Expression Analysis Using DNA Microarrays Demonstrates That E2F-1 Up-Regulates Expression of DNA Replication Genes Including Replication Protein A2

Expression analysis using DNA microarrays demonstrates that E2F-1 up-regulates expression of DNA replication genes including replication protein A2

Yael Kalma1, Lea Marash1, Yocheved Lamed1 and Doron Ginsberg*,1

1Department of Molecular Cell Biology, The Weizmann Institute of Science, Rehovot 76100, Israel

The transcription factor E2F-1 plays a pivotal role in the late G1 phosphorylation of RB family members by regulation of G1/S transition in higher eukaryotes cell cyclin dependent kinases results in release of E2Fs cycle. We used a cell line containing an inducible E2F-1 from the complex, leading to activation and/or and oligonucleotide microarray analysis to identify novel derepression of E2F target genes (Kaelin, 1999). E2F target genes. We show that E2F-1 up-regulates the This, in turn, leads to cell cycle progression. The expression of a number of genes coding for components importance of the RB/E2F pathway in controlling of the DNA replication machinery. Among them is the cell growth is emphasized by frequent inactivation of gene coding for the 32 Kd subunit of replication protein the Rb gene or its upstream regulators, cyclin D, A (RPA2). Replication protein A is the most abundant Cdk4 and p16, in human tumors (Hall and Peters, single strand DNA binding complex and it is essential for 1996; Sherr, 1996; Weinberg, 1995). DNA replication. We demonstrate that RPA2 is a novel Known E2F regulated genes are involved in E2F target gene whose expression can be directly nucleotide synthesis, recognition of origins of replica- regulated by E2F-1 via E2F binding sites in its promoter. tion, DNA replication and control of cell cycle In addition, expression of Topoisomerase IIa and subunit progression (Helin, 1998). In agreement with the IV of DNA polymerase a is also up-regulated upon E2F- regulation of many growth related genes by E2F, 1 induction. Taken together, these results provide novel ectopic expression of E2F-1, 2 or 3 is sucient to links between components of the DNA replication induce S phase entry of quiescent immortal rodent machinery and the cell growth regulatory pathway cells (Johnson et al., 1993; Qin et al., 1994; Shan and involving the Rb tumor suppressor and E2F. Oncogene Lee, 1994; Vigo et al., 1999). Furthermore, over- (2001) 20, 1379 ± 1387. expression of E2F-1 and its heterodimeric partner DP-1 together with activated ras leads to transforma- Keywords: E2F; cell cycle; DNA chips; RPA tion of primary rodent cells (Johnson et al., 1994). More recent studies show that double transgenic mice over-expressing E2F-1 and activated ras in their epidermis develop skin tumors (Pierce et al., 1998). Introduction Thus, E2F-1 contributes to tumorigenesis in vivo and it therefore functions, at least in some settings, as an E2F transcription factors play a pivotal role in the oncogene. control of cell cycle progression and regulate the DNA replication is a complex process involving expression of genes required for G1/S transition numerous proteins yet only four of them, DNA (Dyson, 1998; Muller and Helin, 2000). The DNA polymerase a subunit I and II, proliferating cell binding complex named E2F is a heterodimeric nuclear antigen (PCNA) and DNA polymerase d complex consisting of an E2F component and a catalytic subunit have so far been recognized as the dimerization partner, DP. To date, six E2F genes and products of E2F target genes (Lee et al., 1995; two DP genes have been isolated (Dyson, 1998). E2F Nishikawa et al., 2000; Pearson et al., 1991; Zhao activity is modulated by multiple mechanisms includ- and Chang, 1997). ing negative regulation by interaction with the In this study we used a cell line containing an product of the Rb tumor suppressor gene, RB, and inducible E2F-1 and DNA array hybridization to its related proteins p107 and p130 (reviewed in identify additional E2F target genes. We show that Dyson, 1998). Binding of RB family members to E2F-1 up-regulates the expression of six new genes E2F results in active transcriptional repression of encoding proteins that participate in DNA replication, E2F regulated genes and growth suppression. During among them are the genes encoding for the 32 Kd subunit of replication protein A (RPA2), Topoisome- rase IIa and subunit IV of DNA polymerase a. Their expression is E2F responsive and in the case of RPA2 *Correspondence: D Ginsberg Received 11 October 2000; revised 19 December 2000; accepted 3 this E2F responsiveness depends on the E2F binding January 2001 sites in its promoter. E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1380 Results not described previously as E2F target genes, a most evident functional group of genes included six genes Analysis of up-regulated genes after E2F-1 induction involved in DNA replication, a cellular process In an attempt to identify new E2F target genes we known to be regulated by E2F-1 (Table 1). used a Rat-1a derived cell line containing a stably In the screen of Atlas membrane expression of two integrated Zinc inducible E2F-1, Rat-1-MT-wtE2F-1 known E2F target genes present on the membrane, (Qin et al., 1994). Upon addition of ZnCl2 (100 mM) cyclin E and PCNA, was increased upon E2F-1 to quiescent Rat-1-MT-wtE2F-1 cells, E2F-1 protein induction (Figure 2). In addition, expression of level is elevated and the cells enter S phase and later several genes including the gene encoding for the undergo apoptosis (Qin et al., 1994). When these cells 32 Kd subunit of replication protein A, RPA2, was were kept for 48 h in medium with 0.1% serum they similarly induced (Figure 2). Computerized analysis entered quiescence, as determined by FACS analysis of the data, performed using AtlasImage 1.01 soft- (data not shown). Under these conditions, addition of ware (Clontech, USA), demonstrated that the induc- ZnCl2 (100 mM) resulted in a rapid and signi®cant tion of expression of RPA2, cyclin E and PCNA was elevation of E2F-1 protein level. E2F-1 protein level 3.48-, 2.14- and 1.2-fold, respectively. was increased 3 h after ZnCl2 addition and it remained high for at least 12 h (Figure 1). Total RNA was extracted from Rat-1-MT-wtE2F-1 arrested cells before and after E2F-1 induction in two independent experiments. In one experiment the RNA was extracted 12 h after E2F-1 induction and in the other at two time points after E2F-1 induction: 12 and 16 h. These RNAs were used to prepare probes to screen an Aymetrics Rat DNA microarrays (Rat Genome U34A array) containing 8700 cDNAs and ESTs. RNA extracted from arrested cells before and 12 h after E2F-1 induction was also used to probe an Atlas membrane containing 588 rat cDNAs (Clontech, 7738-1). In the screen of the Aymetrics Rat DNA microarrays, out of the 8700 genes present on each chip expression of 35 genes Figure 1 The kinetic of expression of inducible E2F-1 in and 10 ESTs was reproducibly increased more than quiescent rat cells. Rat-1a-1093 (1093) and Rat-1a-MT-wtE2F-1 twofold upon E2F-1 induction. Seven of the 35 (wt E2F-1) cells were kept for 48 h in medium containing 0.1% serum and then 100 mM ZnCl2 was added. At the times indicated induced genes were known E2F target genes ± Cyclin at the top of each lane (in hours), cells were harvested and cell E, PCNA, Thymidylate synthase, DNA polymerase a extracts were used for Western blot analysis with an anti E2F-1 subunit II, DNA polymerase d catalytic subunit, monoclonal antibody (SQ41). Molecular size markers in kilo- MCM6 and MCM2. Among the other induced genes, daltons are shown on the left

