a role for e2f1 in the induction of arf, p53, and...

A Role for E2F1 in the Induction of ARF, p53, and Apoptosisduring Thymic Negative Selection1

Jing W. Zhu, Deborah DeRyckere, Feng X. Li,Yisong Y. Wan, and James DeGregori2

Departments of Biochemistry and Molecular Genetics and Pediatrics,University of Colorado Health Sciences Center, Denver, Colorado80262

AbstractE2F transcriptional activity controls the expression ofmany of the genes required for G1 to S phaseprogression. E2F1, one member of the E2F family,plays an important role in the induction of apoptosis.We have examined the role of the E2F1 transcriptionfactor in apoptosis during T-cell maturation in thethymus. We show that E2F1 is required for theapoptosis of autoimmune immature T cells duringthymic negative selection in vivo. This T-cell receptor-mediated apoptosis coincides with the E2F1-dependentincrease of p19-ARF mRNA and p53 protein levels. Incontrast, E2F1 is not required for the induction ofapoptosis by glucocorticoids or DNA damage. Theseresults demonstrate a specific role for E2F1, whichtriggers a pathway leading to ARF and p53 induction,in a physiological apoptosis pathway that is uncoupledfrom a normal proliferative event.

IntroductionTo respond to virtually any foreign invader, the immune sys-tem must be capable of recognizing a vast range of antigens.To accomplish this, lymphocytes assemble antigen receptorgenes by the somatic DNA rearrangement of families of genesegments, generating coding sequences with different vari-able (V) regions, which results in an enormous repertoire ofpotential reactivity toward antigens. However, because thisprocess is relatively stochastic, it inevitably leads to thegeneration of lymphocytes with either nonfunctional or self-reactive antigen receptors. To prevent autoimmunity, self-reactive lymphocytes must be eliminated (1). One mecha-nism by which this is accomplished is the induction ofapoptosis, a genetically determined program of cell suicide,

in immature self-reactive T cells during maturation in thethymus.

During the development of T cells, immature lymphocytesfrom the bone marrow migrate to the thymus (1). These cellsthen undergo rearrangement of their genomic b and then a

TCR3 genes, followed by low levels of TCR expression to-gether with the linked CD3 complex and the accessory CD4and CD8 proteins. The CD3 complex mediates TCR signal-ing, in part through the recruitment of the ZAP70 and Srcfamily kinases (2). CD4 and CD8 facilitate the interaction ofthe TCR with the class II MHC and class I MHC, respectively,expressed on APCs. The fate of these immature CD41CD81

(DP) thymocytes is determined by interactions between theirTCR and peptide/MHC complexes presented by thymicAPCs, including epithelial and dendritic cells. Antigens areprocessed in these cells into peptides, and selected peptidesare presented by the MHC on the cell surface. Thymocytesthat are strongly reactive toward self-peptides in associationwith MHC proteins and are therefore potentially self-reactiveare eliminated by apoptosis (negative selection; Refs. 1 and3). DP thymocytes that either fail to express a functional TCRor express a TCR with insufficient affinity or avidity for self-MHC presented in the thymus die, due to the lack of signal-ing via the TCR. In contrast, T cells with moderate affinity forself-MHC (less than 5% of thymocytes) are induced to dif-ferentiate into mature CD41CD82 or CD42CD81 T cells ex-pressing high levels of TCR and CD3 (positive selection).These mature T cells migrate out of the thymus to form theperipheral T cell repertoire. The observation that type I dia-betes appears to result in part from the failure to properlyeliminate autoimmune T cells in the thymus underscores theimportance of negative selection to normal physiology (4).

E2F activity controls the transcription of a group of genesthat are normally regulated at the G1-S-phase transition andencode proteins important for S-phase events including cy-clin E, B-Myb, dihydrofolate reductase, DNA polymerase a,and Cdc6, a limiting component of the prereplication com-plex (5). E2F transcriptional activity is composed of a varietyof heterodimers formed by the association of one of at leastsix different E2F family members with one of at least threedifferent DP proteins. E2F1, E2F2, and E2F3 associate spe-cifically with Rb, and the expression of these E2Fs is growth-regulated in fibroblasts. E2F4 and E2F5 appear to associatewith all three Rb family members, Rb, p107, and p130. Over-expression of E2F1, E2F2, E2F3, E2F4, or E2F5 indicates

Received 9/17/99; accepted 11/1/99.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to indi-cate this fact.1 D. D. is supported by a NIH postdoctoral fellowship. J. D. is supportedby grants from the V Foundation, the Howard Hughes Medical Institute,and the NIH (Grant RO1 CA77314-01).2 To whom requests for reprints should be addressed, at Department ofBiochemistry and Molecular Genetics, University of Colorado Health Sci-ences Center, BRB802, Mail Box C229, 4200 East Ninth Avenue, Denver,CO 80262. Phone: (303) 315-5792; Fax: (303) 315-3244; E-mail:[email protected].

3 The abbreviations used are: TCR, T-cell receptor; vSAG, viral superan-tigen; pi, postinjection; DP, double positive; Rb, retinoblastoma protein;OVA, ovalbumin peptide; Vb, TCR b chain variable region; RPA, Ribonu-clease Protection Assay; GC, glucocorticoid; Dex, dexamethasone; PI,propidium iodide; Adr, Adriamycin; LN, lymph node; APC, antigen-presenting cell; NF-kB, nuclear factor kB; cdk, cyclin-dependent kinase;TNF-R, tumor necrosis factor receptor; GAPDH, glyceraldehyde-3-phos-phate dehydrogenase.

