Current insights into the mechanism of mammalian immunoglobulin class switch recombination

Abstract

Immunoglobulin (Ig) class switch recombination (CSR) is the gene rearrangement process by which B lymphocytes change the Ig heavy chain constant region to permit a switch of Ig isotype from IgM to IgG, IgA, or IgE. At the DNA level, CSR occurs via generation and joining of DNA double strand breaks (DSBs) at intronic switch regions located just upstream of each of the heavy chain constant regions. Activation-induced deaminase (AID), a B cell specific enzyme, catalyzes cytosine deaminations (converting cytosines to uracils) as the initial DNA lesions that eventually lead to DSBs and CSR. Progress on AID structure integrates very well with knowledge about Ig class switch region nucleic acid structures that are supported by functional studies. It is an ideal time to review what is known about the mechanism of Ig CSR and its relation to somatic hypermutation. There have been many comprehensive reviews on various aspects of the CSR reaction and regulation of AID expression and activity. This review is focused on the relation between AID and switch region nucleic acid structures, with a particular emphasis on R-loops.

Keywords: DNA recombination; DNA repair; R-loop; RNase H; activation-induced deaminase; immunoglobulin gene rearrangement; isotype switching.

Figure 1.. Diagram of Class Switch Recombination.

The exons (the constant regions have been simplified to a single rectangle) are depicted with the nearly square blue symbols. The tan circles represent class switch recombination (CSR) sequences, which are often repetitive with numerous AGCT sites and G-clusters (GGGGT or GGGCT)(Dunnick et al., 1993). A single class switch recombination event utilizes the Sμ region (often called a switch donor sequence) and one of the downstream “acceptor” switch regions, in this case Sα. The deleted region is shown as a circular DNA molecule. The VDJ exon is symbolized as a red symbol labeled with a V. The actual promoter of the gene is upstream of this V. There are sterile transcript promoters upstream of each of the switch regions, and all of these are inactive, except for the ones upstream of the Sμ and Sα switch regions (red arrow) that are, in this case, activated for recombination. A detailed diagram of the organization of the sterile transcript promoter and the Iexons is shown in Suppl. Figure 1.

Figure 2.. The R-Loop:Deaminase Model for Class Switch Recombination.

A putative CSR event to IgG3 is shown. Switch region RNA transcripts (green lines) were paired with the C-rich DNA template strand to form an R-loop structure (Yu et al., 2003; Yu and Lieber, 2003). The entire displaced G-rich DNA strand and part of the C-rich DNA at the edges of the R-loop are single-stranded, and therefore, serve as targets for AID. AID deaminates C residues located in the single-stranded region to convert them to uracil (U). UDG removes uracils in the DNA and leaves behind an abasic (apyrimidinic) site, which is cleaved by APE. The sum of nicks on both strands results in double-strand DNA breaks in the switch region, which are repaired by the NHEJ pathway to complete CSR. Coding regions (VDJ and constant region exons) are indicated by rectangles. Small dashes in the switch region indicate base pairing between the switch transcript and the C-rich DNA template.

Figure 3.. Collapsed R-Loops Resulting from RNase H Action on R-loops.

AID may be unable to recognize the single-stranded character of the displaced G-rich DNA strand because that strand may be too close to the RNA:DNA helix of the R-loop. In this model, an RNase H activity is proposed to remove the RNA in the R-loop. During the degradation of the switch region RNA, the annealing of the G-rich DNA strand with the C-rich DNA strand may result in the top strand of switch region repeat number 9 annealing with the bottom strand of repeat number 6. That is, the top strand of one repeat may misalign and anneal to the bottom strand of another repeat, resulting in ssDNA loops on both the top and bottom strands in the switch region. The misaligned repeats (6 versus 9) would, therefore, have many mismatches shown by red asterisks (Yu et al., 2003; Yu and Lieber, 2003). The looped-out DNA may be the actual target of AID-mediated cytidine deamination. The rest of this process (removal of uracil by UDG, cleavage by APE and joining by NHEJ) would be similar to the R-loop:Deaminase model shown in Figure 2.

Figure 4.. Competition Between the mRNA and the Nontemplate DNA Strand for Binding to the Template DNA Strand.

The G-clusters initiate R-loops in mammalian switch regions. G-clusters are important for efficient R-loop initiation within cells (Zhang et al., 2014b).

Figure 5.. Initiation of Thread Back and Elongation of the R-Loop.

When the RNA base pair with the template DNA strand at G-clusters, we refer to this as the R-loop initiation zone (RIZ). Natural mammalian switch regions have many possible locations for such initiation, but for experimental cases, we often create one (Roy and Lieber, 2009; Roy et al., 2008; Roy et al., 2010; Zhang et al., 2014a; Zhang et al., 2014b). After the R-loop has initiated, the R-loop elongates, and we refer to this as the R-loop elongation zone or REZ.

Figure 6.. High affinity AID binding to collapsed R-loop.

A. Bifurcated substrate binding of AID. Adapted from a published graphic abstract (Qiao et al., 2017). B. AID binding to the displaced non-template strand in an R-loop is a low affinity binding to ssDNA. Upon RNase H treatment, S region repeats misalign to form collapsed R-loop that contains branched DNA (red arrows) for high affinity AID binding.

Figure 7.. Diagram of the Human c-MYC Translocation.

The diagram illustrates the translocation of the c-myc gene on human chromosome 8 with the IgH switch regions on chromosome 15. Breaks at both locations are AID-mediated. In addition to evidence for R-loops at the Ig CSR regions, there is also evidence for R-loops at c-myc.