Differential Regulation of Expression of the Mammalian DNA Repair Genes by Growth Stimulation

Differential regulation of expression of the mammalian DNA repair genes by growth stimulation

Ritsuko Iwanaga1, Hideyuki Komori1 and Kiyoshi Ohtani*,1

1Human Gene Sciences Center, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan

During DNA replication, DNA becomes more vulnerable did not change significantly during the cell cycle (Meyers to certain DNA damages. DNA repair genes involved in et al., 1997). However, previous finding that hMSH2 repair of the damages may be induced by growth protein was expressed in proliferating cells of the gut stimulation. However, regulation of DNA repair genes (Wilson et al., 1995) and recent findings that some of by growth stimulation has not been analysed in detail. In repair genes such as MSH2 and Rad51 were induced by this report, we analysed the regulation of expression of overexpression of the transcription factor E2F (Ishida mammalian MSH2, MSH3 and MLH1 genes involved in et al., 2001; Polager et al., 2002), which plays crucial mismatch repair, and Rad51 and Rad50 genes involved in roles in cell growth control, suggest that some of the homologous recombination repair, in relation to cell repair genes may be regulated by growth stimulation. In growth. Unexpectedly, we found a clear difference in spite of the expectation, the regulation of DNA repair regulation of these repair gene expression by growth genes by growth stimulation such as serum stimulation stimulation even in the same repair system. The expres- of quiescent fibroblasts has not been analysed in detail. sion of MSH2, MLH1 and Rad51 genes was clearly During DNA duplication, the DNA becomes more growth regulated, whereas MSH3 and Rad50 genes were vulnerable to certain damages. Genes involved in the constitutively expressed, suggesting differential require- repair machinery may be induced by growth stimula- ment of the repair gene products for cell proliferation. tion. Recent studies identified five major DNA repair MSH3 gene is located in a bidirectionally divergent systems; the nucleotide excision repair (NER), base manner with DHFR gene that is regulated by growth excision repair (BER), mismatch repair (MMR), homo- stimulation, indicating that bidirectionally divergent logous recombinational repair (HRR) and nonhomolo- promoters are not necessarily coordinately regulated. gous end joining (NHEJ). Among these repair systems, Promoter analysis showed that the growth-regulated MMR is mainly involved in repair of DNA lesions that expression of MLH1 and Rad51 genes was mainly are generated during DNA replication by misincorpora- mediated by E2F that plays crucial roles in regulation of tion of nucleotides and by slippage of DNA polymerases DNA replication, suggesting close relation between some (reviewed in Peltomaki, 2001). Base–base mismatch and of the repair genes and DNA replication. a base insertion/deletion mispair are recognized by the Oncogene (2004) 23, 8581–8590. doi:10.1038/sj.onc.1207976 MSH2/MSH6 heterodimer, and short insertion/deletion Published online 27 September 2004 mispairs are recognized by the MSH2/MSH3 heterodimer. The MLH1/PMS2 heterodimer interacts with the Keywords: DNA repair gene; cell growth; gene expres- MSH complexes and plays important roles in the repair sion; E2F processes. As the mismatches are generated during DNA replication, it is possible that the expression of the genes involved in MMR is induced by growth stimulation. Although induction of MSH2 and MSH6 Introduction by overexpression of E2F has been reported (Polager et al., 2002), it does not necessarily indicate regulation of The genomic DNA is exposed to a variety of genotoxic the genes in physiological condition and the regulation stresses such as UV light, ionizing radiation (IR), of MMR gene expression in relation to cell growth has oxidative stress, as well as chemicals. As the DNA is not been analysed in detail. continuously exposed to the genotoxic stresses, the HRR and NHEJ are involved in the repair of double- DNA repair systems must continuously monitor and strand breaks of the DNA caused by IR and radio- repair the damaged DNA. In this regard, DNA repair mimetic chemicals. Double-strand breaks are also genes can be viewed as housekeeping genes. Indeed, a produced when a replicative DNA polymerase encoun- previous report indicated that expression of hMSH2, ters DNA single-strand breaks or other types of DNA hMLH1 and hPMS2 genes involved in mismatch repair lesions during DNA replication. The relative contribution of each system depends on the state of the cell cycle, *Correspondence: K Ohtani; E-mail: [email protected] with NHEJ dominating in G1 and HRR making a Received 2 April 2004; revised 9 June 2004; accepted 16 June 2004; greater contribution in the S and G2 phases (reviewed in published online 27 September 2004 West, 2003). Rad51 plays an essential role in HRR by Growth regulation of DNA repair genes R Iwanaga et al 8582 catalysing strand exchange reaction interacting with other factors such as Rad52, Rad54 and BRCA2 (Clever et al., 1997; Mizuta et al., 1997; Benson et al., 1998). Rad50 is a component of Rad50–Mre11–Nijmegen breakage syndrome (Nbs1) complex that plays an important role in HRR by interacting with other factors such as BRCA1 (Zhong et al., 1999). As double-strand breaks occur more frequently during DNA replication, the genes involved in HRR may be induced by growth stimulation. However, regulation of Rad50 gene expression in relation to cell growth has not been analysed. Although cell cycle-regulated expression of mouse Rad51 mRNA in proliferating cells has been reported (Yamamoto et al., 1996), the molecular mechanism of the regulation has not been analysed. To gain insight into the mechanism of control of DNA repair gene expression, we examined the expression of the DNA repair genes involved in MMR and HRR in relation to growth stimulation of quiescent fibroblasts by serum and analysed the molecular mechanism of the regulation. We selected MSH2, MSH3 and MLH1 genes from the MMR system and Rad50 and Rad51 genes from the HRR system. Our results show that some of the DNA repair genes examined were indeed expressed in a growth-regulated manner. Unexpectedly, however, others were constitutively expressed, indicating that, even in the same repair system, expression of the repair genes is differentially regulated by growth stimulation. Promoter analyses indicated that the growth-regulated expression of the DNA repair genes was mainly mediated by the transcription factor E2F.

