Introduction

The E2F family of transcription factors is known to play a key role in the timely expression of the genes required for cell cycle progression and proliferation (Dyson, 1998). An important function of E2F is the recruitment of the retinoblastoma (pRb) tumor suppressor family of proteins pRb, p107, and p130 (Harbour and Dean, 2000). The pRb family, in turn, inhibits the transcriptional activation of E2F. Currently, seven members of the E2F family (E2F1–7) and two members of the DP family are known, and accumulated evidence suggests that the different members of the protein families have distinct physiological roles (Stevaux and Dyson, 2002). The E2F family can be functionally subdivided into activators (E2F1, E2F2, and E2F3) and repressors (E2F4 and E2F5). The expression level of endogenous E2F1–3 transiently increases near the G1/S boundary (Moberg et al., 1996), and overexpression of E2F1 can induce chromosomal DNA synthesis in quiescent cells (Johnson et al., 1993). Furthermore, the dominant-negative E2F1 molecule inhibits the entry of cells into the S phase (Dobrowolski et al., 1994). E2F-inducible genes include a number of genes whose products function to regulate the initiation of DNA replication (Dyson, 1998; Helin, 1998; Harbour and Dean, 2000).

Initiation of DNA replication is controlled by a regulated assembly of a pre-replicative complex (pre-RC) at each replication origin (Diffley et al., 1994; Diffley, 1998). The pre-RC initially involves the association of Cdt1 and Cdc6 with the origin recognition complex (ORC) proteins 1–6 and, followed subsequently by the assembly of minichromosome maintenance (MCM) proteins 2–7 onto chromatin (Bell and Dutta, 2002). Association of these proteins with chromatin during the G1 phase renders the pre-RC competent to respond to the second set of proteins, the concomitant of which in the late G1 phase is thought to trigger initiation. In the second step, Cdc7/Dbf4 kinase and S-phase cyclin-dependent kinases (S-Cdk) activate the pre-RC and trigger DNA replication by loading Cdc45 onto each origin with programmed timing (Bell and Dutta, 2002). Cdc45 facilitates assembly of the replication machinery by recruiting replication protein A and DNA polymerase (Mimura and Takisawa, 1998; Zou and Stillman, 2000). In addition to S-Cdk and Cdc7, other factors, such as MCM10 and TopBP1, have also been shown to recruit Cdc45 (Van Hatten et al., 2002; Wohlschlegel et al., 2002).

Mcm10 was originally identified in Saccharomyces cerevisiae as an essential DNA replication factor, playing an important role in the pre-RC assembly (Lei and Tye, 2001). Mcm10 interacts with the mammalian ORC2 and mediates the loading of the MCM 2-7 complex onto the replication origins (Homesley et al., 2000; Izumi et al., 2000), and interacts genetically with Cdc45 and DNA polymerases (Kawasaki et al., 2000). In Xenopus, MCM10 depletion affects a later step in replication initiation, blocking the chromatin association of Cdc45 (Wohlschlegel et al., 2002). Likewise, topoisomerase II-binding protein (TopBP1), a protein with eight BRCA1 C-terminus repeat domains, is also required for DNA replication (Makiniemi et al., 2001). In Xenopus, the TopBP1 protein is required for loading of Cdc45 onto the origin, and this it does in a manner distinct from MCM10 (Van Hatten et al., 2002). TopBP1 association with chromatin is dependent on ORC, and independent of S-Cdk and MCM 2-7. TopBP1 is also involved in the cellular response to UV irradiation and DNA damage, indicating that it may be a functional homolog of yeast Cut5 (Yamamoto et al., 2000; Makiniemi et al., 2001; Yamane et al., 2002).

Although considerable progress has been made towards identifying direct and physiologically relevant downstream targets of E2F and pRb, much work still remains to be done in elucidating the mechanism underlying the transcriptional regulation of the newly identified DNA replication factors. The initiation of DNA replication is tightly regulated so that the origins of replication are normally utilized only once per cell cycle. If this tightly coordinated process of pre-RC assembly is deregulated, cells potentially proceed into aberrant proliferation. Therefore, it is important to identify the complete set of physiologically important targets of E2F involved in the initiation of DNA replication. This would provide a valuable foundation for a further understanding of the roles of these factors in the regulation of the pRb/E2F pathway.

In the present study, we focused on understanding the mechanisms of regulation of MCM10 and TopBP1 expression during the cell cycle and DNA damage. For this purpose, we isolated the promoter region of the human MCM10 and TopBP1 genes and investigated the cis-elements and trans-acting factors involved in the regulation of MCM10 and TopBP1 gene expression. We showed that the E2F family of transcription factors directly activates human MCM10 and TopBP1 expression. Repression of the promoter in quiescent cells is associated with recruitment of E2F4, but by the G1/S phase, E2F4 is replaced by E2F1 in parallel with promoter activation. Moreover, E2F4 was shown to occupy the MCM10 and TopBP1 promoter after UV irradiation, but not in doxorubicin-mediated DNA damage, which resulted in the suppression of MCM10 and TopBP1 gene expression. Taken together, it would appear that human MCM10 and TopBP1 are plausible targets in DNA damage whose expression is mediated by the activation of a pRb/E2F pathway.

Results

Characterization of the human MCM10 and TopBP1 promoter to identify the cis-elements

To obtain a complete overview of the promoter region of human MCM10 and TopBP1, which function as DNA replication initiators, we searched the proximal region of the transcription start site for potential cis-elements using TRANSFAC (ver. 4.0, cutoff 80), and recognized several targets, including Sp1-, p53-, and E2F-binding consensus sequences (Figure 1). The human TopBP1 promoter contained a typical TATA-box (-403/-400) sequence near the transcription start site (Figure 1b), whereas the human MCM10 promoter region did not contain any consensus TATA-box near the transcription start site (Figure 1a).

