JIPB
Journal of Integrative Plant Biology
Polycomb‐group histone methyltransferase CLF is required for proper somatic recombination in Arabidopsis
Free Access
Research Article
Na Chen, Wang‐Bin Zhou, Ying‐Xiang Wang, Ai‐Wu Dong and Yu Yu* State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China. *Correspondence: [email protected]
Abstract Homologous recombination (HR) is a key process during meiosis in reproductive cells and the DNA damage repair process in somatic cells. Although chromatin structure is thought to be crucial for HR, only a small number of chromatin modifiers have been studied in HR regulation so far. Here, we investigated the function of CURLY LEAF (CLF), a Polycomb‐ group (PcG) gene responsible for histone3 lysine 27 trimethylation (H3K27me3), in somatic and meiotic HR in Arabidopsis thaliana. Although fluorescent protein reporter assays in pollen and seeds showed that the frequency of meiotic cross‐over in the loss‐of‐function mutant clf‐29 was not significantly different from that in wild type, there was a lower frequency of HR in clf‐ 29 than in wild type under normal conditions and under bleomycin treatment. The DNA damage levels were comparable between clf‐29 and wild type, even though several DNA damage repair genes (e.g. ATM, BRCA2a, RAD50, RAD51, RAD54,
and PARP2) were expressed at lower levels in clf‐29. Under bleomycin treatment, the expression levels of DNA repair genes were similar in clf‐29 and wild type, thus CLF may also regulate HR via other mechanisms. These findings expand the current knowledge of PcG function and contribute to general interests of epigenetic regulation in genome stability regulation.
INTRODUCTION
tion of the histone H2A variant H2AX (Sharma et al. 2012). Studies in yeast and mammalian cells have shown that phosphorylation of a serine residue at the C terminus of H2AX occurs rapidly after DNA breakage. This phosphorylation helps to recruit the key protein RAD51, histone modifiers such as NuA4 histone acetyltransferase, chromatin remodelers like INO80, and cohesion, the sister chromatid‐pairing protein (van Attikum and Gasser 2005; Sharma et al. 2012). Recently, other histone modifications, including H2B phosphorylation and ubiquitylation, H4 phosphorylation, H3 and H4 acetylations, and even H3 and H4 methylations, which are considered to be the relatively stable chromatin markers, have also been implicated in the DNA damage response (van Attikum and Gasser 2005; Faucher and Wellinger 2010). Studies on mammalian cells and yeast suggested that the methylation of lysine 79 on histone H3 (H3K79) and lysine 20 on histone H4 (H4K20) can control the recruitment of the DNA damage response mediator 53BP1 and its yeast ortholog Crb2, respectively, to DNA damage sites (Huyen et al. 2004; Sanders et al. 2004). The trimethylation of H3K4 (H3K4me3), which is known as a transcription activation marker, is also important for the proper response of budding yeast cells to DNA damaging agents (Faucher and Wellinger 2010). Studies on yeast and mouse cells also revealed the involvement of H3K4me3 in double‐strand break (DSB) formation during the initiation of meiotic recombination (Borde et al. 2009; Buard et al. 2009).
