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E2F Proteins Regulate MYCN Expression in Neuroblastomas

2003; Elsevier BV; Volume: 278; Issue: 5 Linguagem: Inglês

10.1074/jbc.m207596200

1083-351X

Verena Strieder, W. Lutz,

Virus-based gene therapy research

Amplification of the MYCN gene, resulting in overexpression of MYCN, distinguishes a subset of neuroblastomas with poor prognosis. The transcription factors driving MYCN expression in neuroblastomas are unknown. In transient-transfection assays, E2F-1, E2F-2, and E2F-3 activate aMYCN reporter construct dependent on the presence of several putative E2F-binding sites. Using chromatin immunoprecipitation, we show that E2F-1, E2F-2, and E2F-3 bind to the proximal MYCN promoter in vivo, specifically in neuroblastoma cell lines expressing MYCN. Inhibition of E2F activity in MYCN-amplified cells by the overexpression of p16INK4A reduced MYCN expression. In addition, we provide evidence that E2F proteins are involved in the negative regulation of MYCN by TGF-β and retinoic acid. These data suggest that E2F transcription factors are critical for both the full activation and the repression of MYCN in neuroblastomas. Amplification of the MYCN gene, resulting in overexpression of MYCN, distinguishes a subset of neuroblastomas with poor prognosis. The transcription factors driving MYCN expression in neuroblastomas are unknown. In transient-transfection assays, E2F-1, E2F-2, and E2F-3 activate aMYCN reporter construct dependent on the presence of several putative E2F-binding sites. Using chromatin immunoprecipitation, we show that E2F-1, E2F-2, and E2F-3 bind to the proximal MYCN promoter in vivo, specifically in neuroblastoma cell lines expressing MYCN. Inhibition of E2F activity in MYCN-amplified cells by the overexpression of p16INK4A reduced MYCN expression. In addition, we provide evidence that E2F proteins are involved in the negative regulation of MYCN by TGF-β and retinoic acid. These data suggest that E2F transcription factors are critical for both the full activation and the repression of MYCN in neuroblastomas. dimethyl sulfate transforming growth factor β retinoblastoma protein TGF-β-inhibitory element all-trans-retinoic acid Chromatin immunoprecipitation cytomegalovirus hemagglutinin N,N-bis(2-hydroxyethl)-2-aminoethanesulfonic acid cycle threshold The transcription factors encoded by the MYC genes form part of a complex regulatory network implicated in diverse tumorigenesis-relevant processes such as cell-cycle control, growth-factor dependence, response to antimitogenic signals, and apoptosis (1Grandori C. Cowley S.M. James L.P. Eisenman R.N. Annu. Rev. Cell Dev. Biol. 2000; 16: 653-699Google Scholar). Overexpression of the MYC genes as a result of chromosomal translocation, gene amplification, or loss of negative transcriptional control plays a prominent role in the etiology of many types of tumors. The evidence for a contribution of the MYCgenes to tumorigenesis and the functional consequences ofMYC overexpression have been the focus of several recent reviews (2Lutz W. Leon J. Eilers M. Biochim. Biophys. Acta. 2002; 1602: 61-71Google Scholar, 3Eisenman R.N. Genes Dev. 2001; 15: 2023-2030Google Scholar). Support for a critical role of MYC in cancer comes from several transgenic mouse models of MYC-induced tumorigenesis in which MYC expression can be reversibly switched off after tumors have developed (4Pelengaris S. Littlewood T. Khan M. Elia G. Evan G. Mol. Cell. 1999; 3: 565-577Google Scholar, 5Felsher D.W. Bishop J.M. Mol. Cell. 1999; 4: 199-207Google Scholar, 6D'Cruz C.M. Gunther E.J. Boxer R.B. Hartman J.L. Sintasath L. Moody S.E. Cox J.D., Ha, S.I. Belka G.K. Golant A. Cardiff R.D. Chodosh L.A. Nat. Med. 2001; 7: 235-239Google Scholar). Switching offMYC expression in the tumors resulted in tumor regression, suggesting that a tumor cell requires continuous MYCexpression, although secondary genetic changes can obliterate thisMYC dependence. The MYCN gene is found amplified in several types of childhood tumors of mostly neuroendocrine origin, including about 25% of neuroblastomas (7Savelyeva L. Schwab M. Cancer Lett. 2001; 167: 115-123Google Scholar). Amplification results in overexpression ofMYCN and distinguishes a subset of aggressive tumors with a poor prognosis (8Brodeur G.M. Seeger R.C. Schwab M. Varmus H.E. Bishop J.M. Science. 1984; 224: 1121-1124Google Scholar). Together, these observations suggest that blockingMYCN expression may be beneficial for neuroblastoma patients. However, the development of a therapy based on this concept is hampered by our lack of understanding of the transcriptional regulation of the MYCN gene in neuroblastomas (9Strieder V. Lutz W. Cancer Lett. 2002; 180: 107-119Google Scholar). Several signals that trigger neuronal differentiation of neuroblastoma cells, including pharmacological concentrations of all-transretinoic acid, cause a repression of MYCN (10Thiele C.J. Reynolds C.P. Israel M.A. Nature. 