Figure 1.

Schematic representation of the human MCM10 (a) and TopBP1 (b) promoter-luciferase plasmid and their derivatives. Putative transcription-factor-binding sites for E2F (black boxes, represented as A, B, and C), Sp1, p53, and the position of the TATA-box are indicated. Exons (white boxes) and the translational initiator (ATG; bent arrows) are indicated. Clear boxes (Luc) indicate firefly luciferase gene used for promoter activity. The positions of the promoter construct are numbered relative to the transcription initiation site at +1 as described in the NCBI UniGene Database. Primers (arrow head) used for the ChIP experiments are positioned. (c) Comparison between the E2F consensus and putative E2F-binding motifs in the human MCM10 and TopBP1 promoter region. The score for each binding motif was calculated (TRANSFAC software) against a maximum score of 100. The orientations of the E2F-binding motifs are indicated (5' 3')

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The E2F-responsive sequence was originally identified as 'TTTCGCGC' in the adenovirus E2 promoter (Kovesdi et al., 1986), and similar sequences were recognized in the promoters of various other genes. In the promoter region of human MCM10, we found three putative E2F-responsive sequences (named A, B, and C) at positions -213/-220 (A; opposite to the direction of transcription), -18/-11 (B), and +166/+173 (C) relative to the transcriptional initiation site (+1) (Figure 1a and c). In the promoter region of human TopBP1 also, we found three putative E2F-responsive sequences (designated A, B, and C; B and C were partially overlapped) at positions -858/-851 (A), -106/-99 (B), and -95/-102 (C; opposite to the direction of transcription) relative to the transcriptional initiation site (+1) (Figure 1b and c). The presence of multiple E2F- and Sp1-binding motifs has also been recognized in other growth-regulated genes, such as E2F1 (Johnson et al., 1994) and MCM7 (Suzuki et al., 1998). To assess the individual contribution of each of these putative elements to the basal promoter activity, first of all, we generated a human MCM10 promoter-luciferase construct containing the MCM10 promoter with the 5' end residing at position -458 and the 3' end at position +622 relative to the transcriptional initiation site and inserted it into a luciferase reporter construct (designated -458/+622, hereafter called pGL3-1.08 kb) (Figure 1a). To generate a human TopBP1 promoter-luciferase construct, a TopBP1 promoter with the 5' end residing at position -2104 and the 3' end at position +236 relative to the transcriptional initiation site was cloned into a luciferase reporter construct (designated -2104/+236, hereafter called pGL3-2.34 kb) (Figure 1b). These promoter constructs were used to study transient gene expression by transfecting them into HCT116 human diploid colon cancer cells or WI-38 cells consisting of primary human fibroblasts and evaluating the firefly luciferase activities by measuring the chemiluminescence with a luminometer. The transfection efficiency was normalized by the dual luciferase assay, which was used to measure the corresponding Renilla luciferase activity on cotransfection of the pRL-TK plasmid.

As shown in Figure 2a, the full-length MCM10 promoter construct, pGL3-1.08 kb, showed approximately 3.5- and 2.0-fold increase of activity in HCT116 cells and WI-38 cells, respectively, as determined by measuring the relative luciferase activities, when that in the control luciferase vector pGL3-basic was taken as 1. To determine in greater detail the cis-elements essential for the basal promoter activity of MCM10, various deletion reporter constructs were generated (Figure 1a). The MCM10 promoter construct, pGL3-0.80 kb, which lacked the 5' part of pGL3-1.08 kb containing the E2F-binding motif (A), showed slightly reduced promoter activity as compared to that of full-length pGL3-1.08 kb (Figure 2a). In contrast, pGL3-0.63 kb, obtained by further deletion of a sequence from the 5' part of pGL3-0.80 kb, exhibited a dramatically decreased promoter activity. In addition, pGL3-0.45 kb also exhibited equal promoter activity to that of pGL3-basic. These results suggest that the region -188/-9, which contains the E2F-binding motif (B) and the proximal Sp1 site may play critical roles in maintaining the basal human MCM10 promoter activity. To verify this, pGL-35 bp, which contains the E2F-binding motif (B) and the proximal Sp1 site, was cloned and subjected to luciferase assay. The promoter activity was as strong as that of the full-length pGL3-1.08 kb.

Figure 2.

Human MCM10 (a) and TopBP1 (b) promoter activities in asynchronously growing human cells. HCT16 cells (black bar) or WI-38 cells (gray bar) were transfected with 200 ng of a reporter construct together with 0.6 ng of pRL-TK, as described under 'Materials and methods.' At 48 h after transfection, the cells were harvested, and extracts were prepared to measure the firefly and Renilla luciferase activities. Values are represented as relative luciferase activities, with that of pGL3-basic being taken as 1

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Like the human MCM10 promoter constructs, human TopBP1 deletion promoter constructs were also generated based on the position of the E2F-like motif (Figure 1b). As shown in Figure 2b, the full-length TopBP1 promoter construct, pGL3-2.34 kb, showed approximately 13- and sevenfold activity in HCT116 cells and WI-38 cells, respectively, as compared to that of pGL3-basic. The TopBP1 promoter construct, pGL3-0.60 kb, which lacked the 5' part of pGL3-2.34 kb containing the E2F-binding motif (A) and the p53-binding motif, showed slightly reduced promoter activity as compared to full-length pGL3-2.34 kb (Figure 2b). In contrast, pGL3-0.30 kb, obtained by further deletion of a sequence from the 5' part containing E2F-binding motifs (B) and (C) while retaining the Sp1-binding motifs around the transcription initiation site, exhibited a dramatically decreased promoter activity (Figure 2b).

