The Plant Journal (2012) 71, 627–638
doi: 10.1111/j.1365-313X.2012.05019.x
CK2-defective Arabidopsis plants exhibit enhanced double-strand break repair rates and reduced survival after exposure to ionizing radiation Jordi Moreno-Romero1,†, Laia Armengot1, M. Mar Marque`s-Bueno1, Anne Britt2 and M. Carmen Martı´nez1,* Departament de Bioquı´mica i Biologia Molecular, Universitat Auto`noma de Barcelona, 08193 Bellaterra, Barcelona, Spain, and 2 Department of Plant Biology, University of California, Davis, CA 95616, USA
1
Received 21 April 2011; revised 28 March 2012; accepted 2 April 2012; published online 11 June 2012. *For correspondence (e-mail
[email protected]). † Present address: Department of Plant Biology and Forest Genetics, Uppsala BioCenter, Swedish University of Agricultural Sciences and Linnean Center for Plant Biology, PO Box 7080, SE-75007 Uppsala, Sweden.
SUMMARY The multifunctional protein kinase CK2 is involved in several aspects of the DNA damage response (DDR) in mammals. To gain insight into the role of CK2 in plant genome maintenance, we studied the response to genotoxic agents of an Arabidopsis CK2 dominant-negative mutant (CK2mut plants). CK2mut plants were hypersensitive to a wide range of genotoxins that produce a variety of DNA lesions. However, they were able to activate the DDR after exposure to c irradiation, as shown by accumulation of phosphorylated histone H2AX and up-regulation of sets of radio-modulated genes. Moreover, functional assays showed that mutant plants quickly repair the DNA damage produced by genotoxins, and that they exhibit preferential use of nonconservative mechanisms, which may explain plant lethality. The chromatin of CK2mut plants was more sensitive to digestion with micrococcal nuclease, suggesting compaction changes that agreed with the transcriptional changes detected for a number of genes involved in chromatin structure. Furthermore, CK2mut plants were prone to transcriptional gene silencing release upon genotoxic stress. Our results suggest that CK2 is required in the maintenance and control of genomic stability and chromatin structure in plants, and that this process affects several functions, including the DNA damage response and DNA repair. Keywords: protein kinase CK2, Arabidopsis thaliana, DNA damage responses, comet assay, MNAse sensitivity.
INTRODUCTION The integrity of DNA is continuously challenged within the cell. To counteract the severe biological consequences of DNA damage, an intricate network of genome surveillance mechanisms, often referred to as DNA damage responses (DDRs), has evolved. Most of the DDR components identified in animals and yeasts have counterparts in plants (Britt, 1996, 1999; Bray and West, 2005; Kimura and Sakaguchi, 2006). However, DNA repair pathways in animals include some components for which homologues have not been found in plants, and some DDR regulators are unique to plants (Britt, 1999; Kimura and Sakaguchi, 2006; Yoshiyama et al., 2009). The initial stages of the DDR in mammals are governed by a pair of closely related protein kinases, termed ataxia telangiectasia mutated (ATM) and ATM- and RAD3-related (ATR). ATM and ATR function as sensors of DNA damage, and control the phosphorylation of histone H2AX in regions ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd
close to damaged chromatin (Kinner et al., 2008; Mah et al., 2010). Phosphorylated H2AX (c-H2AX) mediates the formation of DNA damage foci, which are large aggregates of proteins and repair factors that surround the lesion sites (Riches et al., 2008). Plants possess ATM and ATR orthologues (Garcia et al., 2003; Culligan et al., 2004). ATM senses double-strand breaks (DSBs), triggering a transcriptional response to ionizing radiation (IR) (Garcia et al., 2003), whereas ATR senses repair intermediates or stalled replication forks and has a prominent role in the UV-induced response (Culligan et al., 2004; Yoshiyama et al., 2009; Furukawa et al., 2010). Both ATR and ATM are involved in IR-induced phosphorylation of H2AX in Arabidopsis, although ATM is responsible for the majority of focus formation in M-phase cells (Friesner et al., 2005). IR-induced DSBs are among the most harmful lesions in DNA, and must therefore be eliminated before chromosome segregation. In 627