Table 1 DNA replication related genes induced by E2F-1 Fold of induction Gene Function 0 ± 12 h (A) 0 ± 12 h (B) 0 ± 16 h (B)

Topoisomerase IIaa DNA replication 3.4 3.1 2.9 2.7 1.9 2 DNA polymerase a subunit IVa DNA replication 3 3.6 4.2 2.1 2.1 NI DNA polymerase a subunit II DNA replication 2.8 3.2 2.6 DNA polymerase d catalytic subunit DNA replication 2.8 9.1 7.5 Replication factor C (RF-C4) (37 Kd)a DNA replication 2.7 3.4 2.8 1.8 2.1 1.9 Replication protein A (RPA2) (32 Kd) DNA replication 2.6 3 2.2 PCNA DNA replication 2.5 2.2 1.9 Fen1 DNA replication 2.3 2.6 2.2 DNA polymerase a subunit IIIa DNA replication 2 2.6 1.8 19.4 3.7 3.4 Cyclin Ea Cell cycle 4.6 4.9 3.2 2.8 2.7 2 Thymidylate synthase Nucleotide synthesis 2.3 3.2 3.3 MCM6 Origin recognition 2.1 2 2 MCM2 Origin recognition 2.1 2.2 2.1

aThese genes were present twice on the DNA chip. Fold of induction in both cases is presented. (A) and (B) are two independent experiments. NI, not informative