Results

Regulation of MSH2, MLH1 and Rad51 gene expression by growth stimulation To examine the need for mammalian repair genes during cell growth, we defined the expression of MSH2, MSH3 and MLH1 genes involved in MMR, and Rad50 and Rad51 genes involved in HRR after growth stimulation. We first used rat embryonic fibroblasts REF52 that were easily rendered quiescent by serum starvation and then stimulated to re-enter into the cell cycle synchronously by addition of serum. Cells were harvested at quiescence and at various time points following serum stimulation, and then analysed by Northern blot experiment (Figure 1a and b). To monitor cell cycle progression, DNA content was examined flow cytometric analysis (Figure 1c). DNA synthesis began between 12 and 16 h Figure 1 Growth-regulated expression of DNA repair genes. (a) after serum stimulation and reached maximal level Growth-regulated expression of DNA repair genes in REF52 cells. around 16 h. Serum-starved REF52 cells were restimulated with serum and were harvested at the indicated time intervals. Expression of the As can be seen in Figure 1a and b, mRNA levels of indicated DNA repair genes were sequentially examined by MSH2 and Rad51 genes were markedly induced by Northern blot analysis. MCM5 and ARPP cDNAs were used as growth stimulation. The levels of mRNAs began to controls. (b) Relative mRNA levels of DNA repair genes after increase at 12 h and showed maximum levels, up to 12- serum stimulation. The signals in Figure 1a were quantified and and 15-fold increase, respectively, at 16 h after serum expressed at 0 h point as 1. (c) Cell cycle progression of REF52 cells after the addition of serum. Aliquots of REF52 cells in Figure 1a stimulation. The expression of MLH1 gene was also were examined for DNA content by staining DNA with propidium significantly induced but with slightly delayed kinetics iodide and flow cytometric analysis

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8583 than MSH2 and Rad51 genes, up to fourfold increase, at clear from these results that in REF52 cells the 20 h after serum addition. In contrast, the increase in expression levels of MSH2, MLH1 and Rad51 genes mRNA levels of Rad50 and MSH3 genes was within are regulated by cell growth, whereas MSH3 and Rad50 twofold range, indicating that Rad50 and MSH3 genes genes are expressed at similar levels in growing cells and are not signiﬁcantly induced by serum stimulation. It is in resting cells.

Activation of MLH1 and Rad51 promoters by growth stimulation To examine the molecular mechanism of growth- regulated expression of MSH2, MLH1 and Rad51 genes, we examined whether the promoter activities of these repair genes were responsible for the growth- dependent accumulation of the endogenous mRNA. As shown in Figure 2a, the promoter activities of Rad51 and MLH1 genes were markedly induced by serum stimulation, up to 11- and 4.5-fold increase, respectively. In contrast, the MSH3 and Rad50 promoter activities were not signiﬁcantly induced by serum stimulation. These results suggested that the growth-regulated expression of MLH1 and Rad51 genes was mainly mediated at the level of transcription. Unexpectedly, the MSH2 promoter activity was barely induced by serum stimulation, in contrast to the marked induction of MSH2 mRNA by serum stimulation.