628 Jordi Moreno-Romero et al. eukaryotes, DSBs can be repaired by two major pathways: homologous recombination (HR) and non-homologous DNA end joining (NHEJ). HR is a high-fidelity mechanism that uses unbroken, homologous sequences to template repair of DSBs. It is mostly used to repair DSB breaks due to DNA replication, using sister chromatid information (Orel et al., 2003). In contrast, NHEJ does not require significant sequence homology for DSB repair, and is a prominent mechanism in eukaryotes (Lieber, 2010). In plants, four major non-homologous recombination pathways have been described: (i) canonical non-homologous end joining (C-NHEJ), which is Ku-dependent, (ii) alternative end joining (A-EJ or A-NHEJ), which is more error-prone and uses microhomologies for recombination (also called MMEJ), (iii) the back-up pathway (B-NHEJ), which is Ku-independent and involves Parp1, Xrcc1 and DNA ligase III, and (iv) an unidentified pathway that confers severe genomic instability (Charbonnel et al., 2011). A hierarchical organization of these pathways during post-S phase has been proposed in Arabidopsis (Charbonnel et al., 2011). DNA damage responses are tightly linked to chromatin structure. In plants, mutations in replication-coupled chromatin assembly factor or chromatin assembly factor 1 (CAF1) result in sensitivity to DNA damage, de-repression of transcriptional silencing, and genome instability (Takeda et al., 2004; Kirik et al., 2006; Schonrock et al., 2006). Chromatin is also a major factor in the regulation of the HR pathway, as exemplified by different Arabidopsis mutants, such as fas1-4, which is defective in the p150 subunit of CAF1, mim, which lacks a chromatin structural component related to the structural maintenance of chromosomes (SMC) family (Mengiste et al., 1999; Kirik et al., 2006), and bru1, which encodes a nuclear protein of unknown function (Takeda et al., 2004). Protein kinase CK2 is an evolutionarily conserved Ser/Thr kinase involved in a wide variety of cellular functions (Issinger, 1993; Allende and Allende, 1995; Meggio and Pinna, 2003), including the DDR (Loizou et al., 2004). Interestingly, most DDR-related CK2 substrates in mammals are proteins that facilitate access to DNA or form a scaffold to support complexes involved in chromatin remodelling. Examples include HP1b, a heterochromatin-binding protein that facilitates access of DDR factors (Ayoub et al., 2008, 2009), XRCC1, a scaffold protein that recruits the machinery for single-strand break (SSB) repair (Loizou et al., 2004; Parsons et al., 2010) and is also involved in DSB repair in Arabidopsis (Charbonnel et al., 2010), and MDC1, an adaptor protein that interacts with the MRN (MRE11–RAD50–NBS1) complex involved in DSB repair (Chapman and Jackson, 2008; Melander et al., 2008; Spycher et al., 2008). Here we have used an inducible dominant-negative allele of CK2 to investigate the role of this protein kinase in damage tolerance and DNA repair in the higher plant Arabidopsis. We demonstrate that lack of CK2 activity
confers hypersensitivity to a variety of genotoxic agents, although CK2-defective plants displayed normal levels of IRinduced histone H2AX phosphorylation and are able to activate a transcriptional response. Our results suggest that mutant plants show preferential use of non-conservative pathways for DNA repair. Moreover, changes in chromatin structure and de-repression of transcriptional gene silencing suggest that CK2 is required in the maintenance of chromatin structure, sustaining the viability of plant cells despite the deleterious effects of genotoxic agents. RESULTS Arabidopsis plants depleted of CK2 activity are hypersensitive to genotoxins In previous studies, we demonstrated that an inducible dominant-negative allele of CK2 (CK2mut) could be successfully used to deplete CK2 activity in Arabidopsis plants (Moreno-Romero et al., 2008; Marques-Bueno et al., 2011). Here we have used the same mutant to study the involvement of CK2 in plant DDRs. We performed transient inductions of the transgene (typically treatment of 5-day-old