829Vol. 10, 829–838, December 1999 Cell Growth & Differentiation

distinct abilities of these E2F family members to activate thetranscription of different target genes, and E2F1 uniquelyinduces apoptosis in serum-starved fibroblasts (6). Interest-ingly, disruption of E2F1 in the mouse results in an excess ofmature T cells due to a maturation stage-specific defect inthymocyte apoptosis (7) as well as the genesis of a diverserange of tumors in older adults (8). DP thymocytes fromE2F12/2 mice exhibit reduced apoptosis when cultured invitro (7), suggestive of a defect in passive cell death. Inaddition, E2F12/2 thymocytes show reduced apoptosis invivo (2.5% versus 3.5% for E2F11/1 mice) in response tointrathymic injection of CD3e-specific antibody. By bindingto the TCR/CD3 complex, anti-CD3e elicits TCR signaling,resulting in apoptosis primarily in DP thymocytes (9).

Previous work has shown that the induction of S phase byE2F1 overexpression in fibroblasts is accompanied by ap53-dependent induction of apoptosis (10–13), althoughoverexpression of E2F1 can in some contexts induce apo-ptosis that is p53 independent (14, 15). The p53 gene ismutated in more than half of human tumors (16). p53 isrequired for the induction of apoptosis in response to DNAdamage from chemotherapeutic agents or radiation, both inT lymphocytes and in oncogenically transformed fibroblasts(17–19). In addition, recent experiments have indicated a rolefor the ARF gene product (p19 in mice; p14 in humans) inoncogene-induced, p53-dependent apoptosis (20, 21). TheARF gene is translated from an alternative reading frame thatoverlaps the p16INK4A reading frame (22) in a locus frequentlydeleted in human tumors (23). Overexpression of E2F1 acti-vates the expression of ARF (6, 24), and in rodent fibroblasts,this activation is specific for the E2F1 and E2F2 family mem-bers (6). Consensus E2F binding sites are present in thehuman ARF promoter, and E2F1 can activate minimal pro-moters containing these sites (24, 25). Recent work hasshown that ARF antagonizes the activity of Mdm2, a proteinknown to target p53 for ubiquitin-mediated degradation.Thus, the activation of ARF leads to the stabilization of p53and the potentiation of its activity (26–29). ARF appears tostabilize p53 in part by blocking the nuclear-cytoplasmicshuttling of Mdm2 and p53 (30–32).

In this study, we demonstrate a specific role for E2F1 in thephysiological apoptotic pathway mediating thymocyte neg-ative selection. The levels of p19-ARF mRNA and p53 proteinincrease during antigen-induced TCR-mediated apoptosis.Importantly, this increase in ARF and p53 levels is dependenton E2F1. These results indicate a specific role for E2F1 andpossibly for ARF and p53 in a physiological apoptosis path-way in vivo, suggesting a common pathway that controls thedeletion of tumorigenic cells as well as autoimmune T cells.

ResultsE2F1 Is Specifically Required for Antigen-induced Apo-ptosis of CD41CD81 Thymocytes in Vivo. The study ofthymocyte maturation has been greatly enhanced by thegeneration of TCR transgenic mice. Transgenic mice havebeen created that express a and b chains for a TCR withknown peptide/MHC specificity in nearly all of their T cells(33). This has greatly facilitated the study of both positive andnegative selection because an entire population of maturing

T cells can be studied, rather than individual cells. In thebackground of the appropriate MHC but in the absence ofantigen, TCR transgene-expressing thymocytes are posi-tively selected. If the antigen recognized by the TCR is pres-ent in the thymus, either as an endogenous antigen or afterits introduction into the thymus, then transgene-expressingthymocytes are eliminated by apoptosis.

We chose to examine the role of E2F1 in antigen-inducedapoptosis using DO11.10 TCR transgenic mice. In these mice,most lymphocytes express a and b chain transgenes encodinga TCR (DO TCR) specific for a chicken OVA (OVA 323–339) inthe context of class II MHC I-Ad or I-Ab (3, 34). The introductionof OVA in vivo results in the elimination of the majority ofthymocytes through the induction of apoptosis in CD41CD81

(DP) T cells. To assess whether E2F1 is required for thymicnegative selection, we crossed the DO TCR transgene into micedisrupted for E2F1 (7) and further backcrossed both DO andE2F1 into the C57Bl/6 (MHC H-2b/b) genetic background forone to three additional generations. Where indicated, mice alsocontained disrupted Rag2 genes (35). Rag2 is an essentialcomponent of the recombinase that mediates the assembly ofeither TCR or immunoglobulin chains from germ-line arrays ofexons (36). The expression of the DO TCR bypasses the mat-uration block imposed by the Rag2 deficiency; consequently,virtually all lymphocytes in DO1 Rag22/2 mice bear the DOTCR exclusively (34). T and B cells are absent in E2F11 andE2F12/2 mice that are Rag22/2 and nontransgenic for DO,indicating that E2F1 is not required for the developmental blockimposed by the failure to rearrange antigen receptors (data notshown).

