B lymphocytes are the immune system cells that recognize and dispose pathogens such as viruses and bacteria though special receptors on their cell surface known as antibodies. How the immune system recognizes and eliminates pathogens via antibody molecules depends to a great extent on three genetic processes targeting B cell antibody genes: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles heavy (H) and light (L) chain antibody genes from variable (V), diversity (D), and joining (J) gene segments. This recombination, which is catalyzed by the RAG1 and RAG2 complex, is tightly regulated during ontogeny. Somatic hypermutation on the other hand introduces random point mutations at the N terminal portion of the antibody gene in activated, mature B cells during the immune response. Mutations coupled to cell selection during increase the binding affinity of the antibody for the pathogen. Lastly, CSR changes the C terminal portion of the antibody gene to diversify how pathogens are eliminated. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced cytidine Deaminase (AID). This protein modifies the chemical nature of DNA, converting cytidines into another base called uracil, a process known as cytidine deamination. Because uracils are mutagenic, AID activity attracts a plethora of repair enzymes to the immunoglobulin loci. These enzymes can either faithfully repair the DNA lesions, or convert them into single or double strand breaks, which are intermediate to hypermutation and CSR respectively. The importance of RAGs and AID in the immune response is highlighted in humans and animals deficient for these enzymes, which are highly susceptible to infection and exhibit gut flora-dependent hyperplasia of intestinal villi. Conversely, complex diseases such as autoimmunity have long been associated with RAG and AID-dependent activity. Moreover, both RAGs and AID are promiscuous by nature, in that they can also target non-immunoglobulin genes, including oncogenes (tumor-inducing genes). This off-targeting activity can lead to DNA mutations and oncogene deregulation, resulting in malignant transformation. In addition, RAG and AID-mediated DNA breaks can also recombine or bring oncogenes into close proximity of the immunoglobulin loci, a chromosomal irregularity known as a translocation. Chromosomal translocations are responsible for the formation of B cell lymphomas in humans. Burkits and multiple myeloma are prime examples. These arguments underscore the important of unraveling how RAG and AID activity is regulated under normal conditions and deregulated during tumorigenesis. This fiscal year we have furthered our understanding of RAG and AID activities in two separate studies: i) The RAG1 and RAG2 enzymes play unique roles in the assembly of V, D, and J segments. RAG1 contains domains that interact with recombination signal sequences of antigen receptor genes, as well as key amino-acids that are essential for DNA cleavage. The functions of RAG2 are less well understood. It interacts with RAG1, enhances the specificity and affinity of DNA binding, and is essential for catalysis. While RAG2 has no detectable DNA binding activity by itself, it contains a protein domain that forms a binding pocket specific for chromatin (the protein scaffold around which DNA is packed). To determine exactly how specific RAG biding is for antigen receptor genes, we made use of a newlydeveloped high-throughput sequencing technique. We found that RAG2 binds in vivo across the entire genome (24,000 sites) in a pattern that would be expected based on its ability to bind to the chromatin scaffold. This extraordinary promiscuity begs the question of how recombination is targeted predominantly to antigen receptor genes. Since the RAG1 and RAG2 enzymes work in pairs, one obvious mechanism would be a tight regulation in RAG1 DNA recruitment. To test this idea, we are currently investigating RAG1 binding in developing T and B cells form mice and humans by the same sequencing technique. We hope these studies will help explain on the one hand the nature of RAGs preference for antigen receptor genes, and on the other how RAGs promiscuity promotes the development of chromosomal translocations in mouse and human leukemias. A manuscript relating these findings was recently published in the journal Cell. ii) In a second set of experiments, we have also mapped AID binding in activated B cells. AID activity only occurs at sites of single-stranded DNA, which is displaced and stabilized by RNA polymerases during transcription. Our sequencing analysis shows AID recruitment to an astonishing 6,000 genes in B-lymphocytes. Curiously, within these genes AID is maximally recruited at sites where the polymerase is stalled. We believe that this feature provides AID with a window of opportunity to attack DNA. Another prominent feature of our results is that AID recruitment to oncogenes in some instances exceeds that measured at antigen receptor gene loci. This is unexpected because the magnitude of the mutation load at the latter is between 10-100 fold higher than the former. Thus, AID activity cannot be fully explained on the basis of AID binding densities alone. What is then the nature of AID specificity? Several lines of evidence indicate that the single stranded DNA binding protein RPA helps AID by further stabilizing single stranded DNA. When we mapped RPA in the mouse genome we found that in contrast to AID, the helper protein is only recruited to antigen receptor genes. The conclusion therefore is that RPA is likely the enzyme that provides specificity. The manuscript describing these results is currently under review.
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