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ARTICLES Oncogene-induced senescence as an initial barrier in lymphoma development Melanie Braig1, Soyoung Lee1, Christoph Loddenkemper2, Cornelia Rudolph3, Antoine H.F.M. Peters4,5, Brigitte Schlegelberger3, Harald Stein2, Bernd Do¨rken1,6, Thomas Jenuwein5 & Clemens A. Schmitt1,6 Acute induction of oncogenic Ras provokes cellular senescence involving the retinoblastoma (Rb) pathway, but the tumour suppressive potential of senescence in vivo remains elusive. Recently, Rb-mediated silencing of growthpromoting genes by heterochromatin formation associated with methylation of histone H3 lysine 9 (H3K9me) was identified as a critical feature of cellular senescence, which may depend on the histone methyltransferase Suv39h1. Here we show that Em-N-Ras transgenic mice harbouring targeted heterozygous lesions at the Suv39h1, or the p53 locus for comparison, succumb to invasive T-cell lymphomas that lack expression of Suv39h1 or p53, respectively. By contrast, most N-Ras-transgenic wild-type (‘control’) animals develop a non-lymphoid neoplasia significantly later. Proliferation of primary lymphocytes is directly stalled by a Suv39h1-dependent, H3K9me-related senescent growth arrest in response to oncogenic Ras, thereby cancelling lymphomagenesis at an initial step. Suv39h1-deficient lymphoma cells grow rapidly but, unlike p53-deficient cells, remain highly susceptible to adriamycin-induced apoptosis. In contrast, only control, but not Suv39h1-deficient or p53-deficient, lymphomas senesce after drug therapy when apoptosis is blocked. These results identify H3K9me-mediated senescence as a novel Suv39h1-dependent tumour suppressor mechanism whose inactivation permits the formation of aggressive but apoptosis-competent lymphomas in response to oncogenic Ras. In response to the activation of mitogenic oncogenes, checkpointmediated failsafe mechanisms such as apoptosis or cellular senescence may be recruited to terminate a pre-malignant condition before a fully transformed stage can develop1,2. Premature senescence provoked by oncogenic Ras is associated with the accumulation of p53 and the D-type cyclin kinase inhibitor p16INK4a that locks the retinoblastoma (Rb) protein in its active hypophosphorylated state3. Active Rb acts as a co-repressor of E2F transcription factors that transactivate genes important for S-phase progression4. Mapping of transcriptionally inactive sites in the vicinity of E2F-responsive promoters to heterochromatin foci enriched for H3K9me in senescent but not in quiescent cells supported the hypothesis that growthpromoting genes might be stably repressed in senescent cells by means of specific and possibly irreversible histone modifications5. The histone methyltransferase Suv39h1 is a candidate in this process, because it might methylate H3K9 as an Rb-bound enzyme in close proximity to E2F target genes6,7, and H3K9me creates binding sites for HP1 proteins to form constitutive heterochromatin locally8,9. Thus, on the basis of an inheritable principle of S-phase gene silencing10, H3K9me-mediated senescence might act as a tumour suppressor programme that limits the transforming capacity of oncogenic Ras. Suv39h1 loss promotes Ras-driven lymphomagenesis To determine the role of Suv39h1 defects in Ras-driven tumorigenesis in vivo, we intercrossed Em-N-Ras transgenic mice, constitutively expressing the activated N-RasG12D oncoprotein in the haematopoietic compartment, to Suv39h1 knockout mice11,12, and monitored offspring survival. Ras transgenic mice carrying no defective Suv39h1 allele, hereafter referred to as controls, entered a terminal disease stage at a median age of 305 days. Suv39h1 2 mice, comprising

