Abstract:
The present invention relates to a protein that is interacting with the minichromosome maintenance complex in eukaryotes. More specifically, the invention relates to the use of a protein, interacting with the minichromosome complex, and the gene encoding this protein in diagnosis, prognosis and treatment of cancer.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This patent application is a national phase entry under 35 U.S.C. §371 of PCT International Patent Application PCT/EP2009/056658, filed May 29, 2009, designating the United States and published in English as International Patent Publication WO 2009/144311 A1 on Dec. 3, 2009, which claims the benefit, under Article 8 of the PCT, of U.S. Provisional Patent Application No. 61/130,552, filed May 29, 2008, the entire disclosure of each of which is hereby incorporated herein by this reference. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present invention relates to a protein that is interacting with the minichromosome maintenance complex in eukaryotes. More specifically, the invention relates to the use of a protein, interacting with the minichromosome complex, and the gene encoding this protein in diagnosis, prognosis and treatment of cancer. 
       BACKGROUND 
       [0003]    All eukaryotic cells replicate their nuclear DNA in a conserved manner, whereby the parent molecule is unwound and each DNA strand becomes the template for nascent DNA synthesis. Coordination of DNA replication with growth and development is essential for survival, as mistakes made during DNA replication can result in apoptosis, growth defects, or oncogenesis. Because of its essential role during development, it is not surprising to see that the molecular machinery controlling DNA replication is highly conserved among organisms. Despite the many years of evolution that separate animals from plants, both organisms use the same E2F/DP pathway to regulate entry into S phase by controlling transcriptional induction of genes required for cell cycle progression and DNA replication. The genome of  Arabidopsis  contains six E2Fs (E2Fa, E2Fb, E2Fc, E2Fd/DEL2, E2Fe/DEL1, and E2Ff/DEL3) and two DPs (DPa and DPb) (Inzé and De Veylder, 2006). Three E2F proteins (E2Fa-c) bind DNA through the consensus E2F binding site by forming heterodimers with DP proteins. Both E2Fa and E2Fb operate as transcriptional activators, whereas E2Fc functions as a repressor (De Veylder et al., 2002; del Pozo et al., 2002). The remaining  Arabidopsis  E2Fs (E2Fd/DEL2, E2Fe/DEL1 and E2Ff/DEL3) contain duplicated DNA-binding domains, allowing binding to consensus E2F sites as a monomer (Kosugi and Ohashi, 2002; Ramirez-Parra et al., 2004; Vlieghe et al., 2005). 
         [0004]    Both in mammals and  Arabidopsis , numerous E2F target genes have been identified using microarrays, chromatin immunoprecipitations, and in silico analyses (Ramirez-Parra et al., 2003; Dimova and Dyson, 2005; Vandepoele et al., 2005). These genes encode proteins active during DNA replication, mitosis, DNA checkpoint control, apoptosis, or differentiation. Remarkably, almost every gene encoding for a protein involved in licensing for DNA replication is transcriptionally controlled by E2F transcription factors, such as the origin recognition complex (ORCs), CDC6, minichromosome maintenance complex (MCMs), and CDT1 genes (Vandepoele et al., 2005). Licensing for DNA replication in eukaryotes is initiated by the formation of the pre-replicative complex (pre-RC) at replication origins (Gillespie et al., 2001; Bell and Dutta, 2002; Diffley and Labib, 2002). First, ORC proteins bind to DNA during the early G1-to-S phase of the cell division cycle. Then, CDC6 binds to these ORC-DNA sites, an event that is followed quickly by binding of CDT1. Finally, replication origins are licensed by loading the MCM complex to form a pre-RC. The MCM complex is a heterohexamer composed of MCM2 to 7 and is likely a component of the helicase that unwinds DNA during replication (Tye and Sawyer, 2000; Labib and Diffley, 2001; Forsburg, 2004). 
         [0005]    Once the formation of the pre-RC has been completed by the loading of MCMs, the DNA is primed for replication through the action of two conserved protein kinases, cyclin-dependent protein kinase (CDK) and Cdc7-Dbf4 (Dbf4-dependent kinase, DDK), resulting into the recruitment of additional replication factors to form the pre-initiation complex (pre-IC) (Kamimura et al., 2001; Masumoto et al., 2002; Takayama et al., 2003; Kanemaki et al., 2006). Loading of the pre-IC onto the origins activates the MCM helicases and recruitment of DNA polymerases, resulting into the initiation of DNA synthesis (Zou and Stillman, 2000). After origin firing, some of the initiation factors move with replication forks to support the elongation step of DNA synthesis (Aparicio et al., 1997; Takayama et al., 2003; Gambus et al., 2006; Kanemaki et al., 2006). Members of the MCM complex, particularly MCM2, have been described as markers for screening, surveillance and prognosis of cancer (Giaginis et al., 2008). 
         [0006]    By studying E2F target genes in  Arabidopsis , we have identified ETG1 (At2g40550) as a novel E2F target gene, being directly controlled by the E2Fa and E2Fb transcription factors. ETG1 null mutants display a slower cell cycle progression. Genetic analysis and transcriptional up-regulation of the PARP2, WEE1 and RAD51 genes indicated that this cell cycle delay originates from the activation of the DNA replication checkpoint. ETG1 is demonstrated to associate with the DNA replication complex, suggesting that the activation of the DNA replication checkpoint in ETG1-deficient plants originates from impaired DNA replication. Surprisingly, the absence of a functional ETG1 allele in a wee1 or atr mutant background has a profound impact on plant development, illustrating that the DNA replication checkpoint aids to the survival of ETG1-deficient plants. 
         [0007]    Even more surprisingly, knock down mutants of the human ETG1 homologue (C10orf119) resulted in a similar phenotype, indicating that the gene is involved in the development of mammalian tumors. This is specifically surprising, as Sakwe et al. (2007) indicated that C10orf119 (MCM-BP) binds to the MCM complex, but only in absence of the most relevant tumor marker MCM2. 
       DISCLOSURE OF THE INVENTION 
       [0008]    A first aspect of the invention is the use of ETG1 or an ETG1 ortholog for the diagnosis and/or prognosis of cancer. An ortholog, as used here, means a sequence with a similar, preferably an identical, function as the reference protein, and a detectable homology (expressed as percentage identity) with the reference sequence. ETG1 orthologs include, but are not limited to, rice ( Oryza sativa ; Os01g0166800), human (C10orf119), mouse (1110007A13Rik),  Xenopus  (CAJ81286),  Drosophila  (CG3430) and fission yeast (SPAC1687.04) orthologs. Preferably, the ETG1 ortholog is the human ortholog C10orf119 (accession NP — 079110). The human ETG1 gene is located on chromosomal position 10q26.11. This particular region shows high frequency of loss of heterozygosity in human meningiomas and colorectal cancers (Mihaila et al., 2003; Karoui et al., 2004). This type of loss of heterozygosity is generally regarded as a hallmark for the localization of a tumor suppressor. 
         [0009]    The “use” as meant herein is any use of the nucleic acid or protein and may be, as a non-limiting example, the genomic DNA for the detection of mutation, the mRNA or derived cDNA for the analysis of the expression, or the protein for the analysis of translated protein. Methods for mutation and snp analysis, expression analysis and detection and quantification of protein are known to the person skilled in the art. Abnormal chromosome content is the most common characteristic of human solid tumors. Preferably, the cancer is a cancer originating from a chromatin cohesion defect. Even more preferably, the cancer is selected from the group consisting of Seminoma, Colon carcinoma, Cervical cancer, Acute Myeloid carcinoma, Wilson tumor, Oligodendroglioma, Renal Carcinoma, Prostate carcinoma and Breast carcinoma. 
         [0010]    Another aspect of the invention is the use of an ETG1 ortholog to treat cancer. Preferably, the use is the modulation of the expression. “Modulation,” as used herein, may be under- or over-expression. A knock down may be realized by, as a non-limiting example, the expression of RNAi. Alternatively, the level of protein may be modulated by a specific interaction, such as the binding of an antibody. Preferably, the ETG1 ortholog is the human ortholog C10orf119. One preferred embodiment is a modulation whereby modulation is a down-regulation in the cancers selected from the group consisting of Seminoma, Colon carcinoma, Acute Myeloid carcinoma, Wilson tumor, and Oligodendroglioma. Another preferred embodiment is a modulation whereby modulation is an up-regulation in the cancers selected from the group consisting of Renal Carcinoma, Prostate carcinoma and Breast carcinoma. 
         [0011]    Still another aspect of the invention is the use of ETG1 or an ETG1 ortholog to screen compounds interfering with the interaction of ETG1 or an ETG1 ortholog with the MCM complex. Preferably, the compound is a compound that interferes with the interaction of ETG1 or an ETG1 ortholog with MCM2, MCM3, MCM4, MCM5, MCM6 and/or MCM7. “Interfering,” as used herein, can be either positive or negative, making the interaction stronger or disturbing the interaction. Methods to study protein-protein interactions (and the effect on compounds on those interactions) are known to the person skilled in the art and include, but are not limited to, two yeast hybrid studies, mappit (Tavernier et al., 2002) and reverse mappit (Lemmens et al., 2006). 
         [0012]    Still another aspect of the invention is the use of a compound interfering with the interaction of ETG1 or an ETG1 ortholog with the MCM complex, isolated according to the invention, to treat cancer. 
     