Regulation of MLH1 and Rad51 promoters by E2F To explore the molecular mechanism of the growth- regulated expression of MLH1 and Rad51 genes, we determined the transcription factor that mediated growth-regulated expression of these genes. The timing of the induction of MLH1 and Rad51 gene expression was around the G1/S boundary, coincided with the expression of MCM5 gene, one of E2F targets (Ohtani et al., 1999) (Figure 1a and b). E2F-like sites were found in MLH1, Rad51 and MSH2 promoters, but not in MSH3 and Rad50 promoters (Figure 2b). We thus examined whether E2F is involved in the regulation of the DNA repair genes by growth stimulation. We ﬁrst examined the effect of activator type E2Fs (E2F-1–E2F-3) on the promoter activities of these genes

Figure 2 Regulation of DNA repair gene promoters by growth stimulation. (a) Activation of MLH1 and Rad51 promoters by serum stimulation. REF52 cells were transfected with indicated human DNA repair gene reporters, serum starved for 48 h, restimulated with serum for 16 h, and examined for luciferase activities. Human MCM5 reporter was used as a control. (b) Nucleotide sequences in the 50 ﬂanking region of the human MSH2, MLH1 and Rad51 genes. Transcriptional start sites of MSH2 gene identiﬁed in this and previous studies are indicated by thick and dotted arrows, respectively. The 50 ends of MLH1 and Rad51 cDNAs are indicated by arrows. Putative E2F recognition sites are marked by frames and mutation of the sites are indicated. (c) Activation of human MLH1 and Rad51 promoters by E2Fs. REF52 cells were transfected with wild-type or E2F-site mutant (mut) of the indicated human DNA repair gene reporters and expression vectors for either E2F-1, E2F-2 or E2F-3. Cells were cultured in the serum-starved condition for 48 h and examined for luciferase activities. (d) Growth-regulated activation of human MLH1 and Rad51 promoters mediated by E2F. Either wild-type or E2F-site mutant (mut) of human MLH1 and Rad51 promoters were tested for serum stimulation as in (a)

