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 two genetic processes targeting B cell antibody genes: somatic hypermutation and class switch recombination. The first mechanism introduces random point mutations at the N terminal portion of the antibody gene. Mutations coupled to cell selection during the immune response increase the binding affinity of the antibody for the pathogen. The second mechanism changes (via gene recombination) the C terminal portion of the antibody gene, which in turns dictates the strategy used by the immune system to eliminate the pathogen in question. Both somatic hypermutation and switch recombination are carried out by a newly discovered enzyme: Activation-Induced cytidine Deaminase or AID. The AID enzyme 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, which attempt to revert all uracils into cytidines. However, while spontaneous cytidine deamination is faithfully repaired, in a manner not fully understood AID-mediated deamination leads to the formation mutations and DNA breaks, which are at the basis of somatic hypermutation and switch recombination respectively.? The importance of AID in the immune response is highlighted in AID deficient humans and animals, 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 AID-dependent hypermutation. Moreover, AID is not only recruited to antibody genes, but increasing evidence indicate that many other genes, including oncogenes (tumor-inducing genes) can be physiological targets of AID. Because, as stated above, AID activity leads to DNA mutations, oncogenes can become deregulated by AID-targeting, resulting in malignant transformation of AID expressing cells in susceptible individuals. In addition, 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 represent another mechanism whereby cellular oncogenes become deregulated in B cell lymphomas in humans, Burkits and multiple myeloma are prime examples of AID-induced translocations leading to tumor development. These important clinical considerations emphasize the need to understand the molecular pathways that regulate AID expression and activity. This fiscal year we have furthered our understanding of AID regulation as published in two separate manuscripts:? ? i) As discussed above, AID induces DNA lesions at Igh genes which are not only critical for recombination of antibody gene constant domains, but are also involved in chromosomal translocations between the cMyc oncogene and the Igh loci. These chromosomal lesions often lead to B cell tumor development both in humans (Burkitts lymphomas) and in mice (plasmacytomas). However, as cMyc translocations are also present in lymphocytes from healthy individuals, it has remained unclear whether AID activity directly influences the dynamics of B cell transformation. Using a tumor mouse model, Makiko Takizawa, a postdoctoral fellow in the lab, has shown that AID expression levels directly define the incidence of B cell tumor development by determining the number of cMyc translocation-bearing cells emerging during tumor induction. These studies predict that in tumor susceptible individuals the actual number of cMyc-translocated cells has clinical significance. ? ii) Because of AID tumor-inducing activity, B lymphocytes have developed a variety of mechanisms to regulate AID during the immune response. Several studies indicate AID is tightly regulated at the transcriptional level, by post-translational modifications, by interaction with specific cofactors, and by trafficking and compartmentalization. In collaboration with F. Nina Papavasiliou from the Rockefeller University we have recently shown AID is also regulated in a less traditional way: by transcriptional downregulation via a microRNA, miR-155. MicroRNAs are a class of non-coding 1830 nt RNAs that function as post-transcriptional regulators of gene expression by targeting their cognate messenger RNAs for degradation or translational repression. Using BAC transgenic mice expressing a fluorescent version of AID (AID-GFP), we showed that the AID is a direct target for miR-155 negative regulation. In mice where the miR-155 target in the AID mRNA was ablated there was enhanced expression of AID protein during the immune response leading to defects in the maturation of antibody molecules, apparently because of AID overactivity. Importantly, in a paper published in the same issue of Immunity, Michel Nunssenzweig and colleagues also showed that miR-155 deficient mice, B cells carry significantly more chromosomal translocations compared to wild type counterparts, indicating that miR-155 protects the genome against AID mutagenic activity.? ? Currently, we are using deep-sequencing techniques to determine the extent of AID mistargeting in both the mouse and human B cell genomes. Our goal is to pinpoint how AID is recruited to and target non-immunoglobulin genes, like cMyc oncogenes.