Suv39h1 2 /2 females and, because of the X-linked Suv39h1 locus, the equally Suv39h1-deficient Suv39h1 2 /y males, succumbed to their condition significantly earlier (Fig. 1a; median time to death 66 days, P ¼ 0.0004). Moreover, the time to death in Suv39h1 þ /2 mice was indistinguishable from the Suv39h1 2 group, indicating insufficient Suv39h1 function in the underlying disease in heterozygous animals. Because p53-deficiency accelerates Ras-mediated tumour progression in vivo13 and disables Ras-induced senescence in vitro14, Em-N-Ras transgenic mice being wild-type or heterozygous for the p53 locus were generated for comparison. Whereas p53 þ /þ mice behaved in the same way as controls, p53 þ /2 mice became terminally ill almost as fast as mice lacking one or both Suv39h1 alleles (Fig. 1a; median time to death 141 days for p53 þ /2 , P ¼ 0.02 compared with controls). No overt signs of disease were observed during more than a year in non-transgenic mice lacking one p53 or at least one Suv39h1 allele. Thus, allelic defects at the Suv39h1 or the p53 locus markedly accelerate disease manifestation in the context of activated Ras. At necropsy, mice that died early (that is, 180 days or less after birth; ‘early death group’) invariably displayed thymic and splenic but not hepatic enlargements due to a clonal lymphoblastic T-cell infiltration accompanied by a massive leukaemic burden and aggressive invasion into non-lymphoid visceral organs including the lungs. In stark contrast, necropsy of mice that died later (that is, more than 180 days after birth; ‘late death group’) revealed a distinct gross pathology with massively enlarged livers and spleens, but only occasional signs of thymic or visceral organ involvement (Fig. 1b, Supplementary Table 1 and Supplementary Fig. S1a, b). Histological examination, immunohistochemistry and immunophenotyping confirmed a myeloid origin of this neoplastic entity in accordance with histiocytic sarcomas previously described in the Em-N-Ras transgenic mouse model11 (Fig. 1c and Supplementary Fig. S1c),

1

Charite´ – Universita¨tsmedizin Berlin/Haematology-Oncology, 13353 Berlin, Germany. 2Charite´ – Universita¨tsmedizin Berlin/Department of Pathology, 12200 Berlin, Germany. Institute of Cell and Molecular Pathology, Hannover Medical School, Carl-Neuberg-Strasse 1, 30625 Hannover, Germany. 4Friedrich Miescher Institute for Biomedical Research, 4058 Basel, Switzerland. 5Research Institute of Molecular Pathology, 1030 Vienna, Austria. 6Max-Delbru¨ck-Center for Molecular Medicine, 13125 Berlin, Germany. 3

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discriminating it from an aggressive T-cell malignancy (Fig. 1c and Supplementary Fig. S1d, e). Altered lymphoid differentiation in the absence of Suv39h1 could be ruled out by a comprehensive immunophenotypic analysis of the cellular compositions in various haematopoietic compartments (Supplementary Fig. S2 and Supplementary Table 2). Taken together, these results show that Suv39h1 or p53 defects disable a tumour-suppressive programme that protects against Ras-driven lymphomagenesis. We next investigated whether oncogenic Ras might select against p53 or Suv39h1 expression in lymphomas that formed in p53 þ /2 or Suv39h1 þ /2 mice, respectively. According to time-to-death data implying that Suv39h1 and p53 limit Ras-induced lymphomagenesis, Suv39h1 transcripts were undetectable in all lymphomas derived from Suv39h1 þ /2 mice (Fig. 2a; 12 of 12 tumours tested), and Em-N-Ras lymphomas arising in p53 þ /2 animals invariably lost the remaining wild-type p53 allele (Fig. 2a; four of five tumours tested), rendering them Suv39h1-null (hereafter referring to all

Figure 1 | Suv39h1- and p53-defects license aggressive lymphomas in response to oncogenic Ras. a, Kaplan–Meier plot showing time-to-death latencies in Em-N-Ras transgenic Suv39h1 2 (Suv39h1 2 /2 females and Suv39h1 2 /y males; n ¼ 7; red), Suv39h1 þ /2 (n ¼ 17; orange), p53 þ /2 (n ¼ 19; blue), and pooled control mice (n ¼ 63; black). b, Representative photomicrographs of tissue sections at terminal disease stages of the indicated organs derived from early-death or late-death group animals (shown are Suv39h1-inactivated (top) and control cases (bottom)). Staining was with haematoxylin and eosin. c, Immunophenotyping of early-death (Suv39h1-inactivated, left) versus late-death (control, right) neoplastic cell populations by two-colour flow cytometry for a T-cell versus a myeloid marker antigen (CD90 versus CD11b).

