β-Catenin induces T-cell transformation by promoting genomic instability Marei Dosea, Akinola Olumide Emmanuela, Julie Chaumeilb, Jiangwen Zhangc, Tianjiao Suna, Kristine Germara, Katayoun Aghajania, Elizabeth M. Davisd, Shilpa Keerthivasana, Andrea L. Bredemeyere, Barry P. Sleckmane, Steven T. Rosenf, Jane A. Skokb, Michelle M. Le Beaud, Katia Georgopoulosg, and Fotini Gounaria,1 aSection of Rheumatology and Knapp Center for Lupus and Immunology Research, University of Chicago, Chicago, IL 60637; bDepartment of Pathology, New York University School of Medicine, New York, NY 10016; cFaculty of Arts and Sciences (FAS) Center for Systems Biology, Harvard University, Cambridge, MA 02138; dSection of Hematology/Oncology and the Comprehensive Cancer Center, University of Chicago, Chicago, IL 60637; eDepartment of Pathology and Immunology, Washington University School of Medicine, St. Louis, MO 63110; fRobert H. Lurie Comprehensive Cancer Center, Feinberg School of Medicine, Northwestern University, Chicago, IL 60611; and gCutaneous Biology Research Center, Massachusetts General Hospital, Charlestown, MA 02129 Edited* by Harvey Cantor, Dana-Farber Cancer Institute, Boston, MA, and approved December 4, 2013 (received for review August 20, 2013) Deregulated activation of β-catenin in cancer has been correlated pathways and mutations in the Wnt signaling pathway are a with genomic instability. During thymocyte development, β-cate- common example (7). In normal development Wnt activity is short- nin activates transcription in partnership with T-cell–specific tran- lived and expression of Wnt target genes oscillates, suggesting scription factor 1 (Tcf-1). We previously reported that targeted that Wnt activity is controlled by inherent negative feedback activation of β-catenin in thymocytes (CAT mice) induces lympho- loops (8). Uncontrolled activation of the central effector of Wnt mas that depend on recombination activating gene (RAG) and signaling, β-catenin, has been causatively linked to genome instability myelocytomatosis oncogene (Myc) activities. Here we show that in multiple cancers, including hematopoietic malignancies (9–12). these lymphomas have recurring Tcra/Myc translocations that However, how uncontrolled β-catenin promotes genomic instability resulted from illegitimate RAG recombination events and resem- in these malignancies is unknown. bled oncogenic translocations previously described in human T- β-Catenin exerts Wnt-mediated transcription functions by ALL. We therefore used the CAT animal model to obtain mecha- interacting with members of the TCF/LEF family of HMG do- – nistic insights into the transformation process. ChIP-seq analysis main DNA binding proteins. The T-cell specific TCF/LEF factor uncovered a link between Tcf-1 and RAG2 showing that the two Tcf-1 (product of the Tcf7 gene), one of the earliest transcriptional proteins shared binding sites marked by trimethylated histone-3 regulators induced in thymus seeding T-cell progenitors, is essential lysine-4 (H3K4me3) throughout the genome, including near the for T-cell commitment (13, 14). Tcf-1 constitutively interacts with translocation sites. Pretransformed CAT thymocytes had increased DNA and is thought to mediate activation of transcription when β DNA damage at the translocating loci and showed altered repair bound by -catenin and repression when bound by Groucho. Earlier studies showed that Tcf-1 is recurrently required during T-cell de- of RAG-induced DNA double strand breaks. These cells were able – to survive despite DNA damage because activated β-catenin pro- velopment (15 17). Tcf-1 is most abundant in DP thymocytes, and its absence compromises DP thymocyte survival (17, 18). moted an antiapoptosis gene expression profile. Thus, activated β – β Here we use a mouse model of -catenin induced T-cell -catenin promotes genomic instability that leads to T-cell lympho- β mas as a consequence of altered double strand break repair and malignancy to address how constitutive activation of -catenin in- duces genomic instability. Previously, we reported that stabilization increased survival of thymocytes with damaged DNA. beta-catenin/Tcf-1 | DNA recombination Tcf7 | Ctnnb1 Significance evelopment of lymphocytes involves recombination of their Understanding molecular mechanisms that underlie genomic Dgenomic DNA to allow for expression of antigen receptor instability will remove a major obstacle to effective treatment genes. Thymocytes first rearrange the T-cell receptor (Tcr) β, γ, of cancer. Here we characterize a unique animal model that − − and δ loci at the CD4 CD8 double-negative-3 (DN3) stage of allows insight into mechanisms of genomic instability leading α + + to oncogenic translocations. We show that thymocyte-specific