DO1 mice of the genotypes indicated in Fig. 1 were in-jected i.p. with OVA or control (CON) peptide. Thymuseswere harvested 14 or 24 h pi, the number of cells in thethymus was determined, and thymocytes were analyzed forthe expression of CD4, CD8, and the DO TCR by flow cy-tometry. The injection of CON peptide (ovalbumin residues324–334), which is presented by I-A but does not stimulatethe DO TCR (37), did not result in a significant reduction ofthymic cellularity in comparison with PBS-injected mice (Ref.3; Fig. 1; data not shown). As expected, the injection of OVAcaused a dramatic decrease in thymic cellularity in DO1

E2F11 mice by 24 h pi. Results obtained from E2F11/1 orE2F11/2 mice were similar for all of the experiments de-scribed in this study. In sharp contrast, no significant de-crease in thymic cellularity was observed in DO1 E2F12/2

mice injected with OVA (Fig. 1). Flow cytometric analysis ofthymocytes isolated from OVA-injected DO1 E2F11 micerevealed a selective loss of DP thymocytes by 14 h pi, andthe remaining DP cells exhibited reduced expression of CD4and CD8 (Fig. 1B), which is characteristic of thymocytesundergoing TCR-mediated apoptosis (38). In contrast, DO1

E2F12/2 thymocytes exhibit normal expression of CD4 andCD8 (Fig. 1B). Thymocytes from E2F11 or E2F12/2 miceexpress similar levels of the DO TCR, as determined bystaining with the clonotypic KJ26.1 monoclonal antibody(data not shown). Thus the absence of E2F1 appears toprevent or at least substantially delay the thymocyte celldeath resulting from peptide-induced negative selection. In-terestingly, OVA injection results in activation of peripheral

830 A Role for E2F1 in Thymic Negative Selection

DO1 CD41 T cells, which is independent of E2F1 genotype(data not shown), indicating that E2F1 is not required for theTCR-activated proliferative response of mature T cells.

E2F1 Mutant Mice Are Partially Deficient in the Deletionof T Cells Reactive with Endogenous Retroviral Superan-tigens. E2F1 deficiency clearly prevents the deletion of DOTCR transgenic thymocytes after I-Ab presentation of OVA. Weasked whether the deletion of nontransgenic T cells mediatedby endogenous antigen might also be influenced by E2F1 gen-otype. In mice, a variety of superantigens are encoded in the 39long terminal repeat of endogenous mouse mammary tumorviruses (reviewed in Ref. 39). These vSAGs bind to certain classII MHCs, particularly I-E MHC, facilitating their interaction withVb segments on thymocytes and resulting in the negative se-lection of these thymocytes. By monitoring the expression ofparticular Vb chains using monoclonal antibodies, it was shownthat thymocytes that are self-reactive with vSAGs are present atthe developmental stage of low TCR expression but are elimi-nated before they express high levels of TCR (40, 41). AlthoughCD41 T cells are more efficiently deleted due to the associationof vSAGs with class II MHC molecules, CD81 T cells are alsodeleted (39). Although the deletion of vSAG-reactive T cells isimpaired in the absence of gp39-CD40 interaction (42), othermutations that impair peptide-mediated T-cell deletion do notappear to affect vSAG-mediated deletion (see “Discussion”).

Because the H-2b locus does not encode for I-E MHC, webred the E2F1 mutation into the B10.D2 background (H-2d/d

and I-E1, but otherwise congenic with C57Bl/10) for at leastthree generations. The I-Ed MHC efficiently mediates thedeletion of Vb chains reactive with vSAGs, and T cells bear-ing Vb5, Vb7, Vb11, and Vb12 are normally deleted inB10.D2 mice, which express mouse mammary tumor provi-ruses Mtv 8, 9, and 17 (39). By monitoring Vb expression onT cells from either E2F11 or E2F12/2 mice, we further char-acterized the role of E2F1 in thymic negative selection with-out the expression of a transgenic TCR or the introduction ofexogenous antigen. Lymphocytes were isolated from theperipheral LNs of 1–2-month-old E2F11 or E2F12/2 miceand stained with fluorochrome-linked antibodies againstCD4, CD8, and one of several different Vb chains. The cellswere then analyzed by three-color flow cytometry. E2F1mutant mice exhibited significantly greater numbers of Tcells expressing Vb5, although the deletion of Vb12-bearingT cells was unimpaired (Fig. 2). In comparison, T cells dis-playing Vb6, which are not recognized by the vSAGs ex-pressed in these mice, were not deleted. Similar results wereobtained using thymocytes (data not shown).

It is not clear why the absence of E2F1 differentially impairsthe deletion of T cells bearing different vSAG-reactive Vb

chains. The impaired deletion of Vb5-bearing lymphocytes inE2F12/2 mice is more apparent in the CD8 T cells than in theCD4 T cells. Because vSAG-mediated cross-linking of the TCRand I-E is facilitated by CD4 but not CD8 (39), class I MHCrestricted T cells (CD81) may receive a weaker signal that is

Fig. 1. E2F1 is required for antigen-induced ap-optosis during negative selection in vivo. A, E2F11

