Β-Catenin Induces T-Cell Transformation by Promoting Genomic Instability

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 induces 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 lymphomas 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 translocating 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 translocations 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 survival of cells with damaged DNA and with DNA Tcf-1 at RAG2 sites throughout the genome (Fig. 2 A and B). repair errors. Moreover, Tcf-1 and RAG2 peaks had a similar shape and summit location (Fig. 2C), suggesting that they bind in close Results proximity. Approximately 80% of all RAG2 binding sites were T-Cell Lymphomas Induced by β-Catenin Activation Have Recurrent shared with both Tcf-1 and H3K4me3 (“trimethylated Tcf-1 Translocations. Constitutive activation of β-catenin as seen in sites” in the following) (Fig. 2D). Conversely, 84% of all trime- human cancer can be modeled in mice by Cre-mediated deletion thylated Tcf-1 sites were bound by RAG2 (compared with only of its proteolytic degradation domain (20). We have previously 65% of all H3K4me3 sites). These data identify trimethylated reported that CD4Cre-mediated activation of β-catenin in this Tcf-1 sites as bona fide RAG2 binding sites. They further suggest manner (CAT mice) leads to DP T-cell lymphomas, targeting that Tcf-1 is present at most DNA sites that are vulnerable to off- ∼90% of CAT mice with a latency of 99 d (19). Transformation target RAG-mediated DSBs. required RAG-mediated DNA damage, and RAG activity was We next examined whether the cobinding of Tcf-1 and RAG2 not simply needed for progression to the DP stage (19). Based could predict expression levels of the target genes. To this end on this finding we speculated that tumorigenesis resulted from we assessed the average expression of genes in WT, CAT, and β-catenin–induced genomic instability. Indeed, spectral karyo- lymphoma-derived DP cells, stratified by Tcf-1 and RAG2 type (SKY) analyses of eight independent lymphomas identified binding and by the presence or absence of H3K4me3 marks. On a wide range of chromosomal translocations and amplifications average, cooccupied genes did not change expression between (Table S1) indicating genomic instability. Six samples had a re- WT, CAT, or lymphoma DP cells. Genes bound by Tcf-1 showed curring translocation of Tcra to the myelocytomatosis oncogene higher expression than the average gene expression of all genes (Myc) locus, t (13, 14) (C2;D1), and one had a translocation of in DP thymocytes. Cobinding of RAG2 and Tcf-1 marked genes Tcrb to the Myc locus (Fig. 1A and Fig. S1A) suggesting error- with significantly higher expression compared with genes only prone repair of RAG-induced DSBs. Fluorescence in situ hy- bound by Tcf-1 (Fig. 2E). Thus, Tcf-1 and RAG2 bind actively bridization (FISH) showed that in the recurrent translocation the transcribing genes that have an increased probability to sustain joining (J), constant (C), and Eα enhancer regions of the Tcra DNA damage as a result of transcription initiation. locus were placed downstream of Myc into the Plasmacytoma variant translocation 1 (Pvt1) locus (Fig. 1 B and C). Chimeric Pvt1/ Trimethylated Tcf-1 Sites Are Preserved upon Activation of β-Catenin. Tcra transcripts were also detectable in these lymphomas (Fig. The dependence of CAT lymphomas on RAG-mediated DNA S1B). The translocation places the Myc locus under the control of damage and the overlapping binding of Tcf-1 and RAG2 suggest the Tcra Eα enhancer, explaining the overexpression of Myc in that Tcf-1 may have a role in DNA recombination and/or repair CAT lymphomas (19). A similar translocation, targeting TCRA to that is influenced by β-catenin, leading to genomic instability. We the MYC/PVT1 locus, marks ∼2% of human T-ALL (21). Se- therefore determined the effect of β-catenin activation on tri- quencing translocation breakpoints from such T-ALL samples methylated Tcf-1 binding sites. By extending our ChIP-seq identified cryptic RAG recombination signal sequences at the analyses to CAT thymocytes, we observed that Tcf-1 peak shape PVT1 side of the breakpoint and lead to suggestions that they and summit location at shared Tcf-1 sites were similar in CAT represented illegitimate RAG-mediated events (22). The Pvt1 and WT DP thymocytes (Fig. 3A). Ward’s clustering of tag locus is also frequently targeted by proviral integrations in murine density distributions filtered on WT Tcf-1 sites showed that leukemia virus-induced T-cell lymphomas in mice (23) and in rats similarities between Tcf-1 and H3K4me3 patterns persist, al- (24). It is also the site for breakpoints in ∼20% of human Burkitt’s though some Tcf-1 sites are lost or have reduced enrichment in

Fig. 1. β-Catenin activation causes genomic instability. Representative images of (A)SKYmeta- phase from a CAT lymphoma with Tcra/Myc translocations (see Table S1 for full karyotypes) and (B) FISH analysis. (C) Cartoon of the translocation as predicted by FISH and the presence of a fusion transcript (Fig. S1B). BACs used for FISH analysis are indicated. Not drawn to scale.