Homologous recombination (HR) is a powerful mechanism in biological processes (Schuermann et al. 2005). During meiosis, HR produces new combinations of DNA sequences by chromosome pairing and exchange, hence generating genetic variation in offspring (Wijeratne and Ma 2007). In somatic cells, HR maintains genome integrity and stability through the accurate repair of DNA lesions, thus contributing to the normal growth and development of organisms (Krejci et al. 2012). Multiple molecular mechanisms have evolved to regulate HR to ensure it occurs at the right time, in the proper place, and in an appropriate manner. One of the central players in HR is the RAD51 protein, which binds the ends of DNA breaks to form RAD51‐DNA nucleoprotein filaments, thereby mediating homologous pairing and strand exchange (Krejci et al. 2012). Other factors, such as BRCA1 and RAD54, are also involved in promoting the assembly and stabilization of nucleoprotein complexes and are crucial for HR (Heyer et al. 2010; Krejci et al. 2012). Chromatin is a highly ordered structure that packages genomic DNA, histones, and other molecules. Modifications to chromatin such as DNA methylation and histone modifications are important for all DNA‐related processes, including transcription, replication, recombination, and repair of DNA damage. In recent years, an increasing number of studies have focused on HR regulation in the context of chromatin. One crucial histone modification involved in HR is the phosphorylaJune 2014 | Volume 56 | Issue 6 | 550–558
Keywords: Arabidopsis; CLF; DNA repair; homologous recombination; meiosis Citation: Chen N, Zhou WB, Wang YX, Dong AW, Yu Y (2014) Polycomb‐ group histone methyltransferase CLF is required for proper somatic recombination in Arabidopsis. J Integr Plant Biol 56: 550–558. doi: 10.1111/jipb.12157 Edited by: John J. Harada, University of California, Davis, USA Received Sept. 9, 2013; Accepted Dec. 30, 2013 Available online on Jan. 7, 2014 at www.wileyonlinelibrary.com/ journal/jipb © 2014 Institute of Botany, Chinese Academy of Sciences
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Role of CLF in homologous recombination Methylations of H3K9 and H3K27 on the N‐terminal tail of histone H3 are well‐known epigenetic markers associated with repressive chromatin. A study on histone markers of transcriptional silencing after ionizing irradiation showed that H3K27me3, but not H3K9me2, accumulated in chromatin regions labeled by g‐H2AX (phosphorylation of H2AX at Ser 139) in mammalian cells (Seiler et al. 2011). The mammalian Polycomb‐ group (PcG) protein, H3K27 methyltransferase EZH2, was found to be recruited to sites of DNA damage (Chou et al. 2010). In Arabidopsis thaliana, genome‐wide analysis showed that H3K27me3 is highly enriched in a large number of genes within euchromatin (Zhang et al. 2007). Three EZH2 homologs, MEDEA (MEA), CURLY LEAF (CLF), and SWINGER (SWN), are known to be responsible for catalyzing H3K27 methylation in Arabidopsis (Yu et al. 2009). MEDEA functions specifically in the female gametophyte and during seed development (Grossniklaus et al. 1998). CLF is widely expressed, and controls leaf and flower morphology, as well as flowering time, via repressing floral homeotic gene AGAMOUS (AG) and the Class I KNOX gene SHOOT MERISTEMLESS (STM) by H3K27me3 (Goodrich et al. 1997; Schubert et al. 2006). CLF is also required for WUSCHEL (WUS) repression, which is important in controlling the fate of the floral meristem (Liu et al. 2011), and to maintain root meristem activity (Aichinger et al. 2011). SWN has a partially redundant function with CLF in regulating both vegetative and reproductive development; the clf swm double mutant showed a loss‐of‐cell differentiation after germination, causing callus formation on seedlings (Chanvivattana et al. 2004). In this study, we used previously reported HR reporter constructs (Melamed‐Bessudo et al. 2005; Francis et al. 2007;
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Schuermann et al. 2009) to investigate the function of CLF in both somatic HR and meiotic HR. Although the ratio of meiotic cross‐over (CO) in clf‐29 was not significantly different from that in wild type, somatic HR was impaired in the clf‐29 mutants under normal growth conditions as well as under bleomycin treatment. We analyzed DNA damage levels and the expression levels of DNA repair genes in clf‐29 and wild type. Our results provide insights into the function of CLF in somatic HR, and therefore, increase the current state of knowledge about the biological functions of PcGs.
RESULTS Somatic intra‐ and inter‐molecular HR frequencies are reduced in clf‐29 First, we investigated the role of CLF in somatic HR using both the intramolecular HR reporter line 1406 and the intermolecular reporter line IC9C (Schuermann et al. 2009). This substrate for recombination consists of two parts of the b‐glucuronidase (GUS) gene, which partially overlap. The two parts can recombine to form a functional GUS gene after a HR event (Figure 1A, D). As shown in Figure 1B, the restored GUS activity can be visualized as a blue spot after histochemical staining. We introduced the reporter lines 1,406 and IC9C into the clf‐ 29 mutant background by genetic crosses, and the corresponding homozygous lines were analyzed to detect GUS activity. Wild‐type plants segregating from the crosses were used as the controls. For each genotype, we analyzed 50 plants to determine the proportion showing substrate recombination.
