Footprint analyses of AID cytosine deamination In antigen-stimulated B cells, activation-induced cytidine deaminase (AID) initiates immunoglobulin (Ig) gene somatic hypermutation (SHM), gene conversion (GCV) and class switch recombination (CSR) by inflicting DNA lesions at specific genomic loci via DNA cytosine deamination (converting cytosine to uracil). While essential for the B cells' capacity to produce optimized antibodies, these lesions are also significant threats to genomic integrity in B cells undergoing these processes. In fact, AID-associated oncogenic mutations and chromosomal translocations are frequently observed in tumors of B cell origin. Although it is firmly established that DNA deamination is the mechanistic basis of AID's function during both SHM and CSR, precisely how AID-generated uracils lead to error-prone repair at Ig variable (V) region, but DNA double strand breaks (DSB) at Ig heavy (H) chain switch (S) regions remains an unanswered question. In sum, post-AID events during SHM and CSR are largely unknown. Current models that attempt to explain these events are mostly hypothetical. This major gap of knowledge can be at least partially attributed to the lack of knowledge of the frequency and positions of AID-generated uracils at the Ig loci. This application focuses on the mechanism of CSR and aims to delineate AID footprints at switch regions in stimulated B cells undergoing CSR. My laboratory has established a robust cellular CSR model that allows genetic dissection of CSR using a cell line (CH12F3.2A) capable of efficient cytokine-induced CSR in vitro. We have constructed a large collection of gene knockout strains, including the ones that are defective for uracil repair. Loss of uracil repair causes retention of AID- generated uracils, allowing detection of uracils as C to T mutations after PCR amplification. In addition, we have established an efficient knock-in method to replace the endogenous S regions with short synthetic regions (~1kb) that can be easily PCR amplified, yet still support efficient CSR. These genetic manipulations plus the monoallelic nature of CSR in CH12F3.2A cells permits, for the first time, a delineation of AID footprints on both DNA strands of individual switch regions across its entire length. This will allow characterization of precise switch region footprints of AID activity in individual single cells. These ?snapshots? of AID footprints on single molecules should provide unprecedented insight into the mechanism by which AID-generated uracils are processed into DSBs (which are important CSR immediates) during CSR.
AID is a protein that mutates DNA and trigger error-prone repair to diversify antibody genes. We will study AID 'footprints' in genetically engineered cells that are incapable of repair AID-induced DNA damage to elucidate the in vivo function of AID during the process of antibody isotype switching.
