The rapidly proliferating cells in plant meristems must be protected from genome damage. Here, we show that the regulatory role of the Arabidopsis RETINOBLASTOMA RELATED (RBR) in cell proliferation can be separated from a novel function in safeguarding genome integrity. Upon DNA damage, RBR and its binding partner E2FA are recruited to heterochromatic γH2AX-labelled DNA damage foci in an ATM- and ATR-dependent manner. These γH2AX-labelled DNA lesions are more dispersedly occupied by the conserved repair protein, AtBRCA1, which can also co-localise with RBR foci. RBR and AtBRCA1 physically interact in vitro and in planta Genetic interaction between the RBR-silenced amiRBR and Atbrca1 mutants suggests that RBR and AtBRCA1 may function together in maintaining genome integrity. Together with E2FA, RBR is directly involved in the transcriptional DNA damage response as well as in the cell death pathway that is independent of SOG1, the plant functional analogue of p53. Thus, plant homologs and analogues of major mammalian tumour suppressor proteins form a regulatory network that coordinates cell proliferation with cell and genome integrity.

Department of Medical Chemistry Molecular Biology and Pathobiochemistry Semmelweis University Budapest Hungary

Department of Molecular Genetics Utrecht University Utrecht The Netherlands

Department of Plant Sciences Wageningen University Research Centre Wageningen The Netherlands

Institute of Microbiology CAS v v i Laboratory of Cell Reproduction Prague 4 Czech Republic

Institute of Plant Biology Biological Research Centre Szeged Hungary

Laboratory of Proteomic Research Biological Research Centre Szeged Hungary

School of Biological Sciences Centre for Systems and Synthetic Biology Royal Holloway University of London Egham UK

Technical Analytical Research Group of HAS Budapest Hungary

Komentář v

doi: 10.15252/embj.201694571 PubMed

Zobrazit více v PubMed

Amiard S, Charbonnel C, Allain E, Depeiges A, White CI, Gallego ME (2010) Distinct roles of the ATR kinase and the Mre11‐Rad50‐Nbs1 complex in the maintenance of chromosomal stability in Arabidopsis . Plant Cell 22: 3020–3033 PubMed PMC

Amiard S, Gallego ME, White CI (2013) Signaling of double strand breaks and deprotected telomeres in Arabidopsis . Front Plant Sci 4: 405 PubMed PMC

Aprelikova ON, Fang BS, Meissner EG, Cotter S, Campbell M, Kuthiala A, Bessho M, Jensen RA, Liu ET (1999) BRCA1‐associated growth arrest is RB‐dependent. Proc Natl Acad Sci USA 96: 11866–11871 PubMed PMC

Avni D, Yang H, Martelli F, Hofmann F, ElShamy WM, Ganesan S, Scully R, Livingston DM (2003) Active localization of the retinoblastoma protein in chromatin and its response to S phase DNA damage. Mol Cell 12: 735–746 PubMed

Bao Z, Hua J (2015) Linking the cell cycle with innate immunity in Arabidopsis . Mol Plant 8: 980–982 PubMed

Berckmans B, Lammens T, Van Den Daele H, Magyar Z, Bogre L, De Veylder L (2011a) Light‐dependent regulation of DEL1 is determined by the antagonistic action of E2Fb and E2Fc. Plant Physiol 157: 1440–1451 PubMed PMC

Berckmans B, Vassileva V, Schmid SP, Maes S, Parizot B, Naramoto S, Magyar Z, Alvim Kamei CL, Koncz C, Bogre L, Persiau G, De Jaeger G, Friml J, Simon R, Beeckman T, De Veylder L (2011b) Auxin‐dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins. Plant Cell 23: 3671–3683 PubMed PMC

Bester AC, Roniger M, Oren YS, Im MM, Sarni D, Chaoat M, Bensimon A, Zamir G, Shewach DS, Kerem B (2011) Nucleotide deficiency promotes genomic instability in early stages of cancer development. Cell 145: 435–446 PubMed PMC

