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

Abstract

Secondary diversification of the Ig repertoire occurs through somatic hypermutation (SHM), gene conversion (GCV), and class switch recombination (CSR)—three processes that are initiated by activation‐induced cytidine deaminase (AID). AID targets Ig genes at orders of magnitude higher than the rest of the genome, but the basis for this specificity is poorly understood. We have previously demonstrated that enhancers and enhancer‐like sequences from Ig genes are capable of stimulating SHM of neighboring genes in a capacity distinct from their roles in increasing transcription. Here, we use an in vitro proteomics approach to identify E‐box, MEF2, Ets, and Ikaros transcription factor family members as potential binders of these enhancers. ChIP assays in the hypermutating Ramos B cell line confirmed that many of these factors bound the endogenous Igλ enhancer and/or the IgH intronic enhancer (Eμ) in vivo. Further investigation using SHM reporter assays identified binding sites for E2A and MEF2B in Eμ and demonstrated an association between loss of factor binding and decreases in the SHM stimulating activity of Eμ mutants. Our results provide novel insights into trans‐acting factors that dictate SHM targeting and link their activity to specific DNA binding sites within Ig enhancers.

Helix-Loop-Helix Proteins in Adaptive Immune Development

“…E2A/E2-2 DKO mice have impaired CSR to IgE, due to loss of activation of the 3’RR enhancer and IgE GLT ( 85 ). Further, E-box binding sites within Ig enhancers promote efficient SHM ( 96 – 99 ). E2A may help direct AID to DNA, and genome wide E2A occupancy is associated with AID targeting ( 99 ).…”

“…Further, E-box binding sites within Ig enhancers promote efficient SHM ( 96 – 99 ). E2A may help direct AID to DNA, and genome wide E2A occupancy is associated with AID targeting ( 99 ). Finally, we suggest that E2A proteins act to promote CSR and SHM by initiating loop extrusion across the switch regions and V gene segments and note that E2A likely plays an additional role in promoting phase separated droplets to orchestrate CSR and SHM.…”

Common light chain chickens produce human antibodies of high affinity and broad epitope coverage for the engineering of bispecifics

“…This location also ensures that gene conversion in the B cell lineage will be focused on the light chain transgene. 40,41 The variable region is a pre-rearranged VK3-15/JK1 region made by joining the germline gene segments together, with no chewing back of the ends. This rearranged sequence is expressed in humans, as it is found in the National Center for Biotechnology Information's Expressed Sequence Tags database.…”

Disease-associated CTNNBL1 mutation impairs somatic hypermutation by decreasing nuclear AID

“…20). Comparisons were made by studying other EBV-immortalized B cell lines derived from 3 different healthy donors, an AID-deficient patient (AID -/-), and a uracil N-glycosylase-deficient (UNG-deficient) patient (UNG -/-), as well as unmodified CTNNBL1 M/M Ramos B cells and CRISPR/ Cas9-edited AID -/-Ramos B cells that lack AID expression ( Fig 23,24). We found that CTNNBL1 expression in both the patient EBV BLCLs and the CTNNBL1 466 V/V Ramos B cells was similar to expression in control EBV-derived BLCLs and Ramos B cells, showing that the M466V missense mutation does not appear to alter the CTNNBL1 cellular pool ( Figure 2).…”