Oncogene (1999) 18, 2299 ± 2309 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc Cell growth-regulated expression of mammalian MCM5 and MCM6 genes mediated by the transcription factor E2F Kiyoshi Ohtani1, Ritsuko Iwanaga1, Masataka Nakamura*,1, Masa-aki Ikeda2, Norikazu Yabuta3, Hiromichi Tsuruga3 and Hiroshi Nojima3 1Human Gene Sciences Center, Tokyo Medical and Dental University, Tokyo 113-8510, Japan 2Department of Developmental Biology, Graduate School of Dentistry, Tokyo Medical and Dental University, Tokyo 113-8549, Japan; 3Department of Molecular Genetics, Research Institute for Microbial Diseases, Osaka University, Suita 565-0871, Japan Initiation of DNA replication requires the function of family (MCM2-7) that have been identi®ed in yeast, MCM gene products, which participate in ensuring that Xenopus, and human. Mcm proteins seem to regulate DNA replication occurs only once in the cell cycle. the initiation at the replication origin where the loading Expression of all mammalian genes of the MCM family of the proteins onto the origin recognition complex is induced by growth stimulation, unlike yeast, and the (ORC) is regulated by Cdc6 and cyclin-dependent mRNA levels peak at G1/S boundary. In this study, we kinases (Donovan et al., 1997; Tanaka et al., 1997). examined the transcriptional activities of isolated human However, the mechanism(s) by which Mcm proteins MCM gene promoters. Human MCM5 and MCM6 control the initiation of DNA replication remains promoters with mutation in the E2F sites failed in unclear. promoter regulation following serum stimulation and Xenopus Mcm proteins seem to be able to access exogenous E2F expression. In addition, we identi®ed a replicated DNA only after the breakdown of nuclear novel E2F-like sequence in human MCM6 promoter envelope in M phase. The Mcm proteins in S. which cooperates with the authentic E2F sites in E2F- cerevisiae are in the nucleus during G1 phase but dependent regulation. Forced expression of E2F1 could disappear from the nucleus upon the initiation of S induce expression of all members of the endogenous phase, whereas Mcm proteins in ®ssion yeast and MCM genes in rat embryonal ®broblast REF52 cells. mammalian cells are in the nucleus throughout the Our results demonstrated that the growth-regulated mitotic cell cycle. Unlike yeast, in which expression of expression of mammalian MCM5 and MCM6 genes, the Mcm proteins and messages is stable during the cell and presumably other MCM members, is primarily cycle, mammalian cells show a dramatic induction of regulated by E2F through binding to multiple E2F sites MCM mRNAs at G1/S boundary coupled with a in the promoters. signi®cant rise in protein levels upon growth stimula- tion such as stimulation of resting ®broblasts with Keywords: mammalian MCM; E2F; promoter; cell serum (Tsuruga et al., 1997a). Thus, in mammalian cycle; DNA replication cells, the transcriptional mechanism of expression of the MCM genes is, at least in part, central to the regulation of Mcm activities. To address this issue, we previously isolated the 5' ¯anking regions of human Introduction MCM5 (HsMCM5) and MCM6 (HsMCM6) genes (Tsuruga et al., 1997a,b). Both sequences were found In the cell cycle of eukaryotic cells, replication of DNA to have putative E2F binding consensus sites, occurs once per cell cycle to ensure the integrity of the suggesting E2F-dependent regulation of the genome. The mechanism that inhibits re-replication of HsMCM5 and HsMCM6 genes. the DNA that had already replicated in the current S Overexpression of E2F has been shown to induce phase seems to be conserved from yeast to human. A cellular DNA synthesis in serum starved ®broblasts model in Xenopus hypothesized a trans-acting factor (DeGregori et al., 1997; Johnson et al., 1993; Qin et al., (licensing factor), which distinguishes replicated DNA 1994; Shan and Lee, 1994), suggesting that genes from unreplicated one (Blow and Laskey, 1988). On critically involved in regulation of DNA replication are the other hand, the MCM family genes have been targets of E2F. Indeed, E2F is known to regulate a identi®ed in Saccharomyces cerevisiae as essential genes class of genes involved in DNA replication (La involved in the initiation of DNA replication. MCM Thangue, 1994; Nevins, 1992). These include genes gene products are reminiscent of a putative licensing for enzymatic machinery for DNA synthesis, such as factor. A series of experiments with Xenopus egg dihydrofolate reductase (DHFR) and DNA polymerase extract identi®ed Mcm molecules as components of a a, and regulatory molecules for the initiation of DNA putative licensing factor (Chong et al., 1995; Kubota et replication, such as HsOrc1 and HsCdc6 (Ohtani et al., al., 1995, 1997; Madine et al., 1995; Thommes et al., 1996, 1998; Yan et al., 1998). HsOrc1 and HsCdc6 may 1997). At present, there are six genes of the MCM be necessary for the initiation