seedlings with dexamethasone for 48 h), followed by genotoxic treatment on the 7th day. Unless otherwise indicated, uninduced CK2mut plants were used as controls. Our results show that mutant seedlings are hypersensitive to IR at 100 Gy (Figure 1a), exhibiting anthocyanin accumulation, cotyledon necrosis and developmental arrest. The effects are specifically due to accumulation of CK2mut protein, as dexamethasone treatment had no effects on c-irradiated wild-type or atm plants. The Arabidopsis atm mutant is a well-known IR-hypersensitive mutant that is defective in the DDR response. Moreover, CK2mut plants were hypersensitive to IR when dexamethasone treatment was performed before or after c irradiation (Figure 1b). As Arabidopsis mutants affected in DNA repair processes show defects in root growth under genotoxic stress (Garcia et al., 2003; Culligan et al., 2006), we measured daily root growth in postIR CK2mut seedlings. Root growth was permanently arrested upon 80 Gy treatment in CK2mut seedlings (Figure 1c, left; note that CK2mut roots were initially shorter, as previously described by Moreno-Romero et al., 2008), whereas the arrest was transient in Arabidopsis wild-type. Moreover, root viability appeared highly compromised in CK2mut plants (Figure 1c, right). We have recently shown that depletion of CK2 activity alters polar auxin transport (Marques-Bueno et al., 2011). In order to ascertain whether the IR hypersensitivity was an indirect effect of auxin transport impairment, we treated CK2mut and control seedlings with either indole-3-acetic acid (IAA) or N-1-naphtylphtalamic acid (NPA) (an inhibitor of polar auxin transport) prior to IR exposure. Quantification of plant fresh weight in post-IR 20-day-old plants showed that the IR hypersensitivity of CK2mut seedlings was not
ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 627–638
CK2 in DNA damage responses in Arabidopsis 629
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reversed by exogenous IAA (Figure S1), in contrast to other CK2mut phenotypes previously reported by Marques-Bueno et al. (2011). Moreover, NPA slightly affected the growth of post-irradiated control plants, but did not have deleterious effects. Altogether, these data led us to conclude that the hypersensitivity phenotype shown by CK2mut plants is not an indirect consequence of impaired auxin transport. We also tested the sensitivity of CK2mut plantlets to UV-C and methyl methanesulfonate (MMS). CK2mut plants were hypersensitive to UV-C radiation, showing significant growth inhibition at 30 000 J m)2 (Figure 1d), and to MMS over a range of 25–100 ppm (Figure 1e). Taken together, these results strongly suggest that CK2 is required to successfully recover from different genotoxic treatments in Arabidopsis. The genotoxins tested are known to produce different kinds of lesions into DNA, and thus CK2 may act upstream of different signalling pathways necessary for DNA repair. Alternatively, CK2 may be required at multiple points or may play a more general role in plant survival. For the subsequent studies, we focused on IR-induced responses using c irradiation. IR-induced responses have been well characterized in Arabidopsis wild-type, facilitating their study in mutant genotypes. Moreover, c irradiation has a DSB production ratio (number of DSBs/base pair versus dose) that is similar in all eukaryotes (Su, 2006). CK2 expression and activity do not significantly change after exposure to IR
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Figure 1. Hypersensitivity of CK2mut plants to genotoxins. (a–c) Responses to ionizing radiation (IR). Five-day-old seedlings were treated with dexamethasone (Dex) for 48 h and then c-irradiated. (a) Phenotype of 21-day-old seedlings after receiving 100 Gy. (b) Dose–response experiment. IR was applied either after dexamethasone treatment (+Dex +c) or before dexamethasone treatment (+c +Dex). (c) Daily root growth (left panel), and cell death detected by Evan’s blue staining (right panel) after 80 Gy IR. Scale bar = 0.3 mm. (d) Dose–response to UV-C. Experiments were performed as in (a). (e) Dose–response to methanesulfonate (MMS). Images show 14-day-old seedlings.
