Arabidopsis RETINOBLASTOMA-RELATED PROTEIN ... - Springer Link

Plant Mol Biol (2008) 66:259–275 DOI 10.1007/s11103-007-9268-2

Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 is involved in G1 phase cell cycle arrest caused by sucrose starvation Hiroto Hirano Æ Hirofumi Harashima Æ Atsuhiko Shinmyo Æ Masami Sekine

Received: 9 July 2007 / Accepted: 18 November 2007 / Published online: 7 December 2007 Ó Springer Science+Business Media B.V. 2007

Abstract Although sucrose availability is crucial for commitment to plant cell division during G1 phase by controlling the expression of D-type cyclins, it has remained unclear how these factors mediate entry into the cell cycle. Here we show that Arabidopsis RETINOBLASTOMA-RELATED PROTEIN 1 (AtRBR1) is involved in G1-phase cell cycle arrest caused by sucrose starvation. We generated estrogen-inducible AtRBR1 RNA interference (RNAi) Arabidopsis suspension MM2d cells, and found that downregulation of AtRBR1 leads to a higher frequency of arrest in G2 phase, instead of G1-phase arrest in the uninduced control, after sucrose starvation. Synchronization experiments confirmed that downregulation of AtRBR1 leads to a prolonged G2 phase and delayed activation of G2/M marker genes. Downregulation of AtRBR1 also stimulated the activation of E2F-regulated genes when these genes were repressed in the uninduced cells under the limited sucrose conditions. We conclude that AtRBR1 is a key effector for the ability of sucrose to modulate progression from G1 phase. Keywords Arabidopsis MM2d cells RB Sucrose starvation

H. Hirano H. Harashima A. Shinmyo Graduate School of Biological Sciences, Nara Institute of Science and Technology, Takayama 8916-5, Ikoma, Nara 630-0101, Japan M. Sekine (&) Department of Bioproduction Science, Faculty of Bioresources and Environmental Sciences, Ishikawa Prefectural University, Suematsu 1-308, Nonoichimachi, Ishikawa 921-8836, Japan e-mail: [email protected]

Abbreviations ACT Actin ChIP Chromatin immunoprecipitation cDNA Complementary DNA CDC Cell division control CDK Cyclin-dependent kinase DP DNA-binding heterodimerization partner protein E2F Adenovirus E2 promoter-binding factor GST Glutatione S-transferase HA Hemagglutinin IP Immunoprecipitation LSC Laser scanning cytometer ORF Open reading frame MBP Maltose-binding protein MCM Mini chromosome maintenance MCS Multi-cloning site PCR Polymerase chain reaction PCNA Proliferating cell nuclear antigen RB Retinoblastoma RBR Retinoblastoma-related RNAi RNA interference RNR Ribonucleotide reductase RTReverse transcriptase polymerase chain reaction PCR WCE Whole cell extract

Introduction Development and growth of multicellular organisms are dependent on the spatial and temporal control of cell proliferation and differentiation (Coffman 2004). Plants respond flexibly to environmental conditions, modulating their growth rate and developmental pattern in response to

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innate genetic cues and external environmental signals (Gutierrez 2005). One of the most important mediators of these signals is sucrose, the major transported carbon source in most plants. Sucrose not only regulates photosynthesis and respiration, serving as a key component of storage compounds, but also acts as an important signaling molecule that controls gene expression and developmental processes (Gupta and Kaur 2005; Rolland et al. 2006). Moreover, sucrose molecules mimic morphogens by providing positional information to the cell cycle machinery and various developmental programs (Rolland et al. 2006). In eukaryotes, cyclin-dependent kinases (CDKs) play central roles in cell cycle control (Pines 1999). Two major types of plant CDKs participate principally in this process (Joubes et al. 2000; Inze and De Veylder 2006). A-type CDKs (CDKAs) can complement mutations in yeast CDC28/cdc2, while B-type CDKs (CDKBs) cannot. CDKBs have plant-specific features including cell cycleregulated expression that peaks during the G2 phase. Based on sequence similarity and expression patterns, three major classes of cyclins have been identified in plants (Dewitte and Murray 2003; Wang et al. 2004). A-type cyclins (CYCAs) are broadly involved during S to M phase, but B-type cyclins (CYCBs) control the G2/M transition. D-type cyclins (CYCDs) are thought to act as mediators linking extracellular and developmental signals to the cell cycle. The expression of Arabidopsis CYCD2;1 and CYCD3;1 is regulated by plant hormones and/or sucrose (Hu et al. 2000; Riou-Khamlichi et al. 2000; Healy et al. 2001). As CYCD3;1 is rate-limiting for G1/S transition (Menges et al. 2006), sucrose availability is thought to act as a major determinant of cell division by controlling these two genes. CYCD3;1 is a highly unstable protein whose levels decline rapidly in response to sucrose depletion, its proteolysis mediated by a proteasome-dependent pathway (Planchais et al. 2004). GeneChip analysis revealed that expression of genes involved in S-phase entry decreases during sucrose starvation, a process which may be linked to G1-phase arrest (Contento et al. 2004). E2F transcription factors are key components of cell cycle control in higher eukaryotes (Attwooll et al. 2004; Korenjak and Brehm 2005). In animals, the promoter regions of numerous cell cycle genes that are active during S phase contain multiple E2F-binding sites. E2F and DP proteins interact to form active transcription factors that are regulated by the retinoblastoma (RB) protein, which contains the A and B pocket domains that are involved in binding of most of its associated proteins. Although RB represses transcription by binding E2Fs, CDK-mediated phosphorylation of RB during G1 phase releases a functional E2F-DP heterodimer to activate target genes and allow cell cycle progression to S phase. RB-E2F complexes also actively repress transcription to maintain the

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differentiated state, which has led to the notion that the RB-E2F pathway is one of the most important regulators of cell proliferation and cell differentiation. A number of cDNAs encoding E2Fs and RETINOBLASTOMA-RELATED (RBR) proteins have been identified in plants. In Arabidopsis, the E2F/DP family consists of six E2F (E2Fa to E2Fc, DEL1 to DEL3) and two DP (DPA and DPB) proteins (Gutierrez et al. 2002; Shen 2002; Vandepoele et al. 2002). E2Fa to E2Fc possess the typical organization found in animals, including a DNA binding domain, a DP heterodimerization domain, an RBR binding domain, and a transactivation domain. By contrast, DEL1 to DEL3 contain only a duplicated DNA binding domain that allows DNA binding without requiring the DP proteins (Gutierrez et al. 2002; Kosugi and Ohashi 2002a; Shen 2002; Ramirez-Parra et al. 2004). E2Fa and E2Fb can bind with DPA to transcriptionally activate target genes (De Veylder et al. 2002; Sozzani et al. 2006). Although transient expression experiments have shown that E2Fc lacks transcriptional activation properties (Kosugi and Ohashi 2002b), its overexpression delays cell division and represses S-phase genes (del Pozo et al. 2002). The abundance and stability of E2Fb are increased by exogenously applied auxin, and overexpression with DPA of E2Fb, but not E2Fa, is sufficient to support cell proliferation in the absence of auxin (Magyar et al. 2005). E2Fs and RBR also play important roles in plant development (Gutierrez 2005; Inze and De Veylder 2006). Overexpression of E2Fa-DPA (De Veylder et al. 2002) or CYCD3;1 (Dewitte et al. 2003) in Arabidopsis results in uncontrolled cell proliferation and delayed differentiation, and has severe effects on plant development. Additionally, particle bombardment of tobacco BY-2 cells with the maize ZmRBR1 gene suppresses cell division, while overexpression of RepA, encoding a viral protein that sequesters RBR from its physiological binding proteins, stimulates cell division (Gordon-Kamm et al. 2002). Inhibition of RBR function by virus-induced gene silencing prolongs cell proliferation and also delays cell differentiation in the leaves and stems, indicating that RBR positively regulates cell cycle exit (Park et al. 2005). The RepA-inducible system to inactivate RBR function revealed that cells respond differently to RBR inactivation in regulating their cell division and endoreplication properties during Arabidopsis leaf development (Desvoyes et al. 2006). A T-DNA tagging mutant of Arabidopsis AtRBR1 caused female gametogenesis abnormality prior to fertilization (Ebel et al. 2004), and Wildwater et al. (2005), using conditional loss-of-function RNA interference (RNAi) approaches, showed that AtRBR1 regulates the size and state of the root stem cell. Nevertheless, the protein levels of RBR during these regulatory processes are largely unknown in plants.