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1381

Figure 2 Dierential hybridization of atlas rat array. Total RNA prepared from growth arrested Rat-1a-MT-wtE2F-1 cells before and after 12 h induction with 100 mM ZnCl2 was used to generate cDNA. Radiolabeled cDNA was hybridized to the Figure 3 RPA2 expression is upregulated by E2F-1 and by Clontech Atlas membranes (7738-1). An enlargement of part of serum. (a) RPA2 mRNA levels are upregulated by E2F-1. Rat-1- the membranes is presented. Each cDNA is present on the ®lter MT-wtE2F-1 (wtE2F-1) and Rat-1-MT-E2F-1dlTA (E2F-1dlTA) on two adjacent spots. Three of the genes which were upregulated cells were kept in medium with 0.1% serum for 48 h and then upon induction of E2F-1 are indicated by arrows cells were treated with 100 mM ZnCl2 for 12 h (+) or not treated (7) prior to RNA extraction. Total RNA was used for RT ± PCR analysis using RPA2 and GAPDH speci®c oligonucleotides. (b) Cells were treated as in a and then lysed and subjected to Western blot analysis using an anti E2F-1 monoclonal antibody (Santa Analysis of RPA2 induction by E2F-1 Cruz, sc-251). (c) RPA2 mRNA levels are upregulated by serum in both Rat and Human cells. Rat-1a-1093 (1093), Rat-1a-MT- Replication protein A (RPA) is the most abundant wtE2F-1 (wt E2F-1) and T98G cells were kept in medium with ssDNA binding protein in eukaryotic cells and it plays 0.1% FCS for 48 h and then serum was added to a ®nal an essential role in DNA replication (Erdile et al., concentration of 15%. Cells were lysed at the indicated times (in hours) after serum addition, RNA was extracted and subjected to 1990; Kenny et al., 1990). RPA is a heterotrimer RT ± PCR analysis using RPA2 and GAPDH speci®c oligonu- composed of 70 (RPA1), 32 (RPA2) and 14 Kd cleotides (RPA3) subunits (Fairman and Stillman, 1988; Wold and Kelly, 1988). The DNA chip analysis indicated that expression of the RPA2 gene was reproducibly elevated upon E2F-1 induction (Table 1) and similar by FACS analysis (data not shown). Under these elevation was detected using the Atlas membrane. conditions, RPA2 mRNA levels were induced in all Therefore we further investigated the regulation of its three cell lines 12 h after serum addition (Figure 3c), expression by E2F-1. Elevation of RPA2 mRNA levels demonstrating that RPA2 gene expression is growth upon E2F-1 induction was detected also by RT ± PCR regulated. analysis (Figure 3a) and Northern blot analysis (data Induction of E2F-1 expression in quiescent Rat-1- not shown). To investigate whether this E2F-1 induced MT-wtE2F-1 cells leads to S phase entry ((Qin et al., activation of RPA2 expression requires a transcription- 1994) and Figure 4). Therefore, up-regulation of RPA2 ally active E2F-1 we utilized a Rat-1 derived cell line expression may be an indirect consequence of E2F-1- containing a stably integrated Zinc inducible E2F-1 induced cell cycle progression. To address this possibi- mutant, Rat-1-MT-E2F-1dlTA. This E2F-1 mutant lity we tested whether RPA2 up-regulation precedes lacks the transactivation domain and, therefore, can E2F-1-induced S phase entry. As can be seen in Figure not transactivate gene expression. Upon addition of 4, RPA2 mRNA levels are increased already 6 h after Zinc to this cell line protein levels of E2F-1dlTA were E2F-1 induction while cell cycle distribution of the cells comparable to those of wtE2F-1 in Rat-1-MT-wtE2F-1 at this time does not dier signi®cantly than that of un- following Zinc addition (Figure 3b). However, this did induced cells. At 12 h post induction S phase entry is not lead to an increase in RPA2 expression (Figure 3a). evident and RPA2 mRNA levels are further increased. This result demonstrates that RPA2 gene expression is Thus, RPA2 induction precedes S phase entry indicat- not induced in response to the Zinc addition itself and ing that the former is not an indirect consequence of the that a transcriptionaly functional E2F-1 is required for latter. The RPA2 gene and its upstream sequence are this induction. To test whether expression of RPA2 available as part of the Human Genome project. To changes as arrested cells resume growth, cells of Rat-1- further study the regulation of RPA2 expression by 1093 (containing the empty vector), Rat-1-MT-wtE2F- E2F-1 we determined the position of its transcription 1 cell lines and the human glioblastoma cell line T98G start site by performing a 5' rapid ampli®cation of were growth arrested at G1 by serum starvation and cDNA ends (race) using Gibco ± BRL 5'RACE kit then allowed to resume growth by addition of serum. (18374-054). As shown in Figure 5a, a PCR reaction Growth arrest and resumption of growth were veri®ed using an oligonucleotide located 588 bp downstream to

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1382

Figure 4 RPA2 induction precedes S phase entry. Rat-1-MT- E2F-1 cells were serum starved (0.1% serum) for 48 h prior to the addition of 100 mM ZnCl2. At the times indicated at the top of each graph (in hours), cells were harvested. Cell cycle distribution was measured by the Cell-Cycle Flow Cytometry Assay. The graphs depict cell cycle distribution of living cells. The lower right panel depicts PRA2 mRNA levels. Cells were treated same as for FACS analysis and then lysed and total RNA was extracted. RNA was used for RT ± PCR analysis using RPA2 and GAPDH speci®c oligonucleotides

the 5' end of the previously published RPA2 cDNA (Erdile et al., 1990) gave rise to a single fragment of 631 bp. Sequencing of this PCR product demonstrated Figure 5 Sequence and schematic representation of the human that it contains 53 additional bases upstream to the 5' RPA2 promoter. (a)5' race employed primers at position +588 end of the published human RPA2 cDNA. Comparison of the longest available cDNA (left lane). RNA was extracted of the sequence of the PCR product to the genomic from MCF-7 cells and subjected to 5' race analysis. Sizes of DNA fragment containing the RPA2 gene clearly indicated markers, in base pairs, are depicted on the right. (b) Nucleotide sequence of the human RPA2 5' end and upstream region. The that the additional 53 bases are positioned immediately transcription start site, as determined by the 5' RACE is marked upstream to the 5' end of the published cDNA. The 5' by an arrow. The E2F binding sites are boxed. (c) Schematic end of our 5'-race product is therefore used as the +1 representation of putative binding sites of transcription factors in reference point (Figure 5) and the RPA2 promoter lies the promoter of human RPA2. The transcription start site is upstream to it. An examination of the RPA2 promoter marked by an arrow. The four E2F binding sites (including a bi- directional E2F binding site at 7313) and putative sites for region revealed four putative E2F DNA binding sites at binding of ATF-1 and SP-1 (GC Box) are indicated positions 7125/7118,7320/7313,7309/7316 and 7406/7399. The site at 7320/7313 matches perfectly with the E2F consensus binding sequence and the other three sites dier from the consensus at one position but fragment is indeed a functional promoter (Figure 6). are identical to E2F sites in known E2F responsive The role of E2F-1 in controlling the RPA2 promoter promoters (Figure 5). Computer analysis of the 700 bp activity was then assessed by co-transfection of sequence upstream to the transcription start site RPA2(7627/+55)Luc and E2F-1 and DP1 expression identi®ed additional potential binding sites of SP1 and plasmids, into H1299 cells. This co-transfection resulted ATF1. As for many of the known E2F target genes, the in an additional 2.5-fold increase in luciferase activity RPA2 promoter is TATA-less. A 682 bp fragment over the basal promoter activity (Figure 6), indicating spanning the RPA2 promoter from +55 to 7627 was that the RPA2 promoter is an E2F responsive synthesized by PCR using human placenta genomic promoter. No such increase was detected upon co- DNA as a template and was cloned in pA3Luc resulting transfection of pA3Luc together with E2F-1 and DP1 in RPA2(7627/+55)Luc. Transient transfection of (Figure 6). To test whether the E2F DNA binding sites RPA2(7627/+55)Luc into cells of the human lung present in the RPA2 promoter are required for its E2F carcinoma cell line H1299 resulted in a luciferase responsiveness, three of these sites were mutated using activity which was 34-fold higher than that of the PCR-site directed mutagenesis to generate RPA2(Mu- empty vector, pA3Luc, indicating that the cloned t)Luc. Unlike the result obtained using RPA2(7627/