(E2F11/1 or E2F11/2) and E2F12/2 DO TCR trans-genic littermates (H-2b/b; 20–24 days old) wereinjected i.p. with 0.5 mg of OVA (ISQAVHAA-HAEINEAGR) or CON (SQAVTAAHAEI) peptides.Thymuses were isolated 24 h pi, and thymic cel-lularity was microscopically determined using ahematocytometer. For each set of littermates, thenumber of T cells in the thymus relative to E2F11

mice injected with CON peptide is shown with theSE indicated. The data shown were derived fromfive experiments (in two of the experiments, thelittermates were all Rag22/2), except for the CON-injected E2F1 mutant sample, which was derivedfrom two experiments. The average cellularity forthe CON-injected DO1 E2F11 mice was 4.34 3107. The unpaired Student t test value for OVA-injected wild-type versus E2F1 mutant mice is P 50.00054. B, DO TCR transgenic, Rag22/2 litter-mates of the indicated E2F1 genotypes were in-jected i.p. with CON or OVA, and thymus cellswere isolated 14 h pi. Thymocytes were stainedwith fluorescent-labeled a-CD4 and a-CD8 anti-bodies and analyzed by flow cytometry. The per-centage of DP thymocytes is indicated in the up-per right corner of each panel. The total number ofcells in each thymus is also shown.

831Cell Growth & Differentiation

more easily impeded by E2F1 deficiency. In addition, although2-fold more CD81 T cells bearing Vb5 are present in the LNs ofE2F12/2 mice, it does not appear that E2F1 deficiency com-pletely eliminates the deletion of these self-reactive T cells. InC57Bl/6 mice lacking I-E expression, approximately 5% and7% of CD41 and CD81 peripheral T cells express Vb5, respec-tively (data not shown). Thus, the absence of E2F1 reduces butdoes not eliminate negative selection mediated by endogenoussuperantigens. Our results do not imply that E2F1 is required fornegative selection mediated by all MHC/antigen/TCR interac-tions. E2F1 deficiency may substantially increase the thresholdfor the affinity or avidity of MHC/antigen/TCR interactions thatare required to induce apoptosis, such that T cells that wouldnormally undergo negative selection survive. Nonetheless, Tcells bearing TCRs that are highly reactive with self may still beeliminated in the absence of E2F1. Indeed, we have observed areduced E2F1 requirement for negative selection induced bymore robust TCR signals (data not shown). Perhaps strongerTCR signals induce an E2F1-independent apoptotic pathway(s)that can compensate for the absence of E2F1.

E2F1 Is Not Required for Thymocyte Apoptosis In-duced by DNA Damage or GCs. Either DNA damage orGCs can also stimulate thymocyte apoptosis, with only theformer being dependent on p53 (17, 18). We asked whetherE2F1 is required for thymocyte cell death mediated by eitherDNA damage or GC induced by treatment with Adr or Dex,respectively. Thymocytes from either E2F11/1 or E2F12/2

mice (DO2; C57Bl/6 background) were cultured in the pres-

ence of Dex, Adr, or control DMSO and then assayed forapoptosis by staining with FITC-linked annexinV and PI.Apoptotic cells display phosphatidylserine in the outer cellmembrane, which is bound by annexin V (43). Either Dex orAdr induced apoptosis in both E2F11/1 and E2F12/2 thy-mocytes (Fig. 3A). Whole body irradiation (50–500 cGy) ofeither E2F11 or E2F12/2 mice also resulted in the selectivedeletion of DP thymocytes by 24 h, which was independentof the E2F1 genotype at all doses (Fig. 3B). Thus, the induc-tion of cell death by either DNA damage or GC treatment isindependent of E2F1 and is therefore distinct from the anti-gen-induced apoptosis that occurs during negative selec-tion. These data suggest that E2F1 is not required for apop-tosis in general but plays a specific role in the T-cellapoptotic signaling pathway activated by antigen.

E2F1 Is Required for the Induction of p19-ARF and p53upon TCR Stimulation. To better understand the mecha-nism by which E2F1 is required for TCR-dependent negativeselection, we analyzed the expression of various knownmodulators of apoptosis during negative selection. DO1

E2F11 mice were injected i.p. with either CON or OVA, andthymuses were harvested at various time points for the anal-ysis of cell number; CD4, CD8, and DO TCR expression; andmRNA/protein levels. At 10 h pi or earlier, OVA-stimulatedthymocytes were indistinguishable from CON-treated thy-mocytes in terms of both cell number and CD4/CD8/DO TCRexpression. OVA-stimulated thymocytes displayed the re-duced expression of CD4 and CD8 characteristic of thymo-

Fig. 2. E2F1 mutant mice are partially defective inthe deletion of T cells bearing TCRs reactive withendogenous retroviral superantigens. Lymphocyteswere isolated from the peripheral LNs of 1–2.5-month-old E2F11 or E2F12/2 H-2d/d mice. Lympho-cytes were stained with fluorescent-labeled antibod-ies against CD4, CD8, and either TCR Vb5, Vb6, orVb12. The cells were then washed and analyzed bythree-color flow cytometry. Cells were gated first forthe expression of CD4 or CD8 and then analyzed forthe expression of the indicated Vb chain. The num-ber of lymphocytes isolated from LNs and the rela-tive percentages of CD41 and CD81 T cells in LNswere similar in E2F11 and E2F12/2 mice. A, flowcytometric profiles of Vb5 and Vb6 expression onCD81 T cells from two 76-day-old E2F11/2 orE2F12/2 siblings. B, results obtained from at leastfour E2F11 and five E2F12/2 mice (sets of litter-mates) expressed as the percentage of Vb1 cellsamong CD41 or CD81 cells. The SE is indicated. p,the unpaired Student t test value for the percentageof CD81 T cells expressing Vb5 in wild-type versusE2F1 mutant mice is P 5 0.011.


cytes undergoing TCR mediated apoptosis by 12 h pi, andreduced cellularity was evident by 16 h (data not shown).