Identification of the E2F-regulatory region of the MCM10 and TopBP1 promoter

Next, we sought evidence to show that members of the E2F family transcriptionally regulate the MCM10 and TopBP1 genes. As shown in Figure 3a, exogenous coexpression of E2F1 caused up to an eightfold increase and that of E2F2 and E2F3 caused up to a 2–3-fold increase in the MCM10 pGL3-1.08 kb promoter activity, whereas coexpressions of E2F4–7 was associated with no increase in promoter activity as compared to that of the pcDNA3 control vector. To determine the E2F-responsive site in the MCM10 promoter, we employed putative E2F-responsive-sequence-mutated pGL3-1.08 kb promoter constructs (named mutA, mutB, mutC, and mutBC) (Figure 1a), which were coexpressed with E2F1 or the pcDNA3 control expression vector. As shown in Figure 3b, the MCM10 promoter construct, mutA, which mutated the E2F-binding motif (A), showed equal promoter activity as compared to that of wild-type pGL3-1.08 kb. To confirm that the E2F-binding motif (B) or (C) alone confers the E2F1-mediated MCM10 promoter activity, we introduced mutations in the E2F-binding motif (B) or (C) and generated mutB or mutC. As expected, pGL3-1.08 kb mutB or mutC exhibited a reduced promoter activity when as compared to that of wild-type pGL3-1.08 kb. In contrast, mutBC, prepared by double mutation of E2F-responsive sequences (B) and (C) from the pGL3-1.08 kb, exhibited a dramatically decreased promoter activity, comparable to that of pGL3-basic (Figure 3b). These results suggest that both E2F-binding motifs (B) and (C) play critical roles in the E2F1-mediated human MCM10 promoter activity.

Figure 3.

The E2F-binding motif of the MCM10 promoter is sufficient to confer responses to ectopic E2F expression. (a) Activation of the MCM10 promoter by members of the E2F family. HCT116 cells were transfected with 200 ng of pGL3-1.08 kb and 200 ng of CMV-promoter-driven expression vectors for E2F1–7, together with 0.6 ng of pRL-TK. The parental CMV vector, pcDNA3, was used as a negative control. Transfected cells were treated as described under 'Materials and methods' to determine the extent of stimulation by E2F. Values are represented as relative luciferase activities, with that of the control vector pcDNA3 being taken as 1. (b) Mutation analyses of the human MCM10 promoter to identify the cis-elements required for the responses to ectopic E2F1 expression. HCT116 cells were transfected with 200 ng of pGL3-1.08 kb wild-type (WT) or E2F-binding-site-mutated, mutA, mutB, mutC, or mutBC MCM10 promoter constructs, and 200 ng of CMV-promoter-driven expression vectors for E2F1, together with 0.6 ng of pRL-TK. The parental CMV vector, pcDNA3, was used as a negative control. Transfected cells were treated as described under 'Materials and methods,' to determine the extent of stimulation by E2F1. Values are represented as relative luciferase activities, with that of the control vector pcDNA3 being taken as 1

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Similarly, to identify the critical E2F-responsive sequences, which activate the TopBP1 promoter construct, first of all, we checked the responsiveness of the full-length TopBP1 promoter construct pGL3-2.34 kb to various E2E transcription factors. As shown in Figure 4a, exogenous coexpression of E2F1 caused up to a fivefold increase, and that of E2F2 and E2F3 caused up to a twofold increase in the TopBP1 pGL3-2.34 kb promoter activity, whereas coexpression of E2F4–7 was associated with no increase of promoter activity as compared to that of the pcDNA3 control vector. Next, we employed various deletion mutants as indicated in Figure 1b, and examined whether or not they were activated by E2F1. As shown in Figure 4b, pGL3-2.34 kb mutA, which mutated the E2F-responsive motif (A), was still activated by E2F1. On the other hand, the pGL3-0.60 kb mutB and mutC promoter construct, which had mutations in the E2F-responsive motifs (B) and (C), respectively, showed reduced response to E2F1. The pGL3-0.60 kb mutBC promoter construct, which did not contain any E2F-responsive sequence other than (A), showed defective E2F response (Figure 4b). These results suggest that while the two overlapping E2F-responsive motifs (B) and (C) located in the vicinity of the transcription initiation site are critical for the full responsiveness of the promoter to E2F1, the 5' E2F-responsive motif (A) is not essential for the response to E2F.

Figure 4.

The E2F-binding motif of the TopBP1 promoter is sufficient to confer responses to ectopic E2F expression. (a) Activation of the TopBP1 promoter by members of the E2F family. HCT116 cells were transfected with 200 ng of pGL3-2.34 kb and 200 ng of CMV promoter-driven expression vectors for E2F1–7, together with 0.6 ng of pRL-TK. The parental CMV vector, pcDNA3, was used as a negative control. Transfected cells were treated as described under 'Materials and methods,' to determine the extent of stimulation by E2F. Values are represented as relative luciferase activities, with that of the control vector pcDNA3 being taken as 1. (b) Mutation analyses of the human TopBP1 promoter to identify the cis-elements required for the responses to ectopic E2F1 expression. HCT116 cells were transfected with 200 ng of pGL3-2.34, -0.60 or E2F-binding-site-mutated, pGL3-2.34 mutA and -0.60 kb mutB, mutC, or mutBC TopBP1 promoter constructs, and 200 ng of CMV-promoter-driven expression vectors for E2F1, together with 0.6 ng of pRL-TK. The parental CMV vector, pcDNA3, was used as a negative control. Transfected cells were treated as described under 'Materials and methods,' to determine the extent of stimulation by E2F1. Values are represented as relative luciferase activities, with that of the control vector pcDNA3 being taken as 1

