Mouse Model of Endemic Burkitt Translocations Reveals the Long-Range Boundaries of Ig-Mediated Oncogene Deregulation
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Mouse model of endemic Burkitt translocations reveals the long-range boundaries of Ig-mediated oncogene deregulation Alexander L. Kovalchuka,1, Camilo Ansarah-Sobrinhob,1,Ofir Hakimc,1, Wolfgang Reschb, Helena Tolarováb, Wendy Duboisd, Arito Yamaneb, Makiko Takizawab, Isaac Kleinf, Gordon L. Hagerc, Herbert C. Morse IIIa, Michael Potterd,1, Michel C. Nussenzweige,f,2, and Rafael Casellasb,g,2 aLaboratory of Immunogenetics, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Rockville, MD 20852; bGenomics and Immunity, National Institute of Arthritis and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD 20892; cLaboratory of Receptor Biology and Gene Expression, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; dLaboratory of Cancer Biology and Genetics, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892; eLaboratory of Molecular Immunology, and fHoward Hughes Medical Institute, The Rockefeller University, New York, NY 10065; and gCenter of Cancer Research, National Cancer Institute, National Institutes of Health, Bethesda, MD 20892 Edited by Klaus Rajewsky, Max-Delbrück-Center for Molecular Medicine, Berlin, Germany, and approved May 22, 2012 (received for review January 5, 2012) Human Burkitt lymphomas are divided into two main clinical human Burkitt lymphomas (BLs), mouse plasmacytomas (PCTs), variants: the endemic form, affecting African children infected with and rat immunocytomas (15, 16). malaria and the Epstein-Barr virus, and the sporadic form, distrib- Human BLs are divided into two main clinical variants: the uted across the rest of the world. However, whereas sporadic endemic form, affecting African children infected with malaria translocations decapitate Myc from 5′ proximal regulatory ele- and the Epstein-Barr virus, and the sporadic form, distributed ments, most endemic events occur hundreds of kilobases away across the rest of the world (17). Although both tumors are from Myc. The origin of these rearrangements and how they de- driven primarily by Igh/Myc rearrangements (18), the precise regulate oncogenes at such distances remain unclear. We here location of the breakpoints differs in the two subsets. In sporadic recapitulate endemic Burkitt lymphoma-like translocations in BLs (and mouse PCTs), translocations occur within Myc exon – ′ IMMUNOLOGY plasmacytomas from uracil N-glycosylase and activation-induced 1 intron 1, and as such they decapitate the oncogene from its 5 cytidine deaminase-deficient mice. Mapping of translocation regulatory elements (19). These canonical rearrangements result – breakpoints using an acetylated histone H3 lysine 9 chromatin from AID activity at IgS (during CSR) as well as at Myc (20 23). immunoprecipitation sequencing approach reveals Igh fusions up In contrast, endemic BL translocations are noncanonical in that ∼ they map hundreds of kilobases upstream from Myc basal to 350 kb upstream of Myc or the related oncogene Mycn.A – comprehensive analysis of epigenetic marks, PolII recruitment, promoters (24 28). Because these lesions leave Myc upstream and transcription in tumor cells demonstrates that the 3′ Igh en- promoter elements intact, it is unclear how they bring about hancer (Eα) vastly remodels ∼450 kb of chromatin into translo- oncogene deregulation. The etiology of these events is also un- cated sequences, leading to significant polymerase occupancy known, primarily because to date endemic BL-like translocations and constitutive oncogene expression. We show that this long- have not been systematically reproduced in the mouse. Because of their proximity to V(D)J genes, endemic BL range epigenetic reprogramming is directly proportional to the rearrangements have been proposed to arise as by-products of physical interaction of Eα with translocated sites. Our studies thus SHM but not of CSR (19, 27, 29). On the basis of this idea and in uncover the extent of epigenetic remodeling by Ig 3′ enhancers an attempt to recapitulate endemic translocations in the mouse, and provide a rationale for the long-range deregulation of translo- we combined a UNG null allele (30) with the Bcl-xL transgene − − cated oncogenes in endemic Burkitt lymphomas. The data also shed that produces plasma cell tumors (31). In UNG / B cells, AID light on the origin of endemic-like chromosomal rearrangements. hypermutation remains active as evidenced by high levels of C-to-T mutation (32). In contrast, CSR (32, 33) and canonical chromosome conformation capture | chromosome translocations | Igh/Myc translocations (34) are impaired because uracyl deglyco- activation-induced cytidine deamination | epigenetics sylation is an important intermediate step in double-strand DNA − − (dsDNA) break formation (12). The UNG / mouse thus might be uring B-cell responses to antigen, somatic hypermutation an ideal model to recapitulate rare, noncanonical translocations. D(SHM) introduces point mutations at variable (V) genes, a Consistent with a primary role for AID and UNG in the CSR