The goal of this proposal is to identify the molecular mechanisms responsible for targeting somatic hypermutation (SHM) to antibody variable regions. We and others have shown that activation induced cytidine deaminase (AID) is probably the only B cell specific protein that is required for SHM, but the mechanisms that target AID and the other ubiquitous factors involved in SHM to cytidines in hot spots in the V region have not been identified. We will determine if other macromolecules are associated with AID or are required to activate its enzymatic activity and examine their role in targeting AID to the heavy chain V region. In the previous grant period we have shown that this targeting is associated with the hyperacetylation of the histones associated with the V region but not with the C region that is protected from mutation. We propose to extend these findings by identifying any other chromatin modifications that occur and by determining the role of these modifications in remodeling chromatin to make the V region accessible to mutating factors or in recruiting the mutational factors to the V region. We will also identify the histone acetylases and deacetylases that carry out these modifications and use RNAi or gene replacement to determine if these enzymes are required for targeting of SHM to hot spots and to the V region. We will use gene replacement of mutated and deleted regulatory and other DNA elements to identify the cis-acting sequences in the Ig gene that play a role in the targeting of mutation either through the specific recruitment of the mutational factors or by mediating the changes in chromatin structure. Most of these experiments will be carried out Burkitt's lymphoma cells lines but we will confirm the major findings with normal B cells.
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