    
     
       BRIEF DESCRIPTION OF THE FIGURES 
         [0013]      FIG. 1 : Molecular and phenotypic analysis of ETG1-deficient plants. (A) The exon (boxes) and intron (lines) structure of ETG1. Coding and non-coding regions are shown as black and white boxes, respectively. White triangles indicate T-DNA insertion sites. Arrows indicate primer positions used for real-time RT-PCR analysis. (B) Real time RT-PCR analysis of ETG1 expression in wild-type (WT), etg1-1 and etg1-2 plants. RT-PCR was performed using total RNA prepared from first leaves of nine-day-old plants. All values were normalized against the expression level of the ACTIN2 gene. (C) Seedling phenotypes of 21-day-old wild-type (WT) and etg1-1 plants. (D) Ploidy level distribution of the first leaves of three-week-old wild-type (col-0), etg1-1 and etg1-2 plants as measured by flow cytometry. Data represent average ±SD (n=5). (E) Drawing-tube image of the first leaves of three-week-old wild-type (WT; left) and etg1-1 (right) plants. Bar indicates 100 μm. (F-H) Leaf growth of the first leaf pair of wild-type (WT), etg1-1 and etg1-2 plants. Leaf blade area (F), Epidermal cell number on the abaxial side of the leaf (G), and epidermal cell size on the abaxial side of the leaf (H). Data represent average ±SD (n=5). (I-J) Adult phenotype of wild-type (WT) and etg1-1 plants. The plants were photographed five weeks after germination. (J) shows magnification of leaves in wild-type and etg1-1 plants. 
           [0014]      FIG. 2 : Kinematic analysis of first leaf pair of wild-type (WT) and etg1-1 plants. (A) Leaf blade area; (B) average cell area on abaxial side of the leaf; (C) number of cells on abaxial side of leaf; and (D) cell division rate. Leaves were harvested at the indicated time points. Data represent average ±SD (n=5). 
           [0015]      FIG. 3 : ETG1-depletion leads to a G2 arrest. (A) Ploidy level distribution of the first leaves of eight-day-old wild-type (WT) and etg1-1 plants as measured by flow cytometry. (B) Ratio of 4C/2C cells by flowcytometry using first leaves of eight-day-old wild-type (WT) and etg1-1 plants. Data represent average ±SD (n=3). (C) Real time RT-PCR analysis of CDKB1;1 and CYCB1;1 expression in wild-type (WT) and etg1-1 plants. RT-PCR was performed using total RNA prepared from nine-day-old seedlings. All values were normalized against the expression level of the ACTIN2 gene. 
           [0016]      FIG. 4 : ETG1 gene expression is regulated by the E2Fa transcription factor. (A) Sequence of the ETG1 promoter showing the presence of two E2F binding sites at −158 by (I) and -136 (II) from the ATG translation start codon (SEQ ID NO:40). (B) ChIP assays in eight-day-old  Arabidopsis  plants using antibodies specific for E2Fa, E2Fb, E2Fc and DELL Semiquantitative PCR was used to estimate the relative enrichment of genomic fragments of ETG1 promoter. (C) Real time RT-PCR analysis of ETG1 expression in wild-type (WT) and E2Fa/DPa overexpressing plants. RT-PCR was performed using total RNA prepared from six-day-old plants. All values were normalized against the expression level of the ACTIN2 gene. (D) Histochemical localization of GUS activity in transgenic six-day-old seedlings carrying the wild-type (col-0), either one (ΔI or ΔII) or both (ΔI,II) of the E2F elements ETG1 promoters fused to GUS gene. The shoot (upper) and the root apical regions (bottom). 
           [0017]      FIG. 5 : ETG1 assembles into the replisome and is essential for DNA replication. (A) Subcellular localization of ETG1. The full length ETG1-eGFP fusion protein is localized in nucleus. (B) ETG1 interacts with MCM5 in yeast. Yeast PJ69-4a cells were transformed with a plasmid encoding a GAL4 DNA binding domain-ETG1 and -MCM5 fusion (GAL4-DBD-ETG1 and -MCM5), respectively. Yeast PJ69-4-alfa cells were transformed with GAL4 activation domain-ETG1, -MCM5 and -GUS fusion as negative control (GAL4-AD-ETG1, -MCM5 and -GUS). Interactions between fusion proteins were assayed by mating method. Diploid strains were spotted on plates with (+His, positive control) or without (-His) histidine. (C) BiFC assay of ETG1 interaction with MCM5 in plant. Tobacco epidermal cells were transfected with combination of constructs encoding indicated fusion proteins. YFPN, the fragment containing amino acid residues 1-154 of YFP; YFPC, the fragment containing amino acid residues 155-238 of YFP. Arrowheads show nuclei. (D) Subcellular localization of MCM5. The full length GFP-MCM5 fusion protein is localized in nucleus and cytoplasm. (E) BrdU incorporation of the wild-type (col-0) and etg1-1. Three-day-old seedlings were soaked in BrdU solution for the indicated periods, and genomic DNA was extracted from them. The amounts of BrdU incorporation were determined by ELISA using an anti-BrdU antibody. Data represent average ±SD (n=3). 
           [0018]      FIG. 6 : Loss of ETG1 induces expression of DNA replication checkpoint and DNA repair genes. (A) Real time RT-PCR analysis of WEE1 and RAD51 expression in wild-type (white bars) and etg1-1 (black bars) plants. RT-PCR was performed using total RNA prepared from nine-day-old seedlings. All values were normalized against the expression level of the ACTIN2 gene. (*) and (**) indicate significant statistical differences by t-test (p&lt;0.05 and p&lt;0.01, respectively) between wild-type and etg1-1. Data represent average ±SD (n=3). (B-F) Histochemical localization of GUS activity in transgenic six-day-old PARP2::GUS (B), PARP2::GUS crossed with etg1-1 (C), PARP2::GUS grown on MS agar plate with 1 μg/ml bleomycin (D), WEE1::GUS (E) and WEE1::GUS crossed with etg1-1 plants. All seedlings were grown on MS agar plate except for (C). 
           [0019]      FIG. 7 : ETG1 depletion activates ATR/WEE1 DNA replication stress checkpoints. (A-H) Seedlings phenotype of 21-day-old wild-type (col-0) (A), etg1-1 (B), wee1-1 (C), atr-2 (D), etg1-1/wee1-1 (E, F) and etg1-1/atr-2 (G, H) grown on MS plate. (F) and (H) show magnification of etg1-1/wee1-1 and etg1-1/atr-2 plants. Bars: 5 mm (A-E, G) and 1 mm (F and H). (I-Q) Scanning electron micrographs of the 14-day-old whole seedlings (I-K), leaf epidermal cells (L-N) and trichome (O-Q). (I, L, O) wild-type; (J, M, P) etg1-1/wee1-1, and (K, N, Q) etg1-1/atr-2. Bars: 500 μm (A-C), 50 μm (D-F, H, and I) and 100 μm (G). 
           [0020]      FIG. 8 : Up-regulation of mitosis-specific expression genes in etg1. (A) G0 analysis of 121 up-regulated genes in etg1 in the ATH1 microarray experiment. (B) Enrichment of M-phase-specific genes in the etg1 transcriptome dataset. (C) Cell cycle phase comparison of up-regulated genes in etg1, UV-B-treated and bleomycin-treated plants. S-phase (red), G2-phase (blue), M-phase (yellow), and G1 phase (green) specific expression genes. 
           [0021]      FIG. 9 : ETG1 is required for establishment of sister chromatid cohesion. (A) Scheme of chromosome 1 with a sequence cloned in BAC T2P11/T7N9, BACF11P17 and a 178-bp centromere-specific sequence (pAL). (B-F) One FISH signal (B, pairing of both homologs) or two FISH signals (C, D) per BAC (T2P11/T7N9) were regarded as positional alignment at corresponding region, indicating sister chromatids are aligned. Three (E) or four (F) signals were considered to indicate sister-chromatid separation. (G) Percentage of sister-chromatid alignment/separation frequencies analyzed in wild-type (col-0) and etg1 after FISH with labeled BAC from chromosome 1. BAC T2P11/T7N9 were tested in 4C leaf nuclei. 
           [0022]      FIG. 10 : Morphology of sub-confluent cultures of wild-type MCF7 cells. 
           [0023]      FIG. 11 : Aberrant phenotypes (multi-nucleated and giant cells) observed in hETG1 knocked-down (pictures above) and over-expressing (pictures below) MCF7 cell cultures. 
           [0024]      FIG. 12 : Examples of giant and multi-nucleated cells observed upon over-expression of hETG1. The hETG1 protein is in the green channel and β-catenin protein is in the red channel. 
           [0025]      FIG. 13 : Flow cytometry analysis of wild-type and hETG1 knock-down (158067-Const) cell lines. Knock-down cell line shows a depletion of 11% of G1 cells, correlated with a 10% increment in G2 cells. A minor group of cells (1%) displayed a DNA content higher than 4C. 
           [0026]      FIG. 14 : Up-regulation of G2-M markers upon hETG1 knock-down. Cells were harvested after three days of growing, total RNA was extracted and cDNA was synthesized. Relative expression values were normalized against TBP and UBC. The expression levels in control MCF7 cells were arbitrarily set to 1. 
           [0027]      FIG. 15 : Up-regulation of MAD3 kinetochore marker upon hETG1 knock-down. Cells were harvested after three days of growing, total RNA was extracted and cDNA was synthesized. Relative expression values were normalized against TBP and UBC. The expression levels in control MCF7 cells were arbitrarily set to 1. 
           [0028]      FIG. 16 : Karyotypes for sister chromatids problems, detecting chromosomes with totally detached sister chromatids (enclosed in green circles). 
           [0029]      FIG. 17 : Expression analysis of a series of 56 primary human breast cancers. Relative ETG1 expression levels (average of ten samples with low expression set to 1) were depicted ranking low to high. 
           [0030]      FIG. 18 : Distribution of primary breast cancers according to ER status and ETG1 expression. Tumor samples were grouped in quartiles based on ETG1 expression levels from low Q1 to high Q4. 
       
    
    