Given that the overexpression of E2F1 in quiescent fibro-blasts results in stabilization of p53 and induction of apo-ptosis, which is usually p53 dependent (44, 45), we examinedwhether p53 up-regulation might be involved in TCR-medi-ated thymocyte apoptosis. Western blot analysis of cell ly-sates revealed increased p53 protein levels in OVA-treated

DO1 E2F11 thymocytes relative to CON-treated cells by 16 hpi (Fig. 4A). No change in p53 mRNA is evident (data notshown), indicating that changes in p53 levels are transla-tional or posttranslational. Importantly, we do not observeany increase in p53 levels in OVA-treated DO1 E2F12/2

thymocytes (Fig. 4B). These results indicate that the accu-mulation of p53 protein in response to TCR signaling isdependent on the presence of E2F1. g-Irradiation of thymo-

Fig. 3. E2F1 is not required for thy-mocyte apoptosis induced by DNAdamage or GCs. A, thymus cellswere isolated from wild-type andE2F1 mutant mice (H-2b/b) and cul-tured in the presence of 20 mM Adr(Sigma) for 6 h; 0.05 (L), 0.1 (M), or0.2 mM (H) Dex (Sigma) for 3 h; orDMSO for 3 or 6 h (Con, left andright panels, respectively). Cellswere harvested, stained with fluo-rescence-labeled annexin V (A) andPI, and examined by flow cytometry.The percentage of A1 (PI1 or PI2, asindicated) cells is shown. Adr fluo-rescence prevented the determina-tion of PI incorporation. B, E2F11 orE2F12/2 mice (1–2-month-old mice)were either mock-treated or irradi-ated in a Nordion Cobolt 60 gammairradiator with 50, 100, 250, or 500cGy. The mice were sacrificed 24 hlater, and thymocytes were ana-lyzed for the expression of CD4 andCD8 by flow cytometry (left panel).The percentage of DP thymocytesand thymic cellularity are indicated.The graph in the right panel showsthe thymic cellularity (relative to themock-treated E2F11 littermate) andthe percentage of DPs at the indi-cated radiation doses. Each datapoint is derived from the average ofat least two experimental mice.

Fig. 4. E2F1 is specifically required for the induction of p53 protein levels following TCR stimulation. DO TCR transgenic littermates (C57Bl/6, except inA) of the indicated E2F1 genotypes were injected with 0.5 mg of OVA or CON peptides, and thymus cells were isolated at the indicated times pi. A, p53protein levels increase on TCR activation. Cell lysates were prepared from E2F11, DO TCR transgenic mice (Balb/C background) and analyzed by Westernblot using a-p53 and a-actin antibodies. B, p53 accumulation after TCR activation is E2F1 dependent. Thymus cells were isolated 24 h pi. Cell lysates wereprepared and analyzed by Western blot as described in A. The levels of p53 normalized for actin expression and relative to that of the CON-injected mouseare shown. C, p53 accumulation after DNA damage is E2F1 independent. Thymocytes were isolated and cultured as described in Fig. 1C. Cell lysates wereprepared and subjected to Western blot analysis as described in A.


cytes has been previously shown to result in the accumula-tion of p53 protein (17). Consistent with the fact that DNAdamage-induced apoptosis is E2F1 independent, the accu-mulation of p53 in response to DNA damage was not de-pendent on E2F1 (Fig. 4C). As expected, p53 does not ac-cumulate in response to treatment with Dex.

Recent work has suggested a pathway involving the ARFgene product, leading to p53 accumulation and apoptosis inresponse to oncogene activation (46). ARF may be a tran-scriptional target of E2F1 that is critical for the accumulationof p53 and the induction of E2F1-dependent apoptosis. Toinvestigate this possibility, we examined the expression ofARF in OVA-stimulated thymocytes by RPA. Indeed, in-creased ARF expression is observed by 6 h pi, with peakexpression at 10 h pi (Fig. 5A), preceding any observableeffect on thymocyte viability or CD4/CD8 expression. ARFexpression returns to basal levels by 14 h pi. There is anotable delay between the induction of ARF message andthe increase in p53 protein levels, which we do not presentlyunderstand. Importantly, whereas the expression of ARF wasactivated approximately 7-fold by 10 h in OVA-stimulatedDO1 E2F11 thymocytes, no increase was observed in DO1

E2F12/2 thymocytes (Fig. 5B). In OVA-treated E2F12/2 thy-

mocytes, ARF induction is also not observed by 15 h, a timewhen apoptosis is already apparent in the OVA-treatedE2F11/1 thymocytes, and ARF expression has returned tobasal levels. These data provide evidence that ARF expres-sion is regulated during apoptosis in vivo and indicate thatARF is a physiological target of E2F1. Interestingly, OVAstimulation does not result in increased expression of otherE2F target genes, such as cyclin E (Fig. 5C) and cyclin A (Fig.5D). Increased expression of cyclin A has been shown tocoincide with increased proliferation in immature CD42CD82

T cells (47). Thus, TCR-stimulated thymocyte apoptosis ispreceded by a specific, E2F1-dependent induction of ARF inthe absence of a general increase in E2F-dependent tran-scription. These results demonstrate a role for E2F1 in ap-optosis that is not associated with a proliferative event.

