A cis-Acting Diversification Activator Both Necessary and Sufficient for AID-Mediated Hypermutation

Abstract

Hypermutation of the immunoglobulin (Ig) genes requires Activation Induced cytidine Deaminase (AID) and transcription, but it remains unclear why other transcribed genes of B cells do not mutate. We describe a reporter transgene crippled by hypermutation when inserted into or near the Ig light chain (IgL) locus of the DT40 B cell line yet stably expressed when inserted into other chromosomal positions. Step-wise deletions of the IgL locus revealed that a sequence extending for 9.8 kilobases downstream of the IgL transcription start site confers the hypermutation activity. This sequence, named DIVAC for diversification activator, efficiently activates hypermutation when inserted at non-Ig loci. The results significantly extend previously reported findings on AID-mediated gene diversification. They show by both deletion and insertion analyses that cis-acting sequences predispose neighboring transcription units to hypermutation.

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

“…However, none of the known transcriptional control elements in mouse B cells appears to be absolutely required for SHM, suggesting a redundancy or unidentified elements (48). Recent studies discovered novel cis-acting elements in the IgL locus of the DT40 chicken B cell line that are both essential and sufficient for AIDmediated sequence diversification (59,60). In addition, it was shown that CAGGTG cis elements in the context of Ig enhancers were sufficient and essential to target mutations to a nearby transcribed gene in transgenic DT40 cell lines (61).…”

Balancing AID and DNA repair during somatic hypermutation

“…Thus far, germline deletion of Ig enhancers has resulted in either normal SHM, or impaired SHM accompanied by reduced transcription. Interestingly, recent studies in the chicken DT40 B-cell line identified a large DNA region 3 0 of the Igl constant region with apparent SHM targeting activity [56,57].…”

Technologies of directed protein evolution in vivo

“…In the future, transgene diversification in DT40 may be accomplished by addition of gene conversion on top of hypermutation if homologous non-immunoglobulin conversion donor sequences are inserted upstream of the transgene, thus resembling the natural process of antibody diversity generation in avian species. Studies on AIDdependent gene diversification have revealed that natural restriction of hypermutation and gene conversion activities to the immunoglobulin loci is controlled by cis-regulatory sequences usually overlapping with immunoglobulin enhancers [62][63][64][65]. These findings pave the way to creation of non B cell hypermutating cell lines as platforms for directed protein evolution by means of modifying a cell line of interest.…”