     DETAILED DESCRIPTION OF THE INVENTION 
     Examples 
     Example 1 
     The Loss of ETG1 Suppresses Cell Division 
       [0031]    Previously, we identified 70 conserved plant E2F target genes (Vandepoele et al., 2005). This list holds 40 known regulators of DNA replication and chromatin dynamics, as well as 21 genes with unknown function. To identify among the latter novel S phase regulatory genes, we performed a phenotypic screening using T-DNA insertion lines obtained from the Salk Institute Genomic Analysis Laboratory (http://signal.salk.edu/cgi-bin/tdnaexpress). One of these T-DNA insertion mutants showed an endoreduplication phenotype. We designated this T-DNA insertion mutant as E2F target gene 1 (etg1; At2g40550). 
         [0032]    To address the role of ETG1 in plant growth and development, we analyzed the effect of loss of function of ETG1 in detail using two independent T-DNA insertion lines. Plants were grown under long-day conditions (16 hours/light, 8 hours/darkness) at 22° C. on 0.5×MS agar plate (Valvekens et al., 1988). The etg1-1 (SALK — 071046) and etg1-2 (SALK — 145460) alleles were found in the Salk Institute Genomic Analysis Laboratory engine (http://signal.salk.edu/cgi-bin/tdnaexpress). These seeds were acquired from the ABRC. 
         [0033]    To screen for homozygous insertion alleles, the following primer pairs were designed: 5′-AGACCAAGATGGTCAGAGGATC-3′ (SEQ ID NO:1) and 5′-ACTGGAACACAGTAAAGCAAGC-3′ (SEQ ID NO:2) for etg1-1, and 5′-AAATTAACCGGAATGGGTTTG-3′ (SEQ ID NO:3) and 5′-ATGACTCAGATTGATGCCTGG-3′ (SEQ ID NO:4) for etg1-2. The T-DNA was inserted in the first intron (etg1-1; SALK — 071046) or last exon (etg1-2; SALK — 145460) of the ETG1 gene ( FIG. 1 , Panel A), respectively. ETG1 transcripts were not detected in the etg1-1 mutant, whereas 80% reduction in transcript level was observed in etg1-2, compared to control plants ( FIG. 1 , Panel B). In etg1 mutant seedlings, plant growth appeared macroscopically normal ( FIG. 1 , Panel C). However, by comparing the ploidy level of wild-type plants with etg1-1 and etg1-2 mutants, a significant change in the distribution of the C values was found. etg1 mutants leaves contained an increased population of cells with an 8C and 16C DNA ploidy level, demonstrating that deficiency for ETG1 stimulated endoreduplication ( FIG. 1 , Panel D). When comparing the first pair of leaves from wild-type and etg1 mutant plants at maturity, the leaf blade area was found to be almost identical for both genotypes ( FIG. 1 , Panel F). By contrast, a significant increase in the average abaxial pavement cell area was observed in the mutant plants ( FIG. 1 , Panels E and G), accompanied with a decrease in the number of cells per leaf ( FIG. 1 , Panel H). 
         [0034]    At the stage of bolting, younger leaves showed a slightly elongated and serrated leaf phenotype ( FIG. 1 , Panels I and J), resembling the phenotype observed for plants in which cell division is inhibited by ectopic expression of the CDK inhibitory KRP2 gene (De Veylder et al., 2001). In addition, the root growth rate of the mutant plants was significantly reduced, suggesting an inhibition of the cell cycle in etg1 mutant plants. 
         [0035]    To study the effect of loss of ETG1 function on cell cycle progression in more detail, we performed a leaf kinematic growth analysis. Kinematic analyses of leaf growth was performed as described by Boudolf et al. (2004) and De Veylder et al. (2001). Leaf growth of etg1 and Col-0 was analyzed on five plants from 5 to 22 DAS by measuring the total leaf blade area of all cells from the abaxial epidermis drawn with a drawing tube attached to the microscope, the total number of cells. The average cell area was determined from the number and total area of drawn cells, and the total number of cells per leaf was calculated by dividing the leaf area by the average cell area (averaged between the apical and basal positions). 
         [0036]    Finally, the average cell division rate for the whole leaf was determined as the slope of the log 2-transformed number of cells per leaf, which was done using five-point differentiation formulas (Erickson, 1976). Subsequently, seedlings were fixed in 100% ethanol overnight, replaced by lactic acid for microscopy. When wild-type and etg1-1 were compared, significant differences were observed ( FIG. 2 ). Leaf blade area was similar in the wild-type and etg1-1 plants during the whole period of leaf development ( FIG. 2 , Panel A). However, the average cell area, which initially was about 100 μm 2  in both plants, increased significantly faster in the etg1-1 mutant. The average surface area of etg1-1 cells was 155% of those of wild-type cells at maturity (3,880±320 versus 2,500±255 μm 2 , respectively;  FIG. 2 , Panel B). Simultaneously, the number of epidermal cells of etg1-1 was only about 60% of these of wild-type (6,650±530 versus 11,170±1,017 cells, respectively;  FIG. 2 , Panel C). 
         [0037]    Until nine days after sowing, the average cell division rate for the whole leaf, calculated on the basis of the increase of cell numbers over time, were constantly lower in the etg1-1 mutant than wild-type leaves ( FIG. 2 , Panel D). The average cell cycle duration time between days 5 and 9, estimated as the inverse of the cell division rate, was significantly longer in the etg1-1 mutant compared to wild-type plants (25.3 hours versus 21.1 hours, respectively). In summary, these data illustrate that ETG1-deficient plants suffer from a cell cycle delay, resulting in a reduction in total cell number. This reduction in cell number is offset by an increase in cell size, resulting into a nearly identical leaf size. 
       Example 2 
     Loss of ETG1 Function Causes a G2 Cell Cycle Arrest 
       [0038]    To pinpoint the cell cycle arrest point, we measured the ratio of 4C/2C cells by flow cytometry using eight-day-old leaves. As at this time point, leaf cells of both genotypes are dividing ( FIG. 2 , Panels C and D); consequently, 2C and 4C cells represent G1 and G2 cells, respectively. The flow cytometry was performed on plants grown in Petri dishes filled with 0.5×MS agar as described by Boudolf et al. (2004). Three biological and two technical replicates were used. By comparing the ploidy level of wild-type and etg1-1, a significant increase in the ratio of 4C/2C cells was observed in etg1-1 plants (0.79±0.04 versus 0.29±0.06 in etg1-1 and wild-type plants, respectively;  FIG. 3 , Panels A and B). These data indicate an inhibition of the G2-to-M transition in the etg1-1 mutant. 
         [0039]    As a confirmation, the expression levels of a number of cell cycle marker genes were analyzed by real-time RT-PCR. RNA was extracted from  Arabidopsis  tissues with RNEASY® Plant Mini Kit (Qiagen). First-strand cDNA was prepared from total RNA with the Superscript™ III First-Strand Synthesis System (Invitrogen) and according to the manufacturer&#39;s instructions. Quantitative PCR was performed with the LIGHTCYCLER® 480 SYBR Green I Master (Roche) with 100 nM primers and 0.1 μg of RT reaction product. Reactions were run and analyzed on the LIGHTCYCLER® 480 Real-Time PCR System (Roche) according to the manufacturer&#39;s instructions. All quantifications were normalized to ACTIN2 cDNA fragments amplified under the same conditions. Quantitative reactions were done in triplicate and averaged. Primers used were 5′-GGCTCCTCTTAACCCAAAGGC-3′ (SEQ ID NO:5) and 5′-CACACCATCACCAGAATCCAGC-3′ (SEQ ID NO: 6) for ACTIN2, 5′-TTGCAACCAGGCACCTTGAA-3′ (SEQ ID NO:7) and 5′-CAAATCGGCGGGCATTATGT-3′ (SEQ ID NO:8) for ETG1, 5′-TGGTGCTGGACATTTCAGTCGG-3′ (SEQ ID NO:9) and 5′-CAAGAGCTTGCACTTCCATCATAG-3′ (SEQ ID NO:10) for WEE1, 5′-CGAGGAAGGATCTCTTGCAG-3′ (SEQ ID NO:11) and 5′-GCACTAGTGAACCCCAGAGG-3′ (SEQ ID NO:12) for RAD51, 5′-CTCAAAATCCCACGCTTCTTGTGG-3′ (SEQ ID NO:13) and 5′-CACGTCTACTACCTTTGGTTTCCC-3′ (SEQ ID NO:14) for CYCB1;1, and 5′-CTCGAGATGGACGAAGAAGG-3′ (SEQ ID NO:15) and 5′-CGACGCAGAGTAATCGAACA-3′ (SEQ ID NO:16) for CDKB1;1. A substantial increase in the transcript level of the G2-to-M-phase-specific cyclin CYCB1;1 and CDKB1;1 genes were seen in the etg1-1 mutant ( FIG. 3 , Panel C). These data confirmed that the observed increase in the number of 4C cells is specifically due to a G2 arrest rather than a consequence of cells prematurely entering the endoreduplication cycle. 
       Example 3 
     ETG1 Transcript is Regulated by E2Fa and E2Fb Transcription Factors 
       [0040]    The ETG1 gene was originally identified by microarray analysis, comparing the transcriptome of wild-type  Arabidopsis  plants with that of plants ectopically expressing the heterodimeric E2Fa-DPa transcription, showing a strong up-regulation of ETG1 into the latter (Vandepoele et al., 2005). This induction was confirmed by quantitative real-time PCR analysis ( FIG. 4 , Panel C). To analyze whether the ETG1 gene is directly regulated by E2F transcription factors, we searched for the presence of consensus E2F binding sites in its putative promoter region. Two consensus E2F binding elements were found, ATTCCCGC (158 by upstream from the putative start codon) and TTTCCCGC (136 by upstream), both in a reverse orientation ( FIG. 4 , Panel A). 
         [0041]    To address whether ETG1 is an E2F target gene in vivo, we performed chromatin immunoprecipitation (ChIP) experiments. Immunoprecipitations were performed using anti-E2Fa, -E2Fb, -E2Fc, and DEL1 antibodies. ChIP was performed according to Bowler et al. (2004) with few modifications. Briefly, 1 g of eight-day-old seedlings was harvested, rinsed in ddH 2 O, and cross-linked in 1% formaldehyde for ten minutes. Cross-linking was stopped by addition of glycine to a final concentration of 0.125 M. Tissue was grinded and chromatin extracted. The chromatin solution was sonicated using a Branson 1200 sonifier. After preclearing, 10 μl of the appropriate antibodies was added to the chromatin solution and incubated overnight at 4° C. After collection of the immunoprecipitate with protein A agarose beads, beads were washed and immunocomplexes eluted. Cross-linking was reversed by incubation at 65° C. overnight. Proteinase K digestion was followed by phenol/chloroform extraction and ethanol precipitation. Recovered DNA was used in 25 cycle PCR reaction. 
         [0042]    As shown in  FIG. 4 , Panel B, ETG1 promoter sequences were not recovered from the immunoprecipitates with either anti-DEL1 or anti-E2Fc antibodies. However, promoter fragments of the ETG1 gene were specifically amplified from the anti-E2Fa and -E2Fb immunoprecipitates. These results indicate that E2Fa and E2Fb can bind directly to the ETG1 promoter in vivo, likely participating in the regulation of its expression. 
         [0043]    The regulation of the ETG1 promoter activity through its E2F consensus sites was further analyzed using transgenic plants expressing the β-glucuronidase (GUS) reporter gene under control of the ETG1 promoter. To define the contribution of each of the E2F binding sites, we deleted either one (ΔI or ΔII) or both (ΔI,II) of the E2F elements. The ETG1 promoter sequence was amplified from  Arabidopsis  genomic DNA by PCR with the FP-ETG1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATATGAAAACCTAATTCCTCTG-3′ (SEQ ID NO:17)) and RP-ETG1 (5′-GGGGACCACTTTGTACAAGAAAGCTGGGTCG GTCAGACAATCGTAAGCTGGT-3′ (SEQ ID NO:18)) primers. Each E2F element in the ETG1 promoter were mutated by two-step overlap extension PCR with FP-ETG1, RPETG1 primers and promoter-specific primers, FPΔI-ETG1 (5′-ATGGATAATGAACCTAGGAGATATG-3′ (SEQ ID NO:19)) and RPΔI-ETG1 (5′-CTCCTAGGTTCATTATCCATGCCCATTC-3′ (SEQ ID NO:20)) for mutation of the first E2F element (I), and FPΔII-ETG1 (5′-AGGAGATATGGGCCCAACTATACACACTTG-3′ (SEQ ID NO:21)) and RPΔII-ETG1 (5′-TAGTTGGGCCCATATCTCCTAGGTT-3′ (SEQ ID NO:22)) for mutation of the second E2F element (II), respectively ( FIG. 4 , Panel A). Both E2F elements in the ETG1 promoter were mutated by PCR using PCR fragments of ΔI-ETG1 promoter with FP-ETG1, RP-ETG1, FPΔII-ETG1 and RPΔII-ETG1 primers. 
