Three dimensional architecture of the Igh locus facilitates class switch recombination

Abstract

Immunoglobulin (Ig) class switch recombination (CSR) is responsible for diversification of antibody effector function during an immune response. This region specific recombination event, between repetitive switch (S) DNA elements, is unique to B lymphocytes and is induced by activation induced deaminase (AID). CSR is critically dependent on transcription of noncoding RNAs across S regions. However, mechanistic insight regarding this process remained unclear. New studies indicate that long range intra-chromosomal interactions among Igh transcriptional elements organize the formation of the S/S synaptosome, as prerequisite for CSR. This three dimensional chromatin architecture simultaneously brings promoters and enhancers into close proximity to facilitate transcription. Here, we recount how transcription across S DNA promotes accumulation of RNA polymerase II leading to the introduction of activating chromatin modifications and hyperaccessible chromatin that is amenable to AID activity.

Introduction

B lymphocytes undergo a set of three programmed gene alterations that diversify immunoglobulin (Ig) antigen binding capability or effector function. Antigen binding specificity is encoded by the combinatorial joining of V_H, D_H and J_H or V_L and J_L gene segments on the heavy (H) and light (L) chain, respectively. H and L chain assembly via V-D-J joining occurs in an orderly stepwise process during early B cell development in the bone marrow. Somatic hypermutation (SHM), a mutational process, is focused to rearranged V_HDJ_H and V_LJ_L region genes and leads to increased affinity of antibody binding to antigen, in germinal center B cells. Class switch recombination (CSR) provides for diversification of C_H effector function while maintaining the original V(D)J rearrangement in mature B cells. IgH effector function is encoded by eight constant (C_H) region genes (μ, δ, γ3, γ1, γ2b, γ2a, ε and α) that span a 220 kb genomic region located near the right telomere of chromosome 12. CSR involves an intra-chromosomal deletional rearrangement that focuses on repetitive switch (S) DNA located upstream of each C_H gene (with the exception of Cδ) (reviewed in [1,2]).

Germline or noncoding transcript (GLT) promoters associated with each of the paired S-C_H regions target CSR to specific S regions by selective transcriptional activation (reviewed in [1,2]). Germline transcripts (GLTs) are so termed because they lack an open reading frame and are not translated [3]. Combinations of cytokines and B cell activators induce transcription from specific GLT promoters [4]. Gene targeting experiments indicate the critical requirement for transcription and transcriptional elements to enable CSR (reviewed in [1,5]). Activation induced deaminase (AID) activity deaminates dC residues in transcribed S regions and is essential for CSR and SHM [6,7]. The mechanism by which AID induced DNA lesions are repaired and culminate in SHM or CSR have been extensively reviewed elsewhere [1,3,8–14].

S regions separated by as much as 150 kb can be targeted for recombination. This distance represents a topological challenge to formation of the S/S synaptosome, a requirement for CSR. However, the mechanism by which two distant S regions targeted for recombination are brought into close proximity has been unclear. Recent studies indicate that regulatory elements act over large genomic distances to bring widely separated elements into close spatial proximity with their target genes or other functional elements [15–21]. The chromatin conformation capture (3C) assay was developed to determine whether distant promoters and enhancers come into contact by long range looping or whether they serve as entry sites or nucleation points for factors that ultimately communicate with the gene [22]. Here, we explore recent findings regarding the influence of GLT transcription on three dimensional chromatin architecture, and its relationship to epigenetic chromatin modifications to suggest a model of how these processes integrate to regulate the CSR reaction.

The looping out and deletion mechanism of CSR

The organization of the C_H region of the Igh locus is illustrated in Figure 1A. The Eμ intronic enhancer is located at the 5′-end of the C_H subregion. The 3′ regulatory region, 3′Eα, contains DNase hypersensitive sites (hs) 3a, hs1,2, hs3b and hs4 and functions as a locus control region LCR (reviewed in [23]). Further downstream there are additional hs sites 5–7 [24]. Sequestered between the two enhancers are eight C_H region genes each paired with a S region (with the exception of Cδ). S region transcription is a defining feature of CSR. C_H genes are organized in transcription units consisting of a noncoding “intronic” (I) exon, the S region and the C_H coding regions. GLTs emanate from an intronic (I) promoter located upstream of each I exon, run through the S region and then terminate at the 3’ end of it’s neighboring C_H coding region (Fig. 1A) [4]. Downstream S regions are selectively targeted for recombination with Sμ by directed activation of the isotype specific I exon promoters in response to combinations of antigen or mitogen, cytokines and costimulatory signals [2,4]. Targeted deletions of I exon promoters abolish transcription and severely affect CSR frequency [25–27].