Biedermann S, Harashima H, Chen P, Heese M, Bouyer D, Sofroni K, Schnittger A (2017) The retinoblastoma homolog RBR1 mediates localization of the repair protein RAD51 to DNA lesions in Arabidopsis . EMBO J 36: 1279–1297 PubMed PMC

Block‐Schmidt AS, Dukowic‐Schulze S, Wanieck K, Reidt W, Puchta H (2011) BRCC36A is epistatic to BRCA1 in DNA crosslink repair and homologous recombination in Arabidopsis thaliana . Nucleic Acids Res 39: 146–154 PubMed PMC

Bolte S, Cordelieres FP (2006) A guided tour into subcellular colocalization analysis in light microscopy. J Microsc 224: 213–232 PubMed

Carnevale J, Palander O, Seifried LA, Dick FA (2012) DNA damage signals through differentially modified E2F1 molecules to induce apoptosis. Mol Cell Biol 32: 900–912 PubMed PMC

Chen Z, Higgins JD, Hui JT, Li J, Franklin FC, Berger F (2011) Retinoblastoma protein is essential for early meiotic events in Arabidopsis . EMBO J 30: 744–755 PubMed PMC

Ciccia A, Elledge SJ (2010) The DNA damage response: making it safe to play with knives. Mol Cell 40: 179–204 PubMed PMC

Cimprich KA, Cortez D (2008) ATR: an essential regulator of genome integrity. Nat Rev Mol Cell Biol 9: 616–627 PubMed PMC

Cook R, Zoumpoulidou G, Luczynski MT, Rieger S, Moquet J, Spanswick VJ, Hartley JA, Rothkamm K, Huang PH, Mittnacht S (2015) Direct involvement of retinoblastoma family proteins in DNA repair by non‐homologous end‐joining. Cell Rep 10: 2006–2018 PubMed PMC

Cools T, De Veylder L (2009) DNA stress checkpoint control and plant development. Curr Opin Plant Biol 12: 23–28 PubMed

Cools T, Iantcheva A, Weimer AK, Boens S, Takahashi N, Maes S, Van den Daele H, Van Isterdael G, Schnittger A, De Veylder L (2011) The Arabidopsis thaliana checkpoint kinase WEE1 protects against premature vascular differentiation during replication stress. Plant Cell 23: 1435–1448 PubMed PMC

Coschi CH, Ishak CA, Gallo D, Marshall A, Talluri S, Wang J, Cecchini MJ, Martens AL, Percy V, Welch I, Boutros PC, Brown GW, Dick FA (2014) Haploinsufficiency of an RB‐E2F1‐Condensin II complex leads to aberrant replication and aneuploidy. Cancer Discov 4: 840–853 PubMed

Cruz‐Ramirez A, Diaz‐Trivino S, Blilou I, Grieneisen VA, Sozzani R, Zamioudis C, Miskolczi P, Nieuwland J, Benjamins R, Dhonukshe P, Caballero‐Perez J, Horvath B, Long Y, Mahonen AP, Zhang H, Xu J, Murray JA, Benfey PN, Bako L, Maree AF et al (2012) A bistable circuit involving SCARECROW‐RETINOBLASTOMA integrates cues to inform asymmetric stem cell division. Cell 150: 1002–1015 PubMed PMC

Cruz‐Ramirez A, Diaz‐Trivino S, Wachsman G, Du Y, Arteaga‐Vazquez M, Zhang H, Benjamins R, Blilou I, Neef AB, Chandler V, Scheres B (2013) A SCARECROW‐RETINOBLASTOMA protein network controls protective quiescence in the Arabidopsis root stem cell organizer. PLoS Biol 11: e1001724 PubMed PMC