392 | www.pnas.org/cgi/doi/10.1073/pnas.1315752111 Dose et al. Downloaded by guest on October 1, 2021 − in the same cell eight times more frequently (P = 1.1 × 10 3; Fig. 4D, i). Moreover, when Tcra and Myc were in close proximity (“paired”), only CAT but not WT thymocytes had DNA damage on both loci (P = 0.02; Fig. 4D, ii). These data suggest that β-catenin activation causes increased DNA damage and/or aberrant repair at off-target DSBs (in this case the Myc locus) resulting in translocations.

β-Catenin Activation Affects Repair of RAG-Mediated DSBs. Having shown a genome-wide overlap of Tcf-1 and RAG2 binding and increased DNA damage, we asked whether CAT cells had compromised DNA repair. Defects in NHEJ DNA repair are reflected in the sequence composition of coding joints (CJs), generated during repair of RAG-mediated DSBs. We therefore sequenced Tcra CJs from thymocytes of three WT and three CAT mice using an established assay (29). Two hundred eleven unique WT and 243 CAT Va2-Ja56 Tcra CJ sequences were annotated with respect to the presence of N- or P-nucleotides (Table S3). N-nucleotides are nontemplated nucleotides added during NHEJ repair by TdT, whereas P-nucleotides result from Fig. 2. Trimethylated Tcf-1 sites predict RAG2 binding. ChIP-seq analyses of WT thymocytes. (A) Similar genome-wide binding patterns of RAG2, Tcf-1, asymmetric opening of a coding end hairpin (Fig. 4E). Tabula- tion of the data showed that TdT failed to add N-nucleotides in a and H3K4me3. Tag density is plotted for a representative area on Chr6. (B) = Similarity analysis using Ward’s clustering of chromatin occupancy by Tcf-1 significantly higher fraction of CAT CJ compared with WT (P and RAG2 (described in ref. 53). (C) Normalized tag density distribution 0.04, t test; Fig. 4F). This defect was not due to reduced TdT centered on shared trimethylated Tcf-1 sites. Solid lines, ChIP; dotted lines, levels as components of the NHEJ machinery, including TdT, control. Lower right shows overlay of ChIP signal for Tcf-1, RAG2, and are expressed at comparable levels in WT and CAT thymocytes H3k4me3. (D) Percentage of peaks that RAG2 shares (■) or that are shared (Fig. S3). The reduced frequency of N-nucleotide addition sug- with RAG2 (□) for the indicated subsets. (E) Log2 expression of all genes gests impaired NHEJ and is reminiscent, albeit milder, of a expressed in DP thymocytes or stratified by the presence of Tcf-1, RAG2, and phenotype that has been described in Ku80-deficient cells (30). H3K4me3 (me3) at the gene promoter. Like Ku80-deficient cells, CAT cells had no significant differences in the number of P-nucleotides (P = 0.7, t test) or the overall number of deletions or insertions at the Tcra locus (Fig. CAT thymocytes (Fig. 3B). This trend excluded trimethylated 4F and Table S3). This finding suggests that activation of Tcf-1 sites, which predict RAG2 binding (Fig. 2). The number of β-catenin in thymocytes may affect repair of DNA DSBs. trimethylated Tcf-1 sites remained unaltered (Fig. 3C), with ∼70% completely identical sites in WT and CAT thymocytes. Activation of β-Catenin Provides a “Survival License” to CAT Thymocytes. These findings indicated that activation of β-catenin did not Studies in mice suggest that genomically unstable leukemia arises markedly influence the deposition of H3K4me3 in the proximity when the balance between DNA repair, cell cycle progression, and of Tcf-1 sites. We thus predicted that RAG2 binding would also survival is disturbed. Unresolved DSBs normally trigger cell cycle be unaltered by stabilization of β-catenin. Indeed, quantitative arrest to allow time for repair or cell death in case repair is not ChIP (ChIP followed by qPCR) of RAG2 at select binding sites promptly accomplished (31). However, failure to coordinate DNA showed that RAG2 binding did not significantly change in CAT DP thymocytes compared with WT (Fig. S2). We conclude that activation of β-catenin does not significantly alter trimethylated Tcf-1 sites or RAG2 binding. Thus, any illegitimate RAG activity would target similar Tcf-1 proximal sites in CAT cells, but high levels of β-catenin may interfere with the protection of these sites.