Suv39h1-deficient lymphomas) or p53-null, respectively. Unlike p53 loss by allelic deletion, lack of Suv39h1 expression probably reflects a selective advantage for progenitor cells in which the remaining Suv39h1 allele maps to the inactivated X chromosome15, because we did not detect gross genomic deletions at this locus in any Suv39h1 þ /2 -derived lymphoma by allele-specific polymerase chain reaction (PCR) (data not shown). Thus, Ras-driven lymphomagenesis is promoted by selective inactivation of Suv39h1 or p53 function. The rapid formation of Suv39h1-null or p53-null lymphomas raises the possibility that they might select for high-level expression of oncogenic Ras. However, immunoblot analysis confirmed remark-

Figure 2 | Oncogenic Ras selects against Suv39h1, ARF or p53 in primary lymphomas. a, Suv39h1 RT–PCR (top; TBP, internal control) and allelespecific p53 genomic PCR to detect the wild-type or knockout (mutant) allele in tumour (T) and corresponding normal (N) tissues (bottom; mouse embryo fibroblast (MEF) DNA, internal control). b, Immunoblot analyses of control (ctrl), Suv39h1-null and p53-null lymphomas for N-Ras, p16INK4a and a-tubulin (loading control). c, Relative changes of the GFP-positive fraction 48 h after infection with MSCV-p16INK4a-IRES-GFP (left) or MSCV-ARF-IRES-GFP (right) in control (n ¼ 3), Suv39h1-null (n ¼ 8) and p53-null lymphomas (n ¼ 5) determined by flow cytometry; error bars denote s.d. d, Suv39h1-RT–PCR (top) and p53 PCR (bottom; as in a) in lymphomas derived from Suv39h1 þ /2 ;p53 þ /2 and Suv39h1 2 ;p53 þ /2 mice.

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able inter-sample variability of Ras protein levels within any given genotype, but having similar ranges between different genotypes (Fig. 2b). Particularly high p16INK4a protein levels, induced by the Ras–Raf–MAP kinase/ERK kinase (MEK) signalling cascade16, were found in Suv39h1-null cells, underscoring the putative role of p16INK4a acting upstream of Suv39h1 (Fig. 2b and Supplementary Fig. S3a). To dissect oncogenic signalling in Suv39h1-null or p53-null lymphoma cells further, we introduced complementary DNAs

Figure 3 | Suv39h1 loss produces chromosomally stable senescencedefective, but apoptosis-competent, Ras lymphomas. a, Spectral karyotypes of individual Suv39h1-null lymphomas; sample no. 3 with a normal karyotype, and sample no. 5 with a T(6;15) translocation (see Supplementary Table 3 for details). b, Growth curve analysis of freshly isolated control (n ¼ 5; black), Suv39h1-null (n ¼ 14; red) and p53-null lymphomas (n ¼ 4; blue). c, Twenty-four-hour viability after exposure to adriamycin; cells as in b. d, Left: SA-b-galactosidase activity in bcl2-infected lymphomas 5 days after no treatment or 0.1 mg ml2 1 adriamycin (n ¼ 3; percentages reflect positive cells (means ^ s.d.)). Insets show, after 3 days, increased cell size (forward scatter, FSC) and granularity (side scatter, SSC) reminiscent of cellular senescence in treated control cells only. Right: growth curve analysis (n ¼ 4); only control cells arrest after treatment with adriamycin (ADR; P ¼ 0.0001). UT, untreated. Relative cell numbers are the proportion of viable cells detectable after 5 days. 662