Figure 1. Somatic homologous recombination (HR) is reduced in the clf‐29 mutant compared to wild type (A) Recombination event in intramolecular HR line 1,406. Constructs consist of two fragments of the GUS gene, which partially overlap and can recombine to form a functional GUS gene after a HR event. (B) Arabidopsis leaf with blue spots, which represent a functional GUS gene (A). Arrows indicate independent HR events. (C) HR capacity in clf‐29 and wild type (WT) in intramolecular HR line 1,406, under normal conditions or bleomycin treatment. (D) Recombination event in intermolecular HR line IC9C. Recombination requires intermolecular interaction to restore a functional GUS gene via an HR. (E) HR capacity in clf‐29 and WT in intermolecular HR line IC9C, under normal conditions or bleomycin treatment. www.jipb.net
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Using the intramolecular HR line 1,406, we found that the HR capacity in the clf‐29 mutant was reduced to approximately half of that in wild type under normal growth conditions (Figure 1C). When the strand‐break generating agent bleomycin was applied at various concentrations, the HR capacity was drastically induced in both the mutant and wild type (Figure 1C). Upon bleomycin treatment, there was also an obvious reduction in the HR frequency in the mutant when compared with that in the wild type. Because the clf‐29 plants were slightly smaller than wild‐type plants, the observed HR reduction could be explained by a possible reduced number of cells in the mutant. To compare HR on an equal cell number basis, we measured the quantity of genomic DNA and determined the relative HR frequencies after normalization to the amount of genomic DNA (Figure S1). In this case, the HR reduction in clf‐29 compared with that in wild type was still significant in both untreated and bleomycin‐treated plants (Figure 1C). In the analysis using the intermolecular reporter line IC9C, clf‐29 also showed a dramatically lower HR capacity than that of wild type under normal growth conditions and under bleomycin treatment (Figure 1E). Therefore, both the intra‐ and the inter‐molecular HR assays showed that there was a lower HR frequency in the clf‐29 mutant than in wild type, thus indicating a role of CLF in somatic HR in plants. DNA damage repair genes are downregulated in clf‐29 We examined GUS reporter gene expression in different genotypes. There were similar levels of GUS expression driven by the 35S promoter in clf‐29 and wild‐type lines (Figure S2), thus excluding the possibility that differences were due to reporter gene suppression. To explore the reason for the decreased frequencies of somatic HR in clf‐29, we performed a comet assay to directly monitor the extent of DNA damage in wild‐type and clf‐29 seedlings. The comet assays were carried out using plants grown under normal conditions and those treated with bleomycin. The typical nuclei comets observed in each genotype are shown in Figure 2A. The percentage of DNA in comet tails was not significantly different between clf‐29 and wild type under both normal and bleomycin‐treated conditions. This result suggested that in somatic HR, the function of CLF is likely to be downstream of DNA damage. Next, we analyzed the expression levels of several key genes involved in HR, including RAD50, RAD51, RAD54, BRCA2a, and PARP2. We included Ataxia Telangiectasia Mutated (ATM), which encodes a signaling kinase expressed in response to DNA damage (Ismail et al. 2005), in this analysis. Consistent with the decreased HR frequencies in clf‐29, all of the tested genes were downregulated in clf‐29 compared with their respective expression levels in wild type under normal growth conditions (Figure 2C). Upon bleomycin treatment, HR genes and ATM were drastically induced in clf‐29 and wild type (Figure 2C), but their expression levels did not significantly differ between clf‐ 29 and wild type. Given that the HR reduction in clf‐29 was still significant after bleomycin treatment, we propose that CLF also may be involved in other aspects of the recombination machinery after the induction of HR. Meiotic CO frequencies are not significantly affected in clf‐29 In addition, we investigated the role of CLF in meiotic recombination using the previously developed Fluorescent June 2014 | Volume 56 | Issue 6 | 550–558