Increased DNA Damage at Paired Tcra/Myc Loci in CAT Thymocytes. To pursue this idea further, we inspected the translocation sites in pretransformed thymocytes. The Pvt1 locus lies within an early replicating chromosome fragile region (27). Such sites have been proposed to sustain frequent DSBs and become acceptor sites for translocations when NHEJ repair is compromised (27). We found several overlapping Tcf-1 and RAG2 binding sites near the translocation breakpoint at the Pvt1 locus (Fig. 4A), supporting the notion that Pvt1 may sustain off-target RAG-mediated DSBs. A prerequisite for translocations to occur is that the translocating loci have DSBs when they are in close proximity. DSBs normally trigger the DNA damage response that involves phos- phorylation of histone H2AX at serine 139 (γ-H2AX) around the damaged region (28). To examine whether proximal Tcra and IMMUNOLOGY Myc loci had increased damage, we compared the presence of γ-H2AX on these loci in WT and CAT thymocytes. Three- Fig. 3. Trimethylated Tcf-1 sites are preserved in CAT thymocytes. ChIP- dimensional immuno-DNA FISH analyses confirmed that in seq analyses of WT and CAT thymocytes. (A) Tcf-1 signal at sites that 25% of DP thymocytes the Tcra and Myc loci exist in close overlap between WT and CAT plotted as a percentage of maximum. (B) proximity (Fig. 4B). However, in WT cells, damage is prevented Ward’s hierarchical clustering of tag density for similarity analysis as in Fig. from spreading to the Myc locus because γ-H2AX is rarely found 2. me3, H3K4me3. (C) Absolute number of WT and CAT Tcf-1 sites that on Myc. Only 0.7% of WT cells with damaged Tcra also had overlap with H3K4me3 and are shared between WT and CAT (black) and damaged Myc (Fig. 4 C and D, i, and Table S2). This differed in overlap with H3K4me3 but are not shared between WT and CAT (gray) and CAT thymocytes where damaged Tcra and Myc loci cooccurred do not overlap with H3K4me3 (white).

Dose et al. PNAS | January 7, 2014 | vol. 111 | no. 1 | 393 Downloaded by guest on October 1, 2021 and up-regulation of survival pathways (Figs. S4 and 6A). Fur- thermore, the antiapoptotic protein Bcl-xL was robustly up- regulated in CAT thymocytes, consistent with earlier suggestions that it is directly targeted by β-catenin (33) (Fig. 6B). Despite up-regulation of Bcl-xL, CAT and WT thymocytes showed similar spontaneous cell death (Fig. 6C). To assess if Bcl-xL provided a survival advantage in response to challenge, we treated WT and CAT thymocytes with genotoxic γ-irradiation (1.25 Gy) and compared their survival. Indeed, CAT thymocytes were more resistant to DNA damage than their WT counterparts (Fig. 6D). This resistance was dependent on increased Bcl-xL levels because treatment of irradiated cells with ABT263, a pharmacologi- cal inhibitor of the Bcl2 protein family with highest affinity for Bcl-xL (34), eliminated the survival advantage of CAT thymocytes (Fig. 6D). Moreover, the resistance of CAT thymocytes was limited to treatment with genotoxic agents, including the top- oisomerase II inhibitor etoposide, and γ-irradiation (Fig. 6E). By contrast, a comparable fraction of WT and CAT thymocytes survived treatment with Brefeldin A, an inhibitor of vesicular traffic (Fig. 6E). β-Catenin thus enhances cellular survival in the presence of DNA damage. Discussion We previously reported that activation of β-catenin at the DP stage of thymocyte development leads to T-cell lymphomas that Fig. 4. CAT thymocytes have increased DNA damage at translocating loci depend on RAG-induced DNA breaks and c-Myc expression and altered repair of DSBs. (A) Cooccupancy of Tcf-1 and RAG2 at the (19). These earlier observations had prompted us to speculate translocating Myc/Pvt1 locus. (B–D) Three-dimensional FISH of DP thymo- that the resulting lymphomas were genomically unstable. Here < μ = = γ cytes. (B) Tcra and Myc loci at 1 m(NWT 304, NCAT 317). (C) -H2AX we confirm this hypothesis by demonstrating that the lymphomas association on Tcra/Myc pairs either exclusively on Tcra (Upper)oronboth have widespread genomic changes including recurrent trans- alleles (Lower). (D) γ-H2AX association on both loci in the same cell, (D, i) locations of TCRα to the Myc/Pvt1 locus, and we provide irrespective of pairing status or (D, ii)inTcra/Myc pairs, as