Target DNA Sequence Directly Regulates the Frequency of Activation-Induced Deaminase-Dependent Mutations

Abstract

Activation-induced deaminase (AID) catalyses class switch recombination (CSR) and somatic hypermutation (SHM) in B lymphocytes to enhance Ab diversity. CSR involves breaking and rejoining highly repetitive switch (S) regions in the IgH (Igh) locus. S regions appear to be preferential targets of AID. To determine whether S region sequence per se, independent of Igh cis regulatory elements, can influence AID targeting efficiency and mutation frequency, we established a knock-in mouse model by inserting a core Sγ1 region into the first intron of proto-oncogene Bcl6, which is a non-Ig target of SHM. We found that the mutation frequency of the inserted Sγ1 region was dramatically higher than that of the adjacent Bcl6 endogenous sequence. Mechanistically, S region-enhanced SHM was associated with increased recruitment of AID and RNA polymerase II, together with Spt5, albeit to a lesser extent. Our studies demonstrate that target DNA sequences influence mutation frequency via regulating AID recruitment. We propose that the nucleotide sequence preference may serve as an additional layer of AID regulation by restricting its mutagenic activity to specific sequences despite the observation that AID has the potential to access the genome widely.

Efficient AID targeting of switch regions is not sufficient for optimal class switch recombination

“…Thus, tethering of AID appears as an intrinsic property of transcribed S regions. However, by contrast to the Bcl6 model where regions flanking the core Sg1 were free of AID targeting 20 , we found a sustained high SHM rate throughout the 2-kb-long Jk region that cannot be fully related to the fact that Igk is a much better AID target than Bcl6 (ref. 29).…”

“…While Igk locus enhancers are likely responsible for AID recruitment onto both promoters of the KIKS locus, the extended SHM shows that the inserted S region structure per se modulates the pattern of AID recruitment. These results are reminiscent to the robust AID targeting recently reported in a model in which the core Sg1 region was inserted in the Bcl6 locus 20 . Thus, tethering of AID appears as an intrinsic property of transcribed S regions.…”

“…While such defects of the potential 'CSR factory' components are absent in our model (where normal IgH CSR occurs), the KIKS locus might then simply demonstrate a defective recruitment of the knocked-in S regions into such CSR factories. Whether the Igk locus may intrinsically dictates differential outcome of AID-induced lesions, as it was recently demonstrated when S regions were knocked-in in other loci 20,32 , is another possibility. But the presence of ISDs, few S-S junctions, 53BP1 recruitment and gH2AX foci, proves that beside AIDinduced single-strand mutations, DSBs occur at the KIKS locus but are not appropriately handled for efficient synapsis and junction of distant breaks.…”

“…This high mutation rate at long distances from the promoter likely involved an increased and/or extended AID recruitment on S sequences containing highly repetitive RGYW motifs, as recently demonstrated for Sg1 (ref. 20). However, a similarly frequent SHM affected the Jk5 intron from the same rearranged loci (8.10 mutations per kb).…”

AID-Initiated DNA Lesions Are Differentially Processed in Distinct B Cell Populations

“…We recently demonstrated that AID’s mutagenic activity depends on its target sequence at a non-Ig locus, Bcl6 (39). Thus, we sought to test whether target DNA sequences could disrupt the protective mechanism by modifying the processing manner of AID-initiated lesions at the c-myc locus.…”