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8584 under serum starvation. The MLH1 and Rad51 Unresponsiveness of the human MSH2 promoter in the promoters showed B4-fold activation by all of the isolated form E2Fs (Figure 2c). In contrast, MSH2, MSH3 and Rad50 promoters were barely activated by any of these E2Fs. Although serum stimulation and E2F-1 induced MSH2 We next examined the effect of mutation of the E2F-like gene expression in REF52 cells, human MSH2 promoter sites on the responsiveness to the E2Fs and serum was not activated by either stimulus in REF52 cells. stimulation. Mutation of the E2F-like sites in MLH1 Several possibilities could explain this discrepancy. and Rad51 promoters elevated the promoter activities First, accumulation of MSH2 mRNA level by serum under serum starvation and rendered both promoters stimulation and E2F-1 in REF52 cells is mediated by unresponsive to E2Fs and to serum stimulation (Figure increase in stability of the mRNA instead of transcrip- 2c and d). Based on these results, we concluded that tional activation. Second, human MSH2 gene is not activation of MLH1 and Rad51 promoters by serum growth regulated. Third, the 50 flanking region analysed stimulation was mainly mediated by E2F. did not contain real promoter sequences. Finally, the human MSH2 promoter is unique among E2F-regulated promoters in that it cannot fulfill its function in the Induction of endogenous DNA repair genes by E2F isolated form. To determine the effect of E2F under more physiologi- To discriminate these possibilities, we first examined cal condition, we examined the effect of E2F on the stability of MSH2 mRNA in REF52 cells. To this endogenous repair gene expression. Quiescent REF52 end, we examined the levels of MSH2 mRNA after cells were introduced with E2F-1 by infection of blocking transcription by actinomycin D in the absence recombinant adenovirus and the expression of each of or presence of serum or E2F-1. As shown in Figure 4a, the DNA repair genes was examined by Northern blot the stability of MSH2 mRNA was not significantly analysis. As shown in Figure 3, the levels of MLH1 and changed by serum stimulation or E2F1, indicating that Rad51 mRNAs were increased 2.5- and 4.3-folds, induction of rat MSH2 gene should be mediated at the respectively, by E2F-1. In contrast, the levels of level of transcription. MSH3 and Rad50 mRNAs were not significantly We next examined whether the human MSH2 gene changed. These results confirmed that E2F regulates was regulated by growth stimulation. For this purpose, MLH1 and Rad51 genes but not MSH3 and Rad50 we examined the MSH2 mRNA levels in serum-starved genes. In spite of unresponsiveness of the human MSH2 HFFs after restimulation with serum. As shown in promoter to E2Fs, MSH2 mRNA was markedly Figure 4b and c, the MSH2 mRNA was clearly induced induced up to eight-folds by exogenously introduced up to fourfolds in HFFs. The endogenous MSH2 gene E2F-1. was also induced by overexpression of E2F-1 in HFFs, up to 4.6-folds (Figure 4c). These results indicated that human MSH2 gene was also growth regulated and could be induced by E2F-1. We next examined whether the region analysed indeed contained transcriptional start sites. For this purpose, we used the modified 50 RACE to identify the full-length 50 cDNA ends using mRNA from asynchronously growing human foreskin fibroblasts (HFFs). Eight PCR products were cloned and sequenced. The 50 ends of all PCR products corresponded to either nucleotide number À36 or À62 in the 50 flanking region analysed (Figure 2b), indicating that the region analysed did contain transcriptional start sites. This prompted us to examine whether the E2F site in the MSH2 promoter could bind E2F. For this, we performed gel mobility shift assay using the typical E2F site from DHFR promoter as a probe and examined whether the E2F site from the MSH2 promoter could compete for typical E2F sites. As shown in Figure 4d, the E2F site from the MSH2 promoter significantly reduced the E2F binding to the DHFR E2F sites, whereas the mutant form of the site did not show any competing activity, indicating that the E2F site of the MSH2 promoter has specific E2F- binding activity. Figure 3 (a) Induction of endogenous DNA repair gene expres- Finally, we examined whether the human MSH2 sion by E2F-1. Serum-starved REF52 cells were infected with either promoter was activated by serum or E2Fs in HFFs. As Ad-E2F1 or Ad-Con and further cultured under serum starvation for 21 h. Expression of the indicated DNA repair genes were shown in Figure 4e, although the human Cdc6 examined by Northern blot analysis. ARPP cDNA was used as a promoter, a typical E2F target, was clearly activated control by serum and E2Fs, the MSH2 promoter was scarcely

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8585

Figure 4 Unresponsiveness of the human MSH2 promoter in the isolated form. (a) Stability of MSH2 mRNA in the presence or absence of serum or E2F-1. The levels of MSH2 mRNA was examined by Northern blot analysis at indicated time points after addition of actinomycin D in the presence or absence of serum or E2F-1 (left panel). The ratio of the mRNAs loaded onto the gel was 7 : 1 : 5 for serum-starved, serum-stimulated and E2F-1-stimulated conditions. Signals were quantified and represented at 0 h time point as 1 (right panel). (b) Induction of MSH2 gene expression in HFFs by serum stimulation. Expression of MSH2 gene was examined after restimulation of serum starved HFFs as in Figure 1a. (c) Induction of endogenous MSH2 gene expression in serum-starved HFFs by overexpression of E2F-1. Serum-starved HFFs were infected with either Ad-E2F1 or Ad-Con, and expression of MSH2 gene was examined as in Figure 4b. (d) E2F-binding ability of the putative E2F sites in human DNA repair gene promoters. Gel mobility shift assays were performed with 293 cell extract and E2F sites of the DHFR promoter as a probe. Competition assays were performed with wild (WT) and mutant (Mut) forms of putative E2F sites from human MSH2, MLH1 and Rad51 promoters. (e) Unresponsiveness of the human MSH2 promoter to serum and E2Fs in HFFs. Human MSH2 or Cdc6 reporter was transfected into HFFs with or without E2F expression vectors, cultured under serum starvation for 72 h and assayed. Serum was added to serum-stimulated samples 24 h before harvest activated by either stimulus in HFFs as in REF52 cells. DHFR gene is a typical E2F target regulated by cell These results indicated that, in spite of regulation of growth (Slansky et al., 1993), whereas rat MSH3 gene endogenous MSH2 genes by growth stimulation and and human MSH3 promoter were not activated by E2F-binding ability of the E2F site, the human MSH2 serum stimulation, suggesting that MSH3 and DHFR promoter could not respond to serum stimulation or genes are differentially regulated by growth stimulation. E2Fs under these assay conditions. To confirm this, we examined the expression of MSH3 and DHFR genes after restimulation with serum in Bidirectionally divergent promoters not coordinately serum-starved HFFs. As shown in Figure 5a, MSH3 regulated in relation to growth stimulation gene expression did not significantly change by serum stimulation. In contrast, DHFR gene was clearly During the course of our study, we noticed that MSH3 induced. The endogenous MSH3 and DHFR genes also gene was located in a bidirectionally divergent manner showed differential response to exogenously introduced with DHFR gene. A common feature of bidirectionally E2F-1 in HFFs, DHFR clearly induced whereas MSH3 divergent promoters is coordinate regulation by sharing not significantly induced (Figure 5b). Human MSH3 the same promoter elements (Adachi and Lieber, 2002). and DHFR promoters were also differentially regulated