percentage of mechanistic insights into the transformation process. total cells (D, i) or of cells with ≥1 Tcra/Myc pair (D, ii). Asterisks indicate β × −3 × −2 Activation of -catenin provides CAT thymocytes with a sur- statistical significance. P values: (D, i)1.1 10 and (D, ii)1.9 10 . nd, vival license as it represses apoptosis and activates survival path- none detected. (Scale bars, 1 μm.) (E) Exemplary CJ sequences along germ- line sequence (top) to illustrate P (blue) and N nucleotides (orange), dele- ways. This pattern also includes BclXL that is critical for survival tions, and insertions identified in the data set (see Table S3 for all sequen- of DP thymocytes. Indeed, CAT thymocytes survive better when ces). (F) CJ sequencing results from three mice per genotype were pooled challenged with DSB inducing agents, a survival advantage that and subjected to statistical analysis (Materials and Methods). is lost when BclXL is pharmacologically inhibited. Survival of cells with damaged DNA is known to facilitate oncogenic translocations. However, increased survival is not sufficient for trans- repair and apoptosis—forexample,inmicedeficientforNHEJ formation because mice that overexpress the antiapoptotic Bcl-2 components as well as p53—induces B-cell lymphomas with or Bcl-xL do not develop T-cell malignancies comparable to our Igh/Myc translocations analogous to the Tcra/Myc translocations model (35). Uncontrolled β-catenin kills two birds with one stone described here. We therefore reasoned that high levels of β-catenin as it simultaneously promotes survival and DNA damage. This β may promote transformation by altering the balance between cell seems to depend on the levels of -catenin because models with β cycle progression and survival. To examine whether CAT thy- moderate up-regulation of -catenin develop lymphomas only mocytes harbored cell cycle defects, we performed BrdU pulse when p53 is also ablated (36). Our model, based on constitutive activation of endogenous β-catenin, expresses high levels of labeling paired with DNA content analyses. We observed no β differences between CAT and WT DP thymocytes with respect to -catenin that evidently suffice to tip the balance toward transformation without the need for additional genetic manipulation. the fraction of cells in the different phases of the cell cycle (Fig. 5 β A and B). Moreover, we had previously shown that CAT thymo- Our studies suggest that high levels of -catenin may influence cytes do not have p53 loss-of-function mutations (19). To further the repair of DSBs. In this study, we mapped the genome-wide rule out cell cycle defects, we determined RAG2 protein levels in DNA binding pattern of Tcf-1 in DP thymocytes. This will be G1 versus the S/G2/M phases of the cell cycle. RAG2 is normally a valuable resource for future investigations regarding the tran- present at high levels in G1 but is degraded during S/G2/M (32). scriptional control of T-cell differentiation. We unexpectedly To evaluate RAG2 levels along the cell cycle, we sorted DP revealed a genome-wide overlap between Tcf-1 and the DNA thymocytes from neonatal WT and CAT mice stained with the cell permeable Hoechst 33342 dye according to their DNA content. RAG2 protein levels in G1 (2N DNA content) versus S/G2/M (>2N DNA content) were similar in CAT and WT DP thymocytes, indicating normal cell cycle control of RAG2 degradation (Fig. 5C). Furthermore, WT and CAT thymocytes had comparable levels of Tcf-1 protein both in G1 and S/G2/M phases of the cell cycle, indicating that there was no imbalance between Tcf-1 and RAG2 (Fig. 5C). In summary, therefore, CAT DP thymocytes have no detectable cell cycle defects. Fig. 5. β-catenin activation does not impair cell cycle checkpoints. (A) Cell We next asked whether CAT thymocytes showed enhanced cycle profiles of WT and CAT DP thymocytes from mice injected with BrdU survival. Gene ontology analysis of our transcriptome data (19) 3 h before analysis. (B) Histograms summarize data from two mice per ge- revealed that pretransformed CAT cells had an antiapoptosis notype. (C) Western blot analysis of sorted DP cells in G1 or S/G2/M states of expression signature with significant down-regulation of apoptotic cell cycle. Thymi from newborn mice were pooled for this experiment.

