DNA Replication Origins in Immunoglobulin Switch Regions Regulate Class Switch Recombination in an R-Loop-Dependent Manner

Abstract

Class switch recombination (CSR) at the immunoglobulin heavy chain (IgH) locus generates antibody isotypes. CSR depends on double-strand breaks (DSBs) induced by activation-induced cytidine deaminase (AID). Although DSB formation and repair machineries are active in G1 phase, efficient CSR is dependent on cell proliferation and S phase entry; however, the underlying mechanisms are obscure. Here, we show that efficient CSR requires the replicative helicase, the Mcm complex. Mcm proteins are enriched at IgH switch regions during CSR, leading to assembly of facultative replication origins that require Mcm helicase function for productive CSR. Assembly of CSR-associated origins is facilitated by R loops and promotes the physical proximity (synapsis) of recombining switch regions, which is reduced by R loop inhibition or Mcm complex depletion. Thus, R loops contribute to replication origin specification that promotes DSB resolution in CSR. This suggests a mechanism for the dependence of CSR on S phase and cell division.

Lingering Questions about Enhancer RNA and Enhancer Transcription-Coupled Genomic Instability

“…Studies have postulated that such a transcription complex–replication complex collision can lead to DNA double-strand breaks, particularly at fragile sites in the genome [62–64]. IgH switch sequences are a special case, as it is here that transcription coupled R loops may be responsible for the formation of origin of replication sites that have a role in synapsis of DNA double-strand breaks [65]. Thus, in many different ways, transcription directionality of RNA polII (including at enhancer sites) at DNA polymerase complex associated sites in the genome may play a role in DNA double-strand break formation and genomic integrity regulation.…”

R Loops in the Regulation of Antibody Gene Diversification

“…Using this approach, studies in the CH12 B cell line did not report any effect on switch region mutation frequency following RNaseH over-expression [42] or via short-term retroviral expression in activated B cells [43,44]. However, whereas one study did not report CSR defects in RNaseH-expressing stable CH12 lines [42], another study reported ~50% decrease in CSR following retroviral infection of RNaseH [43]. Finally, RNaseH over-expression in transgenic mice resulted in a slight increase in mutation on the template strand but did not alter CSR frequency [34].…”

“…In line with alternative roles for R loops in CSR, a recent study has implicated R loops in promoting the long-range synapsis of donor and acceptor switch regions that is essential for the final, end-joining phase of CSR [43]. Specifically, it was reported that CSR efficiency is dependent on, and correlates with, the activity of origins of replication in switch regions and that R loops contribute to the specification of these origins during early S phase [43].…”

“…Specifically, it was reported that CSR efficiency is dependent on, and correlates with, the activity of origins of replication in switch regions and that R loops contribute to the specification of these origins during early S phase [43]. Indeed, defects in origins establishment were observed upon shRNA-based depletion of subunits of the replicative helicase, the Mcm complex, as well as upon depletion of R loops with RNaseH.…”

“…Indeed, defects in origins establishment were observed upon shRNA-based depletion of subunits of the replicative helicase, the Mcm complex, as well as upon depletion of R loops with RNaseH. Furthermore, both perturbations led to impaired long-range switch region synapsis and a CSR defect independent of AID activity [43]. This finding shows for the first time that switch region R loops have an additional function in CSR at a step downstream of mutagenesis.…”