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ZmRBR1 interacts with Arabidopsis CYCD3;1 and other cellular or virus-derived factors through its pocket domains (Ach et al. 1997; Dewitte and Murray 2003). In immunoprecipitation assays, RBR was found to interact with FVE, a component of a complex repressing FLC (a repressor of flowering) transcription, through a histone deacetylation mechanism (Ausin et al. 2004). Transient assays with a plasmid encoding myc-tag-fused ZmRBR1 showed that RBR is localized to the nucleus (Ach et al. 1997). Arabidopsis plants overexpressing CYCD3;1 contain much higher levels of AtRBR1 mRNA and protein than those in wild type, which may reflect a feedback mechanism that normally regulates CYCD3;1 activity, because the CYCD3:1 promoter contains an E2F-binding site (Dewitte et al. 2003). Like the regulation of RB function by phosphorylation in animals, ZmRBR1 undergoes changes in level and phosphorylation state during development of endosperm cells (Grafi et al. 1996). Immunoprecipitationcoupled kinase assays showed that RBR was phosphorylated by CYCD/CDKA complexes during G1 phase (Boniotti and Gutierrez 2001; Nakagami et al. 2002). In transient assays, transfection with tobacco E2F activated a reporter gene, and this activation was repressed by co-transfection with tobacco RBR (NtRBR1) (Uemukai et al. 2005). Using phospho-specific antibodies, we demonstrated that distinct sites in NtRBR1 were phosphorylated by probably different types of cyclin/CDK complexes during the cell cycle (Kawamura et al. 2006). These lines of evidence strongly suggest that the RBR-E2F pathway plays important roles in controlling the G1/S transition in plants (Gutierrez 2005). In this study, we generated MM2d cells inducibly downregulating AtRBR1 to evaluate the role of AtRBR1 in cell cycle arrest after sucrose starvation. Downregulation of AtRBR1 caused a higher frequency of arrest in G2 phase in response to stationary phase and sucrose starvation, but also activation of E2F-regulated genes even when cells were sucrose-starved. Our data provide the first evidence that AtRBR1 plays a key role in G1-phase arrest in sucrose starvation.

Materials and methods Plant materials and growth conditions Arabidopsis MM2d cells were cultured at 27°C in modified Linsmaier and Skoog (LS) medium (Nagata et al. 1992) as previously described (Menges and Murray 2002, 2006). MM2d cells were synchronized with aphidicolin, essentially as described by Menges and Murray (2002, 2006) with minor modifications. Five days after subculture, the cells were transferred to 50-ml tubes, centrifuged to prepare 10-ml cell

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pellets, and resuspended in 50 ml LS medium containing 10 lg ml-1 aphidicolin. Measurements of DNA content were made by the method of Nakagami et al. (2002) for a laser scanning cytometer (LSC) or that of Menges and Murray (2002) for a flow cytometer (PA; Partec GmbH, Mu¨nster, Germany). For flow cytometry, cells were treated with a kit for plant ploidy level analysis (Cystain UV; Partec GmbH), after the cells had been chopped with a razor blade, and nuclear particles were counted. For the re-entry experiment, 10-ml aliquots of stationary-phase culture were subcultured into 90 ml of fresh medium. For sucrose starvation, 2-day-old cell-culture cells (100 ml) were washed in 500 ml LS medium supplemented with 0%, 0.3% w/v sucrose (with added mannitol to adjust the osmolality to that of 3% sucrose) or 3% w/v sucrose and suspended in 95 ml LS supplemented with 0%, 0.3% or 3% w/v sucrose. Cells were incubated at 27°C on a shaker, and samples were taken every 12 h. Cell number and fresh weight measurements were performed in triplicate for each of three samples. Cell number was determined in a counting chamber after cells were treated with 1% Cellulase Onozuka RS (Yakult Pharmaceutical, Tokyo, Japan) and 0.1% Pectolyase Y-23 (Kikkoman, Tokyo) for 1 h at 30°C. After protoplasts were prepared as above, cell areas were measured with NIH ImageJ 1.36b software.

Plasmid construction and transformation of Arabidopsis MM2d cells For AtRBR1 RNAi, two multi-cloning site (MCS) oligonucleotides (MCS-F: 50 -TCGAGTCTAGAGGATCCGATA TCCCCGGGAGGCCTAGATCTGTCGACA-30 ; MCS-R: 50 -CTAGTGTCGACAGATCTAGGCCTCCCGGGGATA TCGGATCCTCTAGAC-30 ) were annealed and subcloned into the XhoI/SpeI sites of pBluescript II SK- (Stratagene, La Jolla, CA, USA), generating pBluescript-MCS. To insert linker sequences, a 812-bp PCR fragment was amplified from the GUS gene with primers GUS-F (50 -CTGAAGA GATGCTCGACTGG-30 ) and GUS-R (50 -TCATTGTTTGC CTCCCTGCT-30 ), and the resulting fragment was inserted into the SmaI site of pBSK-MCS to generate pBSK-MCSGUS. Using a cDNA clone of AtRBR1, a 496-bp PCR fragment was amplified by primers AtRBR1-F (50 -CTCGAG TGAAATATTTATTCCTGCCG-30 ), containing an XhoI site, and AtRBR1-R (50 -ACTAGTCTATGAATCTGTTGG CTCGG-30 ), containing a BamHI site. The PCR fragment was then digested with XhoI and BamHI, and inserted separately into XhoI/BamHI- and BglII/SalI-digested pBSK-MCSGUS, respectively, to generate pBSK-MCS-RBGUSBR. A XhoI/SpeI fragment containing the inverted repeat sequences of AtRBR1 was released from pBSK-MCS-RBGUSBR and

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cloned into XhoI/SpeI-digested pER8 binary vector (Zuo et al. 2000), giving rise to pER8-ATRBR1. To clone the *1-kbp region from the start codon to the stop codon of the E2Fa gene, a PCR fragment was generated by primers E2Fa-F (50 -GGTACCTTTCCGTTCTCCTTCTTTT TTTG-30 ), containing a KpnI site, and E2Fa-R (50 -GGA TCCTCACTCGAGTCTCGGGGTTGAGTCAAC-30 ), containing XhoI and BamHI sites, and then inserted into EcoRVdigested pBluescript II SK- to generate pSK-E2Fa. After the sequences were verified, triple HA tandem repeat sequences were inserted into the XhoI site of pSK-E2Fa, which was then digested with KpnI and BamHI. The resulting DNA fragment was inserted into KpnI/BamHI-digested pGreen vector (Hellens et al. 2000), in which the nos-terminator had already been inserted into the SacII/SacI site, generating pGreen-E2Fa. Transformation of Arabidopsis MM2d cells was performed as previously described (An 1985; Menges and Murray 2002, 2006). Transgenic MM2d cells were selected first by screening for resistance to 35 mg/l kanamycin or 5–10 mg/l hygromycin. Suspension cultures were generated from the antibiotic-resistant calli and maintained in modified LS medium supplemented with appropriate antibiotics.