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To clarify the involvement of the pRb/E2F pathway in the regulation of human MCM10 and TopBP1 expression, we cotransfected cells expressing the pRb family proteins with E2F1, because the major role of pRb and its family members, p107 and p130, in cell growth control is regulation of the E2F transcription factors (Harbour and Dean, 2000). As shown in Figure 5a, overexpression of the pRb family proteins did not affect the promoter activity of the MCM10 pGL3-1.08 kb and TopBP1 pGL3-2.34 kb constructs. Therefore, an expression plasmid of each member of the pRb family of pocket proteins, pRb, p107, or p130, together with E2F1, was cotransfected with the MCM10 pGL3-1.08 kb or TopBP1 pGL3-2.34 kb promoter construct, and the luciferase activities were examined. As expected, coexpression of pRb along with E2F1 decreased the activity of the MCM10 pGL3-1.08 kb and TopBP1 pGL3-2.34 kb promoter constructs. In contrast, neither expression of p107 nor that of p130 decreased the activity of the MCM10 pGL3-1.08 kb and TopBP1 pGL3-2.34 kb promoter constructs (Figure 5a). These results are consistent with the current thinking that while pRb interacts with E2F1, p107 and p130 do not interact with E2F1 (Grana et al., 1998). These observations strongly support the view that a pRb/E2F pathway regulates the MCM10 and TopBP1 promoter activities.

Figure 5.

pRb/E2F pathway specifically regulates the MCM10 and TopBP1 promoter. (a) Regulation of the MCM10 and TopBP1 promoter by the pRb family proteins. HCT116 cells were transfected with 200 ng of the MCM10 pGL3-1.08 kb or TopBP1 pGL3-2.34 kb promoter construct and 200 ng of CMV-promoter-driven expression vectors for pRb, p107, and p130, together with 200 ng of CMV-promoter-driven expression vectors for E2F1 or pcDNA3 plus 0.6 ng of pRL-TK. Transfected cells were treated as described under 'Materials and methods,' to determine the extent of suppression of the MCM10 and TopBP1 promoter by the pRb family proteins. Values are represented as relative luciferase activities, with that of the control vector pcDNA3 being taken as 1. (b) RT–PCR analysis of the transcripts for MCM10 and TopBP1 in HCT116 cells following E2F1, p53, or pcDNA3 overexpression. The housekeeping gene, GAPDH, was used as a control (left panel, bottom). Measurement of protein expression following overexpression. Western blots of the introduced E2F1 or p53 (E2F1 as HA-tagged, p53 as Flag-tagged, and pcDNA3 as control) are shown (right panel)

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To determine whether E2F1 also regulates endogenous MCM10 and TopBP1 mRNA expression, HCT116 cells were transfected with E2F1, and the isolated total RNA was subjected to RT–PCR analysis. As illustrated in Figure 5b, exogenously overexpressed E2F1, but not p53 or the pcDNA3 control vector, increased the endogenous MCM10 and TopBP1 mRNA expression, while the GAPDH mRNA expression level remained unchanged. The promoter activity of TopBP1 pGL3-2.34 kb was not changed by p53 overexpression (data not shown). To check the expression level of the overexpressed E2F1 and p53 proteins, cell lysates were prepared after 48 h of transfection and Western blot analysis was performed. As shown in Figure 5b, HA-tagged E2F1 and Flag-tagged p53 were clearly detected in the lysates.

Activation of the MCM10 and TopBP1 genes by serum stimulation

To determine whether the promoter regions of human MCM10 and TopBP1 are regulated in a growth-dependent manner, we assayed the activity of the promoter in serum-starved-and-thereafter-stimulated HCT116 cells. Although MCM10 and TopBP1 mRNA expression has been reported to be induced by serum stimulation at the G1/S boundary (Izumi et al., 2000; Makiniemi et al., 2001), the precise transcriptional regulation of the MCM10 and TopBP1 genes remains unknown. Transfected cells were brought to quiescence by serum starvation, and then serum was added to stimulate their re-entry into the cell cycle. The cells were harvested, and their extracts were prepared, and assayed for luciferase activity. The cell cycle progression of the HCT116 cells was monitored by measurement of BrdU incorporation, which revealed that the G1 S-phase cells were present at the maximum number at 16–24 h after the serum restimulation, with a subsequent decrease of BrdU incorporation at 32 h after the serum restimulation, indicating that the cells then entered the G2/M phase of the cell cycle (Figure 6a and b). A representative time-course experiment shown in Figure 6a and b demonstrates that the activities of the MCM10 (pGL3-1.08 kb) and TopBP1 (pGL3-2.34 kb and pGL3-0.60 kb) promoters were induced by release from serum starvation during the G1–S phase (16–24 h after serum addition) of the cell cycle, as determined by the reporter assay with the isolated fragments (2.5–4-fold increase of the promoter activity) and as demonstrated by the BrdU incorporation analysis, indicating that the growth-dependent regulatory elements are contained within the region used for these experiments. Furthermore, E2F-responsive-site-mutated MCM10 (pGL3-1.08 kb mutBC) and TopBP1 promoter construct (pGL3-0.60 kb mutBC) showed a decreased activity as compared with that of the wild-type promoter construct, providing strong evidence for a central role of E2F activity in the regulation of cell cycle progression. The pGL3-basic control construct showed similar activity before and after serum stimulation (data not shown).

Figure 6.