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8586 by serum stimulation or E2Fs in HFFs. As shown in promoter was barely activated by serum or E2Fs in Figure 5c, although the human DHFR promoter was HFFs. From these results, we concluded that the human clearly activated by both stimuli, the human MSH3 DHFR and MSH3 genes were not coordinately regulated in relation to growth stimulation and to E2Fs.

Growth-regulated expression of DNA repair genes at protein level To examine the significance of the transcriptional regulation of the DNA repair genes, we examined whether the growth-regulated gene expression is reflected at the protein level. To this end, we used normal peripheral blood lymphocytes (PBLs) that were primar- ily in resting state and cell proliferation could be induced by mitogen stimulation. Cell lysates were prepared from phytohemagglutinin (PHA)-stimulated PBLs and fresh PBLs, and Western blot analyses were performed (Figure 6). Rad50, whose mRNA was not significantly induced by serum stimulation in REF52 cells, was detected in resting PBLs and was slightly induced by PHA stimulation. In sharp contrast, expression of MLH1, MSH2 and Rad51 was not detected in resting PBLs and markedly induced by PHA stimulation. MSH3 was not detected in both resting and PHA- stimulated PBLs, probably due to the low expression level of the protein or inappropriate condition for the antibody used. These results suggested that, at least for MLH1, MSH2 and Rad51, transcriptional regulation of the DNA repair genes by growth stimulation was reflected at the protein level in PBLs.

Discussion

Although recent DNA microarray experiments suggested regulation of some DNA repair genes by E2F, the regulation of the repair genes by growth stimulation has not been analysed. In this study, we clearly showed that expression of MLH1 and Rad51 genes was induced by both growth stimulation and E2F. However, the expression of MSH3 and Rad50 genes was constitutive, indicating that genes involved in the same repair system are differentially regulated by growth stimulation. The transcriptional regulation was reﬂected at the protein level in PBLs, suggesting the signiﬁcance of the transcriptional regulation in overall gene expression. In spite of the general concept that bidirectionally divergent promoters are coordinately regulated, we found that bidirectionally divergent promoters are not necessarily coordinately regulated with regard to growth stimulation. Figure 5 Bidirectionally divergent promoters not coordinately regulated in relation to growth stimulation. (a) Differential expression of MSH3 and DHFR genes after serum stimulation in Differential regulation of DNA repair genes by growth HFFs. The same Northern blot used in Figure 4b was reprobed stimulation with MSH3 and DHFR cDNAs. The arrow indicates DHFR signal. (b) Differential induction of MSH3 and DHFR gene The induction of MLH1, MSH2 and Rad51 genes by expression by overexpression of E2F-1 in HFFs. The same growth stimulation is conceivable because the lesions to Northern blot used in Figure 4c was reprobed with MSH3 and be repaired are produced more frequently during DNA DHFR cDNAs. (c) Differential regulation of MSH3 and DHFR promoters by E2Fs and serum stimulation. Responsiveness of the replication. This is also consistent with in vivo observa- MSH3 and DHFR promoters to serum and E2Fs was examined as tion that expression of hMSH2 protein was detected in Figure 4e in highly proliferating cells but not in nondividing