MBP pull-down experiments The full-length ORF of E2Fa was PCR-amplified using the primers 50 -GGATCCATGTCCGGTGTCGTACGATC-30 and 50 -GTCGACTCATCTCGGGGTTGAGTCAA-30 , and the PCR fragment was subcloned into the BamHI and SalI sites of pMAL-c2. Recombinant proteins were produced in Escherichia coli BL21 (codon plus) at 18°C and purified according to the manufacturer’s protocol (New England Biolabs, Beverly, MA, USA). Total protein was extracted from MM2d cells as previously described (Nakagami et al. 2002) with a minor modification using IPB buffer [IP buffer (25 mM Tris–HCl, 75 mM NaCl, 15 mM MgCl2, 15 mM EGTA, 0.1% NP-40, pH 7.5) containing 10 mM NaF, 20 mM b-glycerophosphate, 2 mM Na3VO4]. Total protein (1 mg) was incubated with MBP or MBP-E2Fa for 1 h at 4°C, and then mixed with amylose resin and incubated overnight at 4°C with gentle agitation. After the resin had been washed three times with IPB buffer, proteins were released by boiling in SDS sample buffer and fractionated by SDS-PAGE.

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primers suitable for ligation into the BamHI/SmaI site of pGEX4T-1. Detailed information for construction is available from the authors. GST fusion proteins were purified from E. coli using glutathione Sepharose 4B beads according to the manufacturer’s instructions. Polyclonal antibodies were developed in rabbits against a mixture of the AtRBR1 peptides EKDYYDGKGELDER (amino acids 328–341) and VRNDRAVEANNKPE (amino acids 866–879). The synthetic peptides were coupled to keyhole limpet haemocyanin via an additional cysteine residue at their C termini. Antibodies were purified by protein A-Sepharose (GE Healthcare, Piscataway, NJ, USA) column, with the immunogenic peptides coupled by means of NHS-activated Sepharose 4FF (GE Healthcare). Proteins were extracted in IPB buffer from MM2d cells as previously described (Nakagami et al. 2002). Protein concentrations were determined by the Bradford (1976) method using BSA as a standard. Western blot analysis was performed as previously described (Nakagami et al. 2002). Antibodies were used as a 1:10,000 dilution of rabbit antiHA antibody (Roche Applied Science, Penzberg, Germany) for 2 h at room temperature and the filters were then washed twice in TBS. Polyclonal anti-AtRBR1 antibody was used at a 1:5,000 dilution. Protein bands were visualized with alkaline phosphatase. Immunoprecipitations were performed as described previously (Nakagami et al. 1999, 2002) with 2 lg antiNtRBR1 antibody (Kawamura et al. 2006) in a 15-ll 50% slurry of Protein A Sepharose 4 Fast Flow for MM2d cell extracts.

Phosphatase treatment After protein extracts were prepared from 3-day-old MM2d cells by sonication in calf intestinal alkaline phosphatase (CIP) buffer (10 mM Tris–HCl pH7.9, 1 mM MgCl2, 0.1% NP-40), CIP (New England Biolabs) was incubated with 250 lg of protein extracts for 45 min at 37°C in a total volume of 50 ll, using 13 units per sample. The reaction was also conducted in the presence of phosphatase inhibitor (50 mM EDTA) as a control. The reaction was stopped by adding loading buffer and boiling the samples for 5 min before separation by SDS-PAGE for western blotting.

RNA extraction, RT-PCR, and real-time PCR Antibody preparation, western blot analysis and immunoprecipitation To produce glutathione S-transferase (GST)-fused AtRBR1, the coding region of AtRBR1 was amplified by PCR with

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Total RNA was extracted using an RNeasy Mini kit (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. RNA samples were treated with RNase-free DNase (Promega, Madison, WI, USA) following the manufacturer’s instructions. Two-microgram

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samples of RNA were used for cDNA synthesis using the GeneAmp RNA PCR system (Applied Biosystems, Foster City, CA, USA) following the manufacturer’s instructions. cDNA samples were then amplified for each set of primers, detected in real-time using SYBR Green Master Mix (Applied Biosystems) and used for real-time quantitative RT-PCR in the 7700 Fast Real-Time PCR System (Applied Biosystems). Each primer was used at 0.2 lM per reaction; the ACT8 gene was used as an internal control to normalize amounts of each template. HistoneH4 F: 50 -CCATTCGAAGATTGGCTCGTA-30 HistoneH4 R: 50 -CCACGTGTCTCCTCGTAGATGA-30 PCNA1 F: 50 -TCTACTGCCGGTGACATTGGA-30 PCNA1 R: 50 -GCATCTTCCGGCTTGTCTACAG-30 ACT2 F: 50 -TGCACCACCTGAAAGGAAGTACA-30 ACT2 R: 50 -ATTTTTACCTGCTGGAATGTGCTG-30 ACT8 F: 50 -CCACCCGAGAGGAAGTACAG-30 ACT8 R: 50 -TCCACATCTGCTGGAAAGTG-30 ORC6 F: 50 -GCTGCTGCTTTCTATTTGTGTGC-30 ORC6 R: 50 -CAGACTCTGATGTACCACAAACCT CA-30 CDC6 F: 50 -GTGTCAAGTGGTCAAGATGGATCA-30 CDC6 R: 50 -GGTGTTGAGGTAGAGACTGGATGG-30 RNR F: 50 -TCCGTTCGATTGGATGGAACT-30 RNR R: 50 -CAAACGCGCCGTTACCATT-30 MCM2 F: 50 -ACGGACAAGGAGTTTCTATAGCGA-30 MCM2 R: 50 -CCTAAGGTGCATCCTAGCATGTG-30 MCM3 F: 50 -AACCTTCTGTAGAGCAATTCAGCG-30 MCM3 R: 50 -TCCTCATGTGCTGCCCAAA-30 CYCA1;1 F: 50 -GGCTAAGAAGCGACCTGATG-30 CYCA1;1 R: 50 -TACAAGCCACACCAAGCAAC-30 CYCB2;3 F: 50 -TAAACCACCTGTGCATCGAC-30 CYCB2;3 R: 50 -ATCTCCTCCAGCATTGCTTC-30 CDKB2;2 F: 50 -AGCCTTCACTCTCCCAATGA-30 CDKB2;2 R: 50 -TCAGAGTCTCCCGCAAAGAT-30 The data were derived from two independent experiments performed in duplicate.