encoding the alternative reading frame product of the INK4a locus ARF or p16INK4a into these cells and monitored whether a particular gene activity was enriched for or selected against by means of coexpressed green fluorescent protein (GFP). Consistent with high endogenous p16INK4a protein levels (Fig. 2b and Supplementary Fig. S3b) was our observation that Suv39h1-null and p53-null lymphoma cells were enriched for the INK4a moiety within a 48-h period, whereas control lymphomas maintained stable p16INK4a levels as indicated by an unchanged GFP fraction (Fig. 2c). Both control and Suv39h1-null lymphomas did not tolerate abovephysiological levels of ARF, whereas p53-null lymphomas, consistent with ARF’s acting upstream of p53, accepted exogenous expression of this moiety. Accordingly, endogenous ARF protein expression was low in Suv39h1-null lymphomas in situ compared with high levels in p53-null lymphomas and was undetectable in control lymphomas; both Suv39h1-null and control lymphomas retained wild-type p53 (five of five cases per genotype sequenced; Supplementary Fig. S3b). Both Suv39h1 2 and Suv39h1 þ /2 Em-N-Ras mice being heterozygous for p53 succumbed—within the time-to-death range known from Suv39h1-inactivated animals—to lymphomas lacking Suv39h1 expression but retaining the remaining p53 wild-type allele (Fig. 2d; three of three tumours tested). Hence, Suv39h1 inactivation alleviates the pressure to co-inactivate p53 in response to oncogenic Ras during lymphoma development, although Suv39h1-null lymphomas remain highly sensitive to exogenous p53-activating triggers such as ARF overexpression. Chromosomal stability and Suv39h1 deficiency Because B-cell lymphomas that formed in a non-transgenic context after long latency in the absence of Suv39h1 and the closely related Suv39h2 genes display hyperdiploid karyotypes and to some extent so-called ‘butterfly’ chromosomes reminiscent of a chromosomal segregation defect12, we next analysed the karyotypes and Suv39h2 expression in Ras-driven T-cell lymphomas retaining or losing Suv39h1 expression. By scanning a large number of metaphases, we observed similarly modest deviations from the normal number of 40 chromosomes in control and Suv39h1-null lymphomas (40.3 ^ 0.7 and 40.1 ^ 1.2 chromosomes, respectively; errors are ^ s.d.) and a tendency towards hyperdiploidy in p53-null lymphomas (43.7 ^ 2.2 chromosomes). In particular, no non-segregated ‘butterfly’ chromosomes were detected in any metaphase. Suv39h2 is expressed in Ras-lymphomas throughout the genotypes, including Suv39h1-null cases (three of three samples tested; Supplementary Fig. S3c). More refined molecular cytogenetic analyses by spectral karyotyping underscored the numeric ranges reported, and confirmed that Suv39h1-null lymphomas display either no overt chromosomal aberrations or similar ones to those detected in control lymphomas (Fig. 3a and Supplementary Table 3). More precisely, gains of chromosomes 10 and 17 as well as chromosome 15 translocations were identified in subsets of both genotypes, possibly indicating a clonal evolution contributing to lymphoma progression. Thus, Suv39h1 deficiency promotes lymphomagenesis in response to oncogenic Ras without mis-segregation-driven chromosomal instability. Availability of drug-inducible apoptosis and senescence Recapitulating their aggressive behaviour and equally high proliferative capacity in vivo (Fig. 1b and Supplementary Fig. S3d, e), isolated control, Suv39h1-null and p53-null Ras transgenic lymphoma cells could be readily established as primary cultures in vitro, where they all grew exponentially (Fig. 3b). Whereas Suv39h1-null lymphomas displayed a slight growth advantage at best in comparison with controls, cell numbers in the p53-null group increased most rapidly, raising the question of whether an apoptotic defect in the absence of p53 might contribute to this phenotype (as revealed for spontaneous apoptosis in situ; Supplementary Fig. S3f). To examine the apoptotic capacity of Ras-driven lymphomas in vitro,