1985; 313: 404-406Google Scholar). This down-regulation of MYCN expression is essential for differentiation because ectopic expression of MYCN blocks differentiation (11Peverali F.A. Orioli D. Tonon L. Ciana P. Bunone G. Negri M. Della-Valle G. Oncogene. 1996; 12: 457-462Google Scholar). Neither the transcription factors nor the regulatory elements mediating the response to these signals have been identified as yet. The E2F transcription factors are important regulators of cell-cycle progression, and their activity is negatively controlled by the pRb pathway, which is deregulated in the majority of human cancers (for review, see Refs. 12Harbour J.W. Dean D.C. Nat. Cell Biol. 2000; 2: E65-E67Google Scholar and 13Trimarchi J.M. Lees J.A. Nat. Rev. Mol. Cell. Biol. 2002; 3: 11-20Google Scholar). Recent DNA microarray analyses have identified a large number of genes that are regulated by one or more of the E2F proteins (14Muller H. Bracken A.P. Vernell R. Moroni M.C. Christians F. Grassilli E. Prosperini E. Vigo E. Oliner J.D. Helin K. Genes Dev. 2001; 15: 267-285Google Scholar, 15Ishida S. Huang E. Zuzan H. Spang R. Leone G. West M. Nevins J.R. Mol. Cell. Biol. 2001; 21: 4684-4699Google Scholar, 16Ren B. Cam H. Takahashi Y. Volkert T. Terragni J. Young R.A. Dynlacht B.D. Genes Dev. 2002; 16: 245-256Google Scholar, 17Wells J. Graveel C.R. Bartley S.M. Madore S.J. Farnham P.J. Proc. Natl. Acad. Sci. U. S. A. 2002; 99: 3890-3895Google Scholar, 18Weinmann A.S. Bartley S.M. Zhang T. Zhang M.Q. Farnham P.J. Mol. Cell. Biol. 2001; 21: 6820-6832Google Scholar, 19Stanelle J. Stiewe T. Theseling C.C. Peter M. Putzer B.M. Nucleic Acids Res. 2002; 30: 1859-1867Google Scholar). The products of these target genes regulate diverse cellular processes including cell-cycle progression, apoptosis, differentiation, and DNA repair. There are six E2F family members that can be classified into different subgroups based on their structure, affinity for different members of the pRb family, and putative function. Members of one of these subgroups, which consists of E2F-1, E2F-2, and E2F-3, associate predominantly with pRb and, when overexpressed in serum-starved immortalized cells, are sufficient to induce progression into S-phase. E2F-4 and E2F-5, which form a second subgroup, bind to all pRb family proteins and are impaired in their ability to induce S-phase in quiescent cells. Whereas members of the first subgroup function predominantly in transcriptional activation, members of the second subgroup have been implicated in transcriptional repression. Although neither mutations in the RB gene nor any of the other genetic alterations known to inactivate the pRb pathway in different types of tumors have been detected in neuroblastomas, recent evidence suggests that E2F activity in neuroblastomas may nevertheless be deregulated (20Takita J. Hayashi Y. Nakajima T. Adachi J. Tanaka T. Yamaguchi N. Ogawa Y. Hanada R. Yamamoto K. Yokota J. Oncogene. 1998; 17: 3137-3143Google Scholar, 21Lasorella A. Noseda M. Beyna M. Yokota Y. Iavarone A. Nature. 2000; 407: 592-598Google Scholar). A region of 200 bp immediately upstream of the multiple transcription start sites is highly conserved in the MYCN genes of man and mouse and controls basal promoter activity (22Hiller S. Breit S. Wang Z.Q. Wagner E.F. Schwab M. Oncogene. 1991; 6: 969-977Google Scholar). DMS1 in vivo-footprinting of this region revealed several sites that were either hypersensitive or protected from modification by DMS in neuroblastoma cells expressing MYCN but were absent in cells that do not express MYCN (23Lutz W. Schwab M. Oncogene. 1997; 15: 303-315Google Scholar). One of the protected regions corresponded to two inversely oriented and overlapping E2F binding sites, indicating that members of the E2F family of transcription factors regulate MYCN expression in neuroblastomas. We therefore tested whether the MYCN gene is indeed regulated by E2F proteins in neuroblastomas. We show that the transcription factors E2F-1, E2F-2, and E2F-3 bind the MYCNpromoter in human neuroblastoma cells in vivo, and that blocking E2F acitivity in neuroblastoma cells via overexpression of p16INK4A reduces MYCN mRNA levels in a neuroblastoma cell line with amplification of MYCN. In addition, we show that TGF-β represses the MYCN gene through the E2F-binding sites, and that all-trans retinoic acid induced down-regulation of MYCN is associated with changes in E2F binding to the MYCN promoter. The human neuroblastoma cell lines SH-EP, IMR-32, LA-N-5, and Kelly were a gift of Manfred Schwab and were cultured as described (23Lutz W. Schwab M. Oncogene. 