Protecting the Genome: Mechanisms Targeting Somatic Hypermutation

Somatic hypermutation (SHM) introduces point mutations into immunoglobulin (Ig) genes to support the generation of high affinity antibodies through affinity maturation. SHM is quite dangerous, having been shown to affect many non-Ig genes in germinal center and malignant B cells. Nonetheless, the reaction exhibits a strong preference for Ig loci over non-Ig loci. The targeting mechanism responsible for this preference has remained elusive despite many years of study. We and others have used the chicken Ig light chain (IgL) locus and the chicken Dt40 B cell line as a model system for the study of SHM targeting. This system has several advantages, including efficient homologous recombination and a compact IgL locus, making the search for targeting sequences easier than in the large, complex mammalian Ig loci. We and others have reported that sequences downstream of the chicken IgL constant region are cis-acting SHM targeting sequences, which we refer to as Diversification Activator (DIVAC). Dissection of the chicken DIVAC region reveals that targeting activity is dispersed among numerous sequence elements that function in an additive, synergistic, and/or redundant manner. We find that the E-box motif (CANNTG) is critical for DIVAC function, but has no detectable DIVAC activity unless embedded in the appropriate sequence context. We have recently developed a more sensitive mutation reporter assay in Dt40 cells that allows us to assess the targeting activity of short DNA sequence elements. Two "core" portions of the chicken DIVAC region, both evolutionarily conserved among avian species, have particularly strong SHM targeting activity: the IgL enhancer, and a downstream region rich in transcription factor binding sites characteristic of Ig enhancers. Remarkably, some human and mouse Ig locus enhancers have even stronger DIVAC function in our Dt40 assay than the chicken core sequences. In the mouse Igλ locus, where the canonical enhancers have relatively weak SHM targeting activity, strongly active "shadow enhancers" work synergistically with the canonical enhancers to create powerful DIVAC elements. DIVAC fragments enhance SHM in a position- and orientation-independent manner, and deletion or mutation of certain E-box, NFκB, Mef2, PU.1, or Ets family binding sites – all known to be important for the transcriptional role of Ig enhancers – impair or abolish DIVAC activity. Importantly, the increase in SHM mediated by DIVAC is not mediated by an increase in transcription. We conclude that Ig enhancers and shadow enhancers are the central components of the cis-acting DNA sequences that target SHM to Ig loci, that targeting relies on cooperation between multiple well-known transcription factor binding motifs, and that the targeting mechanism is likely to be conserved from birds to mammals. We are currently investigating whether these insights can be used to understand the widespread mistargeting of SHM that occurs in both normal and malignant B cells. Since multiple myeloma (MM) cells can be induced to express activation-induced cytidine deaminase (AID), we are also studying the targeting of AID in the MM genome and how AID might contribute to the progression of the disease.