Tagged Line (FTL) tetrad analysis system in Arabidopsis (Francis et al. 2007). The FTL tetrad analysis system is a visual assay based on the expressions of transgenes encoding fluorescent proteins. The transgenes are expressed in the pollen of the quartet mutant (qrt1‐2), and the pollen grains resulting from a meiosis event remain physically attached as a tetrad. We crossed the mutant clf‐29 into the qrt1‐2 background, which contains three genetically linked reporter transgenes on chromosome 5. The reporter genes result in different colors of fluorescence that can be observed in the pollen grains (Figure 3A). As a control, we used wild‐type plants segregating from the cross. We monitored meiotic recombination in the F2 progeny from these crosses that were homozygous for clf‐29 (or homozygotes for CLF in the wild‐ type control) and heterozygous for fluorescent markers. Examples of the fluorescence patterns in the pollen tetrads are shown in Figure 3B. We observed no CO (NCO), single COs (SCOI1 and SCOI2), and one type of double CO (DCO) occurring between two strands. Approximately 10 flower buds from 10 plants were analyzed for each sample in either the clf‐29 or wild‐type background. The ratio of CO frequencies was 8.88% in clf‐29 and 8.90% in wild type (Figure 3C). These results indicate that in our tetrad assay, the meiotic CO frequency ratio in clf‐29 was not significantly different from that in wild type. As an alternative approach to measure meiotic recombination, we used a seed‐based assay (Melamed‐Bessudo et al. 2005) in which green and red fluorescent proteins (GFP and RFP, respectively) were expressed under the control of a seed‐specific promoter (Figure 4A, B). The marker line was introgressed into the clf‐29 mutant. Wild‐type plants segregating from the cross served as the control. For each genotype, thousands of seeds were examined under a fluorescence microscope (Figure 4C). The recombination rate, as measured by the ratio of the number of recombinant seeds showing red RFP or green GFP, was 16.20% in clf‐29 and 16.28% in wild type (Figure 4D). Thus, consistent with the tetrad assay, the seed‐ based assay showed that the meiotic CO frequency was not significantly different between clf‐29 and wild type.
DISCUSSION Chromatin structure has crucial functions in HR. To date, only a few chromatin modifiers have been studied in terms of their roles in regulating HR in plants, including the ATP‐dependent chromatin‐remodeling factor INO80, the H2A/H2B‐type histone chaperone NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) group proteins, and the H3/H4‐type histone chaperone CHROMATIN ASSEMBLY FACTOR‐1 (CAF‐1) (Fritsch et al. 2004; Endo et al. 2006; Kirik et al. 2006; Gao et al. 2012). Although previous studies showed that the repressive PcG complex and H3K27 methylation accumulate on sites of DNA damage, a specific role for H3K27 methyltransferase in HR has not been uncovered in plants so far. Here, we showed that the PcG histone methyltransferase CLF is required for somatic recombination in Arabidopsis. Using the GUS reporter system, we found that HR occurred at significantly lower frequencies in clf‐29 plants than in wild‐type plants under normal conditions and under bleomycin treatment. The levels of DNA lesions were not significantly different between clf‐29 and wild type under www.jipb.net
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Figure 2. Effects of clf‐29 on DNA damage and expression of DNA repair genes (A) Typical nuclei comets of wild type and clf‐29. (B) DNA damage levels in wild type and clf‐29 as measured by percentage of DNA nuclei tails in the comet assay. More than 100 individual nuclei were analyzed for each line. Error bars show SD. (C) Expression levels of DNA repair genes as measured by quantitative reverse transcription polymerase chain reaction. Values are relative to those in untreated wild type (set to 1). Error bars show SD. Three biological repeats were analyzed.
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Figure 3. Meiotic recombination in wild type and clf‐29 mutant as determined by the Fluorescent Tagged Line (FTL) tetrad analysis system (A) Genomic positions of FTL markers along chromosome 5 of Arabidopsis. Red, yellow, and cyan circles show FTL markers dsRED2, eYFP, and CFP, respectively. (B) Examples of tetrad fluorescent patterns with no cross‐over (NCO), two types of single cross‐overs (SCOI1 and SCOI2), and one type of double cross‐over (DCO) occurring in two strands. Schematic representation of corresponding CO events is shown at left of each tetrad class. (C) Number of tetrads observed in wild type and clf‐29; CO frequencies were calculated as follows: ((SCOI1 þ SCOI2 þ DCO 2) / total tetrads) 100%.