1997; 15: 303-315Google Scholar). HaCaT were kindly provided by Norbert Fusenig and cultured in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum. Drugs and growth factors were added to the cell culture medium at the following concentrations: all-trans retinoic acid, 50 μm; TGF-β (R&D Systems), 2.5 ng/ml. Cells were transfected in Dulbecco's modified Eagle's medium with 10% fetal calf serum using calcium phosphate precipitation in BBS buffer (50 mmBES, pH 6.95, 280 mm NaCl, 1.5 mmNa2HPO4). For transient reporter assays, cells in 6-cm dishes were transfected with 10 μg of DNA (4 μg of reporter construct, 0.5 μg of CMV enhancer/promoter-based expression vectors for both DP1 and E2F, 2 μg of an eGFP expression vector, and 3 μg of Bluescript). Cells were lysed in 100 mm potassium phosphate/0.2% Triton X-100, followed by three freeze/thaw cycles. Reporter assays were performed according to standard procedures. Luciferase activity was normalized to the activity of a co-transfected lacZ reporter construct for the assays in HaCaT cells, or to total protein of the lysates as determined by Bradford assay for the experiments involving overexpression of E2F proteins. In these cases, the percentage of cells expressing eGFP was determined microscopically before harvesting the cells to exclude differences in the relative transfection efficiencies. No significant differences in the number of eGFP-positive cells were observed between different samples. The luciferase reporter constructs have been described (23Lutz W. Schwab M. Oncogene. 1997; 15: 303-315Google Scholar). The following CMV-based E2F expression vectors were used: pCMV-E2F-1 (24Lukas J. Petersen B.O. Holm K. Bartek J. Helin K. Mol. Cell. Biol. 1996; 16: 1047-1057Google Scholar); pCMV-E2F-2 (25Lees J.A. Saito M. Vidal M. Valentine M. Look T. Harlow E. Dyson N. Helin K. Mol. Cell. Biol. 1993; 13: 7813-7825Google Scholar); pcDNA3-HA-E2F-3 (26Krek W. Ewen M.E. Shirodkar S. Arany Z. Kaelin W.G., Jr. Livingston D.M. Cell. 1994; 78: 161-172Google Scholar); pcDNA3-HA-E2F-4 (27Ginsberg D. Vairo G. Chittenden T. Xiao Z.X., Xu, G. Wydner K.L. DeCaprio J.A. Lawrence J.B. Livingston D.M. Genes Dev. 1994; 8: 2665-2679Google Scholar); pcDNA3-HA-E2F-5 (28Lindeman G.J. Gaubatz S. Livingston D.M. Ginsberg D. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 5095-5100Google Scholar); pCMV-DP-1 (29Jooss K. Lam E.W. Bybee A. Girling R. Muller R. La Thangue N.B. Oncogene. 1995; 10: 1529-1536Google Scholar). Human IMR-32 cells were transfected with an expression plasmid encoding the ecotropic receptor, and G418-resistant cells were pooled for infection with ecotropic retroviruses based on pBabehygro-p16. Recombinant retroviruses were generated and used as described (30Peukert K. Staller P. Schneider A. Carmichael G. Hanel F. Eilers M. EMBO J. 1997; 16: 5672-5686Google Scholar). Infected cells were selected with 100 μg/ml hygromycin and analyzed immediately without further passaging. The p16 construct was obtained by inserting the p16 cDNA derived from pXp16ink4 into pBabehygro (31Schulze A. Zerfass K. Spitkovsky D. Henglein B. Jansen-Durr P. Oncogene. 1994; 9: 3475-3482Google Scholar). SDS-gel electrophoresis, transfer to polyvinylidene difluoride membranes, and Western blotting were performed according to standard procedures using 50 μg of the whole-cell lysates prepared for reporter assays. Cdk2 was used to control for equal loading. The antibodies used were: α-HA, clone 16B12 (MMS-101P, BABCO); α-E2F1, C-20 (sc-193, Santa Cruz); α-E2F-2, C-20 (sc-633, Santa Cruz); α-Cdk2, M2 (sc-163, Santa Cruz). Chromatin immunoprecipitation was performed according to published procedures with some modifications. Cross-linking of the cells as well as preparation and sonication of chromatin were carried out as described by Boyd (32Boyd K.E. Farnham P.J. Mol. Cell. Biol. 1999; 19: 8393-8399Google Scholar). Immunoprecipitation and reversal of cross-links was done according to Takahashi (33Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref Google Scholar). In brief, twelve 15-cm dishes of subconfluent cells were treated with 1% formaldehyde for 10 min at room temperature. Cross-linking was stopped by the addition of glycine to a final concentration of 125 mm. Cells were washed with cold phosphate-buffered saline, scraped into phosphate-buffered saline containing protease inhibitors, pelleted by centrifugation, resuspended in lysis buffer (5 mm Pipes pH 8, 85 mm KCl, 0.5% Nonidet P-40, and protease inhibitors), and incubated on ice for 20 min. Cells were then ruptured in a Dounce homogenizer, and nuclei were pelleted, washed once in lysis buffer, and resuspended in sonication buffer (10 mm Tris pH 7.5, 150 mmNaCl, 1 mm EDTA, 1% Nonidet P-40, 1% deoxycholate, 0.1% SDS, and protease inhibitors). After 10 min, on ice the solution was sonicated with a Bandelin Sonoplus carrying a MS73 tip at 30% maximum power for six to ten 10-s pulses on ice. The chromatin solution was cleared for 10 min at 14,000 rpm, frozen in liquid