Regulation of the DNA Repair Complex during Somatic Hypermutation and Class-Switch Recombination

Abstract

B lymphocytes optimize Ab responses by somatic hypermutation (SH), which introduces point mutations in the variable regions of the Ab genes and by class-switch recombination (CSR), which changes the expressed C region exon of the IgH. These Ab diversification processes are initiated by the deaminating enzyme activation-induced cytidine deaminase followed by many DNA repair enzymes, ultimately leading to deletions and a high mutation rate in the Ab genes, whereas DNA lesions made by activation-induced cytidine deaminase are repaired with low error rate on most other genes. This indicates an advanced regulation of DNA repair. In this study, we show that initiation of Ab diversification in B lymphocytes of mouse spleen leads to formation of a complex between many proteins in DNA repair. We show also that BCR activation, which signals the end of successful SH, reduces interactions between some proteins in the complex and increases other interactions in the complex with varying kinetics. Furthermore, we show increased localization of SH- and CSR-coupled proteins on switch regions of the locus upon initiation of SH/CSR and differential changes in the localization upon BCR signaling, which terminates SH. These findings provide early evidence for a DNA repair complex or complexes that may be of functional significance for carrying out essential roles in SH and/or CSR in B cells.

ETS1 and PAX5 transcription factors recruit AID to Igh DNA

“…Thus, the ChIP sequencing data support that the increases in protein interactions of ETS1 obtained in PLA and co‐IP are coupled to SH/CSR. The increase in localisation of ETS1 at Sμ and Sγ1 upon SH/CSR is in agreement with the reported increases in localisation of E2A and PAX5 at Sμ and Sγ1 .…”

“…BCR stimulation for 0.5 h or 3 h rapidly increased localisation of E2A and PAX5 at both the Sμ and Sγ1 switch region . In contrast, here we found that BCR stimulation for 0.5 h or 3 h reduced the localisation of ETS1 at both switch regions.…”

“…Neither does the finding that ETS1, E2A, PAX5 and IRF‐4 are together with AID on key sequences of the Igh locus exclude that other transcription factors also participate in recruiting AID. We found that the proximity of the protein pairs with ETS1 increased approximately 3‐fold to 6‐fold after 2 days of induced SH/CSR, which is approximately the level of the increase recently reported for the previously analysed AID‐E2A pair . Why were the increases not larger?…”

“…We have recently performed chromatin immunoprecipitation (ChIP) of B cells from mouse spleens with antibodies against AID, PAX5 and E2A and performed next generation sequencing of the precipitated DNA. We found that each of AID, PAX5 and E2A showed localization in ChIP at both Sμ and Sγ1 over the background of input DNA . The localisation of each of AID, PAX5 and E2A at both Sμ and Sγ1 was higher after the two days of stimulation of CSR and SH compared to before the stimulation.…”

“…B cells purified from spleens of C57BL/6 (B6) mice were cross‐linked with 1% formaldehyde and chromatin was prepared as previously described . Chromatin was sheared with covaris‐E 220 to an average chromatin fragment size of 200–500 bp.…”