Growth-regulated expression of the human MCM10 (a) and TopBP1 (b) genes in HCT116 cells. HCT116 cells were transfected with 200 ng of the MCM10 (pGL3-1.08 kb and its derivative mutBC) or TopBP1 (pGL3-2.34 kb, -0.60 kb and its derivative mutBC) promoter construct, together with 0.6 ng of pRL-TK, followed by serum starvation for 48 h and restimulation for 16, 24, or 32 h. Values are plotted as relative luciferase activities, with that at time 0 being taken as 1. Cells synchronized at the G0–G1 phase by serum starvation (0 h), followed by re-entry into the cell cycle by the addition of serum (16, 24, and 32 h) were subjected to measurement of BrdU incorporation to check for the DNA replication activity. (c) The binding of E2F1 and E2F4 proteins changes during the cell cycle. ChIP was performed with antibodies specific for E2F1, E2F4, or irrelevant IgG as indicated, and the resultant immunoprecipitates were amplified with primer pairs corresponding to MCM10 (E2F-binding sites A, B, and C), TopBP1 (E2F-binding sites A and B/C), and actin. Input corresponds to PCR reactions containing 0.5% of the total amount of chromatin used in the immunoprecipitation reactions. Serum-starved (-); and S-phase cells (+) were stimulated with serum for 24 h

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E2Fs bind to the MCM10 and TopBP1 promoters in vivo

To substantiate the contention that E2Fs can bind to endogenous human MCM10 and TopBP1 promoters, chromatin immunoprecipitation (ChIP) assays were performed using antibodies against E2F1 and E2F4. We examined the binding of E2F1 and E2F4 with the promoters of cells arrested at the G0/G1 phase and entering the S phase (24 h after serum restimulation). The chromatin immunoprecipitated by the antibodies was then amplified by PCR with primers specific for the E2F-responsive sequence of MCM10 and TopBP1 promoters (see Figure 1 for primer location). DNA fragments containing the E2F-responsive motifs (B) and (C) in MCM10, and the E2F-responsive motifs (B) and (C) in the TopBP1 promoter were detected, whereas no genomic DNA fragment was detected when irrelevant antibody (IgG) was used instead of anti-E2F antibodies, as shown in Figure 6c. PCR primers specific for the human actin promoter (negative control) did not yield actin promoter DNA in any of the E2F-ChIP samples, as expected from the previous work demonstrating that E2F does not bind to the actin promoter (Takahashi et al., 2000). Treatment with serum induces the selective recruitment of E2F1 and the concomitant loss of E2F4 on the MCM10 and TopBP1 promoters (Figure 6c). These results indicate that E2Fs associate with the MCM10 and TopBP1 promoters under physiological conditions and the MCM10 and TopBP1 promoters are differentially regulated by E2F1 and E2F4 during cell cycle progression.

E2F4 binds to the MCM10 and TopBP1 promoters after ultraviolet (UV) irradiation

To investigate the relationship between the regulation of endogenous TopBP1 expression and cellular stress, we treated HCT116 cells, which express wild-type p53, with agents inducing DNA damage. First of all, we monitored the protein level of the E2Fs and p53 after UV irradiation and doxorubicin treatment. E2F1, but not other members of the E2F family such as E2F2 and E2F4, accumulates in response to UV irradiation and doxorubicin (data not shown). DNA damage has previously been shown to induce E2F1 and p53 with similar kinetics in human cancer cells (Blattner et al., 1999; Stevens et al., 2003). In agreement with these results, DNA replication activity assessed based on BrdU incorporation was suppressed after UV irradiation and doxorubicin treatment (Figure 7a). The mRNA encoding TopBP1 is known to be downregulated in HaCaT human skin keratinocyte cells in response to UV irradiation (Potter et al., 2000; Herold et al., 2002). We reproduced this phenomenon in HCT116 cells (Figure 7b), and in agreement with the previous report, we found that UV irradiation led to a rapid (after 4 h) decrease in the expression level of TopBP1 mRNA. The decreased expression level of MCM10 was also observed after UV irradiation (data not shown). Doxorubicin treatment was associated with no change in the mRNA expression level of TopBP1 (Figure 7b). The mRNA expression level of E2F1 and GAPDH were constant before and after the induction of DNA damage (Figure 7b). As reported previously, UV irradiation was associated with stabilization of the E2F1 protein but not mRNA level (Blattner et al., 1999).

Figure 7.

E2F4 binds to the MCM10 and TopBP1 promoters after UV irradiation. (a) Cells exposed to 250 Jm^-2 UV-C or 1 M doxorubicin (Doxo) were subjected to measurement of BrdU incorporation. Values are plotted as relative DNA replication activities, with that of the mock being taken as 1. (b) Cells were exposed to UV-C or doxorubicin and total RNA was prepared 24 and 4 h later, respectively. Changes in the gene expression levels were measured by RT–PCR for TopBP1, E2F1, and GAPDH. (c) Time course of changes in the MCM10 and TopBP1 promoter activity after UV irradiation. HCT16 cells were transfected with 200 ng of the MCM10 pGL3-1.08 kb or TopBP1 pGL3-2.34 kb promoter construct, together with 0.6 ng of pRL-TK, as described under 'Materials and methods.' At 48 h after transfection, the cells were subjected to UV irradiation and cultured for 4 or 8 h, and then harvested, and extracts were prepared to measure the firefly and Renilla luciferase activities. Values are represented as relative luciferase activities, with that at time 0 being taken as 1. (d) In vivo detection of promoter occupancy by E2F1 and E2F4 in asynchronously growing and UV-C-irradiated and doxorubicin-treated cells. ChIPs assays were performed with antibodies specific for E2F1, E2F4, p53, or irrelevant IgG as indicated, and the resultant immunoprecipitates were amplified with primer pairs corresponding to MCM10 (E2F-binding sites A, B, and C/p53-binding site), TopBP1 (E2F-binding sites A and B/C), and actin. Input corresponds to PCR reactions containing 0.5% of the total amount of chromatin used in the immunoprecipitation reactions

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We next checked whether the MCM10 and TopBP1 promoter activity was activated in UV-irradiated HCT116 cells. Although E2F1 accumulation occurred in UV-irradiated HCT116 cells, no significant activation of the MCM10 pGL3-1.08 kb and TopBP1 pGL3-2.34 kb promoter constructs was observed (Figure 7c). By ChIP analysis, we found that E2F4 was recruited in vivo on the MCM10 and TopBP1 promoters in UV-irradiated cells, but not in doxorubicin-treated cells (Figure 7d). Occupation of the MCM10 and TopBP1 promoters by the E2F1 protein was dramatically decreased after UV irradiation (Figure 7d). Specific antibody for p53 did not yield MCM10 and TopBP1 promoter DNA in any of the ChIP samples (Figure 7d).