of DNA replication. However, overexpression of HsOrc1 or HsCdc6 is not sucient for the induction of DNA synthesis as shown in rat embryonal ®broblast REF52 cells (Ohtani et al., *Correspondence: M Nakamura Received 6 August 1998; revised 28 October 1998; accepted 29 1998), implying other E2F targets than Orc1 and Cdc6 October 1998 in inducing DNA synthesis in REF52 cells. E2F-mediated expression of mammalian MCM genes KOhtaniet al 2300 In the present study, we show that the cell growth- gene clustered about 15 bp upstream of the 5' end of regulated promoter activity of HsMCM5 and HsMCM5 cDNA (Tsuruga et al., 1997a). There are HsMCM6 is primarily regulated by E2F through two sets of putative E2F binding elements arranged in binding to the multiple E2F recognition sites of an overlapping fashion in the 5' ¯anking sequence of HsMCM5 and HsMCM6 promoters. Furthermore, HsMCM5 cDNA. One set (E2F sites III+IV) is expression of E2F1 induces all genes of the endogen- located about 190 bp upstream of the 5' end of cDNA ous MCM family in REF52 cells. Our results indicate while the other (E2F sites I+II) 2 bp downstream of that the MCM family genes are critical E2F target the 5' end of cDNA (Figure 2B); i.e., each one set lies genes in the regulation of DNA replication. downstream and upstream of the transcriptional initiation sites. Similarly, RNase protection assay of the HsMCM6 5' ¯anking sequence indicated multiple transcriptional start sites in 20 to 1 bp upstream of the Results 5' end of cDNA (Tsuruga et al., 1997b). Our independent search found, in addition to two sets of Cell proliferation-regulated expression of MCM family overlapping E2F sites (E2F sites I ± IV) recognized genes in REF52 cells previously (Tsuruga et al., 1997b), another putative We have recently reported that serum stimulation E2F site (E2F like site V), which overlapped with the induced expression of all genes of the MCM family clustered transcriptional start sites. The 5' ¯anking in human lip ®broblast KD cells and normal rat kidney sequences of the HsMCM5 and HsMCM6 genes ®broblast NRK cells (Tsuruga et al., 1997a,b). Serum- contained multiple Sp1 sites but not typical TATA dependent expression of the MCM family genes was box. These characteristics are similar to those of further examined in rat embryonal ®broblast REF52 previously reported genes, such as B-myb, E2F1 and cells, which were used for a series of transfection HsOrc1, which are primarily regulated by E2F experiments. REF52 cells were made quiescent by (Johnson et al., 1994; Lam and Watson, 1993; Ohtani culture in 0.1% fetal bovine serum (FBS) for 48 h then et al., 1996). stimulated to re-enter the cell cycle by the addition of serum. The level of mRNA expression of each MCM gene post-serum stimulation was monitored by North- ern blotting with poly(A)+ RNA. The latter was puri®ed from cells harvested at quiescence and at various time intervals after the addition of serum. Serum-starved quiescent REF52 cells produced lower levels of mRNAs for all MCM genes compared with asynchronously growing cells (Figure 1). Serum stimulation induced a transient marked inhibition of expression of MCM mRNAs at 4 h, which was followed with a profound increase for up to 12 h. The reduction at 4 h after stimulation may be due to the paucity of general transcription factors which are required for many genes activated at this stage. The levels of MCM mRNAs remained persistently high at least until 28 h post-stimulation. At 16 h post-serum stimulation REF52 cells commenced DNA synthesis which was determined by [3H]thymidine uptake (data not shown). These results indicate that MCM mRNA expression parallels proceeding to G1/S boundary of the cell cycle progression upon the addition of serum, implying the involvement of cell growth regulation in the expression of mammalian MCM genes. HsMCM5 and HsMCM6 promoters Our previous results showed multiple putative E2F sites in 5' ¯anking sequences of the HsMCM5 and HsMCM6 genes (Tsuruga et al., 1997a,b). We wished to determine the functional signi®cance of these sequences in the regulation of HsMCM5 and HsMCM6 genes. Transcriptional start sites were mapped by RNase protection assays. By using RNA Figure 1 Growth-regulated expression of MCM family genes in probes spanning sequences upstream of HsMCM5 and REF52 cells. REF52 cells were serum-starved and re-stimulated HsMCM6 cDNAs, fragments with multiple sizes were with 20% FBS. Cells were harvested at indicated time intervals protected with mRNAs from human T cell lines MT-2 after the addition of serum. Poly(A)+ RNA prepared from and Kit 225 (Figure 2A), indicating multiple transcrip- 660 mg of total RNA was subjected to electrophoresis on a 1% agarose gel and transferred to a nylon membrane. The blot was tional start sites around which no typical splicing sequentially probed with HsMCM2 through HsMCM7 and acceptor consensus sequences were found.