Additional RPA analysis of thymocyte RNA levels revealedan induction of the Bcl-2 family member Bfl-1/A1 by 4 h afterOVA injection (Fig. 6A). The expression of Bfl-1 has recentlybeen shown to be controlled by the Rel/NF-kB transcriptionfactor (48, 49), and the Rel-dependent expression of Bfl-1after antigen receptor ligation is necessary for B-cell survival(49). The induction of Bfl-1 is similar in E2F11 and E2F12/2

mice (Fig. 6B). Thus, antigen activation of immature T cells

Fig. 5. E2F1 is required for the up-regulation of ARF expression on TCR stimulation. DO TCR transgenic littermates (C57Bl/6, except in A) of the indicatedE2F1 genotypes were injected with either 0.5 mg of CON peptide, 0.5 mg of OVA, 5 mg of ovalbumin protein (Sigma), or PBS, as indicated, and thymuscells were isolated at the indicated times pi. A, ARF mRNA levels increase on TCR activation. RNA was isolated from E2F11 DO transgenic mice (B10.D2background), and ARF and GAPDH RNA levels were determined by RPA. The identity of the ARF-specific protected fragments has been verified both bymigration at the expected size (151 nucleotides) and the up-regulation of these fragments in p532/2 fibroblasts (data not shown), consistent with previousobservations (21). Double bands are not uncommon for RPA analysis and are presummed to be due to “breathing” at the ends of the RNA:RNA hybrid, whichallows for limited RNase access. B, induction of ARF expression after TCR activation is E2F1 dependent. Thymus cells were harvested at either 10 or 15 hpi (two separate experiments). RNA was isolated and ARF and GAPDH RNA levels were determined by RPA. The levels of ARF RNA, normalized for GAPDHexpression, relative to that of a CON-injected mouse are shown. At 10 h, we did not observe any reduction in thymic cellularity or CD4/CD8 staining inOVA-treated thymocytes. At 15 h, we observed a 2-fold reduction in the cellularity of the OVA-treated E2F11/1 thymocytes and a reduced percentage ofDP cells, but no reductions were observed in the OVA-treated E2F12/2 thymocytes. C, cyclin E expression is not induced on TCR activation. RNA wasisolated from thymocytes from E2F11 or E2F12/2 mice at the indicated times pi, and cyclin E and GAPDH RNA levels were determined by RPA. The levelsof cyclin E RNA (normalized for GAPDH expression) relative to that of a CON-injected mouse are shown. D, cyclin A expression is not induced on TCRactivation. RNA was isolated from thymocytes from E2F11 mice at the indicated times pi, and cyclin A and GAPDH RNA levels were determined by RPA.


results in the activation of some transcriptional targets inde-pendent of E2F1. These results are important because theyeliminate the possibility that the absence of negative selec-tion in E2F1 mutant mice is due to a failure of OVA toaccumulate and be presented in the thymus. As a furtherindication that apoptosis during negative selection is distinctfrom DNA damage- or GC-induced apoptosis, Bfl-1 mes-sage levels are not induced by either of the latter two stimuli.In fact, a reproducible 2-fold reduction in Bfl-1 mRNA levelswas observed after Adr treatment (Fig. 6C). Although the Baxgene has been shown to be regulated by p53 (50), the in-duction of p53 in the thymus by DNA damage or TCR stim-ulation did not result in substantial Bax activation (Fig. 6).

DiscussionWe have shown that E2F1 functions upstream of ARF andp53 in a thymic apoptotic pathway that contributes to theelimination of autoimmune T cells. ARF mRNA and p53 pro-tein levels are increased during negative selection, and thisincrease is dependent on the E2F1 transcription factor.Given the demonstrated roles for ARF and p53 in the induc-tion of apoptosis, our data suggest that ARF and p53 maycontribute to TCR-mediated thymocyte apoptosis. However,these data do not imply that ARF and p53 are required fornegative selection. Other targets of E2F1 may redundantlycontribute to this apoptosis because overexpression of E2F1can induce apoptosis independent of p53 (14, 15), and ARFactivation by the DMP1 transcription factor induces cell cyclearrest, not apoptosis (51). Nonetheless, given the presenceof potential E2F-responsive elements in the ARF promoter(24, 25) and the demonstrated association of ARF withmdm2/p53, our data, together with the data of others, sup-port the model shown in Fig. 7. TCR stimulation and/orcostimulatory signals result in the activation of E2F1 throughan as yet undefined pathway. E2F1 transcriptionally acti-vates the expression of ARF, which interferes with Mdm2function, resulting in the stabilization and potentiation of p53(46). p53 then contributes to apoptosis by regulating genetranscription and possibly also through transcription-inde-pendent effects (16). Antigen stimulation of immature thymo-cytes can also activate other pathways, such as the NF-kBpathway leading to the transcriptional activation of Bfl-1, thatare independent of E2F1. DNA damage results in p53 accu-mulation and apoptosis (52), independent of E2F1 (this work)and ARF (53). Finally, more robust or prolonged TCR signalsmay also induce negative selection through pathways thatdo not require E2F1. Our data does not address how E2F1activity is controlled during thymic negative selection. E2F1activity may be regulated by Rb and G1 cdk activity analo-gous to its control during G1 to S phase in fibroblasts. Phar-macological inhibition of cdk2 activation blocks thymocyteapoptosis induced by TCR stimulation, irradiation, or GCs invitro (54). Inhibition of cdk2 activity also prevents caspase-mediated cleavage of Rb in response to Dex or irradiation.Further work will be required to delineate the pathway thatcontrols E2F1 activation during thymic negative selection.