ChIP assays Chromatin immunoprecipitation (ChIP) assays and data analysis were performed basically as previously described (Gendrel et al. 2002). Briefly, MM2d cells were treated with 1% formaldehyde for 30 min at 25°C and the crosslinking reaction was then stopped with 0.1 M Gly. Nuclei were extracted, lysed in lysis buffer (50 mM Tris–HCl pH 8.0, 10 mM EDTA, and 1% SDS), and sonicated to shear DNA to an average size of 700–1,000 bp. Crude chromatin lysates were suspended in dilution buffer (1.1%

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Triton X-100, 1.2 mM EDTA, 16.7 mM Tris–HCl pH 8.0, 167 mM NaCl) and precleared with protein A agarose beads (GE Healthcare), and then incubated for 2 h at 4°C with 2 lg each of anti-NtRBR1 (Kawamura et al. 2006), anti-HA (Roche) or anti-histone H3 (Abcam, Cambridge, UK) antibodies. Immunocomplexes were recovered using protein A agarose, extensively washed, and eluted with 100 ll elution buffer (1% SDS, 0.1 M NaHCO3, 10 mM DTT). Cross-links were reversed, the samples treated with proteinase K, and DNA was recovered after phenol/chloroform extraction by ethanol precipitation. DNA was resuspended in 50 ll of water and 1-ll aliquots were used for PCR. The primer sequences used were as follows: PCNA1 promoter proximal F: 50 -GGTTGTCACCGA GACAAGATTCG-30 PCNA1 promoter proximal R: 50 -GTTCCCGCCAATG CGCTA-30 PCNA1 promoter distal F: 50 -AATAACGGTCTTTTA TGTTTTGCTGG-30 PCNA1 promoter distal R: 50 -GCATTTGTTGGTTTGA GAATTTTGA-30 ACT8 promoter F: 50 -TCTGTGACAATGGTACTGG AATGG-30 ACT8 promoter R: 50 -GGTGCTCTTCAGGAGCAATA CG-30

Results AtRBR1 is highly phosphorylated during G1 to S phase Expression of AtRBR1 has been studied at the transcriptional level, but little is known of its regulation at the posttranscriptional level during the cell cycle. For analysis of AtRBR1 protein, rabbit antibodies were generated against two peptides of AtRBR1. To verify that the antiserum specifically detect the intended protein, it was used to probe western blots of Arabidopsis whole cell extract (WCE) and purified GST-fused AtRBR1; anti-AtRBR1 antibody was used both directly and in competition with the purified GST-AtRBR1 (Fig. 1A). Although GSTAtRBR1 appears to be unstable in E. coli, anti-AtRBR1 antibody detected purified GST-AtRBR1 protein of about 138 kDa and a protein of about 112 kDa in WCE of actively growing cells. Both proteins were barely detectable when the antibody was preincubated with purified GST-AtRBR1. The anti-AtRBR1 antibody was then used to determine AtRBR1 levels as partially synchronized MM2d cells exit from stationary phase and re-enter the cell cycle (Fig. 1B). Using LSC, progression of the cell cycle

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Fig. 1 Phosphorylation state of AtRBR1 after MM2d cells were subcultured into fresh medium. (A) WCE of 3- or 4-day-old MM2d cells, as well as purified GST-AtRBR1, were used for western blotting with anti-AtRBR1 antibody. The antibody was used directly (left panel) or in competition with purified GST-AtRBR1 (about 5 lg) (right panel). Asterisks indicate bands that were not detected after preincubation of the antibody with purified GST-AtRBR1. The lower bands in the lane of GST-AtRBR1 may represent partially translated or degraded products. (B) AtRBR1 protein levels were analyzed as partially synchronized MM2d cells exited from stationary phase and re-entered the cell cycle. Stationary-phase MM2d cells were subcultured into modified LS medium, and samples were taken every 2 h

over 12 h for western blot analyses with anti-AtRBR1 antibody. The SDS-PAGE gel was stained with CBB as a loading control. (C) Cytometric analysis after MM2d cells were subcultured into fresh medium. Cytometric analyses were performed on 5 9 103 cells. Each block represents a sample taken at 2-h intervals. A DNA histogram of the cytometry results is shown, and indicates the percentages of cells in G1 (black bars), G2 (grey bars), and S (white bars) phases. (D) Phosphatase experiment. Extracts from cells were incubated with or without (Mock) CIP for 45 min at 37°C and subjected to western blotting (upper panel). A reaction was also conducted with CIP in the presence of 50 mM EDTA. CBB staining of the gel is shown in the lower panel as a loading control

was monitored by measuring the proportion of cells in each phase (Fig. 1C). Stationary-phase MM2d cells (7 days after subculture) were transferred into fresh medium, and samples were taken every 2 h over 12 h for western blot analyses under electrophoretic conditions that would permit distinction between different phosphorylation forms of AtRBR1. Only single bands were observed during the first

6 h of culturing, whereas additional shifted bands were detected between 8 and 12 h (Fig. 1B). To clarify whether the shifted bands corresponded to potentially hyper-phosphorylated forms of AtRBR1, protein extracts were treated in vitro with CIP (Fig. 1D). Phosphatase treatment of extracts from actively growing cells resulted in a shift in AtRBR1 migration (Fig. 1D,

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lanes 1 and 2), but no shift occurred when phosphatase treatment was conducted in the presence of the protein phosphatase inhibitor EDTA (Fig. 1D, lane 3). We conclude that the shifted band is phosphorylated AtRBR1 and that AtRBR1 becomes hyper-phosporylated during G1 to S phase.

non-phosphorylated AtRBR1 could bind to MBP-E2Fa; the shifted band could not (Fig. 2, upper panel). Thus, we conclude that E2Fa can bind to hypo- and/or non-phosphorylated, but not hyper-phosphorylated, forms of AtRBR1.

AtRBR1 is downregulated by inducible RNAi Hyper-phosphorylated AtRBR1 cannot interact with E2F

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AtRBR1 can interact with E2Fb (Magyar et al. 2005), but the form of AtRBR1 to which E2F proteins can bind is not known. To test whether phosphorylated AtRBR1 can interact with E2Fa, we carried out in vitro pull-down assays. E2Fa was fused N-terminally with maltose-binding protein (MBP), and expressed in and purified from E. coli (Fig. 2). Other bands appear to represent truncated translation products or degradation products, because virtually no detectable band was co-purified from E. coli expressing MBP alone (Fig. 2, lower panel). MBP and MBP-fused E2Fa were mixed with WCE of MM2d cells and purified on MBP-affinity (amylose) columns. Complex formation was then analyzed by western blotting with anti-AtRBR1 antibody. Under our experimental conditions, only a fastermigrating band representing hypo-phosphorylated and/or

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Fig. 2 Pull-down assays to analyze interaction between AtRBR1 and E2Fa. Upper panel: E2Fa was fused N-terminally with MBP. MBPE2Fa (middle lane) and MBP (right lane) were purified from E. coli using amylose resin, and mixed with 1 mg crude extracts of 3-day-old MM2d cells. Complex formation was visualized by western blotting with anti-AtRBR1 antibody after the proteins were purified on amylose resin and separated on a 6% SDS-PAGE gel. The total input of corresponding proteins (left lane) was also fractionated. Lower panel: CBB staining of the gel is shown. Dots may represent degraded products