         [0044]    Each PCR fragment was cloned into pDONR201 entry vector by BP recombination reaction and subsequently transferred into the pKGWFS7 destination vector (Karimi et al., 2002) by LR recombination reaction, resulting in a transcriptional fusion between the ETG1 promoter and the eGFP-GUS fusion gene. All constructs were transferred into the  Agrobacterium tumefaciens  C58C1RifR strain harboring the plasmid pMP90. The obtained  Agrobacterium  strains were used to generate stably transformed  Arabidopsis  with the floral dip transformation method (Clough and Bent, 1998). Seedlings were stained on multiwell plates (Falcon 3043; Becton Dickinson). GUS assays were performed as described by Beeckman and Engler (1994). Samples mounted in lactic acid were observed and photographed with a differential interference contrast microscope (Leica). 
         [0045]    More than five independent transgenic lines were analyzed per gene construct, all showing identical results. In six-day-old seedlings, high levels of ETG1 expression were observed in the shoot apical and root meristem ( FIG. 4 , Panel D). This expression pattern matches that one of the E2Fa and DPa genes (De Veylder et al., 2002). Deletion of either one of the E2F binding elements (ΔI or ΔII) led to GUS activity pattern identical to that of plants carrying the wild-type ETG1 promoter ( FIG. 4 , Panel D). In contrast, deletion of both E2F binding elements resulted into a drastic decrease in promoter activity ( FIG. 4 , Panel D). These results suggest that E2Fa/E2Fb bind both E2F consensus elements in the ETG1 promoter and regulates its expression in dividing tissues. 
       Example 4 
     ETG1 is Nuclear Protein Conserved in Eukaryotes 
       [0046]    The ETG1 gene encodes for a protein of 589 amino acid residues (At2g40550; genbank accession AAY25444). ETG1 is a singleton in  Arabidopsis . When searching for similar proteins by sequence comparison, no identification was found with any other functional annotated protein, neither a specific amino acid domain could be identified with the exception for a putative nuclear localization signal, PFKKMKV (SEQ ID NO:23) (amino-acids 184-190), suggesting that ETG1 resides in the nucleus. To investigate the subcellular localization of ETG1, an ETG1:eGFP (GFP for enhanced green fluorescent protein) fusion protein was transiently expressed in tobacco leaf epidermal cells. ETG1:eGFP fluorescence was observed in the nucleus only, illustrating that ETG1 is a nuclear protein ( FIG. 5 , Panel A). 
         [0047]    ETG1 orthologous proteins were found in rice ( Oryza sativa ; Os01g0166800), human (C10orf119), mouse (1110007A13Rik),  Xenopus  (CAJ81286),  Drosophila  (CG3430) and fission yeast (SPAC1687.04) (Supplemental  FIG. 2 ). In every case, a putative E2F consensus element could be identified in the corresponding putative promoter region, indicating that ETG1 is highly conserved E2F target gene. Interestingly, fission but no budding yeast ortholog could be identified. 
       Example 5 
     ETG1 is a Component of the Replisome Complex and Essential for DNA Replication 
       [0048]    Patterns of coexpression can reveal networks of functionally related genes and provide deeper understanding of processes requiring multiple gene products (Stuart et al., 2003; Wei et al., 2006). To predict ETG1 function, we searched for genes coexpressed with ETG1 using the ATTED-II coexpression database (Obayashi et al., 2007). This search revealed that ETG1 is highly coexpressed with genes encoding DNA replication proteins, such as minichromosome maintenance family proteins (MCM2, 3, 4, 5 and 7), proliferating cell nuclear antigen proteins (PCNA1 and 2), DNA primase small subunit protein, and DNA polymerase alpha subunits (Table 1). Moreover, when searching for proteins interacting with orthologous ETG1 proteins, using the BioGRID protein interactions database (http://www.thebiogrid.org/index.php), we found that the  Drosophila  ortholog CG3430 interacted with MCM5 protein. An identical interaction between the  Arabidopsis  ETG1 and MCM5 (At2g07690) proteins was demonstrated using the yeast two-hybrid system ( FIG. 5 , Panel B). Yeast two-hybrid bait and prey vectors were obtained through recombinational GATEWAY cloning (Invitrogen). The ETG1 and MCM5 open reading frames were recombined into the pDEST22 and pDEST32 vectors (Invitrogen) by an LR reaction, resulting in translational fusions between the open reading frames and the GAL4 transcriptional activation and GAL4 DNA binding domains, respectively. Plasmids encoding the baits and preys were transformed into the yeast strain PJ69-4-alfa (MATalpha; trp1-901, leu2-3,112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2::GAL1-HIS3, GAL2-ADE2, met2::GAL7-lacZ) and PJ69-4a (MATa; trp1-901, leu2-3,112, ura3-52, his3-200, gal4Δ, gal80Δ, LYS2TGAL1-HIS3, GAL2-ADE2, met2TGAL7-lacZ) by the LiAc method (Gietz et al., 1992), and plated on SD plates without Leu and on SD plates without Trp for two days at 30° C., respectively. Interactions between fusion proteins were assayed by mating method. Diploid strains were transferred to SD plates without Leu and Trp (as a control) and to SD plates without Leu, Trp, and His. Plates were incubated at 30° C. and scored for growth of yeast and, hence, protein-protein interaction after two days. 
         [0049]    The ETG1-MCM5 protein-protein interaction was confirmed in planta by BiFC experiments (Bracha-Drori et al., 2004; Walter et al., 2004). The full-length open reading frames of ETG1 and MCM5 with/without stop codon were amplified by PCR using F-ETG1 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGGGAGGACCAGCTTACGATT-3′ (SEQ ID NO:24)) and R-ETG1 (5′-GGGGACCACTTTGTACAAGAAAGCTGGG TCTTACTTGAGCCTCTCCTTTCTA-3′ (SEQ ID NO:25)) primers, and F-MCM5 (5′-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCATGTCAGGATGGGACGAAGGAG-3′ (SEQ ID NO:26)) and R-MCM5 (5′-GGGGACCACTTTGTACAAGAAAGCTG GGTCCTCAAGCTTTGCGGACAATAGAA-3′ (SEQ ID NO:27)) primers, respectively, and cloned into pDONR201 ENTRY vector by BP recombination reaction according to the manufacturer&#39;s instructions (Invitrogen). 
         [0050]    Full-length open reading frames of ETG1 and MCM5 were transferred into the pH7FWG2 destination vector (Karimi et al., 2002) by LR recombination reaction, resulting in ETG1:EGFP and MCM5:EYFP fusion proteins, respectively. BiFC assay was performed as described by Walter et al. (2004). The coding region of ETG1 was amplified with 5′-GCCACTAGTGGATCCATGGGAGGACCAGCTTACGATT-3′ (SEQ ID NO:28) and 5′-AGCGGTACCCTCGAGGTACTTGAGCCTCTCCTTTCTA-3′ (SEQ ID NO:29) primers, and cloned via BamHI-XhoI into the plasmid pUC-SPYNE (Walter et al., 2004), resulting in the plasmid expressed as the ETG1:YFPN fusion protein; the coding region of MCM5 was also obtained with 5′-TGGCGCGCCACTAGTATGTCAGGATGGGACGAAGGAG-3′ (SEQ ID NO:30) and 5′-ACCCTCGAGGTCGACGTAAGCTTTGCGGACAATAGAA-3′ (SEQ ID NO:31) primers, and cloned via SpeI-SalI into the plasmid pUC-SPYCE (Walter et al., 2004) to give rise to the plasmid expressed as the MCM5:YFPC fusion protein. 
         [0051]    The  Agrobacterium  strains containing both BiFC constructs were co-infiltrated into tobacco leaves. Epidermal cell layer of tobacco leaves were assayed for fluorescence using confocal laser scanning microscope (Zeiss) two to four days after infiltration. After transfection, YFP fluorescence was observed in the nuclei of cells transfected with the ETG1-YFPN and MCM5-YFPC fusion proteins, demonstrating that the ETG1 protein interacted with MCM5 in the plant nucleus ( FIG. 5 , Panel C; ETG1-YFPN and MCM5-YFPC). As expected, no fluorescence was detected when any combination with empty vectors was introduced into tobacco cells ( FIG. 5 , Panel C; ETG1-YFPN and YFPC, YFPN and MCM5-YFPC). When examining the subcellular localization of MCM5 in plants, with the use of an MCM5:eGFP fusion protein, MCM5:eGFP was found to reside in both the nucleus and cytoplasm ( FIG. 5 , Panel D). 
         [0052]    To identify additional ETG1-associated proteins, tandem affinity purification (TAP) in combination with MALDI-TOF-TOF-MS based protein identification was performed. TAP experiments were done according to Van Leene et al. (2007). In short, the ETG1-coding sequence was cloned by recombination into the pKNTAP vector generating a Pro35S:TAP-ETG1 cassette (pKCETG1TAP).  Arabidopsis  cell suspension cultures were stably transformed by  Agrobacterium -mediated co-cultivation with pKNETG1TAP. Transformed  Arabidopsis  cells were selected and transferred to liquid medium for upscaling. Expression levels of TAP-tagged proteins were checked by protein blotting with an anti-CBP antibody. In a first round of affinity purification, protein extracts of 15 g plant material were incubated with an IgG resin. Bound complexes were released and eluted from the resin by tag cleavage with TEV protease. 
         [0053]    In a second affinity step on a calmodulin agarose column, co-eluting non-interacting proteins and the TEV protease were removed with the flow-through. Finally, both the ETG1 bait and interacting proteins were eluted from the calmodulin agarose via EGTA-mediated removal of calcium. Eluted proteins were separated on 4-12% NuPAGE gels, excised and analyzed by Maldi-TOF/TOF MS as described (Van Leene et al., 2007). To increase the stringency of the data set, contaminating proteins due to experimental background as determined by Van Leene et al. (2007) were systematically subtracted from the lists of co-purified proteins. Next to MCM5, we identified five interacting proteins, including other components of the MCM complex, being MCM2 (At1g44900), MCM3 (At5g46280), MCM4 (At2g16440), MCM6 (At5g44635) and MCM1 (At4g02060) (Table 2). 
         [0054]    Combined with the subcellular localization results, these data indicated that ETG1 assembles into the replisome complex. Therefore, ETG1 depletion is expected to affect the efficiency of DNA replication. To test this hypothesis, a bromodeoxyuridine (BrdU) pulse-labeling experiment was performed. Three-day-old seedlings grown on MS agar plates were incubated in the labeling solution containing 10 μM bromodeoxyuridine (BrdU) (Roche) at room temperature for various time points, and the genomic DNA was extracted with a DNEASY® Plant Mini Kit (QIAGEN). The amounts of BrdU were determined by ELISA using Anti-BrdU-POD antibody (Roche). Three biological and two technical replicates were used at each time point for ELISA. Fifty microliters of the extracted DNA (0.2 μg/ml) was placed in each well. The ELISA procedure was chiefly that of 5-Bromo-2′-deoxy-uridine Labeling and Detection Kit III (Roche). In wild-type, the level of BrdU incorporation into cells gradually increased to a saturation level by three hours ( FIG. 5 , Panel E). By contrast, the rate of BrdU incorporation was lower in etg1-1 plants, illustrating the need for ETG1 for efficient DNA replication. 
         [0000]    
       