Multiple factors that influence apoptosis during thymic neg-ative selection have been identified. TNF-R-mediated signalingappears to serve as a costimulatory signal for negative selection

(55). The interaction of gp39 with CD40, a TNF-R family mem-ber expressed on APCs, is required for negative selection me-diated by either vSAGs or TCR transgene recognition of endo-genously expressed antigen (42). However, inhibition of gp39/CD40 interaction did not affect negative selection mediated byTCR recognition of exogenous antigen. Fas, another TNF-Rfamily member, may also play a role in thymic negative selec-tion (38), particularly at a higher antigen dose (56), although therole of Fas in negative selection is still controversial. The CD30TNF-R family member also contributes to the apoptosis ofautoreactive immature thymocytes. Whereas CD30-deficientmice are resistant to thymocyte negative selection induced bythe endogenous male antigen recognized by the H-Y trans-genic TCR, negative selection of thymocytes that recognizeendogenous vSAGs is unperturbed (57). In contrast, the dele-tion of thymocytes bearing Vb5 chains reactive with vSAGs isreduced in E2F1 mutant mice. E2F1 deficiency does not com-pletely abrogate this deletion, perhaps due to the chronic natureof the antigen or because of the reported differences in the TCRsignaling pathways activated by vSAGs versus peptide/MHC(58, 59).

The involvement of other transcription factors in negativeselection has also been demonstrated. A dominant negativeNur77 transgene, which inhibits all three known members of theNur77 orphan steroid receptor family, reduced negative selec-tion induced by transgenic TCR recognition of a peptide antigenbut did not affect vSAG-mediated thymocyte deletion (60). Incontrast to its traditional role in mediating cell survival, NF-kBappears to be required for thymic negative selection becausethe expression of an IkB transgene abrogates anti-CD3-in-duced apoptosis in the thymus (61). Whereas anti-CD3 canactivate the TCR, antigen presentation by thymic stromal cellsnormally entails multiple interactions between additional cellsurface molecules, such as B7/CD28, LFA-1/intercellular adhe-sion molecule 1, and tumor necrosis factor/TNF-R family mem-bers, that can contribute to apoptotic signaling (42, 62–64).Indeed, whereas TNF-R I/II-deficient mice showed impairedthymocyte apoptosis in response to anti-CD3, antigen-medi-ated clonal deletion was unimpaired (65). Thus, the requirementfor different players in negative selection appears to vary, de-pending on the nature of the recognized antigen, the MHCclass, and the acute versus chronic presence of the antigen(66). Whether deletion occurs in immature DP thymocytes in thethymic cortex or in late-stage DP and immature CD4 singlepositive thymocytes in the medulla may in part underlie thedifferential requirement for particular signaling components.Thus, negative selection is not as simple as each TCR modelimplies.

The important role of the E2F1-ARF-p53 pathway in thecontrol of lymphocyte cell death is underscored by the highsusceptibility of mice disrupted in E2F1 (8), ARF (53), or p53(67, 68) to the development of lymphomas and the frequentmutation of ARF and p53 in human tumors (16, 46). AlthoughE2F12/2 mice are tumor prone, E2F1 deficiency reducespituitary and thyroid tumorigenesis in Rb1/2 mice (69), pos-sibly reflecting the critical role for E2F1 in promoting prolif-eration resulting from Rb inactivation. Recent experimentshave also demonstrated a requirement for E2F1 in p53-dependent apoptosis and excess proliferation resulting from


either the expression of transgenic polyoma virus large Tantigen in the mouse choroid plexus epithelium (70) or theabsence of the Rb gene product during mouse embryonicdevelopment (71). Whereas the other E2F family memberscan largely compensate for the absence of E2F1 duringmouse development, the absence of E2F1 alone appears tosubstantially compromise the aberrant proliferation that re-sults from Rb inactivation. In addition, p19ARF/p16INK4A de-ficiency attenuates the apoptosis that occurs in the Rb2/2

mouse lens without decreasing proliferation (28), suggestingthat E2F1 targets other than ARF contribute to the prolifer-ative role of E2F1. The data presented here suggest that theE2F1-ARF-p53 pathway that functions to eliminate trans-formed cells may also contribute to the elimination of auto-immune T cells. Indeed, E2F12/2 mice that exhibit lympho-proliferation syndrome also display glomerulonephritis of thekidneys (8), which is indicative of autoimmunity. Despite adefect in thymic negative selection, E2F12/2 mice may avoida more severe autoimmune response as a result of peripheralmechanisms that induce tolerance.

Materials and MethodsMice. Mice were housed in the University of Colorado Health SciencesCenter animal resource center in cages with micro-isolator lids. RAG22/2

and DO transgenic mice were created by the F. Alt (Harvard MedicalSchool, Boston, MA) and D. Loh (Nippon Roche Research Center, Kana-gawa, Japan) laboratories, respectively, and obtained from P. Marrack(National Jewish Hospital, Denver, CO). E2F12/2 mice were provided byS. Fields and M. Greenberg (Harvard Medical School). Immunocompro-mised RAG22/2 mice were maintained under sterile conditions. Mice weregenotyped (for E2F1, RAG2, DO TCR, and H-2 genotype) by PCR analysisof DNA extracted from a small ear biopsy. Genomic DNA was isolated asdescribed previously (72). Peptides were produced by the MolecularResources Center at the National Jewish Hospital.