As an estrogen-inducible expression system has been used to generate tobacco BY-2 cells inducibly expressing E2Fb (Magyar et al. 2005), we transformed the pER8 vector (Zuo et al. 2000) into MM2d cells. We found no effect on cellular growth during 7 days after transformed cells were subcultured into medium containing 10 lM b-estradiol or DMSO (data not shown). To downregulate AtRBR1 in MM2d cells, we prepared an RNAi construct having an inverted repeat corresponding to a 496-bp fragment of the 30 -terminal region of AtRBR1 and placed it under the control of an estrogen-inducible promoter. After this plasmid was introduced into MM2d cells via Agrobacterium-mediated transformation, we selected 10 independent cell lines for hygromycin resistance; similar results were obtained with four of these lines, and representative results for one are shown in Fig. 3. To determine the reduction in AtRBR1 levels upon induction of the RNAi construct, we performed protein gel blot analysis on extracts of MM2d cells collected every day after subculturing into fresh medium supplemented with 10 lM b-estradiol or DMSO as a control. AtRBR1 levels were reduced significantly 1 day after estrogen treatment and were depleted to undetectable levels 2 days after induction, whereas AtRBR1 was present in the uninduced cells throughout the culturing period (Fig. 3A). Growth of the uninduced control cells and the induced cells was compared by monitoring cell weight (Fig. 3B) and cell number (Fig. 3C) for 7 days after estrogen induction. While changes in cell number were similar between the induced and uninduced cells, the induced cells weighed much less than the uninduced control cells during 7 days culturing. Consistent with this finding, cell size was reduced significantly after estrogen induction and throughout the culturing period (Fig. 3D, E). Additionally, significant changes in the distribution and progression of cell cycle phases between the induced and uninduced cells were observed during 7 days culturing (Fig. 3F). As cells enter stationary phase, they normally stop dividing in G1, and for the uninduced cells 20% are in G2 phase, while more than 80% of the induced cells are in G2. The prolongation of G2 phase visible in Fig. 3F was further confirmed by calculating cell cycle phase durations (Table 1). At day 3, a decrease of about 55% in the length of G1 phase was evident in the induced cells, and a large

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(B) 0.5 0.4 0.3 0.2 0.1 0

DMSO 0

1

2

3

4

5

6

7

10 7

10 6

estrogen

0

1

Time after subculture (d)

(D)

DMSO

4

5

6

7


(E)

estrogen

1000 800 600 400 200 0

0

1

2

3

4

5

6


(F)

7

DMSO

DMSO

%

estrogen

% 100 80

80 60 40 20 0

estrogen

(n=100)

100

0

1

2

3

4

5

6


increase in the duration of G2 phase was observed, with an apparent tendency for G2 phase to increase in duration at day 4 (Table 1).

Downregulation of AtRBR1 leads to accumulation of G2-phase cells An increase in the proportion of G2-phase cells was clearly observed after downregulation of AtRBR1 during late log phase and stationary phase (Fig. 3F). To examine whether downregulation of AtRBR1 affects the duration of G2 phase, we used aphidicolin block–release experiments to generate cultures that were highly synchronized from G1/S phase (Fig. 4A). Under our experimental conditions, the synchronization efficiency was high, with more than 85%

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estrogen

DMSO Time after subculture (d )

Cell area ( µm2 )

Fig. 3 G2-phase cells increase following RNAi-induced downregulation of AtRBR1. (A) Immunoblot analysis of AtRBR1 in total protein extracts from estrogen-inducible AtRBR1 RNAi MM2d cells. Cells were sampled every day after subculturing into fresh medium supplemented with 10 lM b-estradiol or DMSO as a control. Crude extracts stained with CBB are shown in the lower panels for loading comparison. Changes in fresh weight (B), cell number (C) and cell area (D) of AtRBR1 RNAi MM2d cells in batch-suspension culture are shown. Cell number and cell area were measured after cells had protoplasted. Stationary-phase cells (7 days after subculture) were subcultured into fresh medium and incubated for a further 7 days with 10 lM b-estradiol or DMSO. Fresh weight, cell number and cell area were determined daily; error bars represent the SD (B, C) or SE (D) determined from three samples. (E) Fluorescence images of propidium iodideand Calcofluor-stained cells from estrogen-inducible AtRBR1 RNAi MM2d cells 7 days after subculturing with 10 lM b-estradiol (right) or DMSO (left). Scale bars, 10 lm. (F) The percentages of cells in G1 (black bars), G2 (grey bars), and S (white bars) are shown

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7

S G2

60

G1

20

40 0

0

1

2

3

4

5

6

7


of the cells accumulated at late G1/early S phase, and cell cycle progression was similar in both the induced and uninduced cells at 0 h. We observed that the uninduced cells accumulate higher populations of cells in G1 phase at 12 and 15 h than those in the induced cells, suggesting that downregulation of AtRBR1 leads to a prolonged G2 phase. Although protein levels of AtRBR1are depleted to undetectable levels in the induced cells before aphidicolin treatment, faint bands were visible during the 12 h after aphidicolin removal, while AtRBR1 levels were virtually unchanged in the uninduced cells (Fig. 4B). Since synchronization experiments revealed that downregulation of AtRBR1 leads to an extended G2 phase, we sought to investigate the timing of this delay by examining the expression of G2/M phase marker genes using RT-PCR.

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267

Table 1 Doubling time and phase duration in the induced and uninduced cells of AtRBR1 RNAi during exponential growth Condition

Doubling time (h)

Percentage of cells in cell cycle phase (%)

Duration of each phase (h)

G1

G1

S

G2

S

G2

18.8

52.6a

9.5a

37.9a

9.9a

1.8a

7.1a

Estrogen (induced)

17.6

a

30.8

a

a

a

a

3.3

8.9a

DMSO (uninduced)

18.8

66.3b

9.5b

24.2b

12.5b

1.8b

4.6b

17.6

b

b

b

b

b

10.9b

DMSO (uninduced)

Estrogen (induced)

18.7

31.0

7.1

50.5 61.9

5.4 5.4

1.3

From the exponential phase of the growth curve (day 2–4) under nonsynchronized conditions, the doubling time was calculated. Flow cytometry was used to determine the proportion of exponentially growing cells (day 3a and day 4b) in G1, S, and G2, and the length of each phase was calculated (Granier and Tardieu 1998)

G2/M marker genes include CYCA1;1, CYCB2;3 and CDKB2;2, and these genes are activated in late G2 phase (Menges et al. 2006). The uninduced control cells showed a normal pattern of expression, with a rapid increase of these genes observed between 9 and 12 h, whereas their upregulation was significantly less marked in the induced cells (Fig. 4C). These results suggest that downregulation of AtRBR1 leads to a reduction in the activation of G2/M gene expression and an accumulation of fewer G2-phase cells.

Downregulation of AtRBR1 causes a higher frequency of arrest in G2 phase after sucrose starvation Removal of sucrose from the growth medium of exponentially growing cells leads to a cessation of S-phase entry and a preferential arrest of cells in G1 phase within 12 h (Menges and Murray 2002; Planchais et al. 2004). To investigate the effect of AtRBR1 on the G1-to-S-phase control point, we examined the response of AtRBR1 RNAi MM2d cells to sucrose deprivation. Changes in DNA content in estrogen-inducible AtRBR1 RNAi cells were monitored after 2-day-old cells were transferred into fresh medium supplemented with 0%, 0.3% or 3% sucrose, with or without 10 lM b-estradiol (Fig. 5A). Because AtRBR1 is depleted to undetectable levels 2 days after estrogen induction (Fig. 3A), the distribution and progression of cell cycle phases differed between the induced and uninduced cells at 0 h. Under these experimental conditions, cycling continues in both populations at normal (3%) levels of sucrose. However, the uninduced cells show a slight increase in G1 cells during 36 h of culturing in low (0.3%) levels of sucrose. Progression of the cell cycle is similar in both normal and low levels of sucrose in the induced cells during first 24 h of culturing, whereas the proportion of cells in G1 phase declines, and that of G2-phase cells increases, during the last 12 h in low levels of sucrose compared to control cells with 3% sucrose. A marked increase in G2-phase cells was observed in no (0%)

sucrose, reaching more than 90% after 24 h. In addition, changes in cell number were similar in 3% sucrose in the induced and uninduced cells, but cell number reached a plateau at 24 h after the cells were transferred into induced medium containing 0.3% sucrose. Cell number remained constant in 0% sucrose with or without estrogen induction, indicating that cycling is arrested after sucrose is completely depleted (Fig. 5B). We next examined the response of AtRBR1 levels to sucrose availability by western blot analysis (Fig. 5C). After cells were transferred into the induced medium, AtRBR1 levels were undetectable throughout the 36-h culturing period in 0%, 0.3% and 3% sucrose. In the uninduced medium, AtRBR1 levels remained similar in 3% sucrose. In the presence of 0.3% sucrose, however, AtRBR1 levels did not begin to decline until 12 h, while the hyper-phosphorylated form declined rapidly thereafter and the faster-migrating band also declined between 24 and 36 h. Furthermore, AtRBR1 was depleted to undetectable levels within 12 h after cells were transferred into the uninduced medium containing 0% sucrose.