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primary lymphoma cells of the different genotypes were exposed to the DNA-damaging agent adriamcyin in a short-term cytotoxicity assay. Whereas p53-null lymphomas were highly resistant to adriamycin-induced cell death as expected, Suv39h1-null and control cells underwent massive apoptosis even at low drug concentrations (Fig. 3c; the apoptotic nature of cell death was confirmed by staining with Annexin V; data not shown). Because anticancer drugs can acutely provoke a terminal growth arrest that exhibits morphological, biochemical and genetic features indistinguishable from premature senescence in response to oncogenic Ras3,17,18, we examined drug-inducible senescence in Ras-driven lymphomas that were protected from cell death by expression of the antiapoptotic regulator Bcl2. Adriamycin produced a tight growth arrest accompanied by strongly induced senescence-associated

b-galactosidase (SA-b-gal) activity19 and morphological changes typical for cellular senescence in control lymphomas, whereas both Suv39h1-null and p53-null cells continued to proliferate and remained virtually negative for SA-b-gal activity (Fig. 3d). Moreover, control lymphomas responded to the drug with a sharp decline in 5-bromo-2-deoxyuridine incorporation paralleled by an almost complete accumulation in the G1 fraction, whereas Suv39h1-null cells displayed little change in G1 and S fractions after treatment (data not shown). Irrespective of their ability to enter senescence, lymphomas of all genotypes contained higher amounts of p16INK4a, and Suv39h1-null and p53-null lymphomas induced ARF protein concentrations to various extents in response to adriamycin (compare Supplementary Fig. S3g with Supplementary Fig. S3b). Taken together, these results show that loss of Suv39h1 expression renders Ras-driven lymphomas senescence-incompetent without compromising their apoptotic capacity. Suv39h1 dependence of Ras-provoked senescence To determine whether Suv39h1-controlled cellular senescence might directly limit Ras-induced transformation in the lymphoid compartment, a retroviral construct encoding activated Ras was introduced into primary Suv39h1 þ /þ or Suv39h1 2 /2 splenocytes. The exogenous expression of oncogenic Ras in Suv39h1 þ /þ lymphocytes virtually stalled their net proliferative capacity but allowed the Suv39h1 2 /2 population to increase significantly (Fig. 4a, P ¼ 0.0002). There was no difference in the growth potential between vector-infected Suv39h1 þ /þ and Suv39h1 2 /2 lymphocytes. Accordingly, only Ras-expressing Suv39h1 þ /þ lymphocytes displayed a strong SA-b-gal activity 6 days after Ras induction throughout the population, whereas very little SA-b-gal activity was sporadically detectable in the absence of either or both oncogenic Ras and Suv39h1 (Fig. 4b). In response to Ras only Suv39h1 þ /þ cells displayed a sharp increase in H3K9 trimethylation and produced an intense yet grainy staining pattern for HP1-g, possibly indicating a murine equivalent of Ras-dependent senescence-associated chromatin foci so far described for human cells only5 (Fig. 4c). Thus, Suv39h1-mediated H3K9me-associated senescence serves as an early barrier to mitogenic Ras signalling in primary lymphocytes. Because acetylated H3K9 cannot be methylated by Suv39h1 (ref. 6), and because Suv39h1 might locally reinforce transcriptional repression through association with DNA methyltransferases20,21, we reasoned that a combined treatment of a histone deacetylase inhibitor such as trichostatin A (TSA) with the DNA-demethylating agent 2-deoxy-5-azacytidine (DAC) might at least partly mimic Suv39h1

Figure 4 | Suv39h1/H3K9me-controlled senescence limits Ras-mediated oncogenicity. Primary Suv39h1 þ /þ and Suv39h1 2 /2 splenocytes were stably infected with oncogenic ras or an empty vector as control, and were plated at identical cell numbers immediately on completion of antibiotic selection. a, Growth curve analysis at day 6 (n ¼ 4 independent preparations each); error bars denote s.d. Relative cell numbers are the proportion of viable cells detectable after 6 days. b, c, Cytospin preparations of the day-6 samples assayed for SA-b-galactosidase activity (n ¼ 3; percentages reflect positive cells (means ^ s.d); note that cells may gradually enter an SA-b-galactosidase-positive state) (b), and stained by immunofluorescence for trimethylated H3K9 (medium power-field) and HP1-g (high-power inset) (c).