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8587 (Claij and Te Riele, 2002). It is expected that a higher MMR gene expression is required for checkpoint activation to stop the cell cycle in case of DNA damage during cell growth. This may also explain the induction of MSH2 and MLH1 gene expression by growth stimulation. Rad50 is known to complex with Mre11 and Nbs1 (Dolganov et al., 1996; Carney et al., 1998), which participates in HRR and NHEJ. NHEJ dominates the repair of double-strand breaks in G1 phase. In addition, fractions of Rad50, Mre11 and Nbs1 have been shown to be associated with the telomeric repeat-binding factor TRF2, and Rad50 and Mre11 are present at interphase telomeres (Zhu et al., 2000). These results suggest the requirement of Rad50 in resting cells and may explain the constitutive expression of Rad50 gene. Although expression of MLH1, MSH2 and Rad51 genes was dramatically reduced in quiescent cells, the expression was not totally absent as we observed slight but signiﬁcant expression in serum-starved REF52 cells. The situation may also be true in vivo. For example, whereas the expression of hMSH2 protein was observed in highly proliferating cells in the gut (Wilson et al., 1995), it may not be totally absent in nongrowing differentiated cells. We expect that the low basal level of expression is enough for maintaining genomic integrity in resting cells where DNA replication does not take place.

Regulation of DNA repair genes by E2F Meyers et al. (1997) reported that the expression of Figure 6 Induction of DNA repair gene products by growth hMSH2, hMLH1 and hPMS2 showed only slight stimulation in PBLs. Cell lysates prepared from PHA-stimulated and fresh PBLs were examined for expression of the indicated (within 50%) change during the cell cycle. This is DNA repair gene products by Western blot analysis apparently contradictory to our results that the expression of MSH2 and MLH1 genes was markedly induced by serum stimulation at G1/S boundary. This discrepancy is likely due to the difference between cycling cells differentiated cells in the gut (Wilson et al., 1995). It was and cells entering the cell cycle from quiescence. It is rather unexpected that expression of MSH3 and Rad50 likely that expression of these genes is suppressed in the genes was constitutive. resting state and is induced by growth stimulation when Why some of the DNA repair genes involved in the cells enter into the cell cycle. Consistent with this notion, same repair system are induced by growth stimulation mutation of the E2F-like sites in MLH1 and Rad51 and others are not? One possibility is that the expression promoters raised the basal promoter activities under of DNA repair genes is regulated according to the need serum starvation and rendered the promoters unrespon- of the gene products in the cellular circumstances. A sive to serum stimulation, indicating that the mode of couple of studies indicated that the ratio between the regulation of these genes by E2F is release from the MSH2/MSH6 and MSH2/MSH3 complexes is impor- repression by growth stimulation. This is in agreement tant for efficient base/base mismatch repair (Drummond with previous studies which demonstrated binding of et al., 1997; Marra et al., 1998). Overexpression of endogenous E2F4, the component of E2F4/p130 MSH3 sequesters available MSH2, leading to reduction repressor complex, to MLH1 and Rad51 promoters of MSH2/MSH6 complex and consequently impaired in quiescent cells (Ren et al., 2002; Weinmann et al., base/base mismatch repair (Marra et al., 1998). These 2002). results suggest that the expression of MSH3 gene should Although the activity of the human MSH2 promoter be strictly regulated and may explain induction of in asynchronously growing cells has been analysed MSH2 gene but not MSH3 gene by growth stimulation. (Iwahashi et al., 1998), the same MSH2 reporter did It has been shown that MSH2 and MLH1 are not reflect the regulation of endogenous MSH2 gene involved in check point signaling induced by DNA expression by growth stimulation. To our knowledge, damage (Brown et al., 2003; Hawn et al., 1995), and the this is the first example of E2F-regulated genes whose check point activation requires greater quantities of the promoter does not reflect endogenous gene regulation in MMR proteins than are needed for mismatch correction reporter analyses. The same region of the human MSH2