Activation of E2F-regulated gene expression follows AtRBR1 downregulation Plant E2F/DP proteins regulate the expression of cell cycle and differentiation genes (De Veylder et al. 2002; Ramirez-Parra et al. 2003, 2004; Vlieghe et al. 2003; Vandepoele et al. 2005). To investigate the expression of a set of cell division genes in estrogen-inducible AtRBR1 RNAi MM2d cells, total RNA was extracted from cells in medium supplemented with 0%, 0.3% or 3% sucrose and used for quantitative real-time RT-PCR analyses (Fig. 6). As expected, the induced cells show lower levels of AtRBR1 mRNA in both cultures with 0%, 0.3% and 3% sucrose. In 3% sucrose, levels of PCNA1 (Egelkrout et al. 2002) in the induced cells were higher than those in the uninduced cells during 36 h of culturing. By contrast, a rapid reduction was observed in the levels of PCNA1, as

123

268

(A)

DMSO

%

100

80

80

60 40

S 60 G2 G1 40

20

20 0

3

6

estrogen

%

100

0

9

12

0

15

0

3

Time after aphidicolin removal (h)

6

9

12

15


DMSO

(B)

estrogen

Time after aphidicolin removal (h) 0

kDa

3

6

9

12

15

0

3

6

9

12

15

α-AtRBR1 116 CBB 116

(C)

CYCB2;3

CYCA1;1

35

R e la t i v e e x p r e s s i o n

Fig. 4 Changes in cell cycle progression after estrogen treatment of AtRBR1 RNAi MM2d cells. (A) Cytometric analysis of AtRBR1 RNAi MM2d cells after release of aphidicolin block, showing the population of cells progressing synchronously from G1/S phase. Each block represents a sample taken at 3-h intervals during 15 h culturing. DNA histograms of cytometry results are shown as the percentages of cells in G1 (black bars), G2 (grey bars), and S (white bars). (B) Immunoblot analysis of AtRBR1 in total protein extracts from estrogen-inducible AtRBR1 RNAi MM2d cells. Cells were taken at 3-h intervals after aphidicolin removal and culturing in the presence of 10 lM b-estradiol or DMSO as a control. Crude extracts stained with CBB are shown for loading comparison. (C) Real-time RTPCR analyses of G2/M marker genes are shown, relative to ACT8 expression, in the presence of 10 lM b-estradiol (black bars) or DMSO (grey bars) as a control. Error bars indicate SD; n = 3

Plant Mol Biol (2008) 66:259–275

25

30

20

25 20

15

15

10

10 5

5 0

0

3

6

9

12

15


0

0

3

6

9

12

15


CDKB2;2 R e la t i v e e x p r e s s i o n

50 40 30

DMSO estrogen

20 10 0

0

3

6

9

12

15


well as of two other E2F-regulated genes, ORC6 (DiazTrivino et al. 2005) and RNR (Chaboute et al. 2000, 2002), at 24 h in 0.3% sucrose in the uninduced cells. Reduction was also observed, albeit to a somewhat smaller extent, for CDC6 (Castellano et al. 2001), MCM2 and MCM3 (Stevens et al. 2002) in 0.3% sucrose (Fig. 6). Additionally, a rapid decline occurred in the levels of all these genes at 12 h in 0% sucrose. In the induced cells, however, the expression levels remained similar or increased between 24 and 36 h. Levels of ACT2 varied only subtly during 36 h of culturing, and differences between the induced and uninduced cells in cultures with 0%, 0.3% and 3% sucrose were small, suggesting that downregulation of AtRBR1 resulted in a remarkable activation of E2F-regulated genes, most

123

notably in the induced cells between 0 and 12 h in 0% sucrose and between 24 and 36 h in 0.3% sucrose. We conclude that downregulation of AtRBR1 resulted in failure of repression of E2F-regulated genes in the limited sucrose condition.

AtRBR1 and E2Fa bind to the PCNA1 promoter in vivo We next conducted ChIP assays to determine whether AtRBR1 can bind to the promoter regions of E2F-regulated genes. Because epitopes of the peptide antibody against AtRBR1 are likely to be inaccessible during immunoprecipitations, we used a rabbit polyclonal antibody against

Plant Mol Biol (2008) 66:259–275

0%

0.3%

3%

(A)

DMSO

% 100 80

80

60

60

40

40

20

20

0

0

100

100

80

80

60

60

40

40

20

20

0

0

100

100

80

80

60

60

40

40

20

20 0

12

24

36

0

Time after medium exchange (h)

3%

(B)

× 107 1.2

DMSO

0.3%

S G2 G1 0

12

24

36


(C)

estrogen

estrogen

DMSO


1.0

kDa

0

12

24

36

0

12

24

36

α-AtRBR1 116

0.8

3%

0.6

CBB 116

0.4

α-AtRBR1 116

0.2 1.4

C e ll n u m b e r / m l

estrogen

% 100

0

C e ll n u m b e r /m l

Fig. 5 Altered cell cycle progression in sucrose-starved cells following RNAi-induced downregulation of AtRBR1. (A) Changes in DNA content in estrogen-inducible AtRBR1 RNAi cells after 2-day-old cells were subcultured into fresh medium supplemented with 0%, 0.3% or 3% sucrose, with or without addition of 10 lM b-estradiol. DNA histogram of the percentages of cells in G1 (black bars), G2 (grey bars), and S (white bars) are shown. (B) Cell number was measured daily; error bars represent the SD determined from three samples. (C) Immunoblot analysis of AtRBR1 in total protein extracts from estrogeninducible AtRBR1 RNAi MM2d cells. Cells were taken at 12-h intervals after subculturing into fresh medium supplemented with 0%, 0.3% or 3% sucrose in the presence of 10 lM b-estradiol or DMSO as a control. Crude extracts stained with CBB are shown in the lower panels, for each condition, as a loading comparison