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1383 (Isaacs et al., 1998; Woessner et al., 1991) and we demonstrated that this is true also for Rat-1-1093 and Rat-1-MT-E2F-1 cells. When these cell lines were growth arrested at G1 by serum starvation and then allowed to resume growth by addition of 15% serum, Topoisomerase IIa mRNA levels were induced in both cell lines 12 h after serum addition (Figure 7b) demonstrating that Topoisomerase IIa gene expression is growth regulated.

Discussion

Since the identi®cation of E2F-1 as an RB target a large body of evidence accumulated to support the idea that E2F-1 plays a critical role in regulating S phase entry. First, overexpression of E2F-1 in quiescent cells leads to S phase entry (Johnson et al., 1993; Kowalik et al., 1995). Second, E2F regulates the expression of Figure 6 The RPA2 promoter is activated by E2F-1. H1299 cells genes coding both for enzymes involved in nucleotide were transiently transfected with 0.5 mg of pA3Luc (vector) or a synthesis, such as thymidine kinase, thymidilate RPA2(7627/+55)Luc (wtRPA2) or RPA2(Mut)Luc (mutRPA2), synthase, dihydrofolate reductase (DHFR), and for together with 0.5 mg of pCMV-b-GAL. 0.2 mg pCMV-E2F-1 and 0.2 mg pCMV-DP1 were added where indicated. Forty-two hours proteins which are components of the origin recogni- post transfection cell extracts were prepared and used for b-GAL tion complex such as Orc1, cdc6 and MCM2 to and Luciferase assays. Fold of activation in the luciferase assay, after normalization for b-GAL activity, are depicted in the bar graph. Fold of activation is relative to the sample transected with pA3Luc

+55)Luc, when RPA2(Mut)Luc was transfected into H1299 cells its activity was not changed by co- expression of E2F-1 and DP1 (Figure 6) demonstrating that the eect of E2F-1 on the RPA2 promoter activity is, most likely, a direct eect mediated by the E2F DNA binding sites. Similar results were obtained using another cell line, MCF-7 (data not shown). Interest- ingly, the basal activity of RPA2(Mut)Luc was some- what higher than that of RPA2(7627/+55)Luc (Figure 6) suggesting that the mutated sequences mediate also transcriptional repression.

Induction of Topoisomerase IIa and subunit IV of DNA polymerase a expression by E2F-1 Two of the DNA replication related genes whose expression was upregulated upon E2F-1 induction are the genes encoding for Topoisomerase IIa and subunit Figure 7 Expression of Topoisomerae IIa and DNA polymerase IV of DNA polymerase a. These genes were present a subunit IV is up-regulated by E2F-1 (a) Topoisomerase IIa and twice on the DNA microarray, and they were induced DNA polymerase a subunit IV mRNA levels are upregulated by E2F-1. Rat-1a-MT-wtE2F-1 cells were kept in medium with 0.1% in both cases (Table 1), further validating their serum for 48 h and then cells were treated with (+) or without regulation by E2F-1. Elevation of mRNA levels of (7) 100 mM ZnCl2 for 12 h prior to RNA extraction. Total RNA both genes upon E2F-1 induction was detected also by was used for RT ± PCR analysis using Topoisomerase IIa, DNA RT ± PCR analysis (Figure 7a). Northern blot analysis polymeraes a subunit IV and GAPDH speci®c oligonucleotides. (b) Topoisomerase IIa mRNA levels are upregulated by serum in demonstrated that mRNA levels of Topoisomerase IIa Rat cells. Rat-1a-1093 (1093) and Rat-1a-MT-wtE2F-1 (wtE2F-1) were elevated 12 h after addition of Zinc in Rat-1-MT- cells were kept in medium with 0.1% serum for 48 h and then wtE2F-1 cells but not in Rat-1-1093 cells (Figure 7b) treated by addition of either 100 mM ZnCl2 (upper panel) or thus demonstrating that Topoisomerase IIa gene serum to a ®nal concentration of 15% (lower panel). Total RNA expression is not induced in response to the Zinc was prepared from cells at the indicated times (in hours) after ZnCl2 or serum addition and subjected to Northern analysis using addition itself. It has been previously reported that a Topoisomerase IIa probe. Acidic Ribosomal Protein (ARPP mRNA levels of Topoisomearse IIa increase in S phase PO) was used as a control probe