Discussion

Expression-profiling or ChIP experiments based on microarrays have greatly expanded the spectrum of genes known to be activated at the transcriptional level or bound by E2Fs (Ishida et al., 2001; Kalma et al., 2001; Ma et al., 2002; Ren et al., 2002; Stanelle et al., 2002; Weinmann et al., 2002; Huang et al., 2003; Wells et al., 2003; Young et al., 2003). Among the genes that mediate E2F-dependent DNA replication and DNA repair, some important ones, including MCM10 and TopBP1, have not yet been precisely characterized to be a transcriptional target of E2F. In this study, we isolated the promoter region of the human MCM10 and TopBP1 genes and analysed the mechanism underlying E2F-dependent activation of these important genes. To determine which of the E2Fs modulated the activity of the MCM10 and TopBP1 promoters, several members of the E2F family were tested in luciferase reporter assays. Our results clearly demonstrated that the human MCM10 and TopBP1 promoters were activated by the growth-promoting (E2F1–3) transcription factors. Moreover, ChIP assays performed on native HCT116 cells revealed that the observed regulation by the E2Fs was a physiological event. Previous ChIP experiments have shown that that E2F1, E2F2, and E2F3 associate with the promoters of E2F-responsive genes coincidentally with their activation at the G1/S-phase boundary (Takahashi et al., 2000). Accordingly, growth-regulated expressions of human MCM10 and TopBP1 are mediated mainly via their selective occupancy by E2F1 when the cells enter the G1/S phase and that by E2F4 when they are in the G0/G1 phase. This is consistent with several reports linking the activity of the members of the E2F family to induction of the S phase (DeGregori et al., 1997; Dyson, 1998; Leone et al., 1998).

E2F1 but not the other E2F family proteins is known to be upregulated in response to DNA damage, such as that associated with UV irradiation in a manner analogous to that of p53 (Blattner et al., 1999; Lin et al., 2001). Even though the induced E2F1 was shown to be able to bind to DNA in vitro (Hofferer et al., 1999), it was unable to transactivate a consensus promoter (O'Connor and Lu, 2000). Indeed, stabilized E2F1 in HCT116 cells after UV irradiation could not contribute to the upregulation of MCM10 and TopBP1 promoter activity. When normal human cells are exposed to genotoxic agents, including ionizing radiation (IR) or UV light, they exhibit a number of responses including a transient inhibition of DNA and RNA synthesis and the transcriptional induction or repression of several hundreds of genes, which result in the arrest of cell cycle progression. Thus, we examined whether or not the endogenous expression level of TopBP1 and MCM10 would be affected by DNA damage. Interestingly, we found that the mRNA expression level of TopBP1 and MCM10 was downregulated after the exposure of HCT116 cells to UV irradiation, whereas it was not affected by treatment with doxorubicin. We have applied ChIP assays to study the regulation of MCM10 and TopBP1 genes in vivo in response to DNA damage. UV-irradiated and doxorubicin-treated MCM10 and TopBP1 promoters showed strikingly different behaviors. Both E2F1 and E2F4 bound to the MCM10 promoter in asynchronously growing cells; further, while E2F1 was released and E2F4 was recruited after UV irradiation, it was not after doxorubicin treatment. The TopBP1 promoter was bound by E2F1 but not by E2F4 in asynchronously growing cells and was selectively occupied by E2F4 only after UV irradiation. At present, the precise mechanism underlying the differential status of E2F4 accumulation on the MCM10 and TopBP1 promoters between UV irradiation and doxorubicin treatment remains unclear. Although some functional redundancy exists, doxorubicin and UV irradiation are thought to activate different arms of the DNA-damage-signalling pathway (Durocher and Jackson, 2001). For example, doxorubicin induces DNA double-strand breaks, which are sensed at least in part by the ATM protein kinase (Zhou and Elledge, 2000; Tang et al., 2002). In contrast, UV damages DNA by inducing the formation of pyrimidine dimmers and six to four photoproducts by non-ATM-dependent mechanism likely involving ATR (ATM-Rad3-related protein) (Zhou and Elledge, 2000; Appella and Anderson, 2001). Therefore, the differential abilities to induce E2F4 accumulation on the MCM10 and TopBP1 promoters may be a consequence of the post-translational modification of E2F4 mediated by differential signal transduction pathways.

We have further shown that modulation of a pRb/E2F pathway is directly linked to regulation of the MCM10 and TopBP1 promoter activity, suggesting that disruption of pRb or activation of certain members of the E2F family can lead to overexpression of the MCM10 and TopBP1 proteins. Examination of the expression level of the MCM10 and TopBP1 proteins in tumor specimens may be expected to provide a clearer insight into how aberration of MCM10 and TopBP1 might contribute to the development of various cancers. In conclusion, we have shown that MCM10 and TopBP1 are regulated by a balance of actions between E2F1 and E2F4, during the cell cycle and in response to DNA damage, and we have unveiled a high degree of specificity in the regulation of the MCM10 and TopBP1 gene expressions in response to UV irradiation.