Figure 1. The looping-out and deletion model of Ig switch recombination.

(A) A partial schematic map of IgH locus before CSR is shown not to scale. A productive V(D)J rearrangement has occurred allowing expression of the γ and μ IgH chains. Intact Sμ and Sγ1 are separated by approximately 70kb. Stimulation of B cells with antigen or mitogen induces germline transcription through the Iγ1-Sγ1-Cγ1 region prior to recombination. (B) The Sμ and Sγ1 regions are aligned causing the intervening genomic DNA to form a loop. (C) A reciprocal crossover between Sμ and Sγ1 results in the formation of a new hybrid transcriptional unit containing the original VDJ exons contiguous with Cγ1 and the formation of hybrid Sμ/Sγ1 molecules.

Once transcription targets Sμ and a downstream S region for recombination, CSR occurs through an intra-chromosomal deletional rearrangement that results in the formation of composite Sμ–Sx junctions on the chromosome while the intervening genomic material is looped out and excised (fig. 1B). CSR occurs between two highly repetitive S regions [28]. Concomitant with transcription, AID initiates S region specific double strand breaks (DSBs) [29,30] that are processed through a cascade of events mediated by nonhomologous end joining (NHEJ) [3]. CSR occurs anywhere within the donor Sμ and acceptor S region. Each CSR event produces a new composite S/S junction, which as a population are heterogenous with respect to the position of the recombination crossover points (fig. 1C). However, it has been unclear how distantly located S region–specific DSBs, separated by as much as 150 kb are recruited to partner in a CSR reaction that leads to intra-chromosomal deletion.

The mechanism of CSR is tied to the three dimensional structure of the Igh locus

Long range chromatin interactions over large genomic distances bring widely separated elements into close spatial proximity with their target genes. The mouse β-globin genes and their locus control region (LCR) located more than 50 kb apart engage in higher-order loop structures during transcription, and these interactions are tightly correlated with gene-specific expression [16,17,31]. Looped chromatin structures involved in the regulation of gene expression have also been detected in association with imprinted genes [19,32,33], cytokine clusters [18], estrogen receptor [15], MHC [21], α globin [20] loci and between chromatin boundary elements [34]. In the Igh locus, inducible transcription from the downstream GLT promoters requires the 3′Eα LCR since targeted deletion of hs3b,4 within 3′Eα leads to loss of GLT expression [2,35]. Thus, the direct interaction between GLT promoters and the distant 3’Eα may be mediated by chromatin loop formation. Spatial proximity between the various downstream GLT promoters with 3′Eα may facilitate transcription but would not support S/S synapsis since the S regions would not be juxtaposed with Sμ (Fig. 2A,B). Our chromatin conformation capture (3C) studies indicate that in mature resting B cells, the Eμ and 3′Eα enhancers are in close spatial proximity and form a chromatin loop [36] (fig. 2C). B cell activation leads to cytokine dependent recruitment of the GLT promoters to the Eμ:3′Eα complex and enables transcription of S regions targeted for CSR (Fig. 2C) [36]. Our new data indicate that the GLT promoter (and not the S regions) directly interacts with the 3′Eα element (unpublished observations). Prior to CSR, this looped structure facilitates S-S synapsis since Sμ is proximal to Eμ and a downstream S region is co-recruited with the targeted GLT promoter to Eμ:3′Eα complex. We and others conclude the Igh locus assumes a three dimensional structure that simultaneously supports GLT promoter interactions with the 3′Eα regulatory regions to facilitate transcription of the targeted S regions and juxtaposes the S regions targeted for recombination [36–38].

Figure 2. The Igh locus is configured in chromatin loops in B cells.

(A) A schematic is shown of the linear Igh locus with the VDJ exons (black box), intronic Eμ and 3′Eα enhancers (yellow symbols), C_H regions (gray boxes), S regions (ovals), and I exons (rectangles) indicated. This linear configuration is typical for the Igh locus in splenic T cells [36]. (B) Association of the γ1 locus with the 3′Eα will promote γ1 GLT expression but not S/S synapsis. (C) In resting mature splenic B cells the Igh locus loop is tethered by interactions between Eμ and 3′Eα. Following treatment of resting B cells with LPS and IL4 for 40 hours, Eμ and 3′Eα remain in close proximity and the ©1 locus has been recruited to the 3′Eα regulatory region [36]. Sγ1 is now in close proximity to Sμ.