         
               
             
               
               
               
               
             
               
               
               
               
             
           
               
                 TABLE 1 
               
             
             
               
                   
               
               
                 Genes coexpressed with ETG1 gene. 
               
             
          
           
               
                 Rank 
                 Cor. 
                 Locus 
                 Gene description 
               
               
                   
               
             
          
           
               
                 1 
                 0.92 
                 At5g46280 
                 Minichromosome maintenance family 
               
               
                   
                   
                   
                 protein 3 (MCM3) 
               
               
                 2 
                 0.90 
                 At1g44900 
                 Minichromosome maintenance family 
               
               
                   
                   
                   
                 protein 2 (MCM2) 
               
               
                 3 
                 0.89 
                 At4g02060 
                 Prolifera protein (PRL)/Minichromosome 
               
               
                   
                   
                   
                 maintenance family protein 7 (MCM7) 
               
               
                 4 
                 0.89 
                 At5g41880 
                 DNA primase small subunit 
               
               
                 5 
                 0.88 
                 At1g07370 
                 Proliferating cell nuclear antigen 1 (PCNA1) 
               
               
                 6 
                 0.88 
                 At2g16440 
                 Minichromosome maintenance family 
               
               
                   
                   
                   
                 protein 4 (MCM4) 
               
               
                 7 
                 0.87 
                 At2g07690 
                 Minichromosome maintenance family 
               
               
                   
                   
                   
                 protein 5 (MCM5) 
               
               
                 8 
                 0.84 
                 At1g67630 
                 DNA polymerase alpha subunit B 
               
               
                 9 
                 0.84 
                 At5g67100 
                 DNA polymerase alpha catalytic subunit 
               
               
                 10 
                 0.83 
                 At2g29570 
                 Proliferating cell nuclear antigen 2 (PCNA2) 
               
               
                   
               
               
                 Top 10 ranking genes identified by ATTED-II coexpression database. 
               
               
                 cor.: correlation coefficient 
               
             
          
         
       
     
         [0000]    
       
         
               
             
               
               
               
               
               
               
               
             
               
               
               
               
               
               
               
             
           
               
                 TABLE 2 
               
             
             
               
                   
               
               
                 List of ETG1-copurified proteins identified by Mass Spectrometry 
               
             
          
           
               
                   
                   
                 Protein 
                 Peptide 
                 Sequence 
                 Protein score/ 
                 Best ion score/ 
               
               
                 Locus 1   
                 Gene description 
                 MW 
                 count 
                 coverage % 
                 threshold 
                 threshold 
               
               
                   
               
             
          
           
               
                 At1g44900 
                 Minichromosome 
                 105172 
                 33 
                 43 
                 936/61 
                 104/33 
               
               
                   
                 maintenance 
               
               
                   
                 family protein 2 
               
               
                   
                 (MCM2) 
               
               
                 At5g46280 
                 Minichromosome 
                 86759 
                 14 
                 23 
                 206/61 
                  74/31 
               
               
                   
                 maintenance 
               
               
                   
                 family protein 3 
               
               
                   
                 (MCM3) 
               
               
                 At2g16440 
                 Minichromosome 
                 94168 
                 24 
                 38 
                 696/61 
                 102/28 
               
               
                   
                 maintenance 
               
               
                   
                 family protein 4 
               
               
                   
                 (MCM4) 
               
               
                 At2g07690 
                 Minichromosome 
                 81591 
                 26 
                 38 
                 584/61 
                 107/31 
               
               
                   
                 maintenance 
               
               
                   
                 family protein 5 
               
               
                   
                 (MCM5) 
               
               
                 At5g44635 
                 Minichromosome 
                 93478 
                 20 
                 33 
                 859/61 
                 154/30 
               
               
                   
                 maintenance 
               
               
                   
                 family protein 6 
               
               
                   
                 (MCM6) 
               
               
                 At4g02060 
                 Prolifera protein 
                 80739 
                 26 
                 46 
                 987/61 
                 172/31 
               
               
                   
                 (PRL)/Minichromo- 
               
               
                   
                 some maintenance 
               
               
                   
                 family protein 7 
               
               
                   
                 (MCM7) 
               
               
                   
               
               
                   1 All proteins were detected in the two independent TAP experiments. 
               
             
          
         
       
     
       Example 6 
     ETG1-Deficient Plants Activate the G2 DNA Replication Checkpoints 
       [0055]    Inhibition of DNA replication in  Arabidopsis  results into the simultaneous induction of DNA repair genes and the cell cycle inhibitory WEE1 gene, which arrests cells in the G2 phase of the cell cycle (De Schutter et al., 2007). The decreased rate of DNA replication and observed interaction of ETG1 with replication proteins suggested that the G2-arrest noticed in ETG1-deficient plants might be the consequence of activation of the replication checkpoint. To test this hypothesis, we compared the expression levels of the RAD51 (DNA repair) and WEE1 (cell cycle checkpoint) marker genes by real-time RT-PCR in wild-type versus etg1 mutant plants. Ionizing radiation (γ-irradiation and UV) and radiomimetic drugs (HU, aphidicolin and bleomycin) are known to induce RAD51 and WEE1 expression (Chen et al., 2003; De Schutter et al., 2007). Expression of both RAD51 and WEE1 was significantly up-regulated in the etg1-1 seedlings ( FIG. 6 , Panel A). A similar expression profile was observed in etg1-2 seedlings. 
         [0056]    Activation of the DNA stress checkpoint was confirmed by using plants that carried as transgene the poly(ADP-ribose) polymerase 2 (PARP2) and WEE1 promoter fused to GUS, being markers for DNA stress and activation of the G2 replication checkpoint, respectively (Babiychuk et al., 1998; Doucet-Chabeaud et al., 2001; De Schutter et al., 2007). As shown in  FIG. 6 , Panel B, no GUS activity was observed in PARP2::GUS plants grown under non-stress conditions. By contrast, treatment of the PARP2: GUS reporter line with bleomycin resulted a strong induction of GUS activity ( FIG. 6 , Panel D), demonstrating DNA stress-inducible promoter activity. Similarly, PARP2 promoter activity was induced in a etg1-1 background in the absence of any external DNA stress stimulus ( FIG. 6 , Panel C). Especially, GUS activity was strongly induced in shoot apical meristem and vascular cells. Analogous results were obtained using WEE1::GUS reporter plants. In control plants, WEE1 expression was observed in the shoot apex and vascular cells ( FIG. 6 , Panel E; De Schutter et al., 2007). This expression pattern was intensified in the etg1-1 background ( FIG. 6 , Panel F), confirming the real-time RT-PCR experiments. 
         [0057]    DNA replication stress caused by blocking of the replication fork is mainly sensed by the ATR kinase (Culligan et al., 2004). Previously, we have demonstrated that WEE1 is one of the main targets of the ATR signaling cascade. WEE1 transiently arrests cells in the G2 phase, allowing them to finalize DNA replication before proceeding into mitosis (De Schutter et al., 2007). When assuming that the increased cell cycle duration time observed in the etg1 mutant plants is the result of the activation of the replication checkpoint, it is expected that ETG1-deficiency should have a dramatic impact on the development of plants that are unable to arrest their cell cycle in response to DNA stress. To test this hypothesis, double mutants were constructed between etg1-1 and two DNA stress checkpoint mutants, atr-2 and wee1-1. The atr-2 and wee1-1 mutants have been described previously (Culligan et al., 2004; De Schutter et al., 2007). atr-2 and wee1-1 single mutants are hypersensitive to replication-blocking or DNA damaging drugs plants, but are viable and develop normally in the absence of exogenous DNA-stress treatments ( FIG. 7 , Panels A, C, and D) (Culligan et al., 2004; De Schutter et al., 2007). By contrast, etg1-1/wee1-1 and etg1-1/atr-2 double mutant plants showed a dwarf phenotype under non-stress conditions, illustrating a synthetic interaction between ETG1, and WEE1 or ATR ( FIG. 7 , Panels E-H). 
         [0058]    Scanning electron microscopy revealed severe growth suppression ( FIG. 7 , Panels I-K). Especially, the size of trichomes was reduced in the double mutants ( FIG. 7 , Panels O-Q). No significant difference in leaf epidermal cell shape was observed in etg1-1/wee1-1 double mutants, whereas cells lost their jigsaw-like shape in etg1-1/atr-2 plants ( FIG. 7 , Panels L-N). The double mutants arrested at an early stage of development, indicating a cell cycle arrest. These results unequivocally illustrate that the activation of the DNA replication checkpoint in etg1 mutant plants is essential for their survival. 
       Example 7 
     Up-Regulation of Mitotic-Specific Genes in etg1 
       [0059]    To gain more insight into cell cycle effect in etg1 mutants, we examined transcript levels of 24,000 genes by using Affymetrix ATH1 GeneChip arrays. Triplicate batches of first leaf pairs of nine-day-old wild-type and etg1 plants were harvested for total RNA preparation. The statistical analysis identified a total of 220 differentially expressed genes between wild-type and etg1 at P&lt;0.01, among which 89% were up-regulated and 11% were down-regulated with fold change expression ranging from 1.3 to 14.8 and 0.15 to 0.75, respectively (Tables 3, 4). Interestingly, among the 196 up-regulated genes in etg1, 103 genes (52%) express with a peak in mitosis. Transcription of genes expressed specifically during mitosis is regulated by common upstream cis-acting element, called MSA (mitosis-specific activator; CAACGG). When we checked MSA element in promoter region of up-regulated genes in etg1, significant enrichment of MSA elements was detected. We also calculated the timing of maximal expression of up-regulated genes during the cell cycle. Genes expressed during M phase were clearly more strongly induced than those expressed during the S, G2, and G1 phases ( FIG. 8 , Panel B). To characterize biological processes, the up- and down-regulated genes were analyzed for gene ontology (G0) enrichment (Maere et al., 2005). Among the up-regulated genes in etg1, regulation of progression through cell cycle, mitotic cell cycle, and microtubule-based movement genes were significantly overrepresented ( FIG. 8 , Panel A), indicating that ETG1 is required for proper mitotic cell division. This is surprising, because ETG1 is expressed during S, and its gene product is required for DNA replication. Additionally, we have demonstrated in the previous examples that ETG1-deficient plants suffer from DNA stress, which is expected to result in a cell cycle arrest during S or early G2. We compared the microarray data of etg1 with gene expression changes observed in response to UV-B light and bleomycin treatment, both known to cause DNA breaks (Kilian et al., 2007; Molinier et al., 2005). Surprisingly, the response of cell cycle regulated genes was totally different in the etg1 mutant compared to the other treatments that cause DNA stress. In the latter cases, the set of modified genes was clearly enriched for S phase genes ( FIG. 8 , Panel C). These results suggest that in the etg1 mutant, different checkpoints pathways are activated to suppress cell cycle progression caused by DNA replication defects. 
       Example 8 
     ETG1 is Required for Sister Chromatid Cohesion 
       [0060]    Sister chromatid cohesion is apparently established during DNA replication in S phase and maintained until anaphase onset. To assess the role of ETG1 in the establishment and maintenance of cohesion, we performed FISH analysis, as described by Schubert et al. (2008). To analyze sister chromatid cohesion in interphase nuclei, individual BACs (BAC T2P11/T7N9 and F11P17) from different positions along chromosomes 1 were hybridized to flow-sorted 4C leaf nuclei. The number of FISH signals was taken as a measure for sister chromatid separation. One FISH signal ( FIG. 9 , Panel B; pairing of both homologs) or two FISH signals ( FIG. 9 , Panels C and D) per BAC (T2P11/T7N9) were regarded as positional alignment at corresponding regions, indicating sister chromatids are aligned. Three ( FIG. 9 , Panel E) or four signals ( FIG. 9 , Panel F) were considered to indicate sister chromatid separation. Positional sister chromatid separation occurred in 28.1-32.8% of 4C nuclei in wild-type leaf, whereas etg1 leaf nuclei has 42.7-44.7% of sister chromatid separation ( FIG. 9 , Panel A). By comparing the DNA masses contained three or four FISH signals of wild-type and etg1 when using the BAC T2P11/T7N9, a significant increase in sister chromatid separation was observed in etg1 leaf nuclei ( FIG. 9 , Panel G). These findings reveal that ETG1 protein is required for establishment of sister chromatid cohesion. 
         [0000]    
       
