Targeting Of Somatic Hypermutation By immunoglobulin Enhancer And Enhancer-Like Sequences

Abstract

Immunoglobulin gene enhancers have a conserved function in targeting somatic hypermutation to immunoglobulin genes, thereby supporting the production of high affinity antibodies.

Abstract

Somatic hypermutation (SH) generates point mutations within rearranged immunoglobulin ( Ig) genes of activated B cells, providing genetic diversity for the affinity maturation of antibodies. SH requires the activation-induced cytidine deaminase (AID) protein and transcription of the mutation target sequence, but how the Ig gene specificity of mutations is achieved has remained elusive. We show here using a sensitive and carefully controlled assay that the Ig enhancers strongly activate SH in neighboring genes even though their stimulation of transcription is negligible. Mutations in certain E-box, NFκB, MEF2, or Ets family binding sites—known to be important for the transcriptional role of Ig enhancers—impair or abolish the activity. Full activation of SH typically requires a combination of multiple Ig enhancer and enhancer-like elements. The mechanism is evolutionarily conserved, as mammalian Ig lambda and Ig heavy chain intron enhancers efficiently stimulate hypermutation in chicken cells. Our results demonstrate a novel regulatory function for Ig enhancers, indicating that they either recruit AID or alter the accessibility of the nearby transcription units.

Author Summary

During the B cell immune response, immunoglobulin ( Ig) genes are subject to a unique mutation process known as somatic hypermutation that allows the immune system to generate high-affinity antibodies. Somatic hypermutation preferentially affects Ig genes, relative to other genes, and this is important in preventing catastrophic levels of general genomic mutations that could lead to B cell cancers. We hypothesized that this preferential targeting of somatic hypermutation is assisted by specific DNA sequences in or near Ig genes that focus the action of the mutation machinery on those genes. In this study, we show that Ig genes across species—from human, mouse, and chicken—do indeed contain such mutation targeting sequences and that they coincide with transcriptional regulatory regions known as enhancers. We show that combinations of Ig enhancers cooperate to achieve strong mutation targeting and that this action depends on well-known transcription factor binding sites in these enhancer elements. Our findings establish an evolutionarily conserved function for enhancers in somatic hypermutation targeting, which operates by a mechanism distinct from the conventional enhancer function of increasing levels of transcription. We propose that combinations of Ig enhancers target somatic mutation to Ig genes by recruiting the mutation machinery and/or by making the Ig genes better substrates for mutation.