normal conditions and under bleomycin treatment; this suggested that CLF functions downstream of DNA damage in somatic HR. Consistent with the HR reduction in clf‐29, we found that expressions of all of the tested DNA repair genes were repressed in clf‐29 under normal conditions. It is noteworthy that clf‐29 showed a markedly decreased expression level of ATM, which encodes an essential kinase that specifically responds to DSB DNA damage (Ismail et al. 2005). ATM is a key player in the initiation of the DNA damage June 2014 | Volume 56 | Issue 6 | 550–558
response, and the reduced levels of ATM suggests that CLF may be involved in ATM‐coordinated DNA damage response. The lower recombination frequencies in clf‐29 under normal growth conditions may be strongly correlated with the deficiency of ATM expression. Under bleomycin treatment, the expression levels of the DNA repair genes and the sensitivity to the DNA damaging agent (Figure S3) were comparable between clf‐29 and wild type, but the HR reduction in clf‐29 was still significant. This result suggested that as well www.jipb.net
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Figure 4. Meiotic recombination in wild type and clf‐29 mutant as determined by seed reporter system (A) Distributions of fluorescence markers along chromosome 3 of Arabidopsis. (B) Schematic representation of cross‐over (CO) events. (C) Visualization of Arabidopsis seeds under visible light (left panel), or ultraviolet (UV) light with two different filters, one for green fluorescent protein (GFP; middle panel), and one for red fluorescent protein (RFP; right panel). R, RFP expression only; G, GFP expression only; B, expression of both RFP and GFP; N, no fluorescence markers expressed. (D) Number of corresponding seeds observed in wild type and clf‐29. Recombination rates between GFP and RFP markers are shown as CO frequencies, calculated as follows: (G þ R) / total seeds 100%.
as its role in modulating HR‐related gene expression, CLF may also play roles in other aspects of the recombination machinery after HR induction. Our analyses using the GUS reporter system showed that somatic HR was impaired in the clf‐29 mutant. However, the meiotic CO frequencies in clf‐29 were not significantly different from those in wild type, as revealed in our assays based on fluorescent markers specifically expressed in pollen and seeds. This is possibly because of the differences in sensitivity between GUS and the fluorescent protein reporter assays. Because GUS activity can be monitored with a histochemical assay, subtle changes in signal intensity can be detected in plant tissues. The www.jipb.net
differences in cell cycle and type of damage between meiotic and somatic recombination may also cause the differences of CLF function in the execution of each. Meiotic recombination happens in prophase of meiosis I and is initiated by the programmed DSBs, whereas the scope and diversity of somatic recombination are broader, which happens throughout the cell cycle and is initiated by DSBs including simple breaks, gaps, and ends with damaged based (Andersen and Sekelsky 2010). In fact, by cytological analysis, we found that the chromosome behavior in the clf‐29 mutant was almost the same as that in the wild type through all phases of meiosis (only that in the mutant is shown in Figure S4). However, we cannot exclude the possibility that CLF June 2014 | Volume 56 | Issue 6 | 550–558
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affected meiotic recombination, given that the CO frequency is highly variable along eukaryotic chromosomes. For example, there are fewer COs in centromeric regions and more abundant COs in gene‐dense regions (Drouaud et al. 2007; Giraut et al. 2011). A recent study on the meiotic CO frequency in DNA methyltransferase mutants of Arabidopsis showed that the loss‐of‐function of METHYLTRANSFERASE1 (MET1) resulted in epigenetic remodeling of CO frequencies. The met1 mutant showed increased CO frequencies in the centromeric regions, although the total numbers of COs were similar between the mutant and wild type (Yelina et al. 2012). Therefore, to test the effect of CLF on meiotic recombination, it will be interesting to investigate CO frequencies using more markers with wider chromosome coverage. Our results indicated a role of the PcG H3K27 methyltransferase CLF in somatic recombination in Arabidopsis. In other studies, it was shown that the loss‐of‐Polycomb components such as EZH2 and EZH1 resulted in increased radiation sensitivity of mammalian cells and Caenorhabditis elegans (Chou et al. 2010; Gieni et al. 2011). In contrast to the repressive H3K27me3 modification, methylated H3K4, catalyzed by Trithorax‐group (TrxG) proteins, is associated with active transcription. PcG and TrxG proteins, which catalyze methylation of histone H3K27 and H3K4, respectively, were shown to play antagonistic roles in the maintenance of cell fate in Drosophila and mammals (Simon and Tamkun 2002; Schuettengruber et al. 2009). There are several lines of evidence for the possible antagonistic functions of H3K4 and H3K27 methylation in the control of flowering time and floral identity in Arabidopsis (Alvarez‐Venegas et al. 2003; Saleh et al. 2007). SET DOMAIN GROUP 2 (SDG2) is responsible for the global genome‐wide H3K4me3 deposition (Berr et al. 2010; Guo et al. 2010). Very interestingly, a recent study showed that loss of SDG2 leads to increased DNA damage and activated expression levels of DNA repair genes such as RAD51, RAD54, and PARP1 (Yao et al. 2013). Given that the H3K27 methyltransferase CLF represses gene expression and the H3K4 methyltransferase SDG2 activates gene expression, the decreased expression levels of DNA repair genes in clf‐29 and the oppositely activated levels in sdg2 suggested that these DNA repair genes may not be direct targets of CLF and SDG2. Therefore, the functions of CLF and SDG2 likely differ in terms of their roles in regulating genome integrity and modulating chromatin function, and ultimately, their effects on HR after DNA damage. More detailed analyses should be carried out to explore the molecular mechanisms of these histone modifiers.