development and then the Tcr (Tcra) locus at the CD4 CD8 β double-positive (DP) stage. DNA double strand breaks (DSBs) activation of -catenin induces genomically unstable lympho- mas with Tcra/Myc translocations, reminiscent of human leu- generated during these processes are catalyzed by the recom- β bination activating gene (RAG) recombinase complex. Thus, kemia. Tcf-1, the partner of -catenin, colocalized throughout differentiating T cells sustain programmed RAG-mediated DNA the genome with the RAG2 recombinase at DNA sites thought DSBs, in addition to random DNA damage that results from to be vulnerable to illegitimate recombination. Pretransformed transcription initiation, DNA replication, and spatial reconfigu- thymocytes showed increased DNA damage at the trans- locating loci and altered DNA repair. These cells survived de- ration of the chromatin architecture. An essential component of spite DNA damage. These surprising observations show that the RAG complex is the RAG2 protein, which binds H3K4me3 activated β-catenin promotes genomic instability and cancer by and colocalizes with this histone mark throughout the genome (1– compromising DNA repair and enhancing cell survival. 3). This widespread binding of RAG2 to DNA is puzzling, and it is thought to contribute to off-target generation of DSBs (i.e., DSBs Author contributions: M.D. and F.G. designed research; M.D., A.O.E., J.C., T.S., K. Germar, outside the immune receptor loci) (4). DNA ends generated by K.A., E.M.D., S.K., and F.G. performed research; A.L.B., B.P.S., S.T.R., J.A.S., M.M.L.B., and IMMUNOLOGY the RAG complex recruit nonhomologous end joining (NHEJ) K. Georgopoulos contributed new reagents/analytic tools; M.D., A.O.E., J.Z., M.M.L.B., and proteins, including Xrcc4, Ligase IV, DNA-PKcs, Artemis, and F.G. analyzed data; and M.D. and F.G. wrote the paper. XLF/Cernunnos, that mediate rapid repair (5). The precise mech- The authors declare no conflict of interest. anisms in place to maintain genome integrity in the face of these *This Direct Submission article had a prearranged editor. breaks remain under intense investigation (6). Data deposition: The sequences have been deposited in NCBI Gene Expression Omnibus Failure to repair illegitimate DSBs and/or purge the cells that (accession no. GSE46662). have damaged DNA induces oncogenic translocations and is a se- 1To whom correspondence should be addressed. E-mail: [email protected]. vere impediment to successful therapeutic eradication of cancer This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. cells. Oncogenic events often target conserved developmental 1073/pnas.1315752111/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1315752111 PNAS | January 7, 2014 | vol. 111 | no. 1 | 391–396 Downloaded by guest on October 1, 2021 of β-catenin in thymocytes through Cre-mediated deletion of its lymphomas (25) and in murine plasmacytomas (26). In conclu- proteolytic degradation signal induces RAG-dependent T-cell sion, CAT thymocytes are a useful model to gain insights into the lymphomas (19). This finding supported the notion that geno- contribution of β-catenin to genomic instability because they mic instability may be the underlying cause of transformation. sustain frequent illegitimate RAG recombination events leading Our current findings confirmed this notion by showing that to recurrent oncogenic translocations. lymphomas with activated β-catenin had recurring translo- cations that resembled translocations observed in human T-ALL. Widespread Overlap of Tcf-1 and RAG2 Binding at H3K4me3 Sites. The translocations resulted from illegitimate RAG recom- RAG2 binds H3K4me3-modified histones throughout the ge- bination events as they involved the Tcra locus. At the same nome through its PHD domain, although the physiological sig- time, we linked Tcf-1 and RAG2 by showing that they bound nificance of this widespread distribution is not clear (1–3). To together at chromatin sites marked by H3K4me3 genome-wide. address whether β-catenin may influence RAG activity, we Premalignant CAT DP thymocytes had increased DNA damage mapped the DNA binding pattern of its partner, Tcf-1, in thy- at the translocation sites and showed altered processing of mocytes by ChIP-seq. This experiment revealed that most RAG-mediated DSBs. These cells survived even when they H3K4me3 sites (69%) overlapped with Tcf-1 binding sites. We had damaged DNA because β-catenin activation induced an thus asked if Tcf-1 and RAG2 also had similar genome-wide antiapoptosis expression profile. Thus, our findings show that distribution patterns and compared our Tcf-1 ChIP-seq data to β-catenin activation prompts genomic instability that is associated the public data set for RAG2 (1–3). We observed overlapping with the