Flow Cytometry. Single cell suspensions obtained from thymuses orLNs were strained through nylon mesh and washed in PBS containing 5%fetal bovine serum (FBS/PBS). Cells (5 3 106) were stained in 30 ml of

CD4/CD8/DO TCR or CD4/CD8/Vb antibody solution 1:100 PE-a-CD4(PharMingen #09005B), 1:100 Cy-Chrome-a-CD8 (PharMingen #01048A),1:200 a-Fcg III/II receptor (PharMingen #01241A), and 8 mg/ml FITC-conju-gated KJ1.26 monoclonal antibody or 1:100 FITC-conjugated a-Vb antibody(PharMingen) in FBS/PBS for 45 min on ice. Cells were washed twice with 1ml of FBS/PBS and resuspended in 400 ml of PBS. For annexin V/PI staining,thymocytes were cultured in RP10 [10% FBS (Hyclone) in RPMI 1640 with 0.1mM 2-mercaptoethanol and 1% penicillin-streptomycin (Life Technologies,

Fig. 6. Induction of Bfl-1 expression on TCR stimulation is not E2F1 dependent. DO TCR transgenic littermates (C57Bl/6) of the indicated E2F1 genotypeswere injected with 0.5 mg of OVA or CON peptides, and thymus cells were isolated at the indicated times after injection. A, Bfl-1 expression is inducedon TCR activation. RNA was isolated from E2F11 mice, and Bfl-1, Bax, and GAPDH RNA levels were determined by RPA. The levels of Bfl-1 and Bax mRNAs(normalized for GAPDH expression) relative to that of a CON-injected mouse are shown. The data shown here are from an experiment using Rag22/2 mice.B, Bfl-1 induction after TCR activation is E2F1 independent. Thymus cells were harvested 10 h pi (the same samples as in Fig. 5B), and Bfl-1, Bax, andGAPDH RNA levels were determined as described in A. C, Bfl-1 expression is not induced in thymus cells treated with Adr or Dex. Thymocytes were isolatedand cultured as described in Fig. 3A. Bfl-1, Bax, and GAPDH RNA levels were determined as described in A.

Fig. 7. Model for the E2F1-dependent pathway controlling negative se-lection. TCR stimulation and/or costimulatory signals result in the activa-tion of E2F1, which transcriptionally activates the expression of ARF. ARFinterferes with Mdm2 function, resulting in the stabilization and potentia-tion of p53. DNA damage results in p53 accumulation and apoptosis,independent of E2F1 and ARF. TCR signaling can also induce apoptosisin thymocytes through pathways that do not require E2F1.


Inc.)] in the presence of Dex, Adr, or DMSO; harvested; and washed once inPBS. Cells (1 3 106) were stained in 100 ml of binding buffer [10 mM HEPES(pH 7.4), 140 mM NaCl, and 2.5 mM CaCl2] containing 0.5 mg of PI (BoehringerMannheim) and 1.0 mg of FITC-annexin V (Caltag Laboratories) for 15 min onice. Samples were then diluted with an additional 200 ml of binding buffer.Fluorescence was detected and analyzed using a Coulter Epics XL flowcytometer (Beckman Coulter).

Western Blotting and RPAs. RNA and cell lysates were preparedusing Trizol reagent (Life Technologies, Inc.) according to the manufac-turer’s instructions. a-p53 (#sc6243) and a-actin (#sc1616) antibodieswere purchased from Santa Cruz Biotechnology and used at 0.4 and 0.2mg/ml, respectively. Western blots were performed as per the manufac-turer’s instructions, except that 0.2% Tween 20 was included in theantibody solutions and washes. Quantitation of the resulting autoradio-grams was performed using a Molecular Dynamic densitometer. Levels ofcyclin E RNA, cyclin A RNA, or Bfl-1/Bax RNA were measured using thePharMingen RiboQuant Multi-Probe RPA System and the mCYC-2,mCYC-1, or mAPO-2 template sets, respectively. ARF RNA levels weredetermined by the same method using the pCDNA3 plasmid (Invitrogen)containing ARF exon 1 sequences (nt 4–155 of the ARF ORF) as atemplate for the ARF probe and the mGAPDH probe (PharMingen) as aninternal control. Dried radioactive polyacrylamide gels were exposed toKodak X-OMAT film (shown in figures) or exposed and analyzed on aPhosphorImager to quantitate mRNA levels.

AcknowledgmentsWe thank S. Fields, M. Greenberg, T. Mitchel, D. Hildeman, P. Marrack,

J. Kappler, M. McHeyzer-Williams, R. Leon, K. Murphy, C. Sherr, R.Sclafani, M. Kimbrough, D. Wegmann, and J. Nevins for important re-agents, assistance, and/or advice; K. Helm, P. Schor, and M. Ashton ofthe Cancer Center Flow Cytometry Core (supported by Grant 2 P30 CA46934-09); and P. Skavlen and Center for Laboratory Animal Care forexcellent veterinary care.

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a role for e2f1 in the induction of arf, p53, and...

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