Sequence Dependence of Chromosomal R-Loops at the Immunoglobulin Heavy-Chain Sμ Class Switch Region

Abstract

The mechanism by which the cytidine deaminase activation-induced deaminase (AID) acts at immunoglobulin heavy-chain class switch regions during mammalian class switch recombination (CSR) remains unclear. R-loops have been proposed as a basis for this targeting. Here, we show that the difference between various forms of the S locus that can or cannot undergo CSR correlates well with the locations and detectability of R-loops. The S R-loops can initiate hundreds of base pairs upstream of the core repeat switch regions, and the area where the R-loops initiate corresponds to the zone where the AID mutation frequency begins to rise, despite a constant density of WRC sites in this region. The frequency of R-loops is 1 in 25 alleles, regardless of the presence of the core S repeats, again consistent with the initiation of most R-loops upstream of the core repeats. These findings explain the surprisingly high levels of residual CSR in B cells from mice lacking the core S repeats but the marked reduction in CSR in mice with deletions of the region upstream of the core S repeats. These studies also provide the first analysis of how R-loop formation in the eukaryotic chromosome depends on the DNA sequence.Mammalian immunoglobulin (Ig) genes undergo two types of DNA recombination, in addition to a somatic hypermutation (SHM) process. V(D)J recombination occurs in early lymphocytes and assembles the variable-domain exon so that IgM can be made. Class switch recombination (CSR) occurs only at the Ig heavy-chain locus and is responsible for the change in the heavy-chain isotype from IgM to IgG, IgA, or IgE; this process is also called the heavy-chain isotype switch (6,17,29). CSR occurs at repetitive DNA elements called switch regions, which vary in sequence and length. All of the switch regions are more than 1 kb in length and consist of unit repeats of 25 to 80 bp. All are located downstream of a sterile transcript promoter, which is necessary for CSR. All have a G-rich nontemplate strand, and all are rich in sites at which a cytidine deaminase called activation-induced deaminase (AID) prefers to act, namely, WRC sites (37). The regional nature of CSR, which gave rise to the term regionally specific recombination (15), contrasts with the vast majority of other physiologic recombination systems in biology, which are regarded as site specific. The special features of switch regions (such as being long and repetitive and located downstream of a promoter, having Grich nontemplate strands, and not having sequences conserved among the different switch regions or among vertebrates that carry out CSR) suggested that the mechanism would be unusual relative to other recombination processes in biology.Investigators at the Honjo laboratory discovered the key lymphoid-specific enzyme for both CSR and SHM, AID (22,23). AID is a 26-kDa protein which deaminates C in DNA (5, 25) but only when that DNA is single stranded (2,26,36,38). A key question for CSR and SHM concerns how the DNA becomes single stranded. Because transcription appears to...

Transcription induces strand-specific mutations at the 5′ end of human genes

“…It is important to note that until now it was not fully understood what the conditions for R-loops formations were. A couple of recent studies have suggested that R-loops are initiated from 50-bp-long regions, which contain the motif GGGGC TGGGG and comprise at least 50% Gs (Huang et al 2007;Roy et al 2008). We found ∼300 genes that, in their first 1000 bp, contain such regions.…”

Mechanism of R-Loop Formation at Immunoglobulin Class Switch Sequences

“…The fraction of substrate that forms an R-loop exhibits a mobility difference and runs more slowly than the double-stranded DNA substrate, even on these linear substrates (7). We and others have demonstrated that the DNA in the shifted position is R-looped, based on RNase H1 sensitivity and sodium bisulfite chemical probing for single strandedness (12,31,43).…”

“…Given that AID requires single-stranded DNA, a key question concerns how any single strandedness is exposed within the switch regions (33). We have shown that R-loops are detectable at the S␥3 and S␥2b acceptor switch regions (13,43) and, more recently, at the S switch regions (12). These R-loops can be kilobases in length and provide a ready target for AID at any of the C's within the top strand.…”

AID-induced decrease in topoisomerase 1 induces DNA structural alteration and DNA cleavage for class switch recombination

“…To explore the mechanism by which AID-mediated Top1 reduction augments S region cleavage and CSR, we speculated that aberrant supercoiling by Top1 protein reduction may induce non-B DNA structure in the S region so that Top1 cannot religate, resulting in irreversible cleavage by Top1. DNA structural alterations can be estimated by the DNA's sensitivity to bisulfite modification from dC to dU, which occurs in the single-stranded regions (6,7). The presence of consecutive C bases is suitable for determining the single-strandedness by the bisulfite assay (7).…”

“…The S region transcripts seem to remain on the DNA template for a short period, thus facilitating the formation of R-loop. Lieber and colleagues reported that the bisulfite treatment of nuclear DNA from switch-induced spleen cells converted dC to dU in the S region, indicating the presence of single stranded DNA in the S region in activated B cells (6,7). R-loop may not survive long but lead to the formation of non-B structures including stem-loop, cruciform, and triplex in the S region because of abundant inverted repeats (3,(8)(9)(10).…”