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1384 MCM7 (Leone et al., 1998; Yan et al., 1998). In The RPA2 gene codes for the 32 Kd subunit of addition, genes coding for components of the DNA replication protein A (RPA) which is a heterotrimeric replication machinery itself, such as DNA polymerase protein complex (Fairman and Stillman, 1988; Wold a subunits I and II, PCNA, and DNA polymerase d and Kelly, 1988). RPA is the most abundant ssDNA catalytic subunit are also E2F targets (Lee et al., 1995; binding protein in eukaryotic cells. It can unwind Nishikawa et al., 2000; Pearson et al., 1991; Zhao and dsDNA and it stabilizes DNA in the single strand Chang, 1997). DNA replication is a complex process conformation (Georgaki et al., 1992; Seroussi and and the proper expression and function of many Lavi, 1993; Treuner et al., 1996) and stimulates DNA proteins is required for its initiation as well as its synthesis by DNA polymerase a (Kenny et al., 1990). successful completion. Taking into consideration the RPA plays an essential role in DNA replication and pivotal role played by E2F in controlling S phase entry mutations in RPA2 were found to be lethal to yeast it is reasonable to assume that many other genes (Philipova et al., 1996). Importantly, an anti RPA2 coding for DNA replication proteins will be identi®ed antibody blocks the stimulation of DNA polymerase a as E2F targets. by RPA and two distinct anti RPA2 monoclonal Using RNA from a cell line with inducible E2F-1 as antibodies strongly inhibit SV40 DNA replication a probe for dierential screening of both an Atlas (Erdile et al., 1990; Kenny et al., 1990). These data membrane and DNA chips we detected an increase in clearly demonstrate that the 32 Kd subunit of RPA, the expression of seven known E2F target genes: DNA encoded by RPA2, is essential for DNA replication. polymerase a subunit II, DNA polymerase d catalytic Taken together with our results, these data strongly subunit, PCNA, cyclin E, Thymidylate synthase, suggest that induction of RPA2 expression by E2F-1 MCM6 and MCM2. In addition, we detected an contributes to E2F-1 induced DNA synthesis. increase in the expression of six replication related Little is known about the regulation of expression genes which were not described previously as E2F of the three RPA genes. However, experiments using target genes. These are: (1) Topoisomerase IIa whose S. cerevisia demonstrate that all three RPA genes are primary role is to disentangle interwined chromosomes expressed in a cell cycle dependent manner and their during anaphase (reviewed in Isaacs et al., 1998); (2,3) mRNA levels peak at the G1/S boundary (Brill and Subunits III and IV of DNA polymerase a which Stillman, 1991). The data presented here demonstrates constitute the DNA primase (reviewed in Foiani et al., that expression of at least one component of the RPA 1997); (4) RF-C4, coding for the 37 Kd subunit of heterotrimer, RPA2, is cell cycle regulated also in replication factor C (RF-C). RF-C is a complex higher eukaryotes. In addition to its regulation at the consisting of ®ve proteins which binds the template- transcriptional level, RPA2 is phosphorylated in a cell primer junction thus recruiting DNA polymerases to cycle dependent fashion at the G1/S transition (Din et the site of DNA synthesis (Mossi and Hubscher, 1998); al., 1990; Fang and Newport, 1993), and RPA (5) RPA2 encoding for the 32 Kd subunit of association with origins of replication is regulated by replication protein A which is the most abundant cyclin and Dbf-4 dependent kinases (Tanaka and single strand DNA binding complex (reviewed in Iftode Nasmyth, 1998). Thus, RPA2 is regulated in a cell et al., 1999) and (6) Fen1, a 5'-3' exo/endonuclease cycle dependent manner both at the transcriptional essential for Okazaki fragments maturation (reviewed and at the post-translational levels. Such a dual in Bambara et al., 1997). These genes are depicted in regulation is reminiscent of other E2F target genes Table 1. such as cdc6 (Hateboer et al., 1998; Petersen et al., A comparison of our results with the comprehen- 1999; Yan et al., 1998). The RPA1 gene was present sive analysis of cell cycle-regulated genes of the yeast neither on the Atlas membrane nor on the DNA chip Saccharomyces cerevisiae (Spellman et al., 1998) show used for this study. The RPA3 gene was also present that, in agreement with our ®ndings, most of the on the DNA chip, however, its expression was only yeast homologues of the Rat genes presented in mildly increased upon E2F-1 induction: 2.6, 1.8 and Table 1 are up-regulated in G1 phase of the cell 1.6 in experiments 0-12A, 0-12B and 0-16B depicted in cycle. These include the yeast homologues of DNA Table 1. Thus, it is not clear whether the co-regulation polymerase a subunit II, III and IV, DNA poly- of the three RPA subunits exist also in higher merase d catalytic subunit, PCNA, RF-C4, RPA2 eukaryotes and understanding the regulation of and Fen1. expression of the three RPA subunits requires The E2F-dependent regulation of expression of three additional studies. of the genes shown in Table 1 was further studied, It is noteworthy that RPA has been shown to resulting in the clear identi®cation of RPA2 as a new recognize some types of damaged DNA, to interact E2F-1 target gene. We also demonstrated that E2F-1 with a number of proteins involved in DNA repair and plays a role in the regulation of Topoisomerase IIa and to play a role in the response to DNA damage (He et DNA polymerase a subunit IV. RPA2 mRNA levels al., 1995; Li et al., 1995; Nagelhus et al., 1997). Recent are induced both by E2F-1 and by serum and the reports demonstrate that E2F-1 protein level and DNA RPA2 promoter contains four putative E2F binding binding activity are induced upon DNA damage sites. Our data clearly show that this promoter is (Blattner et al., 1999; Hoerer et al., 1999; O'Connor activated by E2F-1 and mutations in three of the E2F and Lu, 1999). Therefore, it is possible that E2F-1 binding sites abolish this activation. regulates RPA2 expression not only when DNA