Culligan KM, Robertson CE, Foreman J, Doerner P, Britt AB (2006) ATR and ATM play both distinct and additive roles in response to ionizing radiation. Plant J 48: 947–961 PubMed

Culligan KM, Britt AB (2008) Both ATM and ATR promote the efficient and accurate processing of programmed meiotic double‐strand breaks. Plant J 55: 629–638 PubMed

De Schutter K, Joubes J, Cools T, Verkest A, Corellou F, Babiychuk E, Van Der Schueren E, Beeckman T, Kushnir S, Inze D, De Veylder L (2007) Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint. Plant Cell 19: 211–225 PubMed PMC

De Veylder L, Beeckman T, Beemster GT, de Almeida Engler J, Ormenese S, Maes S, Naudts M, Van Der Schueren E, Jacqmard A, Engler G, Inze D (2002) Control of proliferation, endoreduplication and differentiation by the Arabidopsis E2Fa‐DPa transcription factor. EMBO J 21: 1360–1368 PubMed PMC

Dewitte W, Riou‐Khamlichi C, Scofield S, Healy JM, Jacqmard A, Kilby NJ, Murray JA (2003) Altered cell cycle distribution, hyperplasia, and inhibited differentiation in Arabidopsis caused by the D‐type cyclin CYCD3. Plant Cell 15: 79–92 PubMed PMC

Dewitte W, Scofield S, Alcasabas AA, Maughan SC, Menges M, Braun N, Collins C, Nieuwland J, Prinsen E, Sundaresan V, Murray JA (2007) Arabidopsis CYCD3 D‐type cyclins link cell proliferation and endocycles and are rate‐limiting for cytokinin responses. Proc Natl Acad Sci USA 104: 14537–14542 PubMed PMC

Dick FA, Rubin SM (2013) Molecular mechanisms underlying RB protein function. Nat Rev Mol Cell Biol 14: 297–306 PubMed PMC

Dissmeyer N, Weimer AK, Pusch S, De Schutter K, Alvim Kamei CL, Nowack MK, Novak B, Duan GL, Zhu YG, De Veylder L, Schnittger A (2009) Control of cell proliferation, organ growth, and DNA damage response operate independently of dephosphorylation of the Arabidopsis Cdk1 homolog CDKA;1. Plant Cell 21: 3641–3654 PubMed PMC

Flynn RL, Zou L (2011) ATR: a master conductor of cellular responses to DNA replication stress. Trends Biochem Sci 36: 133–140 PubMed PMC

Friesner JD, Liu B, Culligan K, Britt AB (2005) Ionizing radiation‐dependent gamma‐H2AX focus formation requires ataxia telangiectasia mutated and ataxia telangiectasia mutated and Rad3‐related. Mol Biol Cell 16: 2566–2576 PubMed PMC

Fulcher N, Sablowski R (2009) Hypersensitivity to DNA damage in plant stem cell niches. Proc Natl Acad Sci USA 106: 20984–20988 PubMed PMC

Furukawa T, Curtis MJ, Tominey CM, Duong YH, Wilcox BW, Aggoune D, Hays JB, Britt AB (2010) A shared DNA‐damage‐response pathway for induction of stem‐cell death by UVB and by gamma irradiation. DNA Repair 9: 940–948 PubMed

Gehl C, Waadt R, Kudla J, Mendel RR, Hansch R (2009) New GATEWAY vectors for high throughput analyses of protein‐protein interactions by bimolecular fluorescence complementation. Mol Plant 2: 1051–1058 PubMed

Gutzat R, Borghi L, Gruissem W (2012) Emerging roles of RETINOBLASTOMA‐RELATED proteins in evolution and plant development. Trends Plant Sci 17: 139–148 PubMed

Harashima H, Dissmeyer N, Schnittger A (2013) Cell cycle control across the eukaryotic kingdom. Trends Cell Biol 23: 345–356 PubMed