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8588 promoter has been shown to be activated by over- coordinate regulation in terms of basal expression in expression of E2F-1 in combination with DP-1 in U-2 resting state. OS cells (Polager et al., 2002), indicating that the region can respond to E2F at least in certain experimental setting. For the MSH2 promoter to properly regulate Materials and methods expression, it seems necessary to be located on the chromosomes. Chromosomal modifications such as Cell culture acetylation of histones or interaction with nuclear REF52 cells, HFFs and 293 cells were maintained in matrix may be necessary for proper regulation of the Dulbecco’s modified Eagle medium (DMEM) containing promoter activity. 10% fetal calf serum (FCS). For synchronization of the cell It is noteworthy that the expression of some of the cycle, REF52 cells or HFFs were cultured in the medium with DNA repair genes is regulated by growth stimulation 0.1% FCS for 48 or 72 h, respectively, and restimulated by through E2F, which plays critical roles in regulation of addition of serum to 20% final concentration. For the analysis genes involved in DNA replication (Nevins et al., 1997; of MSH2 mRNA stability, serum-starved REF52 cells were Dyson, 1998; Ohtani, 1999). The fact that some of DNA restimulated with serum or infected with the recombinant repair genes are controlled by E2F may indicate that adenovirus expressing human E2F-1, and actinomycin D was some DNA repair machineries are tightly associated added at 8 mg/ml final concentration at 18 h after addition of with the DNA replication machinery during evolutional serum or 20 h after infection of the E2F-1-virus, and harvested at 2, 4 and 8 h after addition of actinomycin D. Normal human processes with regard to the growth-regulated expres- PBLs were cultured in RPMI 1640 medium containing 20% sion of the genes. FCS and were stimulated with 1.5% PHA (Difco) for 72 h. Lin et al. (2001) reported that DNA damage induced accumulation of E2F-1 protein through phosphoryla- Northern blot analyses tion of the protein by ATM. Our findings that expression of some of the DNA repair genes were Northern blot analyses were performed as described (Johnson regulated by E2F prompted us to examine whether et al., 1994). Probes were nucleotides (nt) 225–1593 of human DNA damage stimulated the promoter activity of MSH2 cDNA (GenBank accession number NM_000251), nt MLH1 and Rad51. The reporters were transfected into 272–1139 of human Rad51 cDNA (D13804), nt 76–1431 of human MLH1 cDNA (U07343), nt 653–2608 of human MSH3 REF52 cells, cells were treated with etoposide or cDNA (NM_002439), nt 1596–1879 of rat Rad50 cDNA cisplatin at the same concentration as reported (Lin (NM_022246) and a mouse DHFR cDNA fragment from et al., 2001) and promoter activities were compared with pLTRdhfr26. Human acidic ribosomal phosphoprotein P0 and without the drug treatment. As a control to monitor (ARPP) cDNA was used as a control. the DNA damage, p53 responsive promoter was used as a positive control. While the p53 reporter was activated Plasmids fivefolds, none of the repair gene promoter was significantly activated (data not shown). These results pMLH1-Luc contains nt À1787 to À28 (first nucleotide of suggested that accumulation of E2F-1 protein by DNA ATG translation initiation codon as þ 1) region of human MLH1 50 flanking sequences in pGL3-Basic (Promega) (Ito damage might not be involved in the regulation of these et al., 1999). pMLH1-Luc(À1787) was generated by extending repair gene promoters. 30 end to À1. The E2F-like site mutant, pMLH1-Luc(E2FÀ), was generated by changing E2F-like sites Bidirectionally divergent promoters not coordinately TCTGGCGCCAAA, located at -10 to À1, to TCTGGATC- regulated in relation to growth stimulation CAAA. Similarly, pMSH2-Luc contains À1267 to À17 region of human MSH2 50 flanking sequences in pGL3-Basic Human MSH3 and DHFR genes are located closely in a (Iwahashi et al., 1998). The E2F site mutant, pMSH2- bidirectionally divergent manner separated by only Luc(E2FÀ), was generated by changing the typical E2F site 88 bp (Shimada et al., 1989). One possible function of GCGGGAAA, located at À67 to À60, to ATGGGAAA. bidirectional loci may be to permit two genes to share pHsCdc6-Luc(À570), pHsMCM5-Luc(À1384), pHsMCM5- Luc(E2FÀ), expression vectors for E2F-1–E2F-3 and one CpG island in order to coordinate expression pCMV-b-gal have been described (Johnson et al., 1994; Ohtani utilizing common regulatory elements (Adachi and et al., 1998; 1999). Lieber, 2002). Our finding that the human MSH3 and Human Rad51 promoter-driven luciferase plasmid, pRad51- DHFR genes are differentially regulated by growth Luc(À1493), was generated by cloning À1493 to þ 87 region stimulation and E2F represents an exception to this (50 end of the Rad51 cDNA (D14134) as þ 1) into the KpnI– notion. The differential regulation may be due to the HindIII sites of pGL3-Basic. The E2F site mutant, pRad51- presence of E2F site in close proximity to DHFR Luc(E2FÀ), was generated by changing palindromic E2F-like transcription start site and not to MSH3 transcription sites TTTGGCGGGAAT, located at À31 to À20, to start site, as E2F site can mediate the cell cycle-regulated TTTGGATGGAAT. Similarly, human Rad50 promoter- gene expression when located in close proximity to driven luciferase plasmid, pRad50-Luc(À714), was generated by cloning of À714 to þ 379 region (50 end of the Rad50 transcriptional start sites. To our knowledge, this is the cDNA (U63139) as þ 1) into the KpnI and BglII sites of first indication that bidirectionally divergent genes are pGL3-Basic. Human MSH3 promoter-driven luciferase plas- not necessarily coordinately regulated with regard to mid, pMSH3-Luc(-112), was generated by cloning À112 to growth stimulation. For bidirectionally divergent pro- þ 61 region (50 end of the MSH3 cDNA (J04810) as þ 1) into moters, sharing common regulatory elements may allow the NheI and HindIII sites of pGL3-Basic. Human DHFR