         
               
             
               
               
             
               
               
               
               
               
               
             
           
               
                 TABLE 3 
               
             
             
               
                   
               
               
                 Up-regulated genes in etg1 compared with wild-type 
               
             
          
           
               
                 Fold change 
                   
               
             
          
           
               
                 etg1-1 
                 etg1-2 
                 Gene code 
                 Gene description 
                 Phase 
                 GO category 
               
               
                   
               
               
                 6.91 
                 5.92 
                 At5g60250 
                 Zinc finger (C3HC4-type ring 
                   
                 Biological process unknown 
               
               
                   
                   
                   
                 finger) family protein 
               
               
                 4.19 
                 3.99 
                 At5g23910 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 3.51 
                 3.55 
                 At4g37490 
                 CyclinB1; 1 (CycB1; 1) 
                   
                 Regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 3.39 
                 3.15 
                 At4g02390 
                 Poly(ADP-ribose) polymerase 
                   
                 Protein amino acid ADP- 
               
               
                   
                   
                   
                 (APP) 
                   
                 ribosylation 
               
               
                 3.35 
                 3.20 
                 At5g61070 
                 Histone deacetylase 18 (HDA18) 
                   
                 Chromatin remodeling 
               
               
                 3.32 
                 3.39 
                 At3g44050 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 3.07 
                 3.59 
                 At3g02120 
                 hydroxyproline-rich glycoprotein 
                 M 
                 Biological process unknown 
               
               
                   
                   
                   
                 family protein 
               
               
                 3.05 
                 3.25 
                 At5g45700 
                 NLI interacting factor (NIF) family 
                 M 
               
               
                   
                   
                   
                 protein 
               
               
                 2.89 
                 2.66 
                 At3g27060 
                 TSO2 
                   
                 DNA repair, DNA replication, 
               
               
                   
                   
                   
                   
                   
                 regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 2.80 
                 2.56 
                 At3g23890 
                 TOPII (TOPOISOMERASE II) 
                 M 
                 DNA metabolic process 
               
               
                 2.79 
                 2.65 
                 At5g51600 
                 PLE (PLEIADE) 
                 M 
                 Cytokinesis by cell plate 
               
               
                   
                   
                   
                   
                   
                 formation 
               
               
                 2.78 
                 2.95 
                 At4g35620 
                 CYCB2; 2 (Cyclin B2; 2) 
                 M 
                 Regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 2.75 
                 2.74 
                 At1g76540 
                 CDKB2; 1 (Cyclin-dependent 
                 M 
                 G2/M transition of mitotic cell 
               
               
                   
                   
                   
                 kinase B2; 1) 
                   
                 cycle 
               
               
                 2.68 
                 2.59 
                 At3g51740 
                 IMK2 (Inflorescence meristem 
                 M 
                 Protein amino acid 
               
               
                   
                   
                   
                 receptor-like kinase 2) 
                   
                 phosphorylation 
               
               
                 2.68 
                 2.47 
                 At5g55520 
                 Kinesin related protein 
                 M 
                 Biological process unknown 
               
               
                 2.66 
                 2.94 
                 At3g22880 
                 ATDMC1 (RECA-LIKE GENE) 
                   
                 Meiosis 
               
               
                 2.65 
                 2.54 
                 At3g51280 
                 Male sterility MS5, putative 
                 M 
               
               
                 2.65 
                 2.47 
                 At4g05520 
                 Calcium-binding EF hand family 
                 M 
               
               
                   
                   
                   
                 protein 
               
               
                 2.61 
                 2.58 
                 At5g56580 
                 ATMKK6 ( Arabidopsis  NQK1) 
                   
                 Protein amino acid 
               
               
                   
                   
                   
                   
                   
                 phosphorylation 
               
               
                 2.60 
                 2.47 
                 At2g25880 
                 ATAUR2 (ATAURORA2) 
                 M 
                 Histone phosphorylation 
               
               
                 2.56 
                 2.71 
                 At2g27970 
                 CKS2 (CDK-subunit 2) 
                   
                 Cell cycle 
               
               
                 2.55 
                 2.66 
                 At4g05190 
                 ATK5 ( Arabidopsis thaliana   
                 M 
                 Microtubule cytoskeleton 
               
               
                   
                   
                   
                 kinesin 5) 
                   
                 organization and biogenesis, 
               
               
                   
                   
                   
                   
                   
                 spindle assembly 
               
               
                 2.53 
                 2.76 
                 At2g30360 
                 CIPK11 
                   
                 Protein amino acid 
               
               
                   
                   
                   
                   
                   
                 phosphorylation, signal 
               
               
                   
                   
                   
                   
                   
                 transduction 
               
               
                 2.53 
                 2.57 
                 At5g11510 
                 MYB3R-4 (c-myb-like transcription 
                 M 
                 Cell cycle, regulation of 
               
               
                   
                   
                   
                 factor 3R-4) 
                   
                 transcription 
               
               
                 2.52 
                 2.49 
                 At4g01730 
                 Zinc finger (DHHC type) family 
                 M 
                 Biological process unknown 
               
               
                   
                   
                   
                 protein 
               
               
                 2.51 
                 2.51 
                 At1g76310 
                 CYCB2; 4 (CYCLIN B2; 4) 
                 M 
                 Regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 2.51 
                 2.87 
                 At5g55180 
                 Glycosyl hydrolase family 17 
                 M 
                 Carbohydrate metabolic 
               
               
                   
                   
                   
                 protein 
                   
                 process 
               
               
                 2.47 
                 2.60 
                 At1g08560 
                 KN (KNOLLE) 
                 M 
                 Intracellular protein transport 
               
               
                 2.47 
                 2.58 
                 At4g32830 
                 ATAUR1 (ATAURORA1) 
                 M 
                 Histone phosphorylation 
               
               
                 2.46 
                 2.58 
                 At1g20930 
                 CDKB2; 2 (CYCLIN-DEPENDENT 
                 M 
                 M phase of mitotic cell cycle 
               
               
                   
                   
                   
                 KINASE B2; 2) 
               
               
                 2.44 
                 2.61 
                 At2g26760 
                 CYCB1; 4 
                 M 
                 Regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 2.44 
                 2.30 
                 At5g60930 
                 Chromosome-associated kinesin 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.43 
                 2.57 
                 At1g18370 
                 HINKEL (HIK) 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.43 
                 2.74 
                 At2g37420 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.41 
                 2.71 
                 At2g17620 
                 CYCB2; 1 (CYCLIN B2; 1) 
                 M 
                 Regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 2.37 
                 2.60 
                 At3g25980 
                 Mitotic spindle checkpoint protein, 
                 M 
                 Mitotic cell cycle spindle 
               
               
                   
                   
                   
                 putative (MAD2) 
                   
                 assembly checkpoint 
               
               
                 2.36 
                 2.29 
                 At3g23670 
                 PAKRP1L 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.36 
                 2.46 
                 At1g28290 
                 Pollen Ole e 1 allergen and 
                 M 
                 Biological process unknown 
               
               
                   
                   
                   
                 extensin family protein 
               
               
                 2.35 
                 2.32 
                 At1g44110 
                 CYCA1; 1 (CYCLIN A1; 1); 
                 M 
                 Regulation of progression 
               
               
                   
                   
                   
                   
                   
                 through cell cycle 
               
               
                 2.33 
                 2.22 
                 At3g06030 
                   Arabidopsis  NPK1-related protein 
                   
                 Protein amino acid 
               
               
                   
                   
                   
                 kinase 3 (ANP3) 
                   
                 phosphorylation 
               
               
                 2.32 
                 1.89 
                 At3g60840 
                 Microtubule associated protein 
                 M 
               
               
                   
                   
                   
                 (MAP65/ASE1) family protein 
               
               
                 2.30 
                 2.53 
                 At3g20150 
                 Kinesin motor family protein 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.29 
                 2.34 
                 At5g13840 
                 ccs52B 
                 M 
                 Signal transduction 
               
               
                 2.29 
                 2.71 
                 At1g34355 
                 Forkhead-associated domain- 
                 M 
                 Biological process unknown 
               
               
                   
                   
                   
                 containing protein 
               
               
                 2.27 
                 2.10 
                 At1g02690 
                 Importin alpha-2 subunit, putative 
                 M 
                 Intracellular protein transport 
               
               
                 2.28 
                 2.29 
                 At1g02730 
                 Cellulose synthase-like D5 
                 M 
                 cellulose synthase activity 
               
               
                   
                   
                   
                 (ATCSLD5) 
               
               
                 2.28 
                 2.30 
                 At5g66230 
                 Similar to sugar transporter 
                 M 
                 Biological process unknown 
               
               
                   
                   
                   
                 superfamily 
               
               
                 2.26 
                 2.23 
                 At1g50240 
                 FUSED (FU) 
                   
                 Cellularization of the embryo 
               
               
                   
                   
                   
                   
                   
                 sac, cytokinesis by cell plate 
               
               
                   
                   
                   
                   
                   
                 formation 
               
               
                 2.26 
                 2.00 
                 At1g63100 
                 SCARECROW transcription factor 
                 M 
                 Regulation of transcription 
               
               
                 2.26 
                 2.18 
                 At1g18250 
                   Arabidopsis  thaumatin-like protein 
                 M 
                 Response to other organism 
               
               
                   
                   
                   
                 1 (ATLP-1) 
               
