Regulation of immunoglobulin class-switch recombination: choreography of noncoding transcription, targeted DNA deamination, and long-range DNA repair

Abstract

Upon encountering antigens, mature IgM-positive B lymphocytes undergo class-switch recombination (CSR) wherein exons encoding the default Cμ constant coding gene segment of the immunoglobulin (Ig) heavy-chain (Igh) locus are excised and replaced with a new constant gene segment (referred to as "Ch genes", e.g., Cγ, Cɛ, or Cα). The B cell thereby changes from expressing IgM to one producing IgG, IgE, or IgA, with each antibody isotype having a different effector function during an immune reaction. CSR is a DNA deletional-recombination reaction that proceeds through the generation of DNA double-strand breaks (DSBs) in repetitive switch (S) sequences preceding each Ch gene and is completed by end-joining between donor Sμ and acceptor S regions. CSR is a multistep reaction requiring transcription through S regions, the DNA cytidine deaminase AID, and the participation of several general DNA repair pathways including base excision repair, mismatch repair, and classical nonhomologous end-joining. In this review, we discuss our current understanding of how transcription through S regions generates substrates for AID-mediated deamination and how AID participates not only in the initiation of CSR but also in the conversion of deaminated residues into DSBs. Additionally, we review the multiple processes that regulate AID expression and facilitate its recruitment specifically to the Ig loci, and how deregulation of AID specificity leads to oncogenic translocations. Finally, we summarize recent data on the potential role of AID in the maintenance of the pluripotent stem cell state during epigenetic reprogramming.

Keywords: Activation-induced deaminase; Class-switch recombination; Induced pluripotency; Lymphomagenesis.

Figure 1.1

Secondary immunoglobulin gene diversification. Mature B cells undergo class-switch recombination (CSR) and somatic hypermutation (SHM). During SHM, mutations are introduced into the rearranged variable region genes. CSR is initiated by transcription (grey arrows) through switch (S) regions (ovals) and requires AID and components of several DNA repair pathways. During CSR, the intervening DNA sequence between participating S regions is excised as a switch circle and a new constant region gene is juxtaposed downstream of the variable region exons.

Figure 1.2

Model for SHM. AID deaminates dCs to dUs within RGYW hotspots in variable region genes. Activities of mismatch repair (MMR) and base excision repair (BER) proteins (e.g., UNG of the BER pathway) coupled with DNA replication generate transition and transversion mutations.

Figure 1.3

Germline transcription through S regions. Each constant region gene is comprised of a transcription unit with a cytokine-inducible promoter (P), an intervening (I)-exon, S region, and C_h exons. The primary transcript is spliced and polyadenylated to generate a noncoding mature transcript.

Figure 1.4

Transcription-driven R-loop formation at S regions. Transcription occurs through each participating S region (indicated by arrows) and the nascent transcripts (dotted lines) remain bound to template DNA due to high GC content of the sequence. This displaces the nontemplate strands as ssDNA substrates, forming structures called R-loops.

Figure 1.5

Processing of deaminated DNA during CSR. S regions deaminated by AID are processed by UNG, APE1, and MMR proteins to generate blunt DSBs that are synapsed and ligated to complete CSR.

Figure 1.6

Phosphorylation and DSB-dependent positive feedback loop amplifies DSB formation. Multiple AID molecules are assembled at S region DNA. An initial DNA break due to AID-mediated DNA deamination of cytidines is generated and can result in inefficient CSR. ATM-dependent S38 phosphorylation of AID induces the recruitment of APE1, which increases the number of DNA breaks generated. This induces additional phosphorylation of AID at S38 and APE1 recruitment to generate the high density of DSBs required for efficient CSR. AID phosphorylation at S38 also allows for interaction with RPA, which promotes efficient CSR potentially by recruiting downstream repair factors.

Figure 1.7

Regulation of AID. AID expression, localization and activity are regulated through multiple processes.

Figure 1.8

Transcription factor-binding sites in Aicda locus. The Aicda locus is regulated by four regions of transcription factor-binding sites. Region 1 is just 5’ of the transcription start site (TSS), region 2 is located between exons 1 and 2 (shown in black), region 3 is ~17 kb downstream of the TSS, and region 4 is ~8 kb upstream of the TSS. Adapted from Lee-Theilen and Chaudhuri (2010).

Figure 1.9

AID as an indirect demethylase. The deaminase activity of AID can lead to demethylation through two potential pathways. In one, AID can deaminate methylated cytidines (5mC) to generate thymidine. In the other, AID can deaminate hydroxymethylcytosine (5hmC) that is generated by the oxidation of 5mC by the ten-eleven translocation (TET) proteins. In both pathways, the mismatch is repaired by thymine DNA glycosylase (TDG) and BER components. The end-product is the replacement of methylated cytidines with unmethylated cytidines.