394 | www.pnas.org/cgi/doi/10.1073/pnas.1315752111 Dose et al. Downloaded by guest on October 1, 2021 Wnt target gene expression during somitogenesis lead to suggestions that a negative feedback loop controls the length of Wnt signaling (8). Deregulated β-catenin activation eliminates these oscillations and predisposes to cancer. Therefore, multiple lines of evidence indicate that short-lived bursts and tightly controlled levels of Wnt activity are required for normal development. Our data provide a fresh perspective on β-catenin and Tcf-1 functions and offer unique paradigms for the etiology of human leukemia with translocations involving the TCR loci (43, 44). About 2% of human T-ALL have similar translocations to the ones detected in CAT mice (21). Sequencing identified cryptic RAG recombination signal sequences in the MYC/PVT1 sites of the translocations, leading to suggestions that the translocations resulted from illegitimate RAG recombination events. Murine lymphomas resulting from Pten ablation (45, 46) or Akt activation (47) also show recurrent Tcra/Myc translocations with similar architecture. Notably, β-catenin is required for the generation of Pten-deficient lymphomas (45). Both Pten ablation and Akt activation stabilize β-catenin. Detection of similar translocations in the three animal models prompts us to hypothesize an “axis of genome integrity” with β-catenin/Tcf-1 as its most downstream component. Finally, the organization of the Tcra/Myc translocations in T cells is reminiscent of Igh/Myc translocations in human Burkitt’s lymphomas and B-cell lymphomas arising in mice that are double deficient in NHEJ and p53 (48, 49). This raises the possibility that such translocations share similar etiologies. Altogether, our findings demonstrate that in thymocytes, constitutive activation of β-catenin confers genomic instability. They also provide a unique perspective as to why the levels of β-catenin are so tightly controlled during normal development. Future studies will be necessary to gain further insights into Tcf-1 and β-catenin functions in genome protection and to address Fig. 6. β-Catenin confers a survival profile upon thymocytes. (A)Heatmap how they promote genomic instability in human cancer. depicting average fold change of expression between CAT and WT for the indicated genes in WT and CAT DP thymocytes (biological replicates in col- Materials and Methods umns). (B) β-catenin stabilization increases Bcl-X protein levels. Densitom- L Animals. BALB/c CD4Cre;Ctnnb1Δex3 (CAT) mice (19) were used for SKY and FISH etry was performed on Western blot images, and density is expressed analysis, and C57BL/6 mice (Jackson) were used for all other experiments. Mice relative to β-Actin (Top). Statistical significance was assessed using an unpaired, − were kept in the animal facilities of the University of Chicago according to pro- two-tailed t test (P = 10 5, n = 6 per group). (C–E) Freshly isolated WT and CAT tocol #71880, approved by the local Institutional Animal Care and Use Committee. thymocytes were cultured as follows, and cell viability was assessed by FACS. (C) Spontaneous cell death with DMSO. (D) Specific cell death (i.e., relative to the survival of mock treated cells) upon γ-irradiation and 20 h of SKY Analyses. Sick CAT mice were euthanized, and SKY was performed on culture with ABT263 or DMSO (mock). (E) Specific cell death upon treatment thymic lymphoma cells as described (50). Karyotyping results are in Table S1. with etoposide, γ-irradiation, or Brefeldin A. FISH. Labeled BAC probes for Tcra (RP23-105B7; Spectrum OrangeTM) or Myc (RP23-442F1; Spectrum GreenTM) were labeled with nucleotides (Abbott recombinase RAG2 at trimethylated Tcf-1 binding sites (Tcf-1 Molecular Diagnostics). FISH was performed as described previously (50). sites that are marked by H3K4me3 histone marks). We showed that such sites are predictive of RAG2 binding in thymocytes, Three-Dimensional Immuno-DNA FISH. DNA FISH-immunofluorescence for and although it has been suggested that RAG2 and Tcf-1 pro- γ-H2AX was carried out on interphase nuclei of sorted DPs as in ref. 51. Labeled teins do not directly interact (37), their physical proximity on the BAC probes RP23-255N13 (3′ end of the Tcra locus) and RP24-307D14 (Myc)were used. Distances between alleles were measured between the center of mass of DNA may