Figure 5 | Methylation-targeting therapy mimics Suv39h1 loss in a Ras transgenic context in vivo. Time-to-death observation in Em-N-Ras transgenic mice repeatedly treated with trichostatin A (TSA) and 2-deoxy-5azacytidine (DAC) or vehicle only (mock). Latency indicates the time after initiation (at about 6 weeks of age) of the 4-week protocol until a terminal disease stage was entered or unexpected death occurred (mock-treated, n ¼ 6, black; TSA/DAC-treated (developing leukaemic T-cell lymphomas, inset), n ¼ 11, red).

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inactivation. Whereas Em-N-Ras mice remained free of disease symptoms for more than 8 weeks when exposed to the vehicle only, they all developed clonal CD3þ T-cell lymphomas with leukaemic dissemination—similar, although somewhat more pleomorphic and mature, to Suv39h1-null Ras lymphomas—within 5 weeks under the TSA/DAC protocol (Fig. 5 and Supplementary Fig. S4a, b). Although the combination therapy at the dosage and schedule chosen seemed to be toxic in itself, lethal side effects observed in some of the wild-type mice under the TSA/DAC protocol occurred significantly later (P ¼ 0.0358), with histopathological aspects of myelosuppression and acute hepatic toxicity, but no evidence for lymphoma at necropsy. Thus, pharmacological targeting in vivo of epigenetic modifications including Suv39h1-governed histone and DNA methylation resembles the long-term outcome observed when Suv39h1 is genetically inactivated. The tumourpromoting action of methylation-targeting compounds in this genetic context sharply contrasts with their clinically promising use in myelodysplastic syndrome and some acute myeloid leukaemias22, highlighting the complexity of epigenetic alterations in different neoplastic and pre-neoplastic scenarios. Thus, these results provide evidence that Suv39h1-mediated histone modification controls cellular senescence as a tumour-suppressive programme that both directly limits transformation in response to oncogenic Ras in vitro and abrogates Ras-driven lymphoma formation in vivo. Relevance of oncogene-induced senescence in vivo Cellular senescence provides a key barrier to oncogene-mediated transformation in vitro, but its role in tumour suppression in vivo is poorly demonstrated1,3,23. Our results have now identified H3K9merelated senescence as a mechanism that efficiently cancels Rasinduced lymphomagenesis in vivo, promoting aggressive lymphomas in all Em-N-Ras mice lacking at least one Suv39h1 allele, whereas most transgenic control animals remain free of lymphoma and succumb to non-lymphoid neoplasms much later. Although the translocation breakpoint on chromosome 15 detected in several cases by spectral karyotyping may involve the c-myc locus, alternative alterations sufficient to bypass Ras-induced senescence were found in virtually all lymphomas tested. Ras-provoked senescence is a directly Suv39h1-dependent tumour suppressor mechanism controlled by both ARF and p53 that acts as the prime barrier against lymphoma formation. Accordingly, no lymphoma formed without an expression defect in one of these senescence regulators. Premature senescence has been demonstrated mostly in response to abovephysiological expression levels of oncogenic H-Ras in vitro. In contrast, oncogenic K-Ras expressed from its endogenous promoter has been reported to promote proliferation directly without causing cellular senescence in certain circumstances in vitro, but its immortalizing and transforming capacity in vivo is highly dependent on context and cell type, and seems to rely on a titrated attenuation of the MAP kinase pathway, whose activity is critical for the induction of cellular senescence14,24,25. The implication of Suv39h1 function in Ras-provoked cellular responses might also depend on the cell type, because mouse embryo fibroblasts lacking Suv39h1/2 genes still arrest in response to oncogenic Ras5 and because we did not observe accelerated simultaneous manifestation of histiocytic sarcomas at the time that Suv39h1 2 or p53 þ /2 mice succumbed to their lymphomas. Although progression models for many solid cancers from premalignant tumours to full-blown malignancies have been established, no such precursor lesion has yet been identified in the process of lymphoma development, and it remains technically challenging to detect a putative growth-arrested lesion formed at an unknown level anywhere in the lympho-haematopoietic compartment. Although no Suv39h1 alterations have been reported in manifest human cancers so far, deregulation of Suv39h1 function might also be attributed to defective binding partners, because naturally occurring Rb mutants fail to complex with Suv39h1, thereby disrupting its spatial action on 664