MATERIALS AND METHODS Plant materials and growth conditions All plant lines used in this study were derived from the Columbia ecotype (Arabidopsis thaliana L.). The mutant clf‐29 has been described previously (Xu and Shen 2008). Seeds were surface‐ sterilized (70% ethanol and 0.1% Tween 20 for 10 min) and plated on solid Murashige–Skoog (MS) medium M0255 (Duchefa, Haarlem, the Netherlands) supplemented with 0.9% sucrose and cultivated under a 16:8 h light : dark photoperiod at 21 °C. For bleomycin treatment, sterilized seeds were spread onto solid MS containing 2 mmol/L bleomycin. After 14 d, the effect of the DNA damaging agent on plant growth was evaluated. June 2014 | Volume 56 | Issue 6 | 550–558
Homologous recombination assays in somatic cells The intramolecular HR reporter line 1406 and the intermolecular reporter line IC9C (Schuermann et al. 2009) were each introduced into the clf‐29 background by genetic crosses. Wild‐ type plants segregating from the crosses were used as the control. The homozygous lines for each reporter were used for GUS histochemical staining and HR assessment as described previously (Gao et al. 2012). For bleomycin treatment, 14 d old plants were incubated in liquid MS supplemented with indicated concentrations of bleomycin for 1 d, and were then grown in bleomycin‐free MS medium for 3 d before evaluating HR events. The HR assays were repeated independently at least three times, and mean values and standard deviations were determined. Comet assay We incubated 14 d old seedlings in liquid MS or MS supplemented with indicated concentrations of bleomycin for 6 h, and then harvested the plants for the comet assay. The experimental procedures and evaluations were conducted as described elsewhere (Zhu et al. 2006). The seedlings were sliced and the nuclei suspension was mixed with the same volume of liquid 1% low melting point agarose and spread on a slide precoated with 1% normal melting point agarose. After lysis in high salt buffer (2.5 mol NaCl, 10 mmol/L Tris‐HCl, pH 7.5, 100 mmol/L ethylenediaminetetraacetic acid), the slide was placed on a horizontal gel electrophoresis unit and electrophoresis was carried out for 6 min at 40 V. After clearing, drying, and staining, images of nuclei were captured under an Imager A2 microscope (Zeiss, Jena, Germany). Signals were quantified using CometScore software (http://autocomet.com). Gene expression analysis Total RNA from untreated and bleomycin‐treated seedlings was prepared using the TRIzol kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA was then used for reverse transcription using Improm‐II reverse transcriptase (Promega, Madison, WI, USA). Gene expression was analyzed by quantitative real‐time reverse transcription polymerase chain reaction (RT‐PCR) as previously described (Sui et al. 2012). ACTIN2 was used as the reference gene. Gene‐specific primers for ATM, RAD50, RAD51, RAD54, BRCA2a, and PARP2 used in PCR are listed in Table S1. Measurement of CO frequencies by the FTL tetrad analysis system The clf‐29 mutant was crossed with lines carrying transgenic marker genes encoding fluorescent proteins (dsRED2, eYFP, and CFP), which are expressed specifically in pollen in the qrt1‐2 background; these lines were described previously (Berchowitz and Copenhaver 2008). F2 plants homozygous for clf‐29 and heterozygous for fluorescent markers were selected by monitoring patterns of pollen fluorescence in attached tetrads. We used wild‐type plants segregating from the cross as the control. To assess the three different colors of fluorescence, each tetrad was visualized using under an Imager A2 microscope (Zeiss) using each of three different fluorescent filters (red, yellow, and cyan). Pollen tetrads showing all three colors of fluorescence in two of the pollen grains were considered as NCO pollen tetrads. An SCO yielded a pollen tetrad in which one grain showed all three colors of www.jipb.net
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fluorescence, one grain showed two colors, one showed one color, and one showed no color. Double COs yielded pollen tetrads with two grains with two colors of fluorescence and two grains with one color. One recombinant pollen grain was considered to represent an SCO; two recombinant pollen grains were considered to represent a DCO. The CO frequencies were calculated using the following formula: ((SCOI1 þ SCOI2 þ DCO 2)/total tetrads) 100%.