Harashima H, Sugimoto K (2016) Integration of developmental and environmental signals into cell proliferation and differentiation through RETINOBLASTOMA‐RELATED 1. Curr Opin Plant Biol 29: 95–103 PubMed

Harper JW, Elledge SJ (2007) The DNA damage response: ten years after. Mol Cell 28: 739–745 PubMed

Henriques R, Magyar Z, Monardes A, Khan S, Zalejski C, Orellana J, Szabados L, de la Torre C, Koncz C, Bogre L (2010) Arabidopsis S6 kinase mutants display chromosome instability and altered RBR1‐E2F pathway activity. EMBO J 29: 2979–2993 PubMed PMC

Hinds PW (2014) A little pRB can lead to big problems. Cancer Discov 4: 764–765 PubMed

Hoeijmakers JH (2009) DNA damage, aging, and cancer. N Engl J Med 361: 1475–1485 PubMed

Horvath BM, Magyar Z, Zhang Y, Hamburger AW, Bako L, Visser RG, Bachem CW, Bogre L (2006) EBP1 regulates organ size through cell growth and proliferation in plants. EMBO J 25: 4909–4920 PubMed PMC

Hu Z, Cools T, De Veylder L (2016) Mechanisms used by plants to cope with DNA damage. Annu Rev Plant Biol 67: 439–462 PubMed

Ianari A, Natale T, Calo E, Ferretti E, Alesse E, Screpanti I, Haigis K, Gulino A, Lees JA (2009) Proapoptotic function of the retinoblastoma tumor suppressor protein. Cancer Cell 15: 184–194 PubMed PMC

Johnston AJ, Kirioukhova O, Barrell PJ, Rutten T, Moore JM, Baskar R, Grossniklaus U, Gruissem W (2010) Dosage‐sensitive function of retinoblastoma related and convergent epigenetic control are required during the Arabidopsis life cycle. PLoS Genet 6: e1000988 PubMed PMC

Kobayashi K, Suzuki T, Iwata E, Nakamichi N, Suzuki T, Chen P, Ohtani M, Ishida T, Hosoya H, Muller S, Leviczky T, Pettko‐Szandtner A, Darula Z, Iwamoto A, Nomoto M, Tada Y, Higashiyama T, Demura T, Doonan JH, Hauser MT et al (2015) Transcriptional repression by MYB3R proteins regulates plant organ growth. EMBO J 34: 1992–2007 PubMed PMC

Kuwabara A, Gruissem W (2014) Arabidopsis RETINOBLASTOMA‐RELATED and Polycomb group proteins: cooperation during plant cell differentiation and development. J Exp Bot 65: 2667–2676 PubMed

Lang J, Smetana O, Sanchez‐Calderon L, Lincker F, Genestier J, Schmit AC, Houlne G, Chaboute ME (2012) Plant gammaH2AX foci are required for proper DNA DSB repair responses and colocalize with E2F factors. New Phytol 194: 353–363 PubMed

Liu L, Zhang Y, Tang S, Zhao Q, Zhang Z, Zhang H, Dong L, Guo H, Xie Q (2010) An efficient system to detect protein ubiquitination by agroinfiltration in Nicotiana benthamiana . Plant J 61: 893–903 PubMed

Magyar Z, De Veylder L, Atanassova A, Bako L, Inze D, Bogre L (2005) The role of the Arabidopsis E2FB transcription factor in regulating auxin‐dependent cell division. Plant Cell 17: 2527–2541 PubMed PMC

Magyar Z, Horvath B, Khan S, Mohammed B, Henriques R, De Veylder L, Bako L, Scheres B, Bogre L (2012) Arabidopsis E2FA stimulates proliferation and endocycle separately through RBR‐bound and RBR‐free complexes. EMBO J 31: 1480–1493 PubMed PMC

Nagy SK, Darula Z, Kallai BM, Bogre L, Banhegyi G, Medzihradszky KF, Horvath GV, Meszaros T (2015) Activation of AtMPK9 through autophosphorylation that makes it independent of the canonical MAPK cascades. Biochem J 467: 167–175 PubMed