Materials and methods

Plasmids

The human MCM10 and TopBP1 promoter fragments were generated by PCR from genomic DNA, ligated into the KpnI/XhoI-digested pGL3-basic vector (Promega), and sequenced. Initial PCR primers were designed to amplify 1080-bp (-458/+622) and 2340-bp (-2104/+236) fragments of the MCM10 and TopBP1 promoter sequences, respectively, which are numbered relative to the transcription initiation site at +1 described in the NCBI UniGene Database (Genome View). The genomic clones (GenBank Accession number) used for the designation of the PCR primers were human MCM10 (AL355355) and TopBP1 (AC083905), respectively. The forward (F) and reverse (R) PCR primers for the full-length MCM10 and TopBP1 promoter constructs were, 5'-CATCTACCCTTTCCCTCACTGCACAGATGG-3' (-458F), 5'-A GCCGGGCCAGCCCGCTTCCACCTTCTGGA-3' (+622R), and 5'-GAACTTGCCCCCTAGTGGTGAACTCTTACT-3' (-2104F), 5'-ATCACGGAAGCCACGTCCTCTCGGCGTCAA-3' (+236R), respectively. Deletion mutants of the MCM10 and TopBP1 promoter constructs were generated using the following primers: 5'-TGGCTTCTACCGACAGGAAGGTCCCTTGCC-3' (-188F), 5'-TCAGCTGTGAACGAAGAAGGCGTCCCGCA-3' (-8F), 5'-GCAGCTTGG GACCGCTCGGAGCTGGCATGG-3' (+173F), and 5'-GGAGTATTCCTGGCCTGTGGTCCTACCTCT-3' (-334F), 5'-GTCGGTTCCCGTTCTGGGCAATTTCCGGGT-3' (-34F), 5'-CCCATCTCCAGGAAAAGGCCTGGCTCTGG G-3' (+266R), respectively. Primers for the construction of MCM10 pGL3-35 bp (-43/-9) are follows: 5'-CGGCAGGG GCGGGATCCGGTTGGAATTTTGGCGGGTC-3' and 5'-TCGAGACCCGCCAAAATTCCAACCGGATCCCGCCC CTGCCGGTAC-3'. The forward primer added a KpnI site and the reverse primer added an XhoI site to facilitate subcloning. The E2F-responsive-sequence-mutated promoter construct, MCM10 pGL3-1.08 kb mutA, mutB, mutC, and mutBC, were created using primers -458F and 5'-CTAGCTAGCCCCCTGGTGCCGGGAAGTTTAAG-3', 5'-CTAGCTAGCGACACTCTATTTTACCTAAAGGAGAC-3' and +622R, -458F and 5'-CTAGCTAGCATTCCAACCGGAT CCCGCCCCTG-3', 5'-CTAGCTAGCGGGTTCAGCTGTGAACGAAGAAGG-3' and +622R, and -458F and 5'-CTAGCTAGCTCCAGATCGCGCCCACCCG-3', 5'-CTAG CTAGCGGCAGCTTGGGACCGCT-3' and +622R, respectively. The E2F-responsive-sequence-mutated promoter construct, TopBP1 pGL3-2.34 kb mutA, pGL3-0.60 kb mutB, mutC, and mutBC, were created using primers -2104F and 5'- CTAGCTAGCCTGATTCGTTGTTGCTTCTT-3', 5'-CTAGCTAGCGCTGGTTATCACTAAGGTTTGCA-3' and +236R, -334F and 5'-CTAGCTAGCCGCGCGGCCGCGCTTGC CTTTT-3', 5'-CTAGCTAGCGCGAAAGTCGGTTCCCGT TCT-3' and +266R, -334F and 5'-CTAGCTAGCGCCAACCGCGCGGCCGCGCTT-3', 5'-CTAGCTAGCGTCG GTTCCCGTTCTGG-3' and +266R, and -334F and 5'-CTAGCTAGCAACCGCGCGGCCGCGCTTGCCTT-3', 5'-CTAGCTAGCTTTGTCGGTTCCCGTTCTGGGCA-3' and +266R, respectively. Thus, a part of the E2F-responsive sequences was replaced with NheI sequence (underlined). The following plasmids have been previously described: pcDNA3-HA-E2F1, E2F2, E2F3, E2F4, E2F6, pCMV-HA-E2F5 (where HA denotes hemagglutinin), pRb, p107, p130, pCMV-5xMyc-E2F7 (Yoshida and Inoue, 2004). To construct the C-terminally Flag-tagged p53 expression vector, RT–PCR was performed as described below. The PCR products were digested with BamHI and EcoRI and ligated into their respective sites in the pCMV-Tag4A vector (Stratagene). The primers were 5'-CGGGATCCGCCGCCATGGAGGAGCC GCAGTCAGATCCTAGCGT-3' and 5'-CGGAATTCG TCTGAGTCAGGCCCTTCTGTCTTGAACAT-3'. All the vectors constructed were verified by sequencing.

Cell culture, synchronization and transfections

HCT116 cells were cultured in McCoy's 5A medium (Invitrogen) supplemented with antibiotics and 10% fetal bovine serum (FBS). WI-38 cells were purchased (HSRRB, The Japan Health Sciences Foundation) and grown in Basal Eagle's medium (Invitrogen) supplemented with antibiotics and 10% FBS. For the promoter assay, the cells were transfected with FuGENE6 (Roche) according to the manufacturer's instructions. Briefly, 200 ng of the expression plasmid, 200 ng of the firefly luciferase reporter plasmid (pGL3, Promega), and 0.6 ng of the Renilla luciferase reporter plasmid (pRL-TK, Promega) per 24-well dish were used for each transfection. The cells were harvested 48 h after the transfection, and luciferase assay was performed using the Dual-Luciferase Reporter Assay System in accordance with the manufacturer's protocol (Promega). Experiments were performed at least in triplicate, and the relative activities and s.e. values were determined. To control for transfection efficiency, firefly luciferase values were normalized to the Renilla luciferase values.

To measure growth-dependent induction of the human MCM10 and TopBP1 promoter activities, growth of the transfected HCT116 cells was arrested in the G0 phase by incubation in the presence of 0.1% FBS for 48 h, and the cells were released into the cell cycle by the readdition of 15% FBS. The cells were harvested at 0, 16, 24, and 32 h after the serum stimulation and then assayed for luciferase activity. DNA replication activity was measured by determining the extent of bromodeoxyuridine (BrdU) incorporation into newly synthesized DNA, using Cell Proliferation ELISA (Roche). BrdU labeling was conducted by adding BrdU to the tissue culture medium for a period of 2 h before the cells were harvested.