Chou DM, Adamson B, Dephoure NE, Tan X, Nottke AC, Hurov KE, Gygi SP, Colaiacovo MP, Elledge SJ (2010) A chromatin localization screen reveals poly (ADP ribose)‐regulated recruitment of the repressive Polycomb and NuRD complexes to sites of DNA damage. Proc Natl Acad Sci USA 107: 18475–18480
Seed reporter system for meiotic recombination The clf‐29 mutant was crossed with the previously described marker line Col3‐4/20, which contains GFP and RFP markers on chromosome 3, approximately 5 cM apart (Melamed‐Bessudo et al. 2005). Wild‐type plants segregating from the cross were used as the control. The plants homozygous for clf‐29 (or homozygous for CLF in the wild‐type control) and heterozygous for the fluorescent markers were identified under a fluorescence microscope. Recombination rates between GFP and RFP markers are expressed as CO frequencies, which were calculated as follows: (G þ R) / total seeds 100%.
Endo M, Ishikawa Y, Osakabe K, Nakayama S, Kaya H, Araki T, Shibahara K, Abe K, Ichikawa H, Valentine L, Hohn B, Toki S (2006) Increased frequency of homologous recombination and T‐DNA integration in Arabidopsis CAF‐1 mutants. EMBO J 25: 5579– 5590
ACKNOWLEDGEMENTS We thank Gregory P. Copenhaver, Avraham A. Levy, and Babara Hohn for generously providing us with the FTL tetrad line, seed reporter line, and HR reporter lines (1406 and IC9C), respectively. We thank Wen‐Hui Shen for critical reading of the manuscript and helpful discussion. This work was supported by the National Basic Research Program of China (973 Program, grants nos. 2012CB910500 and 2011CB944600) and the National Natural Science Foundation of China (31371304).
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Melamed‐Bessudo C, Yehuda E, Stuitje AR, Levy AA (2005) A new seed‐ based assay for meiotic recombination in Arabidopsis thaliana. Plant J 43: 458–466 Saleh A, Al‐Abdallat A, Ndamukong I, Alvarez‐Venegas R, Avramova Z (2007) The Arabidopsis homologs of trithorax (ATX1) and enhancer of zeste (CLF) establish “bivalent chromatin marks” at the silent AGAMOUS locus. Nucleic Acids Res 35: 6290–6296 Sanders SL, Portoso M, Mata J, Bahler J, Allshire RC, Kouzarides T (2004) Methylation of histone H4 lysine 20 controls recruitment of Crb2 to sites of DNA damage. Cell 119: 603–614 Schubert D, Primavesi L, Bishopp A, Roberts G, Doonan J, Jenuwein T, Goodrich J (2006) Silencing by plant Polycomb‐group genes requires dispersed trimethylation of histone H3 at lysine 27. EMBO J 25: 4638–4649 Schuermann D, Molinier J, Fritsch O, Hohn B (2005) The dual nature of homologous recombination in plants. Trends Genet 21: 172–181 Schuermann D, Fritsch O, Lucht JM, Hohn B (2009) Replication stress leads to genome instabilities in Arabidopsis DNA polymerase delta mutants. Plant Cell 21: 2700–2714 Schuettengruber B, Ganapathi M, Leblanc B, Portoso M, Jaschek R, Tolhuis B, van Lohuizen M, Tanay A, Cavalli G (2009) Functional anatomy of Polycomb and trithorax chromatin landscapes in Drosophila embryos. PLoS Biol 7: e13 Seiler DM, Rouquette J, Schmid VJ, Strickfaden H, Ottmann C, Drexler GA, Mazurek B, Greubel C, Hable V, Dollinger G, Cremer T, Friedl AA (2011) Double‐strand break‐induced transcriptional silencing is associated with loss of tri‐methylation at H3K4. Chromosome Res 19: 883–899 Sharma A, Singh K, Almasan A (2012) Histone H2AX phosphorylation: A marker for DNA damage. Methods Mol Biol 920: 613–626 Simon JA, Tamkun JW (2002) Programming off and on states in chromatin: Mechanisms of Polycomb and trithorax group complexes. Curr Opin Genet Dev 12: 210–218 Sui P, Jin J, Ye S, Mu C, Gao