nitrogen, and stored at −80 °C. After thawing, the solution was microcentrifuged as before and then pre-cleared by the addition of a mixture of protein A and protein G-Sepharose (previously blocked with 1 mg/ml bovine serum albumin and 1 mg/ml sheared salmon sperm DNA in sonication buffer at 4 °C overnight) for 4 h. Aliquots of the pre-cleared chromatin were incubated with 2 μg of each antibody overnight at 4 °C. One aliquot was incubated without antibody. The supernatant of this sample was later processed in parallel with the immunoprecipitates starting with the RNase A and proteinase K digest (this sample is referred to as "Input"). Immune complexes were captured with 40 μl of blocked protein A/protein G-Sepharose, washed seven times with cold washing buffer (50 mm Hepes pH 7.5, 500 mm LiCl, 1 mm EDTA, 1% Nonidet P-40, 0.7% deoxycholate and protease inhibitors) and once with TE, and then resuspended in 200 μl of TE. Both the immunoprecipitates and the input sample were digested with 10 μg each of RNase A and proteinase K at 55 °C for 3 h. After incubation at 65 °C overnight, the Sepharose was pelleted, and the supernatant containing the co-precipitated DNA was applied to a PCR purification column (Qiagen) according to the instructions of the manufacturer. The purified DNA was eluted in 50 μl of 10 mm Tris. 1 μl of the eluted DNA was used for PCR. The following antibodies were used: anti-Gadd-45 (sc-H165; Santa Cruz), anti-acetylated H3 and H4 (06–599 and 06–866; Upstate Biotechnologies); the antibodies specific for E2F family members and pocket proteins have been used for chromatin immunoprecipitation experiments before (33Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref Google Scholar). The following primers were used:MYCN: 5′-AATGACAAGCAATTGCCAGGC-3′ and 5′-AGGCGCCAAGGCTTTCGCG-3′; GAP-43: 5′-GGGGCTGGGGGAAGTGATTAGTC-3′ and 5′-CCCCCTCTCCACCTCTTTTCTGC-3′;p107 (33Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref Google Scholar); α-prothymosin (34Staller P. Peukert K. Kiermaier A. Seoane J. Lukas J. Karsunky H. Moroy T. Bartek J. Massague J. Hanel F. Eilers M. Nat. Cell. Biol. 2001; 3: 392-399Google Scholar). Thep107 and α-prothymosin PCRs were run for 36 cycles and the GAP-43 PCR for 38 cycles. In the case of theMYCN primers, the PCR was run for 30 cycles with DNA from Kelly and IMR-32, and for 36 cycles for DNA from SH-EP, to compensate for the amplification of the MYCN locus in Kelly and IMR-32. The PCRs for the chromatin immunoprecipitation with pocket protein-specific antibodies were performed on an ABI 7000 using SYBR Green. First, the mean cycle threshold (Ct) values of triplicate reactions of the input samples were analyzed. Chromatin preparations were only used when the input samples showed a Ct difference of 0.5 or less. Then, the Ct values of PCR reactions using the immunoprecipitated chromatin as a template were subtracted from the Ct values of the PCR reactions with the negative control sample (immunoprecipitation of chromatin in the absence of antibody; when the Ct values of the α-Gadd-45 samples were used as negative-control samples, very similar results were obtained). The mean values of these ΔCt values of triplicate PCR reactions were than compared between untreated and retinoic acid-treated samples. Total cytoplasmic RNA was isolated with the RNeasy Kit (Qiagen), and 1 μg was reverse-transcribed using Superscript II and random primers. An aliquot of cDNA first strands corresponding to 50 ng of RNA was used for the PCR amplification. Pilot experiments established that the PCR conditions used are within the linear range of amplification. Selective results were confirmed with real-time PCR on an ABI 7000 using SYBR Green. Primer sequences are available upon request. To test whether transcription factors of the E2F-family are able to activate the MYCN promoter in neuroblastoma cells, we transiently co-transfected human neuroblastoma cells with E2F expression vectors and a reporter construct in which luciferase expression is controlled by 230 base pairs of the humanMYCN gene, which encompass the major transcription start sites and three closely spaced putative E2F-binding sites, two of which overlap (Fig. 1 A). In SH-EP cells lacking amplification and expression of MYCN, this reporter construct was activated 3- to 5-fold by E2F-1, 2- to 3-fold by E2F-2, and 8- to 10-fold by E2F-3 (Fig. 1 A). E2F-4 and E2F-5 did not influence the activity of the MYCN promoter in this assay. Similar results were obtained with two neuroblastoma cell lines with amplified MYCN, IMR-32, and Kelly (data not shown). Western blotting showed high levels of E2F-1, E2F-2, E2F-3, and E2F-4 in the transfected cells (Fig. 1 B). E2F-5 was expressed at about 20% of E2F-3 and E2F-4, respectively. A point-mutant reporter construct lacking the two overlapping E2F-sites was not activated by E2F-1 but was still