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1385 replication is required, i.e. before S phase, but also as numerous proteins and the genes coding for many of part of the DNA damage response. them were not present on the DNA chips analysed Two other genes are demonstrated in the present here. The full spectrum of replication related genes that report to be E2F responsive, namely Topoisomerase are regulated, either directly or indirectly, by E2Fs IIa and subunit IV of DNA polymerase a. awaits additional studies. The latter encodes for the 48 Kd subunit of DNA polymerase a, which is the catalytic DNA primase subunit. It is sucient to synthesize in vitro the short Materials and methods RNA primers required to provide the 3' ends for elongation by DNA polymerase a (reviewed in Foiani Cell culture et al., 1997). DNA polymerase a has four subunits and Rat-1a ®broblasts that had stably integrated the p1093, its subunits I and II are known E2F target genes p1093-E2F-1 or p1093-E2F-1d1TA were grown in Dulbecco's (Pearson et al., 1991; Nishikawa et al., 2000). The gene modi®ed Eagle's medium (DMEM) supplemented with 10% encoding for subunit I was present on the DNA chip, (vol/vol) fetal calf serum (FCS) and G418 (500 mg/ml). T98G however, its hybridization results were not informative. and MCF-7 cells were grown in DMEM supplemented with The genes encoding for subunits II and III were also 10% (vol/vol) fetal bovine serum (FBS). H1299 cells were present on the DNA chip and their expression was grown in RPMI supplemented with 10% (vol/vol) FCS. increased upon E2F-1 induction (Table 1). This raises the possibility that E2F co-regulates the four subunits Western blotting of DNA pol a, that plays a major role in DNA Cells were lysed in lysis buer [20 mM HEPES pH 7.8, replication and is the only enzyme that can initiate 450 mM NaCl, 25% glycerol, 0.2 mM EDTA, 0.5 mM DTT, DNA chains de novo. phenylmethylsulfonyl ¯uoride (43 mg/ml), aprotinin (17 mg/ The Topoisomerase IIa gene codes for a 170 Kd ml), leupeptin (22 mg/ml)]. Twenty mg of protein from each protein whose primary role is to disentangle interwined lysate, as determined by Bradford assay, were resolved by chromosomes during anaphase (Isaacs et al., 1998). electrophoresis in a SDS 10% polyacrylamide gel and The Topoisomerase IIa promoter shares some structur- transferred to ®lters (Protran BA, 85, S&S). Filters were al similarities with other cell cycle-regulated promoters incubated with the indicated antibodies over night after 2 h such as Thymidine kinase, DHFR and DNA poly- incubation in PBS with 0.05% Tween and 5% dry milk. merase a subunit I. However, it does not contain E2F Binding of the primary antibodies was detected using an binding sites, and previous studies have shown that, enhanced chemiluminescence kit (ECL Amersham). unlike many E2F target genes, its mRNA expression peaks in late S rising to about 10-fold higher than in Screening of atlas cDNA expression array G1 (Isaacs et al., 1998; Woessner et al., 1991). These Rat-1-MT-E2F-1 cells were kept for 48 h in DMEM data suggest that the E2F dependent increase detected containing 0.1% FCS. Half of the cells were treated with in expression of Topoisomerase IIa is most probably 100 mM ZnCl2 for 12 h and then cells were harvested and total indirect. This is in agreement with the presence of a RNA was extracted using Tri Reagent method (Molecular, myb binding site within the Topoisomerase IIa Research Center). After DNase treatment radiolabeled cDNA was prepared from 9 mg of total RNA and hybridized to the promoter (Isaacs et al., 1998) and the ability of B- membranes (Clontech, 7738-1) according to the manufac- myb, a known E2F target gene (Lam and Watson, turer's instructions. Computerized analysis of the data was 1993), to up-regulate expression of Topoisomerase IIa. performed using AtlasImage 1.01 software (Clontech, USA). Irrespectively of the mechanism by which Topoisome- The signal intensities were normalized globally using the sum rase IIa is up-regulated in response to E2F induction, method (AtlasImage Clontech, USA). this is an interesting phenomenon, especially since Topoisomerase IIa is the primary cellular target of the DNA chip most widely used anti-neoplastic agents including RNA was extracted as described for the Atlas cDNA doxorubicin, etoposide and mitoxantrone. It has been expression array and double strand cDNA was synthesized documented that Topoisomerase IIa mRNA levels are by Reverse Transcription. Biotin-labeled cRNA was pro- higher in a variety of tumors in comparison to adjacent duced from the cDNA and used to probe Aymetrics Rat normal tissue (Hasegawa et al., 1993; Isaacs et al., DNA micro-array (Rat Genome U34A array). Hybrydization 1998). It would be interesting to test whether tumor- and washes were performed using Aymetrics gene chip related mutations in the RB/E2F pathway result in system according to manufacturer's instructions. changes in the levels of Topoisomerase IIa, and whether this is re¯ected in the responsiveness of the RT ± PCR tumor to anti-neoplastic treatment with inhibitors of Topoisomerase IIa. Reverse transcription-PCR (RT ± PCR) was performed on total RNA prepared by the Tri Reagent method. 7.5 mgof The ability of E2F-1 to up-regulate the expression of RNA were employed for cDNA synthesis using M-MLV a number of genes coding for components of the DNA reverse transcriptase (Promega, 200 u) and oligo dT (0.5 mg, replication machinery provide novel links between Pharmacia). PCR was performed on 1 ml of the 20 ml cDNA components of the DNA replication machinery and sample. Below are indicated, respectively, the number of the cell growth regulatory pathway involving RB and cycles and the sequences of 5' and 3' primers used for each of E2F. DNA replication is a complex process involving the tested genes: for the gene encoding RPA2, 30

