Immunoglobulin class-switch DNA recombination: induction, targeting and beyond

Abstract

Class-switch DNA recombination (CSR) of the immunoglobulin heavy chain (IGH) locus is central to the maturation of the antibody response and crucially requires the cytidine deaminase AID. CSR involves changes in the chromatin state and the transcriptional activation of the IGH locus at the upstream and downstream switch (S) regions that are to undergo S-S DNA recombination. In addition, CSR involves the induction of AID expression and the targeting of CSR factors to S regions by 14-3-3 adaptors, and it is facilitated by the transcription machinery and by histone modifications. In this Review, we focus on recent advances regarding the induction and targeting of CSR and outline an integrated model of the assembly of macromolecular complexes that transduce crucial epigenetic information to enzymatic effectors of the CSR machinery.

Figure 1. CSR entails DNA deletion

CSR exchanges the gene encoding the heavy chain constant region (C_H) with one of a set of downstream C_H genes (depicted is CSR from Sμ to Sα1 in the human IgH locus). This deletion-recombination reaction requires AID and involves the generation of double-strand DNA breaks in S regions (lying upstream of the constant-region gene) followed by DSB repair. This leads to juxtaposition of rearranged VDJ DNA with a downstream C_H exon cluster and deletion of the intervening sequence between S regions as an extrachromosomal circle.

Figure 2. Enzymes and scaffold elements in CSR

CSR is initiated by AID, which is targeted to S regions by 14-3-3 adaptors that specifically bind 5′-AGCT-3′ repeats in the S region core and recruit AID and PKA to S region DNA. AID is phosphorylated at Ser38 of the N-terminal region by PKA, generating a binding site for RPA. AID also binds 14-3-3 proteins through its C-terminal region (inset). RPA enhances AID deamination of deoxycytosines in transcribed S region DNA. The resulting deoxyuracils are removed by UNG, which would be recruited and stabilized on S regions in a fashion dependent on the scaffold functions of 14-3-3, RPA and REV1 (Zan, H., C.A. White, L. Thomas, J. Zhang, G. Li, E.S. Yu, Z. Xu, T. Mai and P. Casali. 2012. Submitted for publication). RPA and REV1 bind to different regions of UNG. UNG is also stabilized by AID, which indirectly interacts with UNG (likely through 14-3-3, Lam, T. and P. Casali, unpublished observation), within a putative macromolecular complex. Excision of UNG-generated abasic sites by APEs and/or the MRE11–RAD50 lyase results in single stranded breaks, which either directly form double stranded breaks (as occurring in the S region core) or are converted to DSBs in an MSH2- and EXO I-dependent fashion (as occurring in the flanking area). DSB resolution by C-NHEJ or A-EJ leads to formation of S-S junctions and CSR.

Figure 3. CSR-inducing stimuli and interplay of transcription factors

CSR entails induction of AID, germline I_H-S-C_H transcription and active histone modifications in S regions by primary CSR-inducing stimuli together with secondary stimuli (cytokines). Both T-dependent (CD40) and T-independent (dual TLR–BCR, TACI–BCR or TLR–TACI engagement) CSR-inducing stimuli — through distinct signal transducers — activate NF-κB for induction of HOXC4 (whose expression is enhanced by estrogen) and HOXC4-dependent induction of AID. The ability of primary CSR-inducing stimuli to induce AID and CSR correlates with their activation of both the canonical and non-canonical NF-κB pathways. Cytokines IL-4, TGF-β and IFN-γ do not activate NF-κB, but activate transcription factors specific for I_H promoters. These in turn bind to specific I_H promoters to initiate germline I_H-S-C_H transcription, thereby specifying the S regions that are to undergo recombination. A combinatorial interplay of primary and secondary CSR-inducing stimuli-activated transcription factors (teal and plum circles, respectively) results in optimal induction of AID and germline I_H-S-C_H transcription. Transcription factor-binding sites in Region I through IV of the AID gene locus that have been shown to regulate AID expression are depicted. Binding sites for MYB and E2F (grey circles) negatively regulate AID expression.

Figure 4. Targeting of the CSR machinery

CSR targeting entails the epigenetic regulation of the S regions that are to undergo recombination (depicted is CSR from Sμ to Sγ1 in the mouse IgH locus), high-avidity binding of 14-3-3 adaptors to 5′-AGCT-3′ repeats and scaffold functions of CSR factors. (a) RNA polymerase II and histone modifying enzymes, such as histone methyltransferase and histone acetyltransferase complexes, are recruited to the Iγ1 promoter to initiate germline Iγ1-S-Cγ1 transcription (red arrow) and catalyze histone modifications, respectively (the Iμ promoter undergoes constitutive transcription initiation and histone modifications). Through SPT5, RNA polymerase II associates with AID at the Iμ and Iγ1 promoter, albeit at low levels. (b) During transcription elongation, RNA polymerase II together with SPT5 and, perhaps, SPT6, PTBP2 and the RNA exosome (all within a putative macromolecular complex), stalls in Sμ and Sγ1, and likely plays a role in recruiting/stabilizing histone modifying enzymes, such as histone methyltransferase and histone acetyltransferase complexes that catalyze H3K4me3 (green ovals) and H3K9acS10ph (orange ovals) modifications, respectively. This and loss of repressive histone modifications, such as H3K9me3 (light blue ovals) and H3K27me3 (grey ovals), reflect the open chromatin state of Sμ and Sγ1 that allows the access of AID and other CSR factors. AID, as “hitched” on the transcription machinery, is enriched in S regions due to RNA polymerase II stalling. (c) AID is further concentrated to Sμ and Sγ1 by 14-3-3 adaptors, which access the same Sμ and Sγ1 regions due to their open chromatin state and are recruited/stabilized there through interactions with 5′-AGCT-3′ repeats and likely H3K9acS10ph. The RNA exosome also interacts with AID and allows AID to deaminate both transcribed and non-transcribed S region DNA. (d) 14-3-3, RPA and enzymatic elements AID and REV1 (Zan, H., C.A. White, L. Thomas, J. Zhang, G. Li, E.S. Yu, Z. Xu, T. Mai and P. Casali. 2012. Submitted for publication) all possess scaffold functions that stabilize each other and other CSR factors, including PKA, UNG and MSH2, on S region DNA within a macromolecular complex.