Naouar N, Vandepoele K, Lammens T, Casneuf T, Zeller G, van Hummelen P, Weigel D, Ratsch G, Inze D, Kuiper M, De Veylder L, Vuylsteke M (2009) Quantitative RNA expression analysis with Affymetrix Tiling 1.0R arrays identifies new E2F target genes. Plant J 57: 184–194 PubMed

Narasimha AM, Kaulich M, Shapiro GS, Choi YJ, Sicinski P, Dowdy SF (2014) Cyclin D activates the Rb tumor suppressor by mono‐phosphorylation. Elife 3: e02872 PubMed PMC

Nowack MK, Harashima H, Dissmeyer N, Zhao X, Bouyer D, Weimer AK, De Winter F, Yang F, Schnittger A (2012) Genetic framework of cyclin‐dependent kinase function in Arabidopsis . Dev Cell 22: 1030–1040 PubMed

Polyn S, Willems A, De Veylder L (2015) Cell cycle entry, maintenance, and exit during plant development. Curr Opin Plant Biol 23: 1–7 PubMed

Preuss SB, Britt AB (2003) A DNA‐damage‐induced cell cycle checkpoint in Arabidopsis . Genetics 164: 323–334 PubMed PMC

Reidt W, Wurz R, Wanieck K, Chu HH, Puchta H (2006) A homologue of the breast cancer‐associated gene BARD1 is involved in DNA repair in plants. EMBO J 25: 4326–4337 PubMed PMC

Ricaud L, Proux C, Renou JP, Pichon O, Fochesato S, Ortet P, Montane MH (2007) ATM‐mediated transcriptional and developmental responses to gamma‐rays in Arabidopsis . PLoS ONE 2: e430 PubMed PMC

Riou‐Khamlichi C, Huntley R, Jacqmard A, Murray JA (1999) Cytokinin activation of Arabidopsis cell division through a D‐type cyclin. Science 283: 1541–1544 PubMed

Rosen EM (2013) BRCA1 in the DNA damage response and at telomeres. Front Genet 4: 85 PubMed PMC

Sadasivam S, DeCaprio JA (2013) The DREAM complex: master coordinator of cell cycle‐dependent gene expression. Nat Rev Cancer 13: 585–595 PubMed PMC

Sakamoto T, Inui YT, Uraguchi S, Yoshizumi T, Matsunaga S, Mastui M, Umeda M, Fukui K, Fujiwara T (2011) Condensin II alleviates DNA damage and is essential for tolerance of boron overload stress in Arabidopsis . Plant Cell 23: 3533–3546 PubMed PMC

Schepers A, Ritzi M, Bousset K, Kremmer E, Yates JL, Harwood J, Diffley JF, Hammerschmidt W (2001) Human origin recognition complex binds to the region of the latent origin of DNA replication of Epstein‐Barr virus. EMBO J 20: 4588–4602 PubMed PMC

Scheres B (2007) Stem‐cell niches: nursery rhymes across kingdoms. Nat Rev Mol Cell Biol 8: 345–354 PubMed

Sherman MH, Bassing CH, Teitell MA (2011) Regulation of cell differentiation by the DNA damage response. Trends Cell Biol 21: 312–319 PubMed PMC

Shiloh Y, Ziv Y (2013) The ATM protein kinase: regulating the cellular response to genotoxic stress, and more. Nat Rev Mol Cell Biol 14: 197–210 PubMed

Shiloh Y (2014) ATM: expanding roles as a chief guardian of genome stability. Exp Cell Res 329: 154–161 PubMed

Su TT (2006) Cellular responses to DNA damage: one signal, multiple choices. Annu Rev Genet 40: 187–208 PubMed