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The CK2a and b subunits are encoded by two small multigene families. The Arabidopsis nuclear genome contains four genes for the catalytic subunit (a) and four genes for the regulatory subunit (b) (Salinas et al., 2006). The IR-induced expression changes of CK2-encoding genes were very small, never reaching twofold, whereas TSO2 (encoding the small subunit of ribonucleotide reductase, which was used as a positive control) was highly over-expressed under the same conditions (Figure S2a). We also analyzed CK2 expression data from the AtGenExpress project, which compiles Arabidopsis data from ATH1 GeneChip arrays (Kilian et al., 2007). CK2a and b transcript levels showed no significant changes in Arabidopsis plants (18-day-old plantlets, Col-0 ecotype) treated with 1.5 lg ml)1 bleomycin (a radiomimetic) plus 22 lg ml)1 mitomycin C (a cross-linking agent)
ª 2012 The Authors The Plant Journal ª 2012 Blackwell Publishing Ltd, The Plant Journal, (2012), 71, 627–638
630 Jordi Moreno-Romero et al. (Figure S2b). Although the plant ages and genotoxins used in the array experiments were different from those employed in our work, both set of data support the idea that CK2 is not transcriptionally regulated in response to DNAdamaging agents. This is consistent with data showing that mammalian CK2 activity is regulated mainly at a posttranslational level (Filhol and Cochet, 2009). However, no significant changes of CK2 activity were found in c-irradiated Arabidopsis wild-type plantlets (Figure S2c). IR-induced global changes of gene expression in CK2mut seedlings Plants subjected to genotoxic stress become impaired in a wide number of cellular functions. Particularly well known is the IR-induced transcriptional burst in Arabidopsis plants, affecting a large number of genes (Chen et al., 2003; Nagata et al., 2005; Culligan et al., 2006; Kim et al., 2007; Ricaud et al., 2007). We analyzed transcript profiles of c-irradiated mutant plants (100 Gy) using Affymetrix ATH1 chips. The study was performed at 1.5 h post-IR, as transcript radiomodulation in Arabidopsis occurs as an early wave after IR and lasts approximately 3 h, with only 10% of the genes still showing changes 5 h post-IR (Ricaud et al., 2007). The experimental design and comparative analysis performed are summarized in Figure 2(a). Pairwise comparisons revealed different numbers of affected genes depending on the variable analyzed (Figure 2a, bottom). For instance, induction of the transgene (pairwise comparison number 2, ‘CK2mut effect’) produced the widest changes, affecting 6614 sequences (P < 0.001) out of the 22 746 sequences present in the array (after subtracting the changes obtained in plants transformed with the empty vector, shown in comparison number 1). IR-induced transcriptional changes in control plants (pairwise comparison number 3, ‘gamma effect’) correlated well with those reported in the literature for Arabidopsis wild-type plants (Table S1a). Genes showing IR-induced transcriptional changes in control and mutant seedlings were grouped into GO functional categories, depicted in Figure 2(b) (‘gamma effect’ and ‘CK2mut + gamma effect’, respectively). The number of
genes within each category is shown in Table S1a. There are four shared categories, but the number of genes within them and/or their statistical significance differ between the two conditions. Ninety genes were present in both datasets (‘CK2mut + gamma effect’ and ‘gamma effect’), 89 showing positive correlation (59 up-regulated and 30 down-regulated in both cases) and one showing negative correlation (upregulated in ‘CK2mut + gamma effect’ and down-regulated in ‘gamma effect’) (Figure 2c, left, and Table S1b). The IRinduced fold changes in expression were lower in mutant than control plants (see the slope of the correlation plot in Figure 2c, right), and this was confirmed by RT-PCR for some of these genes (Figure S3). A time-course study showed that gene under-expression in the mutant was due to weaker induction not induction delay (Figure 2d). To gain insight into the nature of the IR-induced transcriptional response, we further analyzed the genes specifically involved in DNA repair processes. One hundred and fifty genes have a known or predicted function in DNA repair (http://www.uea.ac.uk/~b270/repair.htm). Some are radiomodulated (rapidly IR-induced) (Schonrock et al., 2006; Dohmann et al., 2008), although the majority are not clearly induced after DNA damage (http://bar.utoronto.ca/ or http:// www.weigelworld.org/resources/microarray/AtGenExpress). DNA repair genes with significant changes in our experimental conditions (ANOVA, P value