S regions are specialized targets of CSR

S regions are G/C rich and fall into two general groups, sequences incorporating prevalent pentameric repeats into larger tandem repeats such as found in Sμ, Sε and Sα and a 49 bp repeat that is characteristic of Sγ3, Sγ1, Sγ2b, and Sγ2a [28]. S/S junctions are dispersed throughout S regions and occasionally fall in areas flanking the S regions [39,40]. CSR junctions are distinguished by their lack of consensus sequences or significant homology from site-specific or homologous recombination. S region length influences CSR frequency both in vivo and in vitro [39,41]. Deletion of S regions or their replacement with non-S region sequences by gene targeting methods, reduces CSR indicating that S regions are the specialized targets in this recombination event [41–44].

An intriguing questin is how S regions with their divergent sequences are recognized by AID and the CSR machinery. The degeneracy of the S region repeats and the absence of an identifiable recombination motif have led to models in which higher order structures rather than primary sequence provides the recognition code for the CSR machinery [2,45]. S regions are replete with palindromic sequences that have the potential to form stem-loop structures that are proposed to function as recognition substrates [45–47]. Murine and human S regions are G-rich on the nontemplate strand which contributes to R-loop formation [48,49] and G4 tetraplexes ([50] and Chapter Maizels, this volume). Transcribed S regions contain R loops, comprised of RNA:DNA hybrids in vitro and in vivo, which can provide ssDNA stretches as substrate for AID deamination [48,49]. Targeted inversion of the Sγ1 region, making the G rich strand nontemplate, led to the loss of R-loop formation and a significant reduction of CSR activity, arguing for the importance of R-loops for CSR efficiency [44].

Transcription and asymmetric AID attack

Early studies suggested that AID may function as an RNA deaminase based on similarities to APOBEC1, the catalytic component of a tissue-specific RNA editing complex ([51,52] and chapter Conticello). Identification of a mutational hotspot for AID in SHM indicated a mechanism by which AID functions directly on DNA [53]. This observation prompted Neuberger and coworkers to propose the DNA deamination model for AID action in which AID initiates SHM and CSR by converting deoxycytidine (dC) to deoxyuracil (dU) that can then be processed by one of several mechanisms (Fig. 3) (reviewed in [1,3,8,14]). The DNA deamination model gained substantial support from the direct demonstration that AID dependent dU residues are detected in the 5’Sμ region [54] and that uracil DNA glycosylase (UNG) is required for formation of double strand breaks (DSB) in S regions to enable CSR [29].

Figure 3. AID deamination model and a role for mismatch repair (MMR) in CSR. AID deaminates dC to dU.

(A) The U:G mismatch, can be replicated over to produce T:A, a transition mutation. (B) Using the base excision repair (BER) pathway, dU bases can be excised by a uracil DNA glycosylase (UNG) leaving an abasic site that can then be replicated over by error prone polymerase to produce both transition and transversion mutations. (C) Abasic sites can also be recognized by AP endonucleases (APE) to form ssDNA nicks or double strand breaks (DSBs) when the abasic sites are closely spaced and on complementary strands. (D) Other U:G mismatches could be substrates for mismatch repair (MMR) MSH2/MSH6 binding which in turn recruit PMS2-MLH1 (not shown), and PMS2 nicks the DNA. Exonuclease 1 (Exo I) binds to MSH2 and MLH1, and using the PMS2 induced nick, excises toward the U:G mismatch producing a DSB with a 5′ overhang. An error prone DNA polymerase can fill-in the 5′ overhang and introduces mutations at A:T bp. 3′ overhangs can be excised by Ercc1-XPF. Short overhangs can be used during end joining.

During CSR, AID induced S region mutations begin 150 bp downstream of the I exon transcription start site (TSS) [55] (fig.4). The 3′ boundary for CSR mutations is located downstream of S regions at a distance of up to 10 kb from the TSS [55] (fig. 4). Studies indicate that ectopically expressed AID binds with RNAP II in co-immunoprecipitation assays [56] and interacts with the transcription apparatus in vitro [57] suggesting that AID targeting is linked with transcription. Surprisingly, AID attack is asymmetrically focused within the I-S-C_H region. S regions are substrates for AID activity whereas C_H regions within the same transcription unit are protected [29,30,55] raising the question of how this occurs.

