During this fiscal year we continued our studies to characterize the mechanisms that recruit and restrict the activation induced cytidine deaminase (AID) and its accompanying error-prone DNA repair machinery specifically to the imunoglobulin (Ig) genes. Our model system remains the DT40 cell line, a chicken B-cell line constantly undergoing somatic hypermutation (SHM) and Ig gene conversion (GCV), and showing the unique feature of being modifiable by standard gene targeting strategies. We previously identified an evolutionary conserved mutation enhancer element (MEE) that is critical for SHM/GCV. To identify the importance of individual putative transcription factor binding sites, we used gene targeting to generate DT40 clones in which only individual binding sites (or combinations thereof) are mutated in the context of an otherwise largely intact IGL locus. Preliminary evidence suggests that a putative FOXO site is a negative regulator for SHM/GCV whereas a novel binding site (for which the transacting factor is unknown) might be a positive regulator of SHM/GCV. As none of the mutations completely abrogates these Ig gene diversification processes, we a continuing with our attempts to identify additional important sites within our MEE. Furthermore, we initiated studies to address the important question whether MEEs recruit AID specifically to Ig loci, or whether is is actually the error-prone DNA repair that is the unique aspect of these genes. For this purpose we generated stable DT40 clones that express UGI, a specific inhibitor of UNG, a key enzyme that initiates the DNA repair of U:G mismatches. The clones where generated in different parental DT40 lines that contain/lack the MEEs and will allow us to determine whether the recruitment of AID is one critical function of these novel cis-regulatory elements. Overall, our studies will provide a framework to explain the multiple levels at which the targeted introduction of mutations into Ig genes is controlled to protect the rest of the genome from potentially deleterious and cancer promoting alterations.