269

0.3%

CBB 116

1.2 1.0

α-AtRBR1 116 0%

0.8

CBB 116

0.6 0.4

0%

C e ll n u m b e r / m l

0.2 1.0 0.8 0.6 0.4 0.2

0

12

24

36


the C-terminal portion of tobacco NtRBR1 for ChIP assays to avoid generating false negatives (Kawamura et al. 2006). Specificity of the anti-NtRBR1 antibody was verified and a corresponding band was detected when antiNtRBR1 antibody was used for immunoprecipitations (Fig. 7A). Rather than using anti-E2Fa antibody, the E2Fa coding region was C-terminally ligated to sequences encoding a 3-HA (haemagglutinin) tag, and the resulting

fusion, under the control of the E2Fa promoter, was introduced into MM2d cells. Native HA-tagged E2Fa was then immunoprecipitated from WCE of an exponentialphase culture with anti-HA antibody, and quantitative immunoprecipitation of E2Fa-3HA by the antibody was demonstrated (Fig. 7B). Figure 7C includes a diagram of the PCNA1 gene that shows the positions of the PCR primers used for the ChIP

123

270

Plant Mol Biol (2008) 66:259–275 PCNA1

ORC6

RNR 120

22

25 20

100

18

80

15

12

40

6 5

20

Relative expression

0

0

0

100

25

25

20

20

80

15

15

60

10

10

40

5

20

5 0

0

0

25

25

50

20

20

40

15

15

30

10

10

20

5 0

3%

60

10

12

24

36

0

0%

10

5 0

0.3%

0

12

24

36

0

0

12

24

36


Relative expression

CDC6

MCM2

MCM3

25

25

30

20

20

25

15

15

10

10

5

5

0

0

0

25

25

25

20

20

20

15

15

15

10

10

10

5

5

5

20 15 5

0

0

0

30

18

25

15

18 15

20

12

12

15

9

9

10

6

6

5

3

3

0

0

12

24

36

0

3%

10

0.3%

0%

0 0

12

24

0

36

12

24

36

Time after medium exchange (h) AtRBR1

Relative expression

16

ACT2 20

12

15

8

10

4

5

0

0

16

25

12

20

3%

DMSO

15

8

0.3%

10 4

estrogen

5

0

0

14 12 10 8 6 4 2 0

18 15 12 9

0%

6 3 0 0

12

24

36

0

12

24

36


Fig. 6 Expression analyses in estrogen-inducible AtRBR1 RNAi MM2d cells. After estrogen-inducible AtRBR1 RNAi MM2d cells were treated as described in Fig. 4, total RNA was extracted and used for quantitative real-time RT-PCR analyses. RT-PCR analyses of

123

E2F-regulated genes for estrogen induction (black bars) and DMSO control (grey bars) are shown relative to expression of the ACT8 gene. Error bars indicate the SD; n = 3

kDa

kDa 116

200

200

IP( α-HA)

(B)

E2Fa-3HA WCE

IP(normal IgG) 2µg

IP( α-RBR1) 2µg

kDa

IP(α-RBR1) 1µg

(A) WT WCE

experiments. PCR products are present in the lanes corresponding to immunoprecipitations with anti-HA and antiNtRBR1 antibodies when primer set 2 was used (Fig. 7D). Histone H3 was used as a positive control and PCR products were detected strongly in all the lanes, whereas no product appeared from either primer set 1 for PCNA1 or control primers for ACT8 when anti-HA and anti-NtRBR1 antibodies were used. These results indicate that AtRBR1 and E2Fa-3HA interact with the promoter region of PCNA1, which contains an E2F-binding site. Immunoprecipitation with normal rabbit IgG yielded no detectable band. Thus, we conclude that AtRBR1 and E2Fa can bind to the promoter region of PCNA1.

271 IP( α-NtRBR1) 2µg RNAi WCE

Plant Mol Biol (2008) 66:259–275

97

116

*

.. .. .

* 116

97

97

E2Fa-3HA

IgG 45

α-AtRBR1

α-HA

(C) -1000

-1500

-500

Primer2

Primer1

Discussion

+1

PCNA1 E2F-binding site

Interaction between AtRBR1 and E2F is regulated by phosphorylation Although AtRBR1 has been shown to interact with E2Fb (Magyar et al. 2005), the phosphorylation states of AtRBR1 have not been evaluated. We carried out in vitro pull-down assays to address this question. Under our experimental conditions, E2Fa can bind to hypo- and/or non-phosphorylated forms, but not to hyper-phosphorylated forms, of AtRBR1 (Fig. 2). We have demonstrated previously that transfecting tobacco E2F in transient assays activated a reporter gene, and this activation was repressed by co-transfection with NtRBR1 (Uemukai et al. 2005). These results suggest that plant RBR protein acts like its counterpart, RB, in animals and that the hypo- and/or nonphosphorylated forms are active, binding to repress transcriptional activation activity of E2F.

α-RBR

normal IgG

α-H3

input

α−HA

normal IgG

Primer set

α−H3

(D) AtRBR1 contains 16 putative CDK phosphorylation sites, and treatment of endogenous AtRBR1 with protein phosphatase indicates that slower- and faster-migrating bands represent hyper-phosphorylated and hypo- or non-phosphorylated forms of the protein, respectively (Fig. 1C). Our data thus provide the first evidence that AtRBR1 is highly phosphorylated during G1 to S phase in MM2d cells after partial synchronization in re-entry experiments (Fig. 1A). Although we did not clarify which sites become phosphorylated during G1 to S phase, we have shown using phospho-specific antibodies that distinct sites in tobacco NtRBR1 are phosphorylated during the cell cycle (Kawamura et al. 2006). It will be of interest to determine whether particular site(s) are phosphorylated preferentially during G1 to S phase.

input

AtRBR1 is highly phosphorylated during G1 to S phase

Primer1 Primer2 ACT8 E2FA-3HA

WT

Fig. 7 AtRBR1 and E2Fa interact with the E2F-regulated PCNA1 promoter. (A) WCE of MM2d cells was used for immunoprecipitations with anti-NtRBR1 (RBR) antibody (1 and 2 lg), and the immunoprecipitates were then subjected to western blotting with antiAtRBR1 antibody. MM2d cells were induced by 10 lM b-estradiol to express AtRBR1 RNAi, and immunoprecipitations were also performed with normal IgG as controls. Asterisks indicate immunoprecipitated bands that comigrate with a band detected in WCE of MM2d cells. (B) WCE of MM2d cells or MM2d cells expressing HA-tagged E2Fa under the control of its own promoter were used for western blotting with anti-HA antibody. The lower bands (asterisks) may be degradation products. (C) Graphic representation of the PCNA1 gene promoter region. Arrows and numbers (nt; +1 is the A of the start codon) indicate the positions of the PCR primers used for ChIP experiments. The E2F-binding site is shown. (D) Three-day-old MM2d cells (WT) and those expressing E2Fa-3HA were fixed with formaldehyde for ChIP assays. After immunoprecipitations with anti-NtRBR1 (RBR), anti-HA or anti-histone H3 antibodies, immunocomplexes were used for PCR with PCNA1 promoter-specific primers and ACT8 primers as a control. Input lanes represent 2% of the immunoprecipitated material (50-fold dilution). IgG indicates that ChIP was performed with normal IgG

Downregulation of AtRBR1 leads to accumulation of G2-phase cells We generated estrogen-inducible AtRBR1 RNAi MM2d cells (Fig. 3A). Because AtRBR1 is highly phosphorylated during G1 to S phase (Fig. 1A), we wished to examine whether AtRBR1 is involved in G1-checkpoint control and

