Rad52 mediates class-switch DNA recombination to IgD

Yijiang Xu,Ginell Post,Hang Zhou,Hong Zan,Paolo Casali

doi:10.1038/s41467-022-28576-2

Abstract

In B cells, IgD is expressed together with IgM through alternative splicing of primary VHDJH-Cμ-s-m-Cδ-s-m RNAs, and also through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sμ with σδ. How such DSBs are resolved is still unknown, despite our previous report showing that Rad52 effects the ‘short-range’ microhomology-mediated synapsis of intra-Sμ region DSBs. Here we find that induction of IgD CSR downregulates Zfp318, and promotes Rad52 phosphorylation and recruitment to Sμ and σδ, thereby leading to alternative end-joining (A-EJ)-mediated Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Rad52 ablation in mouse Rad52−/− B cells aborts IgD CSR in vitro and in vivo and dampens the specific IgD antibody response to OVA. Rad52 knockdown in human B cells also abrogates IgD CSR. Finally, Rad52 phosphorylation is associated with high levels of IgD CSR and anti-nuclear IgD autoantibodies in patients with systemic lupus erythematosus and in lupus-prone mice. Our findings thus show that Rad52 mediates IgD CSR through microhomology-mediated A-EJ in concert with Zfp318 downregulation.

Highlights

In B cells, IgD is expressed together with IgM through alternative splicing of primary VHDJHCμ-s-m-Cδ-s-m RNAs, and through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sμ with σδ
homologous recombination (HR) factor Rad[52] competes with non-homologous end-joining (NHEJ) factors Ku70/Ku86 for binding to S region DSB ends and synapses DSB ends by alternative end-joining (A-EJ) through microhomology-mediated end-joining (MMEJ)[20], as inferred from increased NHEJ-mediated IgG, IgA, and IgE CSR events with even fewer S–S junction microhomologies in Rad52−/− B cells in vivo and in vitro[20]
AID introduces DSBs in σδ as it does in Sμ, Sγ, Sα or Sε. In both mouse Rad52−/− B cells and human RAD52 knockdown B cells, ablation or virtual ablation of post-recombination VHDJH−Cδs transcripts resulted in reduced IgD secretion, which occurred in presence of unaltered levels of transmembrane VHDJH−Cδm transcripts, at least within the first 72 h from CSR induction

Summary

Introduction

In B cells, IgD is expressed together with IgM through alternative splicing of primary VHDJHCμ-s-m-Cδ-s-m RNAs, and through IgD class switch DNA recombination (CSR) via double-strand DNA breaks (DSB) and synapse of Sμ with σδ. How such DSBs are resolved is still unknown, despite our previous report showing that Rad[52] effects the ‘short-range’ microhomology-mediated synapsis of intra-Sμ region DSBs. Here we find that induction of IgD CSR downregulates Zfp[318], and promotes Rad[52] phosphorylation and recruitment to Sμ and σδ, thereby leading to alternative end-joining (A-EJ)-mediated Sμ-σδ recombination with extensive microhomologies, VHDJH-Cδs transcription and sustained IgD secretion. Transcription of long primary VHDJH-Cμ-s-m-Cδ-s-m RNA requires the zinc finger ZFP318 repressor of transcriptional termination, which, as shown with genetically modified mouse models, obliterates the effect of the transcriptional termination sites (TTS) intercalated between the Cμ and Cδ exon clusters[10,11]

Methods

Results

Conclusion