Oncogene Growth regulation of DNA repair genes R Iwanaga et al 8589 promoter-driven luciferase plasmid, pDHFR-Luc(À181), was containing 0.1% FCS for 21 or 24 h, respectively, and prepared by cloning of À181 to þ 56 region (50 end of the harvested. DHFR cDNA (BC000192) as þ 1) into NheI and HindIII sites of pGL3-Basic. Gel mobility shift assay Preparation of 293 cell whole-cell extract and gel mobility shift Transfection assay assay were performed as described (Ikeda et al., 1996). The REF52 cells were transfected with 5 mg of reporters with or typical E2F site from the DHFR promoter was used for the without 200 ng of E2F expression plasmids by the calcium- probe and competitors were used at 100-fold molar excess. The phosphate method together with 5 mg of pCMV-b-gal as an competitors were typical E2F site from the DHFR promoter, internal control. HFFs were transfected with 4 mg of reporters typical E2F site from adenovirus E2 promoter and its mutant, with or without 200 ng of E2F expression plasmids along with DNA fragments containing putative E2F site or its mutant 1 mg of pCMV-b-gal using FuGENE 6 (Roche Molecular from MSH2 promoter (nt À158 to À45), MLH1 promoter (nt Systems, Inc.). Luciferase activities were normalized by b- À32 to À1) and Rad51 promoter (nt À115 to þ 3). galactosidase activities. All assays were performed three times in duplicates and the mean7s.d. values are presented. Antibodies and immunoblotting Western blot analysis was performed as described (Iwanaga 50 rapid amplification of cDNA ends (RACE) et al., 2001). Primary antibodies were hMLH1 (1 : 500, 554072, 50 rapid amplification of hMSH2 cDNA ends was performed BD Biosciences), hMSH2 (1 : 200, sc-494, Santa Cruz Biotech- using GeneRacer Kit (Invitrogen). Oligonucleotide corre- nology), hMSH3 (1 : 50, 611390, BD Biosciences), hRad51 sponding to nt 592–572 of the hMSH2 cDNA (NM_000251) (1 : 50, NA71, Oncogene Research Products) and hRad50 was used as a primer for reverse transcription. Oligonucleo- (1 : 200, 611010, BD Biosciences). Secondary antibodies were tides corresponding to nt 545–522 and nt 209–188 of the peroxidase-labeled anti-mouse IgG (1 : 5000, NA9310, Amer- hMSH2 cDNA were used as specific nested primers. sham) (for hMLH1, hMSH3, hRad51 and hRad50) and anti- rabbit IgG (1 : 5000, NA934, Amersham) (for hMSH2). Infection with recombinant adenoviruses Acknowledgements The recombinant adenovirus for expression of human E2F-1, We thank K Maruyama, Y Yuasa, E Ito, Y Iwahashi and Y Ad-E2F1 and control virus, Ad-Con (previously Ad-CMV), Yanagisawa for the reporter plasmids. We also thank S have been described (Schwarz et al., 1995). Quiescent REF52 Morinaga for excellent technical assistance. This work was cells or HFFs were infected with Ad-E2F1 or Ad-Con at supported by a Grant-in-Aid for Scientific Research on multiples of 200 plaque-forming units per cell in 2 ml/150 mm Priority Areas from The Ministry of Education, Culture, plate for 1 h at 371C. The cells were further cultured in DMEM Sports, Science and Technology.

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Oncogene