process that, when coupled to clonal selection, can enhance the breaks mediating Igh/Myc translocations (20, 23, 34), we find that, antibody’saffinity for the antigen (1, 2). Class switch re- in the absence of UNG, the incidence of B-cell transformation is − − combination (CSR) is a second type of B-lymphocyte–specific markedly delayed. Strikingly, the vast majority of UNG / and − − DNA modification reaction that results in diversification of an- AID / tumors that do develop carry endemic BL-like trans- tibody effector functions by replacing the Igh constant domain locations with breakpoints accumulating over hundreds of kilo- Cμ with one of a set of downstream genes: Cγ,Cɛ,orCα (3–5). bases upstream of Myc or its homolog the Mycn oncogene. Both SHM and CSR are initiated by activation-induced cytidine Analysis of these tumors by deep-sequencing techniques sheds deaminase (AID) (6, 7), a B-cell–specific enzyme that deami- nates cytidine residues to produce uracils (8). Multiple repair pathways including uracil DNA glycosylase (UNG), mismatch Author contributions: A.L.K., G.L.H., H.C.M., M.C.N., and R.C. designed research; A.L.K., repair enzymes, and error-prone polymerases process U:G mis- C.A.-S., O.H., H.T., W.D., A.Y., M.T., and I.K. performed research; W.R., M.P., and R.C. matches into SHM or DNA double-strand breaks intermediate analyzed data; and M.P. and R.C. wrote the paper. to CSR (3–5, 9). The authors declare no conflict of interest. Although most AID-mediated lesions result in SHM or CSR, This article is a PNAS Direct Submission. they can occasionally generate chromosome deletions, trans- 1A.L.K., C.A.-S., O.H., and M.P. contributed equally to this work. – locations, and aneuploidy (10 14). These genomic abnormalities 2To whom correspondence may be addressed. E-mail: [email protected] or nussen@ have the potential to deregulate oncogenes and thereby promote rockefeller.edu. cell transformation. Perhaps the best-studied example is the This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. translocation that juxtaposes Igh to the Myc proto-oncogene in 1073/pnas.1200106109/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1200106109 PNAS Early Edition | 1of6 Downloaded by guest on October 2, 2021 light on both the origin of endemic-like translocations and the mouse lines for canonical T(12;15)s by long-distance PCR. As genomic features that impact their tumor capacity. previously described in wild-type mice (31), UNG+/+Bcl-xL and − UNG+/ Bcl-xL accumulated T(12:15)s following pristane injection. Results From 264 DNA samples analyzed for each strain, we isolated 39 − − +/+ UNG / PCTs Lack Canonical Igh/Myc Translocations. UNG deficiency (14.8%) and 36 (13.6%) distinct translocations from UNG +/− has been shown to profoundly impair CSR and canonical Igh/ Bcl-xL and UNG Bcl-xL mice, respectively (Fig. 1C). In con- Myc translocations in LPS+IL-4–stimulated B cells in vitro (34) trast, we failed to detect canonical T(12;15)s in any of the 216 −/− without significantly affecting SHM (32, 33) (Fig. S1). To in- DNAs isolated from UNG Bcl-xL lymphoid tissues or oil vestigate how this phenotype might impact the genesis of chro- granulomas (n = 216, Fig. 1C). This discrepancy, which is con- − − − mosomal translocations in vivo, UNG+/+, UNG+/ , and UNG / sistent with previous ex vivo translocation studies (34), may be mice bearing the Bcl-xL transgene were given one (day 0) or two explained by the fact that dsDNA break formation at switch (days 0 and 60) injections of the mineral oil pristane in the regions is decreased in the absence of UNG (35) (Fig. S2). The peritoneal cavity. Tumor development, which occurs in oil results therefore demonstrate that plasmacytomagenesis in granulomas, was diagnosed by the appearance of transformed Bcl-xL transgenic mice that lack UNG resembles that observed − −/− plasma cells in the ascites. UNG+/+ and UNG+/ mice rapidly in AID mice in that it is not associated with canonical Igh/ developed plasmacytomas with no apparent difference in kinet- Myc translocations. ics (Fig. 1A). In contrast, plasmacytomagenesis was inefficient in − − −/− the absence of UNG. Compared with wild-type controls, UNG / UNG Translocations Preferentially Localize Upstream of Myc Tran- −/− tumors developed with lower frequency (18% vs. 90% at day 220, scription Start Site. To explore whether UNG tumors carried P < 0.0005) and longer mean latency (136 vs. 73 d, Fig. 1A). noncanonical forms of T(12:15)s or other translocations, we next Consistent with previous findings (31), UNG+/+Bcl-xL mice applied FISH and spectral karyotyping (SKY). Despite our in- −/− developed multiple foci of premalignant, hyperchromatic plasma ability to amplify T(12:15)s by PCR, 9 of the 11 UNG tumors cells harboring Igh/Myc translocations [T(12;15)s] as early as 10 d carried rearrangements between chromosomes 12 and 15 (Fig. post pristane injection, whereas their appearance was delayed 2A and Fig. S3). To precisely map the translocation breakpoints, and their number reduced in the absence of UNG [7% (3 of 45 we made use of high-resolution array comparative genomic hy- mice analyzed), Fig.