               
                 2.25 
                 2.08 
                 At1g72250 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.24 
                 2.13 
                 At4g31805 
                 WRKY family transcription factor 
                   
                 Regulation of transcription 
               
               
                 2.24 
                 2.04 
                 At1g03780 
                 Targeting protein-related 
                 M 
                 Biological process unknown 
               
               
                 2.23 
                 2.40 
                 At5g67270 
                 Microtuble-end-binding protein 1 
                 M 
                 Cortical cytoskeleton 
               
               
                   
                   
                   
                 (ATEB1C) 
                   
                 organization and biogenesis 
               
               
                 2.23 
                 2.91 
                 At2g18600 
                 RUB1-conjugating enzyme, 
                   
                 Protein modification process, 
               
               
                   
                   
                   
                 putative 
                   
                 ubiquitin cycle 
               
               
                 2.21 
                 1.99 
                 At3g57860 
                 UVB-insensitive 4-like 
                   
                 Biological process unknown 
               
               
                 2.12 
                 2.06 
                 At1g50490 
                 Ubiquitin-conjugating enzyme 20 
                 M 
                 Cell proliferation 
               
               
                   
                   
                   
                 (UBC20) 
               
               
                 2.12 
                 1.87 
                 At3g17360 
                 Phragmoplast orienting kinesin 1 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                 (POK1) 
                   
                 movement 
               
               
                 2.10 
                 2.32 
                 At5g47500 
                 Pectinesterase family protein 
                 M 
                 Cell wall modification 
               
               
                 2.10 
                 2.14 
                 At2g28620 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.09 
                 1.96 
                 At2g22610 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 2.07 
                 2.03 
                 At4g21820 
                 Calmodulin-binding family protein 
                 M 
               
               
                 2.07 
                 1.98 
                 At1g66620 
                 Seven in absentia (SINA) protein, 
                   
                 Multicellular organismal 
               
               
                   
                   
                   
                 putative 
                   
                 development, ubiquitin- 
               
               
                   
                   
                   
                   
                   
                 dependent protein catabolic 
               
               
                   
                   
                   
                   
                   
                 process 
               
               
                 2.07 
                 1.90 
                 At1g23000 
                 Heavy-metal-associated domain- 
                   
                 Metal ion transport 
               
               
                   
                   
                   
                 containing protein 
               
               
                 2.07 
                 2.33 
                 At4g11080 
                 High mobility group (HMG1/2) 
                 M 
                 Regulation of transcription 
               
               
                   
                   
                   
                 family protein 
               
               
                 2.05 
                 1.95 
                 At1g73620 
                 Thaumatin-like protein, putative 
                   
                 Response to other organism 
               
               
                 2.03 
                 2.21 
                 At1g78430 
                 Tropomyosin-related 
                   
                 Biological process unknown 
               
               
                 2.03 
                 1.87 
                 At2g33560 
                 Spindle checkpoint protein-related 
                 M 
                 Biological process unknown 
               
               
                 2.02 
                 2.05 
                 At3g55660 
                 ATROPGEF6/ROPGEF6 (Kinase 
                 M 
                 Biological process unknown 
               
               
                   
                   
                   
                 partner protein-like) 
               
               
                 2.00 
                 1.79 
                 At4g14330 
                 Phragmoplast-associated kinesin- 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                 related protein 2 (PAKRP2) 
                   
                 movement 
               
               
                 1.99 
                 1.78 
                 At5g40840 
                 Sister chromatid cohesion 1 
                   
                 Mitosis 
               
               
                   
                   
                   
                 (SCC1) protein homolog 2 (SYN2) 
               
               
                 1.98 
                 1.74 
                 At5g27550 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 1.96 
                 2.04 
                 At1g10780 
                 F-box family protein 
                 M 
                 Biological process unknown 
               
               
                 1.94 
                 2.16 
                 At1g59540 
                 Kinesin-like protein 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 1.92 
                 1.99 
                 At1g53140 
                 Dynamin family protein 
                 M 
                 Biological process unknown 
               
               
                 1.92 
                 1.67 
                 At3g10310 
                 Kinesin motor protein-related 
                 M 
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 1.91 
                 1.77 
                 At5g03780 
                 TRF-like 10 (TRFL10) 
                   
                 Response to salicylic acid 
               
               
                   
                   
                   
                   
                   
                 stimulus 
               
               
                 1.90 
                 1.69 
                 At2g36200 
                 Kinesin motor protein-related 
                   
                 Microtubule-based 
               
               
                   
                   
                   
                   
                   
                 movement 
               
               
                 1.90 
                 1.65 
                 At1g57820 
                 Variant in methylation 1 (VIM1) 
                   
                 Regulation of transcription, 
               
               
                   
                   
                   
                   
                   
                 DNA-dependent, centric 
               
               
                   
                   
                   
                   
                   
                 heterochromatin formation, 
               
               
                   
                   
                   
                   
                   
                 DNA methylation on cytosine 
               
               
                 1.89 
                 1.83 
                 At4g03100 
                 Rac GTPase activating protein, 
                 G2 
                 Signal transduction 
               
               
                   
                   
                   
                 putative 
               
               
                 1.87 
                 1.79 
                 At3g27330 
                 Zinc finger (C3HC4-type RING 
                 M 
                 Protein ubiquitination 
               
               
                   
                   
                   
                 finger) family protein 
               
               
                 1.87 
                 1.90 
                 At4g33400 
                 Defective embryo and meristems 
                 M 
                 N-terminal protein 
               
               
                   
                   
                   
                 protein-related (DEM) 
                   
                 myristoylation 
               
               
                 1.86 
                 1.98 
                 At4g21270 
                 ATK1 ( Arabidopsis thaliana   
                   
                 Anastral spindle assembly 
               
               
                   
                   
                   
                 kinesin 1) 
                   
                 involved in male meiosis 
               
               
                 1.85 
                 1.83 
                 At4g28950 
                 ARAC7/ATROP9/RAC7/ROP9 
                 S 
                 Small GTPase mediated 
               
               
                   
                   
                   
                   
                   
                 signal transduction 
               
               
                 1.85 
                 2.08 
                 At2g25060 
                 Plastocyanin-like domain- 
                 M 
                 Electron transport 
               
               
                   
                   
                   
                 containing protein 
               
               
                 1.84 
                 1.97 
                 At4g31840 
                 Plastocyanin-like domain- 
                 M 
                 Electron transport 
               
               
                   
                   
                   
                 containing protein 
               
               
                 1.84 
                 1.87 
                 At5g03870 
                 Glutaredoxin family protein 
                 M 
                 N-terminal protein 
               
               
                   
                   
                   
                   
                   
                 myristoylation 
               
               
                 1.84 
                 2.06 
                 At2g07170 
                 Similar to  TORTIFOLIA  1 (TOR1) 
                 M 
                 Biological process unknown 
               
               
                 1.82 
                 1.73 
                 At5g63920 
                 DNA topoisomerase III alpha, 
                   
                 DNA topological change, 
               
               
                   
                   
                   
                 putative 
                   
                 DNA unwinding during 
               
               
                   
                   
                   
                   
                   
                 replication 
               
               
                 1.80 
                 2.15 
                 At5g55830 
                 Lectin protein kinase, putative 
                 M 
                 Protein amino acid 
               
               
                   
                   
                   
                   
                   
                 phosphorylation 
               
               
                   
               
             
          
         
       
     