imply a functional relationship. CAT thymocytes had γ signs of altered repair as TCR CJ frequently lacked N-nucleo- each BAC signal. Alleles were defined as associated with -H2AX if BAC signals tides. This is also a feature of Ku80-deficient cells, which has led and immunofluorescence foci overlapped by at least one pixel. Statistical sig- to suggestions that Ku80 facilitates access to DSBs for the N- nificance was calculated by a two-tail Fisher exact test in a pair-wise analysis. β Sample sizes were 100 cells minimum per experiment. Data from individual nucleotide adding enzyme TdT (38). -Catenin has been pro- experiments were pooled for the figure but are shown separately in Table S2. posed to outcompete an interaction of KU70 with TCF-4 (39), the epithelial cell counterpart of Tcf-1. In further support of the 8 β ChIP-seq. A total of 10 thymocytes from 4-wk-old mice were formaldehyde- notion that -catenin activation may affect DNA repair, we ob- fixed and sonicated to a size of ∼300 bp. Antibodies to Tcf-1 (kind gift of Hiroshi served increased DNA damage in the Myc/Pvt1 locus of pre- Kawamoto, Kyoto University, Kyoto, Japan) coupled to Protein G Dynabeads malignant CAT DP thymocytes. Taken together, these observations β were incubated overnight with sheared chromatin. ChIP-seq libraries were pre- provide a mechanistic framework for how high levels of -catenin pared from 10 ng of IPed material as before (52) and sequenced on an Illumina IMMUNOLOGY might induce RAG-dependent, genomically unstable lymphomas Genome Analyzer 2. Data were analyzed as described (52). Peaks were called − (19) with recurrent translocations. with model-based analysis of ChIP-Seq (MACS) at a P value cutoff of 10 5.We Wnt signaling relies on β-catenin accumulation for its activity, have previously described ChIP-seq for H3K4me3 in WT thymocytes (52). These and if this is an impediment for genome stability, then strict control data are publicly available under accession GSE32311. of the amplitude and duration of Wnt activity becomes essential. Apparently, hematopoietic development requires precise lineage- RAG2 ChIP. RAG2 ChIP followed by quantitative PCR was performed as described specific dosage of Wnt signaling (40). It has also been suggested (1). PCR data were analyzed as in ref. 53. Primer sequences are in Table S4. that Tcf-1 restrains Lef-1 from uncontrollably activating Wnt signaling and promoting Lef-1–dependent transformation of Tcf-1– Sequencing of Coding Joints (CJ Sequencing). Genomic DNA (50 ng) from deficient early thymocytes (41, 42). Furthermore, oscillations of sorted DPs of three WT and three CAT mice was amplified as in ref. 29 (primer

Dose et al. PNAS | January 7, 2014 | vol. 111 | no. 1 | 395 Downloaded by guest on October 1, 2021 sequences are in Table S4). PCR products were cloned and sequenced at the ACKNOWLEDGMENTS. We thank D. Schatz, G. Teng, and H. Kawamoto for DNA core facility (University of Chicago). Only unique sequences were an- reagents; M. Clark, K. Khazaie, and D. Schatz for advice; and C. Daly, alyzed to avoid PCR duplicates and N- and P-nucleotides were annotated E. Bartom, and M. Maienshein-Cline for technical assistance and data mining. (Table S3). Replicates were considered repeat measures of the population This work was supported by National Institutes of Health Grants R21AI076720 mean [p(noN) = 0.042, p(P) = 0.703, t test]. Alternatively, a two-sided Fisher’s (to F.G.), R01CA158006 (to K. Georgopoulos), F31AI830542 (to K. Germar), and exact test was applied to pooled data [p(noN) = 0.0194, p(P) = 0.726]. R01GM086852 (to J.A.S.), and R01CA158006 (to K. Georgopoulos). Further support came from the American Cancer Society Grant ACS/RSG, LIB-113428 BrdU Uptake Experiment for Cell Cycle State Analysis. Littermates were injected (to F.G.); Chicago Biomedical Consortium (F.G.); The Lady Tata Memorial Trust i.v. with 10 mg BrdU in PBS. Three hours later they were killed, and thymocytes (M.D.); and a Specialized Center of Research of the Leukemia and Lymphoma were stained for CD4, CD8, and TCRβ and then intracellularly with 7-AAD and Society (LLS) [SCOR R7019-04 (M.M.L.B.)]. J.A.S. is an LLS scholar. J.C. is an anti-BrdU and were analyzed on a Fortessa machine (BD Bioscience). Irvington Institute Fellow of the Cancer Research Institute.

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