responsive to E2F-3 (Fig. 1 C). A construct lacking all three E2F-sites was only weakly activated by E2F-3 compared with the wild-type construct. Therefore, the proximal promoter of the MYCN gene in neuroblastoma cells is activated by E2F-1, E2F-2, and E2F-3, but not E2F-4 and E2F-5, and this activation is largely dependent on intact E2F-binding sites. If E2F proteins do play a role in the maintenance of MYCNexpression in neuroblastoma, they should bind to the MYCNpromoter in MYCN-expressing neuroblastoma cells. To test this prediction, ChIP assays with antibodies specific for E2F-1, E2F-2, E2F-3, and E2F-4 were performed with cross-linked chromatin from three neuroblastoma cell lines. Co-precipitated chromatin was used as template for PCR amplification using primers that amplify a 190-base pair fragment of the proximal MYCN promoter, including all three E2F binding sites. In the two cell lines Kelly and IMR-32, which show amplification and overexpression of MYCN, E2F-1, E2F-2, and E2F-3 but not E2F-4 bound to the proximal MYCN promoter (Fig. 2). In contrast, in SH-EP cells lacking detectable MYCN expression, no association of any of the E2F proteins with the MYCN promoter could be detected. Binding of E2F-1, E2F-2, and E2F-3 to the promoter of another E2F-responsive gene, p107 (RBL1), was readily detected in all three cell lines including SH-EP. No product for any of the imunoprecipitated chromatin samples was obtained with primers amplifying part of intron 1 of the PTMA gene encoding α-prothymosin, which harbors a MYC-responsive E-box motif but no E2F-binding site. There were marked differences in the abundance of different E2F proteins at the MYCN promoter in Kellyversus IMR-32 cells, with E2F-1 being the predominant E2F-binding activity in Kelly, whereas E2F-1–E2F-3 were present in equal amounts in IMR-32. From these data, we conclude that members of the E2F family bind the proximal MYCN promoter in vivo specifically in neuroblastoma cells with strongMYCN expression. Having demonstrated binding of E2F proteins to theMYCN promoter in vivo, we next asked whether blocking E2F activity would cause a reduction of endogenousMYCN expression. Overexpression of p16INK4a, an inhibitor of Cdk4 and Cdk6, leads to the accumulation of hypophosphorylated pRb and G1 arrest (35Serrano M. Hannon G.J. Beach D. Nature. 1993; 366: 704-707Google Scholar). IMR-32 cells do not produce detectable amounts of p16INK4a protein (20Takita J. Hayashi Y. Nakajima T. Adachi J. Tanaka T. Yamaguchi N. Ogawa Y. Hanada R. Yamamoto K. Yokota J. Oncogene. 1998; 17: 3137-3143Google Scholar). We generated pools of IMR-32 cells expressing the ecotropic receptor and subsequently infected these cells with pBABEhygro-p16INK4A, an ecotropic retrovirus driving expression of p16INK4a, or an empty control virus. RNA was isolated from hygromycin-selected cell populations and used for RT-PCR analysis. Cells that had been infected with thep16INK4A-containing retrovirus strongly expressed p16INK4A compared with cells that had been infected with the control virus (Fig. 3). p16INK4A caused a down-regulation of both CCNE1 and MYCN (Fig. 3). These results were confirmed by real-time PCR (data not shown). We conclude that E2F activity is required for maximum expression ofMYCN in neuroblastomas. Retinoic acid (RA) triggers neuronal differentiation of some neuroblastoma cell lines including LA-N-5, which carry amplified MYCN. Morphological differentiation, which is completed after about 10 days of treatment, is preceded by a reduction of MYCN expression as early as 6 h after addition of RA (10Thiele C.J. Reynolds C.P. Israel M.A. Nature. 1985; 313: 404-406Google Scholar). During the differentiation process, the amount of E2F-1 protein remains constant and the amount of E2F-4 increases sharply (36Raschella G. Tanno B. Bonetto F. Amendola R. Battista T., De Luca A. Giordano A. Paggi M.G. J. Cell. Biochem. 1997; 67: 297-303Google Scholar). The repression of MYCN through RA has been mapped to the proximal promoter, which, however, lacks a sequence resembling a retinoic acid response element (37Wada R.K. Seeger R.C. Reynolds C.P. Alloggiamento T. Yamashiro J.M. Ruland C. Black A.C. Rosenblatt J.D. Oncogene. 