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 activationinduced deaminase (AID). CSR is critically dependent on transcription of noncoding RNAs across S regions. However, mechanistic insight regarding this process has remained unclear. New studies indicate that long-range intrachromosomal interactions among IgH transcriptional elements organize the formation of the S/S synaptosome, as a 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.

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.

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.

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.

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].