Oncogene (2006) 25, 1786–1798 & 2006 Nature Publishing Group All rights reserved 0950-9232/06 $30.00 www.nature.com/onc ORIGINAL ARTICLE Identification of novel E2F1 target genes regulated in cell cycle-dependent and independent manners R Iwanaga1,3, H Komori1, S Ishida2, N Okamura3, K Nakayama4, KI Nakayama5 and K Ohtani1 1Human Gene Sciences Center, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan; 2Division of Pharmacology, National Institute of Health Sciences, Setagaya-ku, Tokyo, Japan; 3Laboratory of Microbiology and Immunology, Graduate School of Health Sciences, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan; 4Department of Developmental Biology, Center for Translational and Advanced Animal Research on Human Disease, Graduate School of Medicine, Tohoku University, Aoba-ku, Sendai, Japan and 5Department of Molecular and Cellular Biology, Medical Institute of Bioregulation, Kyushu University, Fukuoka, Japan The transcription factor E2F mediates cell cycle-depen- cell cycle progression (Nevins et al., 1997; Dyson, 1998; dent expression of genes important for cell proliferation in Trimarchi and Lees, 2002). The transcriptional ability of response to growth stimulation. To further understand the E2F is cell cycle-regulated mainly through association role of E2F, we utilized a sensitive subtraction method to with the retinoblastoma tumor suppressor family of explore new E2F1 targets, which are expressed at low proteins pRb, p107 and p130. During the progression of levels and might have been unrecognized in previous cells from G1 to S phase, G1 cyclin-dependent kinases studies. We identified 33 new E2F1-inducible genes, (cdks)phosphorylate and dissociate the pRb family including checkpoint genes Claspin and Rad51ap1, and proteins from E2F, resulting in the activation of a group four genes with unknown function required for cell cycle of genes required for progression into the S phase. progression. Moreover, we found three groups of E2F1- Accordingly, expression of typical E2F target genes is inducible genes that were not induced by growth stimula- growth-regulated in a cell cycle-dependent manner. tion. At least, two groups of genes were directly induced Apart from its role in cell cycle progression, E2F is by E2F1, indicating that E2F1 can regulate expression of thought to be involved in other biological processes. genes not induced during the cell cycle. One included Deregulated E2F activity caused by the ectopic expres- Neogenin, WASF1 and SGEF genes, which may have a sion of E2F or by inactivation of pRb not only role in differentiation or development. The other was the facilitates cell cycle progression but also induces cyclin-dependent kinase inhibitor p27Kip1, which was apoptosis (Qin et al., 1994; Shan and Lee, 1994; involved in suppression of inappropriate cell cycle Symonds et al., 1994; Wu and Levine, 1994; Kowalik progression induced by deregulated E2F. E2F1-responsive et al., 1995). The ectopic expression of E2F1 also causes regions of these genes were located more upstream than cell cycle arrest under some cellular circumstance (Dimri those of typical E2F targets and did not have typical E2F et al., 2000). In addition, recent efforts to explore sites. These results indicate that there are groups of E2F1 downstream effectors of mammalian E2F using DNA targets, which are regulated in a distinct manner from that microarrays and chromatin immunoprecipitation of typical E2F targets. (ChIP)have identified a variety of putative E2F target Oncogene (2006) 25, 1786–1798. doi:10.1038/sj.onc.1209210; genes. These targets are thought to be involved not only published online 14 November 2005 in cell cycle progression but also in apoptosis, check- points, DNA repair, metabolism, development and Keywords: cell cycle; E2F; target gene; p27Kip1; growth differentiation (Ishida et al., 2001; Muller et al., 2001; stimulation Polager et al., 2002; Ren et al., 2002; Weinmann et al., 2002; Cam et al., 2004). However, the exact roles and regulatory mechanisms of E2F in biological processes other than cell cycle progression are yet to be elucidated. The induction of E2F targets involved in apoptosis or Introduction checkpoints during the cell cycle would interfere with normal cell growth and some of the genes The E2F family of transcription factors plays a crucial could be differentially regulated from the cell cycle. role in the control of the cell cycle by coordinating the Although E2F might be involved in the regulation of expression of genes involved in DNA replication and genes with a role in development and differentiation, whether such regulation is related to the cell cycle Correspondence: K Ohtani, Human Gene Sciences Center, Tokyo remains obscure. Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo Searching for genes induced by ectopic expression of 113-8510, Japan. E-mail: [email protected] E2F is a powerful technique to identify possible E2F Received 4 August 2005; revised 26 September 2005; accepted 29 targets, and it has been successfully used for DNA September 2005; published online 14 November 2005 microarray experiments (Ishida et al., 2001; Muller Novel E2F1 target genes R Iwanaga et al 1787 et al., 2001; Polager et al., 2002). However, microarrays first and second groups of genes were directly induced have some disadvantages. First, the sensitivity is rather by ectopic expression of E2F1 and were not induced low compared with Northern blotting, raising the during the cell cycle. These results indicate that there are possibility that E2F targets expressed at low levels are groups of E2F1 targets, which are not induced during left unidentified. Second, microarrays cannot detect the cell cycle unlike typical E2F targets. Our results unknown transcripts or discriminate variant transcripts show that p27Kip1 gene plays a role in suppression of from previously characterized transcripts. inappropriate cell cycle progression induced by deregu- To facilitate understanding the roles of mammalian lated E2F, indicating biological significance of the E2F, we attempted to identify new E2F-inducible genes distinct mode of E2F-mediated regulation of the gene. that are expressed at low levels and might remain unrecognized by previous microarray experiments. To this end, we used an improved subtraction method (Balzer and Baumlein, 1994), which is sensitive to Results identify transcripts expressed at low levels. We used E2F1 among the E2F family members, since E2F1 could Sensitive subtraction screening for E2F1-inducible genes significantly change more genes than E2F2 and E2F3 To identify genes induced by the ectopic expression of when ectopically expressed (Muller et al., 2001). Our E2F1, we used an improved subtraction method analyses successfully identified new E2F1-inducible combined with PCR amplification of cDNAs (Balzer genes that were not detected in previous studies, and Baumlein, 1994). E2F1 was ectopically expressed in including those with unknown function required for cell serum-starved rat embryonic fibroblast REF52 cells by cycle progression. Moreover, we found E2F1-inducible infecting a recombinant adenovirus expressing E2F1, genes with a distinct property from that of typical E2F and harvested for mRNA isolation at 21 h postinfection. targets. In contrast to general consensus that expression Induction of a typical E2F target, Cdc6, and little effect of typical E2F targets is growth-regulated, we found six on a p53 target, cyclin G1, were confirmed by Northern transcripts, which are induced by E2F1 but not by blotting (Figure 1a). Double-stranded cDNAs were serum stimulation. Analyses of these transcripts re- synthesized from E2F1-induced and control mRNAs, vealed three groups of genes with different characters. and subtraction was performed for three rounds First included Neogenin, WASF1 and SGEF genes, according to the protocol (Figure 1b). Enrichment of which may have a role in differentiation or development. E2F1-inducible genes was confirmed by Southern Second was the cdk inhibitor p27Kip1, an upstream blotting (Figure 1c). regulator of the RB/E2F pathway. Third included We analysed 2000 subtracted cDNA clones and variant transcripts of dysbindin and cyclin A2. At least, identified 210 independent cDNA fragments. Among Figure 1 Sensitive subtraction screening. (a)Expression of Cdc6 and cyclin G1 genes in mRNAs from control, E2F1-induced and serum-stimulated REF52 cells. Control probe was ARPP cDNA. (b)Scheme of subtraction procedure. ( c)Enrichment of E2F1- induced cDNAs by subtraction. Same amount of cDNAs after each round of subtraction were resolved on agarose gel (top panel)and were probed with þ 6 cDNA (middle panel)or with human DNA polymerase a cDNA (bottom panel). Oncogene Novel E2F1 target genes R Iwanaga et al 1788 Table 1 Summary of E2F1-induced genes ters by serum stimulation is mainly mediated by E2F. Induced Not induced Total Based on these results, we concluded that the Claspin by serum by serum and Rad51ap1 genes are physiological targets of E2F. We also identified new E2F1-inducible genes with roles Known genes 24 (6)3 (1)27(7) ESTs and cDNAs 9 1 10 in DNA replication, cell cycle progression, cellular Unknown transcripts 1 2 3 metabolism, transcription, signal transduction and Total 34 (6)6 (1)40(7) others as shown in Table 2. These results are consistent with the recent reports and add new genes to these Numbers in parenthesis were identified by DNA microarray or ChIP categories. elsewhere. Among 10 ESTs or cDNAs with unknown function induced by serum stimulation, six genes (S25, S27–29, these, CDC2, DNA polymerase a, MCM5 and ribonu- S31 and S32)had significant homology with mouse or cleotide reductase were known E2F targets. To confirm human cDNAs with unknown function (Table 2). Since that the remaining 206 clones in fact represented E2F1- S26, S30 and S33 were homologous only to ESTs, and induced transcripts, we examined expression of all of the S34 was totally unknown, we identified the full-length cDNAs by Northern blotting or by reverse transcription cDNAs to obtain primary information. S26 cDNAs (RT)/PCR. Genes that were induced more than twofold (S26-A and S26-B)were homologous to a mouse cDNA by E2F1 were judged E2F1-inducible.