to specific isotypes. We address this issue in this study by generating mutant mice in which the endogenous Sgamma3 region was replaced with size-matched Sgamma1 sequence. B cell activation conditions are established that support robust gamma3 and gamma1 germline transcript expression and stimulate IgG1 switching but suppress IgG3 CSR. We found that the Sgamma1 replacement allele engages in micro-->gamma3 CSR, whereas the intact allele is repressed. We conclude that S region identity makes a significant contribution to CSR. We propose that the Sgamma1 region is selectively targeted for CSR following the induction of an isotype-specific factor that targets the S region and recruits CSR machinery.

Igh Locus Polymorphism May Dictate Topological Chromatin Conformation and V Gene Usage in the Ig Repertoire.

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 creates the potential for an organism to respond to large numbers of different pathogens. Here, we consider the possibility that genetic polymorphisms with alterations in a vast array of regulatory elements in the immunoglobulin heavy chain (IgH) locus lead to changes in locus topology and impact immune-repertoire formation.

Loop extrusion promotes an alternate pathway for isotype switching.

Class-switch recombination (CSR) involves replacement of the Cµ constant region with another downstream CH region. CSR is initiated by activation-induced cytidine deaminase (AID)-mediated DNA breaks that are targeted to transcriptionally active switch (S) regions. S region promoters (Prs) direct synapsis by associating with the Eµ and 3'Eα enhancers that jointly anchor a chromatin loop. We report that asymmetric loop extrusion allows 3'Eα to track along the locus and form Pr-Pr-E interactions that mediate CSR between downstream S regions, followed by switching to donor Sµ. This alternative pathway bypasses sequential switching and creates immunoglobulin (Ig)E+ B cells in the absence of IgG1 expression. Based on the analysis of diagnostic CSR products in B cell subsets, we identify a BCR-negative cell intermediate that is pivotal to efficient CSR.

Mapping of a functional recombination motif that defines isotype specificity for mu-->gamma3 switch recombination implicates NF-kappaB p50 as the isotype-specific switching factor.

Ig class switch recombination (CSR) requires expression of activation-induced deaminase (AID) and production of germline transcripts to target S regions for recombination. However, the mechanism of CSR remains unclear. Here we show that an extrachromosomal S plasmid assay is AID dependent and that a single consensus repeat is both necessary and sufficient for isotype-specific CSR. Transfected switch substrates specific for mu-->gamma3 and mu-->gamma1 are stimulated to switch with lipopolysaccharide (LPS) alone or LPS and interleukin-4, respectively. An Sgamma3/Sgamma1 substrate containing only three Sgamma3-associated nucleotides reconstituted LPS responsiveness and permitted mapping of a functional recombination motif specific for mu-->gamma3 CSR. This functional recombination motif colocalized with a binding site for NF-kappaB p50, and p50 binding to this site was previously established. We show a p50 requirement for plasmid-based mu-->gamma3 CSR using p50-deficient B cells. Switch junctions from p50-deficient B cells showed decreased lengths of microhomology between Smu and Sgamma3 relative to wild-type cells, indicating a function for p50 in the mechanics of CSR. We note a striking parallel between the affects of p50 and Msh2 deficiency on Smu/Sgamma3 junctions. The data suggest that p50 may be the isotype-specific factor in mu-->gamma3 CSR and epistatic with Msh2.

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.

Chicken MBD4 Regulates Immunoglobulin Diversification by Somatic Hypermutation.

Immunoglobulin (Ig) diversification occurs via somatic hypermutation (SHM) and class switch recombination (CSR), and is initiated by activation-induced deaminase (AID), which converts cytosine to uracil. Variable (V) region genes undergo SHM to create amino acid substitutions that produce antibodies with higher affinity for antigen. The conversion of cytosine to uracil in DNA promotes mutagenesis. Two distinct DNA repair mechanisms regulate uracil processing in Ig genes. The first involves base removal by the uracil DNA glycosylase (UNG), and the second detects uracil via the mismatch repair (MMR) complex. Methyl binding domain protein 4 (MBD4) is a uracil glycosylase and an intriguing candidate for involvement in somatic hypermutation because of its interaction with the MMR MutL homolog 1 (MLH1). We found that the DNA uracil glycosylase domain of MBD4 is highly conserved among mammals, birds, shark, and insects. Conservation of the human and chicken MBD4 uracil glycosylase domain structure is striking. Here we examined the function of MBD4 in chicken DT40 B cells which undergo constitutive SHM. We constructed structural variants of MBD4 DT40 cells using CRISPR/Cas9 genome editing. Disruption of the MBD4 uracil glycosylase catalytic region increased SHM frequency in IgM loss assays. We propose that MBD4 plays a role in SHM.

