Identificaiton and Characterization of Cis-acting DNA Elements Involved in Targeting of Somatic Hypermutation

Diversification of immunoglobulin (Ig) genes in the adaptive immune system generates a large repertoire of antibody receptors required for recognition and elimination of pathogen- associated antigens. Ig genes are diversified and assembled into functional units by V(D)J recombination during B lymphocyte development and are further altered by somatic hypermutation (SI-1M) upon activation. SLIM introduces point mutations at a high rate in the variable region of the Ig genes and these mutations are triggered by targeted deamination of cytosine residues by activation-induced deaminase (AID). Although mis- targeting of AID and SHM can be detrimental to genome integrity, the mechanisms that target SHM specifically to Ig loci are poorly understood.;Here I describe the identification of cis-acting DNA elements responsible for targeting SHM to a reporter transgene inserted into the chicken Ig light chain locus. Using a SHM assay in the chicken B cell line DT40, I have isolated an 1805 base pair (bp) fragment that supports high levels of mutational activity. Systematic deletions of this fragment revealed low levels of targeting activity spread throughout the sequence and two core motifs essential for function; a 150 bp region within the IgL enhancer, and a 350 bp 3' element. These two regions act in a synergistic manner where deletion of either one causes a drastic reduction in SHM activity. The targeting mechanism of the cis-acting DNA elements was also examined by assaying differential protein recruitment in chromatin immunoprecipitation (ChIP) experiments, as well as subnuclear localization of SHM-targeted loci by three-dimensional fluorescent in situ hybridization (3D FISH). Although nuclear localization of alleles was not changed in the presence of SHM-targeting DNA motifs, the ChIP experiments showed that these DNA elements sere necessary for the recruitment of AID. In the end I was not able to identify any particular DNA motif or trans- factor crucial for SHM targeting, but these experiments provide a solid foundation to uncover the mechanism of SHM targeting in future studies.