Cells were seeded in plastic dishes 24 h prior to treatment and were 60–70% confluent by the time of treatment. Cultures were exposed to 250 J/m² UV light (UV-C, 254 nm) using an Amersham UV crosslinker and then incubated for 4–8 h. The topoisomerase inhibitor doxorubicin (Sigma) was added to the cells at a final concentration of 1 M, and was allowed to remain in the cultures for 24 h until they were harvested.

RT–PCR

Total cellular RNA was extracted from HCT1116 cells using Trizol reagent (Invitrogen), according to the manufacturer's instructions. The RT–PCR was performed with the SuperScript One-Step RT–PCR system, according to the manufacturer's instructions (Invitrogen). Briefly, reaction mixtures containing total RNA (500 ng), 0.2 mM dNTPs, 0.8 M of each primer, 2 U of enzyme mixture including SuperScript II RT, Platinum Taq DNA polymerase, and 1 buffer with 1.2 mM MgSO₄ were placed at 50°C for 20 min, followed by 94°C for 2 min, and PCR was then performed as follows: The PCR was conducted over 30 cycles of 94°C for 15 s, 55°C for 30 s, and 70°C for 30 s, followed by 72°C for 10 min. The primers used were follows: TopBP1, 5'-CTCAGCTAGCTTGCTGTTAAA CCACATTGA-3', 5'-TGCGCATTCAAACTTGGCAAACA TCATGGA-3' and E2F1, 5'-TCCAGAGTAGCTCACCTTG TCTCTGCAGCC-3', 5'-GGTCAGAGCACCAGATTCTGG GAGCAGGCA-3'. The GAPDH primer set was purchased (Stratagene). The amplified products were separated on 1.2% agarose gels and visualized under an UV transilluminator.

Western blot analysis

The cells were harvested and lysed in RIPA lysis buffer (50 mM Tris-HCl, pH 7.6, 150 mM NaCl, 1% Nonidet P40, 0.5% sodium deoxycholate, 0.1% SDS, 1 mM EDTA, 1 mM PMSF, and 1 g/ml each of aprotinin, pepstatin, and leupeptin) for 20 min on ice. The cell lysates were centrifuged and the protein concentration was determined using the Bio-Rad protein assay kit (Bio-Rad laboratories). Before being subjected to SDS–PAGE, the reaction was stopped by the addition of Laemmli sample buffer containing 100 mM DTT. Equal amounts of cellular protein (25 g) were electrophoresed on NuPAGE 4–12% Bis-Tris gel with MES running buffer (Invitrogen), and transferred to a Hybond-PVDF membrane (Amersham). The membrane was first blocked in TBS containing 0.1% Tween 20 and 5% nonfat dried milk and then incubated with the following antibodies: anti-Flag-HRP (Sigma), anti-HA-HRP (Santa Cruz Biotechnology) monoclonal antibody, or anti-E2F1 (sc-193), anti-E2F2 (sc-633), anti-E2F4 (sc-866) or anti-p53 (sc-6243) polyclonal antibodies (Santa Cruz Biotechnology), and then with peroxidase-linked anti-rabbit immunoglobulin (Amersham). Enhanced chemiluminescence reagents were used to detect the signals according to the manufacturer's protocol (ECL Plus, Amersham).

ChIP

ChIP assays were performed as previously described (Dhar et al., 2001; Yoshida and Inoue, 2004). Briefly, a 150-cm² dish with subconfluent HCT116 cells was used for each ChIP, and asynchronously growing cells were treated with formaldehyde at 1% final concentration to create protein–DNA crosslinks, and the crosslinked chromatins were then extracted, diluted with RIPA buffer (50 mM HEPES pH7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and protease inhibitor cocktails), and sheared by sonication on ice to an average length of 700 bp. After being precleared with the protein A-Sepharose beads (4°C for 4 h) and blocked with 1% bovine serum albumin, the chromatin was divided into equal samples for immunoprecipitation with 2–5 g of either anti-E2F1 (sc-193), anti-E2F4 (sc-866), or anti-p53 (sc-6243) polyclonal antibodies (Santa Cruz Biotechnology), and rabbit IgG. After incubation at 4°C overnight, a 15-l aliquot of a 50% slurry of preblocked protein A-Sepharose beads was added and the resultant immune complexes were pelleted by centrifugation after 3 h incubation at 4°C, and washed several times with RIPA buffer. The immunoprecipitates were resuspended in TE buffer and the crosslinks were reversed by overnight incubation at 65°C with proteinase K and RNase A. Following phenol/chloroform extraction and ethanol precipitation, the pellets were resuspended in 50 l of distilled water and analysed by PCR. For input control, 1/200th volume of chromatin was amplified. PCR primers (MCM10, -458F and 5'-GTAGAAGCCAGTCTCCTTTAGGTAAAATAG-3', -188F and 5'-CCCGGGACCCCGCGCCTCTAGCCTCGGA-3', and -8F and 5'-CTGAGACTTTCTGGACCACCCTGAGTCGTC-3'; TopBP1, 5'-CAGTGAATAATAAAATGATCCTCATTAG-3' and 5'-CACCGCCTAACACAGCCACAAGATGGCAA-3', -334F and +236R) that generate 280, 330, 510, 360, and 570 bp products, respectively, were used to detect the presence of specific DNA sequences. The PCR primers corresponding to the human actin promoter (negative control) were previously described (Takahashi et al., 2000). The amplified products were separated on 1.2% agarose gels and visualized under an UV transilluminator.

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Acknowledgements

We thank Yoshiko Sakamoto for her technical assistance. This study was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan. Support was also received from CREST of JST (Japan Science and Technology Agency).