Class-switch recombination: after the dawn of AID.

Isotype class switching is central to the humoral immune response. The discovery that mutations in the activation-induced deaminase (AID) gene inhibit class-switch recombination, somatic hypermutation and gene conversion is a major step forward in defining the underlying mechanisms of these gene modification events. The propensity of mutations to occur at dC/dG nucleotides during somatic hypermutation and the homology between AID and cytidine deaminase has resulted in studies demonstrating that AID has the properties of a cytidine-specific mutator and also that elements of the base-excision repair pathway play a central role in class switching and hypermutation. AID is not a promiscuous mutator in the B cell, suggesting that there are specific molecular targeting mechanisms that regulate the accessibility of DNA to AID and differentially regulate class-switch recombination and somatic hypermutation. During class switching, isotype-specific targeting occurs independently of AID and provides another level of specificity to this recombination event.

MBD4 Facilitates Immunoglobulin Class Switch Recombination.

Immunoglobulin heavy chain class switch recombination (CSR) requires targeted formation of DNA double-strand breaks (DSBs) in repetitive switch region elements followed by ligation between distal breaks. The introduction of DSBs is initiated by activation-induced cytidine deaminase (AID) and requires base excision repair (BER) and mismatch repair (MMR). The BER enzyme methyl-CpG binding domain protein 4 (MBD4) has been linked to the MMR pathway through its interaction with MutL homologue 1 (MLH1). We find that when Mbd4 exons 6 to 8 are deleted in a switching B cell line, DSB formation is severely reduced and CSR frequency is impaired. Impaired CSR can be rescued by ectopic expression of Mbd4 Mbd4 deficiency yields a deficit in DNA end processing similar to that found in MutS homologue 2 (Msh2)- and Mlh1-deficient B cells. We demonstrate that microhomology-rich S-S junctions are enriched in cells in which Mbd4 is deleted. Our studies suggest that Mbd4 is a component of MMR-directed DNA end processing.

AID hits the jackpot when missing the target.

Activation induced deaminase is the single B cell specific factor mediating class switch recombination and somatic hypermutation. Numerous studies have shown that AID preferentially targets Ig substrates and also attacks non-Ig substrates to create DNA damage that contributes to lymphomagenesis. AID targeting to Ig loci is linked to transcription but the mechanism governing this process has been obscure. Here we discuss research that illustrates the connection between AID targeting to DNA substrates and transcription processes to reveal rules governing the specificity of AID attack. These observations are woven together to provide a integrated view of AID function and a surprising linkage with global regulation of gene expression.

Extremely Long-Range Chromatin Loops Link Topological Domains to Facilitate a Diverse Antibody Repertoire.

Early B cell development is characterized by large-scale Igh locus contraction prior to V(D)J recombination to facilitate a highly diverse Ig repertoire. However, an understanding of the molecular architecture that mediates locus contraction remains unclear. We have combined high-resolution chromosome conformation capture (3C) techniques with 3D DNA FISH to identify three conserved topological subdomains. Each of these topological folds encompasses a major VH gene family that become juxtaposed in pro-B cells via megabase-scale chromatin looping. The transcription factor Pax5 organizes the subdomain that spans the VHJ558 gene family. In its absence, the J558 VH genes fail to associate with the proximal VH genes, thereby providing a plausible explanation for reduced VHJ558 gene rearrangements in Pax5-deficient pro-B cells. We propose that Igh locus contraction is the cumulative effect of several independently controlled chromatin subdomains that provide the structural infrastructure to coordinate optimal antigen receptor assembly.