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1386 cycles, using 5'-CCGGATCCGCAAGAGCCCGAGCC- tion of the 5' end of human RPA2 mRNA was 5'- CAGC-3' and 5'-CGGAATTCGCACGGTGAGGCCATTT- GGGAATTCGCGCTTAGTACCATGTGTGC-3'. GCTGGC-3'; for the gene encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH), 19 cycles using 5'-ACCA- Isolation of human RPA2 promoter CAGTCCATGCCATCAC-3' and 5'-TCCACCACCCTG- TTGCTGTA-3'. The annealing temperature for both genes A 682 bp fragment of the RPA2 promoter from the +55 to was 508C. For the gene encoding topoisomerase II a,26 7627 was generated by PCR using 100 ng human genomic cycles, using 5'-CAATGCTCAGCCCTTTGGCTCG-3' and placenta DNA as template and 60pm each from the 5' and 5'-GTAGTCCCACTCTCTCTGCC-3'; for the gene encoding 3' primers: 5'-CGGGGTACCGGATGAGACTGGGATG- DNA polymerase a subunit IV, 26 cycles, using 5'- GGG-3' and 5'-CGGAAGCTTCCACTGCGCCGCTCT- CACAACACAGTGAAGCTGGG-3' and 5'-GGGTGAA- GGC-3'. CACTAAAAGGGC-3'. The annealing temperature for both genes was 588C. Plasmids The luciferase reporter plasmid RPA2(7627/+55) Luc was Northern blot analysis constructed by subcloning the 682 bp KpnI/HindIII fragment RNA was extracted from cells by the Tri Reagent method. of the RPA2 promoter in PA3Luc. Mutation in the E2F Twenty mg of total RNA from each sample were separated on DNA binding sites were generated by PCR-site- a formaldehyde gel and then blotted to GeneScreen Plus directed mutagenesis which changed TTTGGCGC(7320 membrane (DU PONT) according to manufacturer's instruc- to 313) to TTTGAAGC, GTTCGCGC(7309 to 7316) tions. Acidic Ribosomal Protein (ARPP PO) was used as a to GTTCGCTT, TTTGGAGC(7125 to 7118) to control probe. TTTGAAGC to generate RPA2(mut)Luc. pCMV-b-Gal, pCMV-E2F-1 and pCMV-DP1 were described previously (Krek et al., 1993; Lindeman et al., 1997). Cell-cycle flow cytometry assays Bromodeoxyuridine (BrdU, ®nal concentration, 10 m ) was M Transfection assay added to cells 30 min prior to harvesting. Cells were trypsinized and ®xed with 70% ethanol (720). After H1299 cells were transfected by the calcium phosphate ®xation, cells were centrifuged for 5 min at 1200 r.p.m. method. Cells were harvested 42 h after transfection, and and incubated for 30 min at room temperature in 1 ml of lysates were tested for luciferase and b-galactosidase activities 2 M HCl/0.5% Triton X-100. After recentrifugation, cells as described previously (Lindeman et al., 1997). were washed with 1 ml of 0.1 M Na2B4O7 (pH 8.5) and reacted with anti BrdU antibody (Becton Dickinson, 347580, 15 ml per test) and then with Fluorescein isothiocyanate-conjugated anti mouse antibody. Then cells were resuspended in PBS containing 5 mg/ml propidium Acknowledgments idodide for 30 min at room temperature. Fluorescence We thank Drs WG Kaelin Jr and PD Adams for cells lines intensity was analysed on a Becton Dickinson ¯ow and Dr D Givol for reagents and critical reading of the cytometer. manuscript. We thank Dr Shirley Horn-Saban for excellent technical assistance with DNA microarray analysis. This work was supported in part by grants from the Israel 5' Rapid amplification of cDNA ends (5' RACE) cancer Association (ICA), Minerva Foundation (Ger- Total RNA was extracted from growing MCF-7 cells using many), the Israel Cancer Research Fund (ICRF) and Yad Tri Reagent method and subjected to 5'-race analysis Abraham Research Center for Diagnostics and Therapy. D according to the manufacture instructions (Gibco ± BRL, Ginsburg is an incumbent of the Recanati Career Devel- 18374-058). The primer used for the ®nal step of ampli®ca- opment chair of cancer research.