Figure 4. Summary of mutation frequency and differential histone modifications in S and C_H regions of activated B cells.

A schematic diagram depicts a generic I-S-CH region downstream of the GLT promoter (Pr). Above the diagram a summary of mutation frequencies 5′ and 3′ of the S region for Ung^−/− Msh2^−/− B cells is shown [56]. A similar distribution of mutations are found for this region in Ung^−/− B cells [82]. Below the I-S-CH schematic a summary of histone modifications and RNA pol II binding is shown. WT B cells were stimulated with LPS or LPS + IL-4 for 48 hours and then analyzed for mutations of by in chromatin immunoprecipitation (ChIP) assays using antisera against H3K9,14Ac, H3K4me3, H3K36me3, H4K20me1, pol II RNA, and pol II p-ser5. Data was amalgamated from published studies [74,75].

Chromatin accessibility to AID attack

Eukaryotic DNA is wrapped around histone octamers and organized into higher order chromatin fibers which regulate access of trans acting factors to DNA [58–60]. Studies suggest that at least two distinct mechanisms are used to achieve efficient transcription through chromatin. One relies on a pathway based on nucleosome loss and the second involves histone acetylation with little or no loss of histones (reviewed in [61]). Our studies indicate that the second pathway involving histone modification occurs during GLT expression during CSR [62,63], as will be described below. The RNA polymerase II (RNAP II) transcription cycle occurs in distinct steps including RNAP II recruitment to the promoter, formation of the preinitiation complex, promoter clearance, processive elongation, and transcription termination [61]. When RNAP II clears the promoter and transits to the promoter proximal pause site it becomes hyperphosphorylated at Ser5 (p-ser5) on the C-terminal domain (CTD). RNAP II then enters into productive elongation, loses p-ser5 and becomes enriched for p-ser2 CTD [61]. An emerging paradigm links transcription regulation with the phosphorylation status of RNAP II C-terminal domain (CTD) and the recruitment of histone modifying enzymes, which in turn introduce histone marks that alter the status of chromatin accessibility [59,64]. This paradigm is relevant to understanding asymmetric AID targeting away from C_H regions and toward S regions.

Transcription activity is strikingly correlated with significant histone acetylation (Ac) at promoters [65,66]. Genome wide studies reveal that promoter proximal sites in transcriptionally active genes are enriched for tri-methyl histone H3 lysine 4 (H3K4me3), hyper-acetylated (Ac) H3K9, and with RNAP II p-ser5 whereas, downstream coding regions are enriched in H3K36me3 and elongating RNAP II p-ser2 [66–69]. Histone methylation can act as a tag for effector proteins containing methyl-binding domains including chromodomains, tudor domains and PHD finger domains [70]. The NuA3 histone acetytransferase (HAT) complex that coordinates transcription activation with histone Ac is directly bound by H3K4me3 (reviewed in [71]). Histone Ac which changes the net charge of nucleosomes and alters chromatin fiber folding properties increases DNA accessibility [72]. Histone Ac may also create binding surfaces for factor-histone contacts that then facilitate recruitment of transcription regulators [59]. Rpd3S, a histone deactylase (HDAC) complex interacts with H3K36me3, which then functions to reduce histone Ac and thereby repress transcription initiation in downstream coding regions [73,74]. The bifurcation of H3K4me3 and H3K36me3 modifications within a transcription unit serve to recruit HATs and HDACs, respectively, which in turn modulate chromatin accessibility.

