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

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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. It remains an open question how AID-mediated gene diversification is targeted to the immunoglobulin loci. Here we define a cis-acting sequence, named DIVAC for diversification activator, which is required for hypermutation of the Ig light chain gene and sufficient to activate hypermutation at various non-Ig loci in the DT40 B cell line. DIVAC is composed of multiple interacting sequences and able to work over considerable distances both upstream and downstream of its target gene. This work provides the first conclusive evidence that AID-mediated gene diversification is targeted to the Ig loci by cis-acting sequences. The conservation of AID-mediated Ig gene diversification during vertebrate evolution suggests that DIVACs also play a role in gene conversion, hypermutation, and switch recombination in mammalian B cells. The findings should be of general interest not only for molecular immunology and the pathogenesis of B cell lymphomas but also the whole field of biology as a unique example of how locus-specific gene diversification is controlled. The described experimental system offers unique advantages to further clarify the molecular mechanism of DIVAC.