E2F-responsive promoters7. Moreover, another Rb-binding H3K9 methyltransferase, Riz1, is inactivated by mutations in human cancers26, and Riz1-deficient mice are lymphoma-prone27, possibly because of a similar H3K9me-related senescence defect. Our results also have important ramifications for therapeutic strategies, because the precise genetic defect ultimately licensing Ras-driven lymphoma formation might interfere with both druginducible apoptosis and senescence programmes. Although Suv39h1null lymphomas are phenotypically similar to those lacking p53 with regard to their early onset, they present with a sensitized p53 machinery susceptible to exogenous apoptotic triggers such as ARF overexpression or DNA-damaging drugs. Interestingly, control lymphomas that formed in response to Ras without explicit defects in Suv39h1 or p53 can bypass oncogene-induced senescence through the loss or critical reduction of ARF expression, but may still recall a senescence programme when triggered by exogenous stimuli such as DNA-damaging anticancer drugs. Because both drug-inducible apoptosis and senescence contribute to the outcome of cancer therapy18, we propose that the identification and functional assessment of genetic defects inactivating oncogene-provoked failsafe mechanisms will be important for optimizing treatment strategies. METHODS Mouse strains and tumour monitoring. Em-N-Ras transgenic mice were intercrossed to mice harbouring targeted deletions in the Suv39h1 or p53 locus. All strains were backcrossed into a C57BL/6 strain background. Genotyping was performed as described28. Time to death was defined as the latency between birth and unexpected death or a terminal disease stage as indicated by more than 20% weight loss or other symptoms of severe sickness. Statistical analysis of Kaplan–Meier survival plots is based on the log-rank (Mantel–Cox) test. After killing of the mice with CO2, single-cell suspensions were isolated from enlarged organs, and tissues were processed as described for histopathology and subsequent staining with haematoxylin and eosin18,28. Blood smears were taken as described28. Spleens from 8–12-week-old non-transgenic mice served as the source for primary splenocyte preparations. For combined TSA (Sigma) and DAC (Sigma) treatments in vivo, 39–45-day-old mice received TSA (0.5 mg kg2 1 body weight) subcutaneously once a day and DAC (2 mg kg2 1 body weight) intraperitoneally three times weekly or mock treatment (solvents only) for up to 4 weeks, and were monitored daily for signs of sickness. Cell culture, gene transfer and analysis of cell growth and integrity. Isolated splenocytes and lymphoma cells were cultured on irradiated NIH 3T3 feeder cells as described28. Numeric karyotypic analysis was performed on more than 200 4 0 ,6-diamidino-2-phenylindole (DAPI)-stained metaphase spreads of at least three lymphoma samples per genotype29, and spectral karyotyping (SKY) was performed as described30. T-cell clonality was assessed by multiplex PCR amplification of genomic DNA isolated from short-term-cultured lymphoma cells, using a panel of V(D)J primers for the T-cell receptor (TCR) b-chain31. Retroviral gene transfer was performed as described29; all retroviruses were MSCV-based constructs that either co-express GFP (namely MSCV-p16INK4aIRES-GFP; MSCV-ARF-IRES-GFP) or a selectable antibiotic resistance gene (namely MSCV-bcl2-puro; MSCV-H-RasV12-puro; MSCV-Suv39h1-hygro), including the corresponding vector-only controls. For enrichment experiments based on GFP co-expressing vectors, freshly infected lymphoma cells were adjusted to about 10% GFP-positive cells by admixing uninfected cells of the same lymphoma population, and the fraction of GFP-positive cells was reassessed by flow cytometry after 48 h in culture. Cell viability and cell numbers were analysed by Trypan blue dye exclusion; 5-bromo-2-deoxyuridine incorporation and cell-cycle analyses were performed as described29. Spontaneous apoptosis in situ was measured as described28 and was quantified by integrated scanning of fluorescence photomicrographs using the NIH image 1.63 software. Apoptosis induced by the DNA-damaging compound adriamycin (Sigma) was quantified by staining with annexin V in accordance with the manufacturer’s protocol (BD Pharmingen). Comparisons of means and standard deviations were performed with the unpaired t-test. Senescence-associated b-galactosidase activity was assessed in cytospin preparations as described18; senescence-associated changes in the cellular morphology were quantified by flow-cytometric lightscatter analysis. All experiments were performed with reproducible results in at least duplicate. Gene sequence and expression analysis. Immunoblotting was performed with whole-cell lysates resolved by 15% SDS–PAGE and transferred to an ImmobilonP membrane (Millipore) by using antibodies against ARF (ab80; Abcam; 1:1000

