This information is current as Target Somatic Hypermutation Identification of Core DNA Elements That

Somatic hypermutation (SHM) diversifies the V region of Ig genes and underlies the process of affinity maturation, in which B lymphocytes producing high-affinity Abs are generated and selected. SHM is triggered in activated B cells by deamination of deoxycytosine residues mediated by activation-induced deaminase (AID). Whereas mistargeting of SHM and AID results in mutations and DNA damage in many non-Ig genes, they act preferentially at Ig loci. The mechanisms responsible for preferential targeting of SHM and AID activity to Ig loci are poorly understood. Using an assay involving an SHM reporter cassette inserted into the Ig L chain locus (IgL) of chicken DT40 B cells, we have identified a 1.9-kb DIVAC (diversification activator) element derived from chicken IgL that supports high levels of AID-dependent mutation activity. Systematic deletion analysis reveals that targeting activity is spread throughout much of the sequence and identifies two core regions that are particularly critical for function: a 200-bp region within the IgL enhancer, and a 350-bp 39 element. Chromatin immunoprecipitation experiments demonstrate that whereas DIVAC does not alter levels of several epigenetic marks in the mutation cassette, it does increase levels of serine-5 phosphorylated RNA polymerase II in the mutation target region, consistent with an effect on transcriptional elongation/pausing. We propose that multiple, dispersed DNA elements collaborate to recruit and activate the mutational machinery at Ig gene variable regions during SHM. T he activation-induced deaminase (AID), encoded by Aicda, plays a pivotal role in Ig gene diversification in developing and activated B cells. AID is required for somatic hypermutation (SHM) and gene conversion (GCV), which diversify the variable (IgV) region exon of Ig H (IgH) and L (IgL) chain genes, and for class switch recombination (CSR), in which recombination between switch regions leads to the replacement of one IgH C region with another (1–4). AID-mediated IgV region mutation can be used both for creation of the primary Ig repertoire in developing B cells (e.g., GCV in chicken and rabbit) and for Ig gene diversification in activated mature B cells in germinal centers (e.g., SHM in mouse and human), where it can facilitate the generation of B cells expressing high-affinity Abs, a process known as affinity maturation (5). SHM, GCV, and CSR can be thought of as occurring in two phases. In the first, AID is recruited to IgV or Ig switch regions, where it deaminates cytosine residues in the DNA, converting them to uracil. Deamination …