A Critical Context-Dependent Role for E Boxes in the Targeting of Somatic Hypermutation

Abstract

Secondary B cell repertoire diversification occurs by somatic hypermutation (SHM) in germinal centers following antigen stimulation. In SHM, activation-induced cytidine deaminase (AID) mutates the variable region of the immunoglobulin (Ig) genes to increase the affinity of antibodies. Although somatic hypermutation (SHM) acts primarily at Ig loci, low levels of off-target mutation can result in oncogenic DNA damage, illustrating the importance of understanding SHM targeting mechanisms. A candidate targeting motif is the E box, a short DNA sequence (CANNTG) found abundantly in the genome and in many SHM target genes. Using a reporter assay in chicken DT40 B cells, we previously identified a 1928-bp portion of the chicken IgL locus capable of supporting robust SHM. Here, we demonstrate that mutation of all 20 E boxes in this fragment reduces SHM targeting activity by 90%, and that mutation of subsets of E boxes reveals a functional hierarchy in which E boxes within "core" targeting regions are of greatest importance. Strikingly, when the sequence and spacing of the 20 E boxes is preserved but surrounding sequences are altered, SHM targeting activity is eliminated. Hence, while E boxes are vital SHM targeting elements, their function is completely dependent on their surrounding sequence context. These results suggest an intimate cooperation between E boxes and other sequence motifs in SHM targeting to Ig loci and perhaps also in restricting mistargeting to certain non-Ig loci.

Transcription factor binding at Ig enhancers is linked to somatic hypermutation targeting

“…Previous studies in mice using deletions of enhancers in Ig transgenes or at the endogenous Ig loci often had difficulty dissociating effects on SHM and transcription . Our previous approaches circumvented these issues by utilizing a knock‐in SHM reporter construct powered by a strong RSV promoter that masked the effect of test sequences on transcription . Our results suggested the possibility that different TF binding sites (and their accompanying TFs) mediated aspects of transcription enhancement and mutation enhancement to varying degrees.…”

“…The abundance of bHLH factors (Supporting Information Table 1A-D) was notable given the numerous studies implicating roles for E-boxes in SHM targeting [50,57,58]. E2A and TFAP4 were the most well-represented bHLH family members in the two Ramos extract conditions (Supporting Information Table 1A and B).…”

Generation and repair of AID-initiated DNA lesions in B lymphocytes

“…Thus, this study clarified a long-standing controversy over the role of cis elements in targeting SHM [59]. Furthermore, novel cis -acting elements were identified in the Igl locus of the DT40 chicken B cell line that directly regulate AID-mediated sequence diversification [87-90]. In addition, it was shown that the E box motif CAGGTG in the context of Ig enhancers were sufficient and essential to target mutations to a nearby transcribed gene in transgenic DT40 cell lines [91].…”

“…In addition, it was shown that the E box motif CAGGTG in the context of Ig enhancers were sufficient and essential to target mutations to a nearby transcribed gene in transgenic DT40 cell lines [91]. In this regard, it has been reported that the context sequences of E box motifs play a more important role in targeting SHM [87]. Overall, these studies highlight an important role of cis regulatory elements in AID targeting to Ig loci [77].…”

Molecular Mechanisms of AID Function

“…In SHM, however, the transcribed V region does not form an R-loop, but generates an abundant single-stranded DNA structure [214,215]. Moreover, SHM is enhanced by the introduction of the transcription factor E2A-binding sequence CAGCTC (E-box motif), which may be prone to forming secondary non-B DNA structures, into the V region or the GFP gene [218][219][220]. Although these triplets are not consecutively arranged as a triplet track, their abundant clusters are frequent targets of mutation in the V region.…”