123

272

whether its phosphorylation state could be linked to this function. We found that the proportion of induced cells decreased in G1 and increased in G2 in stationary phase (Fig. 3F), suggesting that AtRBR1 acts as a regulator controlling the progression of the cell cycle from G1 to S phase. Overexpression of CYCD3;1 in Arabidopsis cell suspension cultures led to a shortened G1 but prolonged G2 phase, whose combined effect is unaltered cell number and doubling time (Menges et al. 2006). Although downregulation of AtRBR1 seemed to accelerate the progression from G1 to S phase, the time course of cell number in the induced cells was similar to that of the uninduced control (Fig. 3C), suggesting that doubling time is similar between the induced and uninduced cells. Given that AtRBR1 is most likely acting downstream of CYCD3;1 in the CYCD/ RBR/E2F pathway, and that AtRBR1 function appears to be regulated by CYCD3;1 activity, a similar compensation mechanism may operate in cell cycle control in cells downregulating AtRBR1. Supporting this scenario, synchronization experiments revealed that downregulation of AtRBR1 leads to reduced activation of G2/M gene expression and accumulation of a smaller population of G2-phase cells (Fig. 4A, C). We also found that downregulation of AtRBR1 led to a smaller cell size, which is probably responsible for a reduced cell weight relative to the uninduced control (Fig. 3B). Inhibition of RBR function by virus-induced gene silencing also resulted in increased numbers of smaller cells in stems (Park et al. 2005). Mutants of the RB homolog mat3 in Chlamydomonas did not have a shortened G1, but impaired size control led to abnormally small cell size (Umen and Goodenough 2001). Thus, AtRBR1 may have a role in cell size control. In mammalian cells, overexpression of cyclin D1 accelerates G1 phase, which leads to a shorter overall cell cycle and smaller cell size (Quelle et al. 1993). We cannot determine the mechanism of the extended G2 phase when AtRBR1 is downregulated, but a similar size control mechanism may be involved.

AtRBR1 is involved in G1-phase arrest during sucrose starvation The Arabidopsis CYCD genes CYCD2;1 and CYCD3;1 are regulated by extrinsic signals, such as sucrose availability (Riou-Khamlichi et al. 2000). CYCD3;1 expression is also regulated by the plant hormones cytokinin (Riou-Khamlichi et al. 1999) and brassinosteroids (Hu et al. 2000). CYCD3;1 is a highly unstable protein that undergoes proteasome-dependent degradation and disappears rapidly when cells are sucrose-starved (Healy et al. 2001; Planchais et al. 2004). In Arabidopsis suspension culture cells

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overexpressing CYCD3;1, CYCD3;1 partially overcomes the G1 arrest induced by sucrose removal, indicating a key role for CYCD3;1 in controlling cell cycle progression in response to sucrose availability. As AtRBR1 is potentially one of the most important target substrates during the transition from G1 to S phase, understanding its role in this process is therefore of central importance to integrating sucrose availability with cell-cycle control. To investigate this, we examined the response of AtRBR1 RNAi MM2d cells when cells were deprived of sucrose (Fig. 5A). Progression of the cell cycle was similar in both 0.3% and 3% sucrose in the induced cells during the first 24 h of culturing, while G1-phase cells were reduced and cells in G2 phase increased within the last 12 h in 0.3% sucrose compared to control cells with 3% sucrose. Moreover, a marked increase in the proportion of G2-phase cells is observed in 0% sucrose, to more than 90% after 24 h. CYCD3;1 drives the G1/S transition, and when cells are sucrose-depleted the decline in CYCD3;1 levels leads to G1 arrest, which is overcome by overexpression of CYCD3;1 (Menges et al. 2006). Downregulation of AtRBR1 has a similar effect on the cellular response to sucrose starvation, suggesting that G1 arrest in sucrosedepleted cells is regulated by levels of CYCD3;1 activity that are mediated through AtRBR1. Additionally, western blot analysis revealed that hyper-phosphorylated AtRBR1 declined rapidly after 24 h, and hypo- or non-phosphorylated AtRBR1 was also reduced between 24 and 36 h in 0.3% sucrose medium; AtRBR1 declined to undetectable levels within 12 h in 0% sucrose (Fig. 5C). The removal of sucrose resulted in a rapid fall in CYCD3;1 protein abundance mediated by a proteasome pathway (Healy et al. 2001; Planchais et al. 2004), whereas levels of CYCD2;1 were unaltered, and the CDK partner of these cyclins, CDKA, declined only after 24 h (Menges and Murray 2002). High levels of AtRBR1 mRNA persisted after 24 h when cells were sucrose-starved (Fig. 6), suggesting that AtRBR1 is degraded post-transcriptionally, but the mechanism involved remains to be clarified. Because the RepA-inducible system revealed that inactivation of RBR activates E2F-regulated genes (Desvoyes et al. 2006), we investigated the expression of a set of cell division genes in estrogen-inducible AtRBR1 RNAi MM2d cells cultured in medium with 0%, 0.3% or 3% sucrose (Fig. 6). In the uninduced cells, a rapid reduction was observed in the levels of PCNA1, as well as other E2F-regulated genes, after 24 h in 0.3% sucrose. Because AtRBR1 was depleted to undetectable levels within 12 h after cells were transferred into the uninduced medium containing 0% sucrose (Fig. 5C), this raises a question about the repression of E2F-regulated genes in the absence of AtRBR1. We found that E2Fa also declined to undetectable levels within 12 h in the

Plant Mol Biol (2008) 66:259–275

same culture conditions (data not shown), which may contribute to the repression of these genes. By contrast, no reduction occurred or levels remained high in the induced cells between 24 and 36 h. Expression of the E2F-regulated genes was reduced moderately in the induced cells between 12 and 36 h in 0% sucrose. These results suggest that downregulation of AtRBR1 resulted in the release of active E2F transcription factors and subsequent activation of E2F-regulated genes. Consistent with the requirement for AtRBR1 for repression of E2Fregulated genes, ChIP assays revealed that AtRBR1 interacted with the promoter region of PCNA1 (Fig. 7D). Many studies have shown that E2F transcription factors possess DNA-binding activity, by electrophoretic mobility shift assay using oligonucleotides containing a consensus E2F-binding site (Kosugi and Ohashi 2002a, b; RamirezParra et al. 2003; 2004; Uemukai et al. 2005; Desvoyes et al. 2006). In this study, ChIP assays revealed that AtRBR1 and E2Fa can bind in vivo to the promoter region of PCNA1, which contains an E2F-binding site (Fig. 7D). Using polyclonal antibodies against carrot DcE2F, ChIP assays indicated that the E2Fb promoter is regulated by an activating E2F, possibly E2Fa (Sozzani et al. 2006). Taken together, these data lead us to conclude that AtRBR1 is functioning to repress E2F-regulated genes, whose inactivation is a key determinant of G1 arrest after sucrose starvation. In conclusion, we show here that Arabidopsis AtRBR1 is highly phosphorylated during G1 to S phase, and that only hypo- or un-phosphorylated AtRBR1 can bind to E2F. Moreover, downregulation of AtRBR1 in MM2d cells leads to a higher frequency of arrest in G2 phase in stationary phase and after sucrose starvation, and also stimulates the activation of E2F-regulated genes. AtRBR1 is therefore one of the key regulators, most likely acting downstream of D-type cyclins, involved in determination of cell cycle arrest in G1 phase after sucrose starvation. Acknowledgements The authors wish to thank Drs. Ko Kato and Kazuya Yoshida for their helpful discussions and suggestions throughout this work, and Dr. Ian Smith for critical reading of the manuscript. We also thank Dr. Masaaki Umeda for providing access to a flow cytometer. This research was supported by a Grant-in-Aid for Scientific Research from the Japanese Ministry of Education, Culture, Sports, Science and Technology and by a Grant-in-Aid for Creative Scientific Research.

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