1992; 7: 711-717Google Scholar). To test whether the E2F-binding sites contribute to the RA-mediated down-regulation ofMYCN, LA-N-5 cells were treated with RA or the solvent control ethanol for 12 days and subjected to chromatin immunoprecipitation with a panel of E2F-specific antibodies. At this time, most of the cells had extended long cellular processes indicative of neuronal differentiation, and MYCN mRNA levels were reduced (data not shown). ChIP assay showed that in untreated LA-N-5 cells, like in Kelly and IMR-32, E2F-1, E2F-2, and E2F-3, were associated with the MYCN promoter (Fig. 4 A). In contrast to Kelly and IMR-32 cells, E2F-3 was the predominant E2F species in LA-N-5 (compare Figs. 2 and 4 A). Furthermore, unlike Kelly and IMR-32, E2F-4 was detected at the MYCN promoter in untreated LA-N-5 cells. In differentiated cells, there was virtually no binding of E2F-2 and E2F-4 to the MYCN promoter, and the binding of E2F-3 was drastically reduced. In contrast, the binding of E2F-1 to theMYCN promoter was unaltered. The loss of binding of E2F-2, E2F-3, and E2F-4 to the MYCN promoter was accompanied by a reduction in the acetylation of histones H3 and H4, consistent with a drop in transcriptional activity of the MYCN gene (Fig. 4 A). To exclude the possibility that the chromatin from the RA-treated cells in general produced less signal despite a similar intensity of the input lanes, the proximal promoter of theGAP-43 gene, which is up-regulated during neuronal differentiation, was also analyzed. Acetylation of the histones H3 and H4 at the GAP-43 promoter was unchanged upon RA treatment. As expected, no binding of any of the E2F proteins was detected. E2F proteins can repress target genes by associating with pocket proteins and recruiting a variety of co-repressor complexes (38Ferreira R. Naguibneva I. Pritchard L.L. Ait-Si-Ali S. Harel-Bellan A. Oncogene. 2001; 20: 3128-3133Google Scholar). To test whether pocket proteins are bound to the MYCN promoter in undifferentiated or differentiated LA-N-5 cells and to exclude the possibility that masking of the antibody epitopes on the E2F proteins by associated pocket proteins in RA-treated cells prevented their detection in the ChIP assay, we performed another chromatin immunoprecipitation with a panel of pocket-protein-specific antibodies, which have previously been shown to detect repressive E2F complexes in ChIP assays (33Takahashi Y. Rayman J.B. Dynlacht B.D. Genes Dev. 2000; 14: 804-816Crossref Google Scholar). We detected low levels of pRb, p107, and p130 at theMYCN promoter in undifferentiated cells (Fig. 4 B). In differentiated cells there were no changes in binding of p107 and p130; in contrast, binding of pRb increased upon differentiation. Thus, down-regulation of MYCN transcription in the course of RA-triggered differentiation of neuroblastoma cells is accompanied by a loss of binding of E2F-2, E2F-3, and E2F-4, a reduction in histone acetylation, and recruitment of pRb. TGF-β has been shown to down-regulate MYCNexpression (39Serra R. Moses H.L. Development. 1995; 121: 3057-3066Google Scholar). E2F-binding sites can confer TGF-β responsiveness to a promoter (40Zhang H.S. Postigo A.A. Dean D.C. Cell. 1999; 97: 53-61Google Scholar). In addition to several E2F-binding sites, theMYCN promoter also contains a putative TGF-β-inhibitory element (TIE; consensus sequence: GnnTTGGnG) (41Kerr L.D. Miller D.B. Matrisian L.M. Cell. 1990; 61: 267-278Google Scholar) that partly overlaps the third E2F-binding site (Fig. 5 A). To determine whether either the E2F-binding sites or the TIE are required for the regulation of MYCN by TGF-β, HaCaT human keratinocytes were transiently transfected with wild-type and mutant MYCNpromoter-dependent luciferase constructs and then either treated with TGF-β for 26 h or left untreated. The wild-typeMYCN promoter was repressed 5-fold by TGF-β in HaCaT cells (Fig. 5 B). A promoter lacking the two overlapping E2F-binding sites showed a reduction in basal activity compared with the wild-type construct, and this residual activity was not reduced further by TGF-β. In contrast, a promoter with a mutation destroying both the TIE and the third E2F-binding site was almost as active as the wild-type promoter and was repressed by TGF-β to the same extent as the wild-type promoter. Thus, in HaCaT cells, the two overlapping E2F-binding sites of the MYCN promoter mediate the major part of the promoter activity as well as its responsiveness to TGF-β, whereas neither the putative TIE nor the third E2F-binding site contribute to promoter activity or TGF-β responsiveness. We have shown here that E2F proteins regulate MYCNtranscription and are required for full activity of the MYCNpromoter in neuroblastomas. This conclusion is based on the following observations: first, the activating members of the E2F family, E2F-1, E2F-2, and E2F-3, activate the MYCN promoter in transient transfections in an E2F-site-dependent manner; second, E2F-1, E2F-2, and E2F-3 bind to the MYCN promoter in vivo specifically in neuroblastoma cells exhibitingMYCN expression; third, inhibition of E2F activity through overexpression of p16INK4A reduces MYCN mRNA levels; fourth, several signals known to down-regulate MYCNexpression either require intact E2F-binding sites for their regulation of the MYCN gene or are associated with changes in the binding of E2F proteins to the MYCN promoter. These data support the existence of a positive feedback loop linking E2F and N-Myc, which may at least in part explain the maintenance ofMYCN expression in aggressive neuroblastomas (for discussion, see Ref. 9Strieder V. Lutz W. Cancer Lett. 2002; 180: 107-119Google Scholar). The status of the pRb pathway in neuroblastomas and its role in neuroblastoma tumorigenesis is far from clear. Mutations inRB itself or other genes implicated in the pathway are rare in neuroblastomas. There is, however, evidence that the pRb pathway may become inactivated in neuroblastomas by other means. One is the inactivation of CDKN2A (p16ink4A) by promoter methylation in some neuroblastomas (20Takita J. Hayashi Y. Nakajima T. Adachi J. Tanaka T. Yamaguchi N. Ogawa Y. Hanada R. Yamamoto K. Yokota J. Oncogene. 