Trapp O, Seeliger K, Puchta H (2011) Homologs of breast cancer genes in plants. Front Plant Sci 2: 19 PubMed PMC

Vanstraelen M, Baloban M, Da Ines O, Cultrone A, Lammens T, Boudolf V, Brown SC, De Veylder L, Mergaert P, Kondorosi E (2009) APC/C‐CCS52A complexes control meristem maintenance in the Arabidopsis root. Proc Natl Acad Sci USA 106: 11806–11811 PubMed PMC

Velez‐Cruz R, Manickavinayaham S, Biswas AK, Clary RW, Premkumar T, Cole F, Johnson DG (2016) RB localizes to DNA double‐strand breaks and promotes DNA end resection and homologous recombination through the recruitment of BRG1. Genes Dev 30: 2500–2512 PubMed PMC

Waterworth WM, Drury GE, Bray CM, West CE (2011) Repairing breaks in the plant genome: the importance of keeping it together. New Phytol 192: 805–822 PubMed

Welch D, Hassan H, Blilou I, Immink R, Heidstra R, Scheres B (2007) Arabidopsis JACKDAW and MAGPIE zinc finger proteins delimit asymmetric cell division and stabilize tissue boundaries by restricting SHORT‐ROOT action. Genes Dev 21: 2196–2204 PubMed PMC

Wildwater M, Campilho A, Perez‐Perez JM, Heidstra R, Blilou I, Korthout H, Chatterjee J, Mariconti L, Gruissem W, Scheres B (2005) The RETINOBLASTOMA‐RELATED gene regulates stem cell maintenance in Arabidopsis roots. Cell 123: 1337–1349 PubMed

Xiao H, Goodrich DW (2005) The retinoblastoma tumor suppressor protein is required for efficient processing and repair of trapped topoisomerase II‐DNA‐cleavable complexes. Oncogene 24: 8105–8113 PubMed PMC

Xiong Y, McCormack M, Li L, Hall Q, Xiang C, Sheen J (2013) Glucose‐TOR signalling reprograms the transcriptome and activates meristems. Nature 496: 181–186 PubMed PMC

Yi D, Alvim Kamei CL, Cools T, Vanderauwera S, Takahashi N, Okushima Y, Eekhout T, Yoshiyama KO, Larkin J, Van den Daele H, Conklin P, Britt A, Umeda M, De Veylder L (2014) The Arabidopsis SIAMESE‐RELATED cyclin‐dependent kinase inhibitors SMR5 and SMR7 regulate the DNA damage checkpoint in response to reactive oxygen species. Plant Cell 26: 296–309 PubMed PMC

Yoshiyama K, Conklin PA, Huefner ND, Britt AB (2009) Suppressor of gamma response 1 (SOG1) encodes a putative transcription factor governing multiple responses to DNA damage. Proc Natl Acad Sci USA 106: 12843–12848 PubMed PMC

Yoshiyama KO, Kobayashi J, Ogita N, Ueda M, Kimura S, Maki H, Umeda M (2013a) ATM‐mediated phosphorylation of SOG1 is essential for the DNA damage response in Arabidopsis . EMBO Rep 14: 817–822 PubMed PMC

Yoshiyama KO, Sakaguchi K, Kimura S (2013b) DNA damage response in plants: conserved and variable response compared to animals. Biology 2: 1338–1356 PubMed PMC

Zalmas LP, Coutts AS, Helleday T, La Thangue NB (2013) E2F‐7 couples DNA damage‐dependent transcription with the DNA repair process. Cell Cycle 12: 3037–3051 PubMed PMC

Zebell SG, Dong X (2015) Cell‐cycle regulators and cell death in immunity. Cell Host Microbe 18: 402–407 PubMed PMC

MDF is a conserved splicing factor and modulates cell division and stress response in Arabidopsis

γ-Tubulin in microtubule nucleation and beyond

Microtubular and Nuclear Functions of γ-Tubulin: Are They LINCed?