References

Bambara RA, Murante RS and Henricksen LA. (1997). J. Hall M and Peters G. (1996). Adv. Cancer Res., 68, 67 ± 108. Biol. Chem., 272, 4647 ± 4650. Hasegawa T, Isobe K, Nakashima I and Shimokata K. Blattner C, Sparks A and Lane D. (1999). Mol. Cell. Biol., (1993). Biochem. Biophys. Res. Commun., 195, 409 ± 414. 19, 3704 ± 3713. Hateboer G, Wobst A, Petersen BO, Le CL, Vigo E, Sardet C Brill SJ and Stillman B. (1991). Genes Dev., 5, 1589 ± 1600. and Helin K. (1998). Mol. Cell. Biol., 18, 6679 ± 6697. Din S, Brill SJ, Fairman MP and Stillman B. (1990). Genes He Z, Henricksen LA, Wold MS and Ingles CJ. (1995). Dev., 4, 968 ± 977. Nature, 374, 566 ± 569. Dyson N. (1998). Genes Dev., 12, 2245 ± 2262. Helin K. (1998). Curr.Opin.Genet.Dev.,8, 28 ± 35. Erdile LF, Wold MS and Kelly TJ. (1990). J. Biol. Chem., Hoerer M, Wirbelauer C, Humar B and Krek W. (1999). 265, 3177 ± 3182. Nucleic Acids Res., 27, 491 ± 495. Fairman MP and Stillman B. (1988). EMBO J., 7, 1211 ± Iftode C, Daniely Y and Borowiec JA. (1999). Crit. Rev. 1218. Biochem. Mol. Biol., 34, 141 ± 180. Fang F and Newport JW. (1993). J. Cell. Sci., 106, 983 ± 994. Isaacs RJ, Davies SL, Sandri MI, Redwood C, Wells NJ and Foiani M, Lucchini G and Plevani P. (1997). Trends Hickson ID. (1998). Biochim. Biophys. Acta, 1400, 121 ± Biochem. Sci., 22, 424 ± 427. 137. Georgaki A, Strack B, Podust V and Hubscher U. (1992). Johnson DG, Cress W, Jakoi L and Nevins JR. (1994). Proc. FEBS Lett., 308, 240 ± 244. Natl. Acad. Sci. USA, 91, 12823 ± 12827.

Oncogene E2F-1 up-regulates expression of DNA replication genes Y Kalma et al 1387 Johnson DG, Schwarz JK, Cress WD and Nevins JR. (1993). Philipova D, Mullen JR, Maniar HS, Lu J, Gu C and Brill SJ. Nature, 365, 349 ± 352. (1996). Genes Dev., 10, 2222 ± 2233. Kaelin WJ. (1999). Bioessays, 21, 950 ± 958. Pierce AM, Fisher SM, Conti CJ and Johnson DG. (1998). Kenny MK, Schlegel U, Furneaux H and Hurwitz J. (1990). Oncogene, 16, 1267 ± 1276. J. Biol. Chem., 265, 7693 ± 7700. Qin XQ, Livingston DM, Kaelin WG and Adams P. (1994). Kowalik TF, DeGregori J, Schwarz JK and Nevins JR. Proc. Natl. Acad. Sci. USA, 91, 10918 ± 10922. (1995). J. Virol., 69, 2491 ± 2500. Seroussi E and Lavi S. (1993). J. Biol. Chem., 268, 7147 ± Krek W, Livingston DM, Shirodkar S. (1993). Science, 262, 7154. 1557 ± 1560. Shan B and Lee W-H. (1994). Mol. Cell. Biol., 14, 8166 ± Lam EW and Watson RJ. (1993). EMBO. J., 12, 2705 ± 2713. 8173. Lee HH, Chiang WH, Chiang SH, Liu YC, Hwang J and Ng Sherr CJ. (1996). Science, 274, 1672 ± 1677. SY. (1995). Gene Expr., 4, 95 ± 109. Spellman PT, Sherlock G, Zhang MQ, Iyer VR, Anders K, Leone G, DeGregori J, Yan Z, Jakoi L, Ishida S, Williams Eisen MB, Brown PO, Botstein D and Futcher B. (1998). RS and Nevins JR. (1998). Gene Dev., 12, 2120 ± 2130. Mol. Biol. Cell., 9, 3273 ± 3297. Li L, Lu X, Peterson CA and Legerski RJ. (1995). Mol. Cell. Tanaka T and Nasmyth K. (1998). EMBO J., 17, 5182 ± Biol., 15, 5396 ± 5402. 5191. Lindeman GJ, Gaubatz S, Livingston DM and Ginsberg D. Treuner K, Ramsperger U and Knippers R. (1996). J. Mol. (1997). Proc. Natl. Acad. Sci. USA, 94, 5095 ± 5100. Biol., 259, 104 ± 112. Mossi R and Hubscher U. (1998). Eur. J. Biochem., 254, Vigo E, Muller H, Prosperini E, Hateboer G, Cartwright P, 209 ± 216. Moroni MC and Helin K. (1999). Mol. Cell. Biol., 19, Muller H and Helin K. (2000). Biochim. Biophys. Acta, 1470, 6379 ± 6395. M1 ± M12. Weinberg RA. (1995). Cell., 81, 323 ± 330. Nagelhus TA, Haug T, Singh KK, Keshav KF, Skorpen F, Woessner RD, Mattern MR, Mirabelli CK, Johnson RK and Otterlei M, Bharati S, Lindmo T, Benichou S, Benarous R Drake FH. (1991). Cell Growth Dier., 2, 209 ± 214. and Krokan HE. (1997). J. Biol. Chem., 272, 6561 ± 6566. Wold MS and Kelly T. (1988). Proc.Natl.Acad.Sci.USA, Nishikawa NS, Izumi M, Uchida H, Yokoi M, Miyazawa H 85, 2523 ± 2527. and Hanaoka F. (2000). Nucleic Acids Res., 28, 1525 ± Yan Z, DeGregori J, Shohet R, Leone G, Stillman B, Nevins 1534. JR and Williams RS. (1998). Proc. Natl. Acad. Sci. USA, O'Connor DJ and Lu X. (1999). Oncogene, 19, 2369 ± 2376. 95, 3603 ± 3608. Pearson BE, Nasheuer HP and Wang TS. (1991). Mol. Cell. Zhao L and Chang LS. (1997). J. Biol. Chem., 272, 4869 ± Biol., 11, 2081 ± 2095. 4882. Petersen BO, Lukas J, Sorensen CS, Bartek J and Helin K. (1999). EMBO J., 18, 396 ± 410.

Oncogene