1998; 17: 3137-3143Google Scholar). However, other neuroblastomas contain large amounts of p16INK4A (42Easton J. Wei T. Lahti J.M. Kidd V.J. Cancer Res. 1998; 58: 2624-2632Google Scholar, 43Diccianni M.B. Omura-Minamisawa M. Batova A., Le, T. Bridgeman L. Yu A.L. Int. J. Cancer. 1999; 80: 145-154Google Scholar). This observation may be explained by the overexpression in neuroblastomas of Id2, which has been suggested to sequester and inactivate pRb and thus bypass p16INK4A (21Lasorella A. Noseda M. Beyna M. Yokota Y. Iavarone A. Nature. 2000; 407: 592-598Google Scholar). IMR-32 is one of the cell lines that, because of the direct transcriptional activation of ID2 by N-Myc, produce large amounts of Id2 (21Lasorella A. Noseda M. Beyna M. Yokota Y. Iavarone A. Nature. 2000; 407: 592-598Google Scholar). However, IMR-32 also lacks detectable p16INK4Aprotein (20Takita J. Hayashi Y. Nakajima T. Adachi J. Tanaka T. Yamaguchi N. Ogawa Y. Hanada R. Yamamoto K. Yokota J. Oncogene. 1998; 17: 3137-3143Google Scholar). If overexpression of Id2 were indeed a way of blocking pRb function in neuroblastomas, one would expect that overexpression of p16INK4A, which controls pRb upstream of Id2, would not be able to reestablish negative control over E2F activity. In U2OS osteosarcoma cells, Id2 can indeed reverse the cell-cycle arrest induced by p16INK4A (44Lasorella A. Iavarone A. Israel M.A. Mol. Cell. Biol. 1996; 16: 2570-2578Google Scholar). We tested whether this is also true for neuroblastoma cells by introducing p16INK4A into IMR-32 by retroviral infection and found that p16INK4A repressed both CCNE1 andMYCN, suggesting that overexpressed Id2 is not able to relieve negative control of E2F activity in neuroblastoma cells. This is consistent with the observation that the majority of primary neuroblastomas do not express detectable amounts of p16INK4A protein and that the lack of p16INK4Ain primary neuroblastomas is associated with a poor prognosis (20Takita J. Hayashi Y. Nakajima T. Adachi J. Tanaka T. Yamaguchi N. Ogawa Y. Hanada R. Yamamoto K. Yokota J. Oncogene. 1998; 17: 3137-3143Google Scholar). The MYC promoter is the target of multiple mitogenic and antimitogenic signals, some of which regulate MYC expression via an E2F site in the MYC promoter (45Johansen L.M. Iwama A. Lodie T.A. Sasaki K. Felsher D.W. Golub T.R. Tenen D.G. Mol. Cell. Biol. 2001; 21: 3789-3806Google Scholar, 46Wong K.K. Zou X. Merrell K.T. Patel A.J. Marcu K.B. Chellappan S. Calame K. Mol. Cell. Biol. 1995; 15: 6535-6544Google Scholar). Our results provide evidence that the E2F-binding sites in the MYCNpromoter are also involved in the response to different signals. The conservation of E2F-binding sites in the proximal promoter regions of both MYC and MYCN and the shared role of these E2F-binding sites in repressing promoter activity in response to external signals points to similarities in the regulatory logic of the two genes despite their very different spatiotemporal expression profiles. We hypothesize that after duplication of the ancientMYC gene into MYC and MYCN, the preservation in both genes of the regulatory elements required for tight negative control was essential, given their oncogenic potential. If true, the E2F-binding sites in the MYC genes could be regarded as central negative control elements to restrictMYC/MYCN expression in a dominant fashion. Down-regulation of MYCN by TGF-β may be mediated by E2F-4-containing repressor complexes, which have been shown to form at E2F target promoters in response to TGF-β in keratinocytes (47Iavarone A. Massague J. Mol. Cell. Biol. 1999; 19: 916-922Google Scholar). Yet, the finding that TGF-β represses MYCN via the E2F binding sites is unexpected in light of several previous reports mapping the repressive effect of TGF-β on the MYC promoter to a TGF-β inhibitory element (TIE), because a putative TIE is also present in the MYCN promoter (48Chen C.R. Kang Y. Massague J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 992-999Google Scholar, 49Yagi K. Furuhashi M. Aoki H. Goto D. Kuwano H. Sugamura K. Miyazono K. Kato M. J. Biol. Chem. 2002; 277: 854-861Google Scholar). Very recently, the TIE in the MYC promoter was redefined as a