       Example 9 
     Constitutive Knock-Down and Overexpression of hETG1 Causes Substantial Changes in Cellular Morphology 
       [0061]    The human ETG1 cDNA (hETG1) was PCR amplified from the 2961492 clone (openbiosystem) using PFU turbo (Stratagene) and the following primers 5′-CACCATGCCGTGTGGGGAGG-3 (SEQ ID NO:32) and 5′-TCTAGAAAGTTCATTTCCATTCACACATTT-3′ (SEQ ID NO:33); following the manufacturer&#39;s instruction. The PCR product was ligated in the pENTR/D/Topo vector (InVitrogen) according to the manufacturer instruction. 
         [0062]    To study the role of human hETG1, transgenic MCF7 cell cultures were generated that either overexpress or silence the hETG1 gene. MCF-7 cell lines were obtained from the American Cell Type Culture Collection (Rockville, Md.) and maintained in DMEM supplemented with 5% FCS, 5% newborn bovine serum, 2 mmol/L-glutamine, 0.4 mmol/L sodium pyruvate, 100 units/mL penicillin and 100 μg/mL streptomycin and in DMEM supplemented with 5% FCS, 2 mmol/L L-glutamine, 0.4 mmol/L sodium pyruvate, 100 units/mL penicillin, 100 μg/mL streptomycin, 6 ng/ml bovine insulin (Sigma-Aldrich, St Louis, Mo.), respectively. All recombinant lentiviruses were produced by transient transfection of HEK293T cells according to standard protocols. Briefly, 0.6 million cells of the packaging cell line HEK293T were seeded in two wells of six-well plates. After 24 hours, 3 μg of the lentiviral knock-down vector pGIZ V2HS-158067 purchased from Open-Biosystems (Huntsville, Ala.), 3 μg of the packaging plasmid pCMV-ΔR8.91 (Zufferey et al, 1997), and 1.5 μg of the envelope plasmid pMD2G-VSVG (Zufferey et al, 1997) were first ethanol-precipitated together and then transfected in the presence of chloroquine (25 μM) into the HEK293T cells using the calcium phosphate precipitation method. 
         [0063]    Transduction of the MCF7 cells was performed in triplicate by resuspending 25,000 cells with 200 ml viral supernatant and plating them in a 96-well plate. The plate was centrifuged for 1.5 hours at 32° C. and 1500 rpm and incubated at 37° C. in a water-saturated incubator under a 5% O 2 /95% CO 2  atmosphere. After 96 hours, the cells were trypsinized, pooled and amplified. Transduction efficiencies were determined by measuring EGFP expression using FACS analysis (Epics Altra from Beckman Coulter, Fullerton, Calif., USA). For the overproduction of hETG, we modified the pWPI (addgene ref 12254) lentiviral vector (Pham et al., 2004) to include a C-terminal tag (Myc) at the end of the gene of interest, a Tet operon sequence in front of the promoter sequence of the vector to allow conditional control of the expression cassette and finally a Gateway® cloning cassette located between the promoter and a C-terminal tag (Myc or V5His) to allow rapid transfer of the genes of interest from Gateway-compatible entry vectors. Co-transduction with a suitable lentiviral vector, pLV-tTR-KRAB-Red (Wiznerowicz and Trono, 2003), allows controlling the expression of the transgene by addition of doxycycline. Finally, the vector also bears an EGFP selection marker driven by an IRES sequence following the Gateway cassette to follow infection efficiency and eventually enrich the population by FACS sorting. 
         [0064]    Overexpression and silencing of the hETG1 gene were confirmed by RT-PCR analysis. RNA was extracted from MCF-7 cells with the RNeasy Plus Mini Kit (Qiagen, Hilden, Germany). cDNA was prepared from 1 μg total RNA with the iScript cDNA synthesis kit (Bio-Rad, Hercules, Calif.) according to the manufacturer&#39;s instructions. For quantitative PCR, PCR reactions were run in triplicate on a LightCycler® 480 Real-Time PCR system (Roche) using the SYBR Green I master Mix (Roche), 100 nM primers and 20 ng of cDNA according to the manufacturer&#39;s instructions The CT (threshold cycle when fluorescence intensity exceeds ten times the SD of the baseline fluorescence) values for the target amplicon and endogenous control (TBP) were determined for each sample. Quantification was performed using the comparative C T  method (DDC T ) Primers used were 5′-ACTCTCCACGAAATACCACTTTG-3′ (SEQ ID NO:34) and 5′-GTAGGATGTTGAGGGACTGACTCG-3′ (SEQ ID NO:35) for hETGJ and 5′-CGGCTGTTTAACTTCGCTTC-3′ (SEQ ID NO:36) and 5′-CACACGCCAAGAAACAGTGA-3′ (SEQ ID NO:37) for TBP. For both the overexpressing and silencing lines appropriate cell lines were selected. The V5-C10 overexpressing line showed an 17-fold induction of hETG1 expression levels. The 158067-Const knock-down cell line showed a depletion of 80% in transcription of hETG1. 
         [0065]    In both the hETG1 overexpression and knock-down lines substantial changes in the cellular morphology were observed. These changes were characterized by the appearance of multi-nucleated and/or giant cells ( FIGS. 10 and 11 ). To assess more clearly the severity of the phenotype observed after hETG1 over-expression or depletion, co-stainings of hETG1 with the membrane marker β-Catenin were performed. Twenty thousand MCF-7 cells were grown for two days at 37° C. Cells were then washed with 1 ml of PBS three times and fixed with 500 μl of ice-cold 100% methanol. Fixed cells were first incubated 1 hour at room temperature in 200 PBS supplemented with 0.04% gelatin, then for 1 hour with primary antibodies (Anti-V5-His-Tag antibody, Santa Cruz Biotechnology INC, and Anti-β-Catenin antibody, Santa Cruz Biotechnology INC) diluted in PBS. Cells were next washed three times with PBS and incubated for 1 hour at room temperature with secondary antibody (Alexa-488 anti-mouse, Ig, 1:5000 dilution; Invitrogen, Carlsbad, Calif. for V5-His Tag and Alexa-594 anti-rat, Ig, 1:5000 dilution; Invitrogen, Carlsbad, Calif. for (3-Catenin). After three washes with PBS, coverslips were mounted on microscopic glass slides using vecatshield supplemented with DAPI (Vector Laboratories, Burlingame, Calif.) to prevent photobleaching. Coverslips were examined with an Olympus BX61 fluorescent microscope under a magnification of 40×. Examples of giant multinucleated cells can be observed in  FIG. 12 . These data indicate a clear cytokinesis defect upon modulation of the hETG1 expression level. 
       Example 10 
     Constitutive Knock-Down of hETG1 Causes a G2 Cell Cycle Arrest 
       [0066]    Two hundred thousand hETG1 knocked-down and control MCF-7 cells (non infected MCF-7) were plated into six-well plates and grown two days in 4 ml of MCF-7 medium. Cells were next trypsinized, centrifuged at 2000 rpm for 5 minutes re-suspended in 1 ml of FAC&#39;s buffer (PBS pH 7.2, 0.5% BSA and 2 mM EDTA) and filtered on a 40 μm strainer (Becton Dickinson, San Jose, Calif.). Cells were incubated on ice in the dark during 15 minutes in FACS buffer supplemented with DAPI. The DNA content was analyzed in triplicate by flow cytometry. Compared to the control cultures, the knocked-down cultures are characterized by a depletion of the 2C (G1-phase) cell population, correlated with an increment in the population of cells with a DNA content equal to 4C (G2-phase) or greater (polyploidy) ( FIG. 13 ), indicating an arrest in their G2 cell cycle phase. 
         [0067]    This cell cycle arrest was further demonstrated by transcriptional up-regulation of G2-M marker genes (such as Cyclin B1, Cyclin B2 and Cyclin H) ( FIG. 14 ), illustrating the importance of functional h-ETG1 for normal cell cycle progression. The combined appearance a G2-M arrest and multi-nucleated cell phenotype indicated defects during sister chromatid organization and/or separation. Recent work has shown that the spindle checkpoint inhibits cell-cycle progression by direct binding of components of the spindle checkpoint pathway or kinetochore-associated proteins (like Mad2 and/or Mad3) to components of the anaphase-promoting complex, resulting into an M-phase arrest (Malureanu et al. 2009; Kulukian et al. 2009). Knocked-down h-ETG1 cells display a clear up-regulation of Mad2 and Mad3 transcripts, corroborating the view that specific problems during sister chromatid separation are at least partly responsible for the observed phenotypes ( FIG. 15 ). 
         [0068]    To further investigate this issue, metaphases in MCF7 wild-type and h-ETG1 knock-down karyotypes were characterized by counting metaphase chromosomes with totally detached chromatids. Briefly, upon h-ETG1 knock-down, cells were cultivated during 2 days at 37° C. To enrich for mitotic chromosomes, subconfluent cells were treated with KaryoMax colcemid (Sigma) for five hours before harvesting. Cells were trypsinized, pelleted and resuspended in hypotonic solution (60 mM KCl) for 30 minutes at room temperature. Cells were repelleted, the hypotonic solution was removed and cells were resuspended in freshly made methanol: glacial acetic acid (3:1) added drop-wise. Two or three drops of suspended cells were applied to precleaned blood smear glass slides and finally chromosomes were counterstained with VectaShield containing DAPI (Vector Laboratories). Microscopic analysis illustrated an increment of chromosomes with totally detached metaphase chromosomes upon h-ETG1 knock-down ( FIG. 16 ), clearly indicating that h-ETG1 plays a fundamental role during chromosome segregation, and that reduced sister chromatids cohesion is a main reason for the observed multi-nucleated cell phenotype. 
       Example 11 
     ETG1 Expression Analysis in Human Primary Breast Cancer 
       [0069]    cDNA synthesis on RNA samples was performed on 1.5 μg total RNA using the Iscript cDNA synthesis kit (Bio-Rad). Subsequently qPCR on the LC480 (Roche) was done for ETG1 and different reference genes (Vandesompele et al. 2002) using LCS480 Sybr Green I master kit (Roche), Fast SYBR master mix kit (Applied Biosystems) and Taqman fast univ. PCR Mastermix (Applied Biosystems). Using GeNorm (Vandesompele et al. 2002) we determined the most accurate set of reference genes for normalisation (HMBS, ACTB, HPRTI, RPL13A, SDHA, TBP and UBC). The average threshold cycle of triplicate reactions was used for all subsequent calculations using the delta Ct method. Relative ETG1 expression levels (average of ten samples with low expression set to 1) were depicted ranking low to high ( FIG. 17 ). 
         [0070]    The expression of ETG1 was further compared with different clinicopathological parameters available for the different analyzed tumors. High ETG1 expression was correlated with a negative estrogen receptor (ER) status ( FIG. 18 ). For breast cancers, mRNA expression profiling has shown that one of the most powerful denominators in determining the gene expression signatures and prognostic groups of breast cancer is estrogen receptor (ER) and ER-related genes. Breast cancers have been separated by gene expression profiles into luminal, basal like, ERBB2, and normal breast-like subgroups (Sorlie et al., 2001). Basal-like tumors express many of the genes characteristic of breast basal epithelial cells and the most typical feature of basal like breast cancers is the lack of expression of ER and genes usually co-expressed with ER (Perou et al., 2000). This negative ER status is a well established prognostic and predictive marker in breast cancer. Microarray studies have shown that basal-like tumors have poor prognosis when compared with ER-positive luminal tumor groups (Sorlie et al., 2003). This finding supports the importance and usefulness of assessing the protein status of ETG1 in human cancer samples. 
       Example 12 
     ETG1 as a Pre-Cancer Marker 
       [0071]    Early detection of cancer by screening remains an important effective method for improving cancer survival. ETG1 is tested for potential clinical practice by the generation of diagnostic antibodies. A full-length human ETG1 cDNA clone was used as a template for PCR amplification with Pfu polymerase to generate an ETG1 coding cassette. The primers used for amplification were hETG1-Inf-Fw 5′-CAAGGTACCAAGCTTAATGCCGTGTGGGGAGG-3′ (SEQ ID NO:38) and hETG1-Inf-Rv 5′-TGCGGCCGCATGCATTTAAAGTTCATTTCCAT-3′ (SEQ ID NO:39). The resulting PCR product was inserted by fusion cloning into the HindIII pLHX32 plasmid downstream of the His 6  tag, to generate pLHXhETG1. Plasmid insert is controlled by DNA sequencing. Plasmid is transformed in MC 1061 bacteria containing a pICA2 plasmid. Exponentially growing  E. Coli  bacteria are induced overnight with 1 mM isopropyl β-D-thiogalactopyranoside at 20° C. The cells are harvested by centrifugation and the cell paste is frozen until required. Frozen MC1061 cell pellets are suspended in buffer A, comprising 20 nM Tris-HCl, pH 7.5, 10% glycerol, 1 mM oxidized glutathione, 200 mM NaCl, 1 mM phenylmethylsulfonyl fluoride, 50 μM leupeptin and 20 μg/ml aprotinin, and are lysed by sonication or French press. Insoluble proteins are removed by centrifugation. Bacterial DNA is removed over a DEAF column equilibrated by buffer A. The flow through is applied on a Co +2  metal chelate column which is washed with buffer A for 4 to 16 hours. Low strength metal binding proteins are removed by a short washing with buffer B, consisting of 20 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM oxidized glutathione, 200 mM NaCl and 10 mM imidazole. His 6 -tagged ETG1 is eluted from the column by buffer C, containing 20 mM Tris-HCl pH 7.5, 10% glycerol, 1 mM oxidized glutathione, 50 mM NaCl and 100 mM imidazole. The purity of the preparation is checked by SDS-PAGE and further purified if needed by Q-resource or monoQ columns. Purified proteins are used for immunization of rabbits for the production of polyclonal antibodies (Eurogentec). In addition purified ETG1 protein is provided to the VIB Nano-body service facility for lama immunization. Generated anti-ETG1 polyclonal antibodies and nanobodies are used for a wide range of applications like immunohistochemistry, immunomodulation, intracellular expression (intrabodies), biosensor-applications, etc. Collaborations are initiated with different pathology departments from which Tissue MicroArrays (TMAs) are utilized for high throughput molecular pathology characterization of various kinds of tumors (e.g. skin, colon, breast). TMAs containing samples with long-term and complete clinical follow-up data are available within this framework, allowing us to obtain prognostic and predictive information. 
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