B lymphocytes are cells of the immune system that recognize and get rid of viruses and bacteria though special receptors on their cell surface called antibodies. The affinity and specificity of these receptors for pathogens depends to a great extent on three genetic processes that assemble and refine these proteins: V(D)J recombination, somatic hypermutation, and class switch recombination (CSR). The first mechanism assembles the antibody gene by combining related DNA segments. The recombination is catalyzed by the RAG1 and RAG2 enzymes. Somatic hypermutation on the other hand introduces random point mutations to increase the binding affinity of the antibody for the pathogen in question. Lastly, CSR introduces further changes to dictate how pathogens are eliminated. Both somatic hypermutation and switch recombination are carried out by a B cell specific enzyme: Activation-Induced Cytidine Deaminase (AID). 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, 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. A typical chromosomal irregularity induced by RAGs and AID (chromosomal translocations) are responsible for the formation of B cell lymphomas in humans. Burkitt lymphomas and multiple myeloma are prime examples. Thus, unraveling how RAG and AID activities are regulated under normal conditions and deregulated during tumorigenesis is key. This fiscal year we have furthered our understanding of AID biology in several important ways: i) Human Burkitt lymphomas (BL) are divided into two main clinical variants: the endemic form, affecting African children infected with malaria;and the sporadic form, distributed across the rest of the world. However, while sporadic translocations decapitate the Myc oncogene regulatory elements that control its expression, most endemic events occur hundreds of kilobases away from Myc. The origin of these rearrangements and how they deregulate oncogenes at such distances has been unclear. To solve this problem we have recapitulated endemic BL-like translocations in mouse plasmacytomas. In the study published in the Proceedings of the National Academy of Sciences we show that long-range deregulation of Myc is directly proportional to the physical interaction of immunoglobulin regulatory domains with translocated sites. Our studies thus uncover the extent of remodeling by these regulators and provide a rationale to the transformation of B cells in endemic Burkitt lymphomas. 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. We therefore 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 published in Nature 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.

Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2012
Total Cost
$3,102,470
Indirect Cost
Name
National Institute of Arthritis and Musculoskeletal and Skin Diseases
Department
Type
DUNS #
City
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Zip Code
Juan, Aster H; Wang, Stan; Ko, Kyung Dae et al. (2017) Roles of H3K27me2 and H3K27me3 Examined during Fate Specification of Embryonic Stem Cells. Cell Rep 18:297
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