noncanonical Smad-binding site, 5′-GGCT-3′, adjacent to the E2F-binding site (50Chen C.R. Kang Y. Siegel P.M. Massague J. Cell. 2002; 110: 19-32Google Scholar). It was shown that both the E2F and Smad-binding sites are required for repression of MYC transcription by TGF-β and that they form a composite regulatory element that binds a repressor complex containing Smad3, Smad4, E2F-4/5, DP1, and p107. Notably, the overlapping E2F sites in the MYCN promoter are flanked by putative Smad-binding sites. Recently, advanced-stage primary neuroblastomas were reported to express strongly reduced levels of the TGF-β type III receptor (51Iolascon A. Giordani L. Borriello A. Carbone R. Izzo A. Tonini G.P. Gambini C. Della Ragione F. Br. J. Cancer. 2000; 82: 1171-1176Google Scholar). In addition, whereas ganglioneuroblastomas, which show a more differentiated phenotype than neuroblastomas, express TGF-β, a majority of primary neuroblastomas do not (52McCune B.K. Patterson K. Chandra R.S. Kapur S. Sporn M.B. Tsokos M. Am. J. Pathol. 1993; 142: 49-58Google Scholar). Together, these observations indicate that neuroblastomas may escape negative growth control by TGF-β. E2F-binding sites, for example in the cdc25A gene, have previously been implicated in mediating regulation by TGF-β (47Iavarone A. Massague J. Mol. Cell. Biol. 1999; 19: 916-922Google Scholar, 53Bouzahzah B., Fu, M. Iavarone A. Factor V.M. Thorgeirsson S.S. Pestell R.G. Cancer Res. 2000; 60: 4531-4537Google Scholar). Neither this site nor the E2F-binding sites in other E2F target genes such as B-myb and E2F1 contribute to the activity of the promoter in the context of transient assays in HaCaT cells, indicating that these E2F-binding sites function exclusively in transcriptional repression (54Li J.M., Hu, P.P. Shen X., Yu, Y. Wang X.F. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 4948-4953Google Scholar). In sharp contrast, the E2F-binding sites of the MYCN promoter are important for promoter activation, accounting for roughly 80% of the promoter activity in HaCaT cells in the absence of TGF-β. TGF-β blocks only the E2F site-dependent activity but not the residual E2F site-independent activity of the promoter. In contrast to the down-regulation by TGF-β, the negative regulation of MYCN by retinoic acid cannot involve an E2F-4/p107 complex because we observed a reduction rather than an increase of binding of E2F-4 to the MYCN promoter during differentiation. The reduction in binding of E2F-4 to theMYCN promoter could simply be a result of reduced protein levels in the course of differentiation. However, the amount of E2F-4 in LA-N-5 cells has previously been shown to increase during RA-induced differentiation (36Raschella G. Tanno B. Bonetto F. Amendola R. Battista T., De Luca A. Giordano A. Paggi M.G. J. Cell. Biochem. 1997; 67: 297-303Google Scholar). The observation that E2F-1, unlike E2F-2 and E2F3, remained associated with the MYCN promoter during differentiation together with the increased binding of pRb suggests that in the course of differentiation an E2F-1/Rb complex rather than an E2F4/p107 complex contributes to the repression of theMYCN gene. This would indicate that in response to different signals, distinct E2F/pocket protein complexes mediate down-regulation of MYCN. The ChIP results show differences in the relative binding of different E2F proteins to the MYCN promoter in various neuroblastoma cell lines, with E2F-1 being predominant in Kelly cells, whereas E2F-3 predominated in LA-N-5. Because MYCN is strongly expressed in all of these cell lines, E2F-1, E2F-2, and E2F-3 appear to be redundant with regard to the activation of the MYCNpromoter. We have no explanation for the association of E2F-4 with theMYCN promoter in undifferentiated, MYCN amplified LA-N-5 cells. The results with the other MYCN-amplified neuroblastoma cell lines Kelly and IMR-32, however, show that E2F-4, although it can be found associated with the MYCN promoter, is not required for the expression of MYCN in these cells. In summary, we have identified E2F-1, E2F-2, and E2F-3 as the first transcription factors known to regulate MYCN expression in neuroblastomas. An understanding of the events that accompany activation and repression of the MYCN gene at these E2F-binding sites may eventually suggest means of blocking expression of MYCN in the tumor cells. We thank Kristian Helin, Nick La Thangue, Rene Bernards, Nick Dyson, and Stefan Gaubatz for providing plasmids, Mary Lou Zouzarte for fluorescence-activated cell sorting analyses, and Manfred Schwab and Norbert Fusenig for the kind gift of cell lines. We thank Matthias Dobbelstein, Caroline Bouchard, Guntram Suske, Holger Christiansen, Joseph B. Rayman, Stefan Gaubatz, Steffi Hauser, and Alfred Lutz for helpful discussions. W. L. expresses sincere thanks to Martin Eilers and Manfred Schwab for continuous support.