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dilution), Bcl2 (no. 554087; BD Pharmingen; 1:2000), H-Ras (OP23; Oncogene; 1:1000), N-Ras (OP25; Oncogene; 1:500) and p16INK4a (M-156; Santa Cruz; 1:500), with antibodies against a-tubulin (T5168; Sigma; 1:8000) as a loading control28. To perform immunohistochemistry on formalin-fixed, paraffinembedded tissue sections and immunocytochemistry on formalin-fixed cytospin preparations for p16INK4a (F-12; Santa Cruz; 1:2000), ARF (ab80; Abcam; 1:20), Ki67 (Tec-3; DakoCytomation; 1:2000), CD3 (no. 1580; Dako; 1:100), CD11b (M1/70; BD Pharmingen; 1:50) and terminal deoxynucleotidyl transferase (DakoCytomation; 1:20), slides were immersed in sodium citrate buffer solutions at pH 6.0, heated in a high-pressure cooker for 5 min, rinsed in cold water and washed in Tris-buffered saline (pH 7.4), and were then treated with a peroxidase-blocking reagent (DakoCytomation) before incubation for 1 h with the respective primary antibody. Binding was detected by the EnVision peroxidase kit using diaminobenzidine as the chromogenic substrate (K 4010; DakoCytomation). For immunofluorescence, cells were fixed in 2% paraformaldehyde, permeabilized with 0.2% Triton X-100 in PBS, preincubated in rabbit normal serum and incubated with primary antibodies against HP1-g (no. 07-332; Upstate; 1:1000), and trimethylated H3K9 (ab8898; Abcam; 1:1000), followed by the secondary anti-rabbit IgG antibody (Alexa Fluor 594; Molecular Probes; 1:2000). For immunophenotyping by flow cytometry, directly fluorescence-conjugated antibodies against B220 (CD45R), surface IgM, Thy1.2 (CD90), TCR-b, TCR-g/d, GR-1, NK1.1, CD3, CD4, CD5, CD8, CD19, CD43, CD68, CD86, CD117, CD138 (all BD Pharmingen) and Mac-1 (CD11b; Acris; 1:400) were used as described28. Suv39h1 and Suv39h2 transcript expression levels, compared with TATA-boxbinding-protein (TBP) expression as an internal control, were analysed after gene-specific reverse transcription (Superscript; Invitrogen) and PCR amplification of lymphoma cell total RNA. RT–PCR products of exons 4–8 of the p53 gene were sequenced as described28.

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Received 19 January; accepted 26 May 2005.

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Supplementary Information is linked to the online version of the paper at www.nature.com/nature. Acknowledgements We thank A. Harris, T. Jacks and M. Serrano for mice; S. W. Lowe for retroviral constructs; B. Teichmann and S. Spieckermann for technical assistance; and A. Lee, M. Reimann and P. Kahlem for discussions and editorial advice. This work was supported by grants from the European Union (to B.S.) and from the Deutsche Krebshilfe (to C.A.S.). S.L. is a postdoctoral fellow of the Jose´ Carreras Leukemia Foundation. Author Information Reprints and permissions information is available at npg.nature.com/reprintsandpermissions. The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to C.A.S. ([email protected]).

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