Consistent with the observation that S regions and not C_H regions are modified by AID, markers of accessible chromatin are associated with S regions but not with C_H regions. In vivo, I-S regions undergoing active transcription are nuclease hypersensitive whereas C_H regions are comparatively inaccessible [62,63]. Accordingly, antisense RNA transcripts are found selectively in S regions and not in C_H regions indicating the reciprocal areas of accessible or repressed chromatin, respectively [75]. Transcriptionally active I-S regions accumulate H3K4me3 and histone Ac modifications while C_H regions become enriched for the repressive countermarks, H3K36me3 and H4K20me1 [56,62,63,76] (Fig. 4). Treatment with tricostatin A, an inhibitor of HDACs, increases H3Ac at S regions concomitant with greater chromatin accessibility and higher frequency CSR demonstrating a direct link between histone Ac and switching frequency [63]. Furthermore, PTIP (PAX interaction with transcription activation domain protein) deficiency displays reduced H3K4me3 and histone Ac marks and impaired transcription initiation at a subset of downstream S regions [77]. PTIP is a component of several histone methyl transferases (HMTases) including MLL3 (mixed lineage leukemia 3) – MLL4 complex that interact with RNAPII p-Ser5 [77,78]. PTIP also indirectly associates with p300/CBP HAT suggesting that PTIP regulates both histone Ac and Me [79]. Interestingly, long range looping interactions between GLT promoters and the 3′Eα enhancer are impaired in PTIP deficient B cells indicative of deficient GLT transcription initiation [80]. Collectively, these observations support the conclusion that histone Ac status and chromatin accessibility in S regions are determined by means of H3K4me3 modifications linked to transcription.

S regions, R-loops and AID targeting

S regions span 1–10 kb beyond the I exons and the TSSs [28], and when transcribed are enriched along their entire lengths with H3K4me3, H3Ac, and H4Ac, activating histone modifications [62,63]. This contrasts with genome wide analyses in which these marks are largely relegated to promoter proximal locations [66,67]. What directs the spread of these modifications long distances from the GLT TSSs? This unusual distribution of H3K4me3 in S regions is linked to stalled RNAP II p-ser5 in S DNA. ChIP studies showed accumulation of RNAP II p-ser5 throughout the Sμ and Sγ3 regions indicating that RNAP II has stalled at multiple sites [63]. In contrast, RNAP II p-ser5 occupancy and H3K4me3 marks remained promoter proximal in Sμ deleted B cells, indicating that it is the S region sequence that stalls RNAP II leading to the unusual H3K4me3 distribution [63,81]. S regions are highly G/C rich and when transcribed develop R-loops over long stretches that enhance CSR efficiency in vivo ([48,49] and chapter Maizels). It is likely that R-loops which demonstrably impede transcription elongation [82,83] are responsible for RNAP II accumulation detected in S regions. Further investigation is required to ascribe a causal relationship between R-loop formation and RNAP II accumulation in S regions.

Is there a connection between RNAP II stalling and focused AID activity on S regions? Recent evidence suggests that AID interacts with the transcription elongation factor Spt5. Spt5 was identified in an shRNA screen as a critical regulator of CSR [84]. Upon clearance from the promoter, RNAP II pausing occurs 25–50 nucleotides downstream of the TSS and is mediated by DSIF (5,6-dichloro-1-b-d-ribofuranosyl-benzimidazole (DRB) sensitivity inducing factor) comprised of Spt4 and Spt5, and NEF (negative elongation factor) [85]. Spt5 interacts with AID and mediates its association with RNAP II [84]. ChIP-seq experiments indicate overlapping positions of Spt5 binding with RNAP II stalling [84,86] and between sites of Spt5 and AID binding genomewide [84]. Spt5 also interacts with several co-transcriptional factors including splicing factors [87], capping enzyme [88] and the RNA exosome [89] that have been functionally associated with CSR [90–92]. Targeted deletion of the GLT splice donor element dramatically impaired CSR [25]. These observations highlight the highly integrated aspect of transcription elongation and RNA processing. Together, these findings suggest that AID is targeted to sites of high occupancy RNAP II by means of its association with Spt5. The unique character of S regions which are prone to forming R-loops, in turn impedes RNAP II elongation. It now appears that Spt5, a cofactor of stalled RNAP II, associates with AID and may function to target it to specific loci. Because C_H regions lack R-loops, stalled RNAP II does not accumulate and Spt5 does not focus AID to these regions.

Summary

Transcription is intrinsic to the mechanism of CSR. The unique structure of the S regions appears to be mechanistically linked to generating open chromatin. These advances highlight the nexus between transcription, long range looping interactions with chromatin remodeling and histone modifications which permit initiation of CSR by AID. Emerging new technologies that allow visualization of higher order chromatin structures are likely to provide new insights into transcription regulation by regulatory elements functioning at great distances from their target genes. Ig CSR is an excellent model to study the intersection of long range chromatin interactions, transcription and recombination.