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) To date, the study of chromosomal aberrations has been primarily limited to events identified in tumors and tumor cell lines. Although we have learned a great deal about the importance of genomic rearrangements in cancer, it has not been possible to develop an understanding of the cellular and molecular requirements that govern their genesis. To examine genomic rearrangements in primary cells in short term cultures (under non-selective conditions), we developed a technique to catalog these events by deep sequencing, TC-seq. Our results and analysis reveal the importance of transcription and physical proximity in recombinogenesis, and identifies hotspots for AID-mediated translocations in mature B cells. These findings are published in the 2011 September issue of Cell. ii) The origin of lymphocyte chromosomal translocations has been ascribed to selection of random rearrangements, targeted DNA damage (RAG and AID activity), or frequent nuclear interactions between translocation partners. However, the individual contributions of these processes have not been measured directly or at a large scale. In a second set of experiments we have examined the role of global nuclear architecture and frequency of DNA damage in the genesis of chromosomal translocations by measuring these parameters simultaneously in cultured B lymphocytes. In the absence of recurrent DNA damage, translocation between Igh or c-myc and all other genes is directly related to their contact frequency. In contrast, translocations associated with recurrent site-directed DNA damage are proportional to the rate of DNA double strand break formation, as measured by accumulation of replication protein A (RPA) at the site of damage. Our findings demonstrate that translocations are not simply random events but that nuclear organization determines which gene pairs translocate and that DNA break formation governs the rate of recurrent chromosomal rearrangements. The manuscript describing these results is currently under review.

Project Start
Project End
Budget Start
Budget End
Support Year
7
Fiscal Year
2011
Total Cost
$3,274,017
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
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Zip Code
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