J, Feng H, Shen WH, Yu Y, Dong A (2012) H3K36 methylation is critical for brassinosteroid‐regulated plant growth and development in rice. Plant J 70: 340–347 van Attikum H, Gasser SM (2005) The histone code at DNA breaks: A guide to repair? Nat Rev Mol Cell Biol 6: 757–765 Wijeratne AJ, Ma H (2007) Genetic analyses of meiotic recombination in Arabidopsis. J Integr Plant Biol 49: 1199–1207 Xu L, Shen WH (2008) Polycomb silencing of KNOX genes confines shoot stem cell niches in Arabidopsis. Curr Biol 18: 1966–1971 Yao X, Feng H, Yu Y, Dong A, Shen WH (2013) SDG2‐mediated H3K4 methylation is required for proper Arabidopsis root growth and development. PLoS ONE 8: e56537
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Yelina NE, Choi K, Chelysheva L, Macaulay M, de Snoo B, Wijnker E, Miller N, Drouaud J, Grelon M, Copenhaver GP, Mezard C, Kelly KA, Henderson IR (2012) Epigenetic remodeling of meiotic crossover frequency in Arabidopsis thaliana DNA methyltransferase mutants. PLoS Genet 8: e1002844 Yu Y, Bu Z, Shen WH, Dong A (2009) An update on histone lysine methylation in plants. Prog Nat Sci 19: 407–413 Zhang X, Clarenz O, Cokus S, Bernatavichute YV, Pellegrini M, Goodrich J, Jacobsen SE (2007) Whole‐genome analysis of histone H3 lysine 27 trimethylation in Arabidopsis. PLoS Biol 5: e129 Zhu Y, Dong A, Meyer D, Pichon O, Renou JP, Cao K, Shen WH (2006) Arabidopsis NRP1 and NRP2 encode histone chaperones and are required for maintaining postembryonic root growth. Plant Cell 18: 2879–2892
SUPPORTING INFORMATION Additional supporting information can be found in the online version of this article: Figure S1. Amount of genomic DNA in wild type and clf‐29 mutant per plant Figure S2. Relative GUS expression levels in wild type and clf‐29 mutant lines under both normal and bleomycin‐treated conditions Values are relative to the untreated wild type (set to 1). Figure S3. Sensitivity of clf‐29 seedlings to bleomycin treatment The m123‐1 seedlings are as a positive control showing hypersensitivity to bleomycin treatment, whereas clf‐29 plants do not display a significant sensitivity when compared with the wild type. Figure S4. Chromosome behavior of clf‐29 mutant Chromosomes became condensed into visible thin lines at leptotene (A). Pairing of the homologous chromosomes began at zygotene (B) and fully synapsed into thick threads at pachytene (C). Further chromosome condensation occurred at diplotene (D) and diakinesis (E), and chiasmata corresponding to cross‐overs formed at pachytene became visible. At metaphase I, all five condensed bivalents were aligned along the equatorial plate (F), and after that, homologous chromosomes were separated and moved to the opposite direction sat anaphase I (G) and telophase I (H). From prophase II to telophase II, the sister chromatids of each chromosome segregated like in mitosis, resulting in the formation of four haploid set of chromatids (I–L). Table S1. Primers for RT‐PCR.
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Journal of Integrative Plant Biology
Polycomb‐group histone methyltransferase CLF is required for proper somatic recombination in Arabidopsis
Free Access
Research Article
Na Chen, Wang‐Bin Zhou, Ying‐Xiang Wang, Ai‐Wu Dong and Yu Yu* State Key Laboratory of Genetic Engineering, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 200433, China. *Correspondence: [email protected]
Abstract Homologous recombination (HR) is a key process during meiosis in reproductive cells and the DNA damage repair process in somatic cells. Although chromatin structure is thought to be crucial for HR, only a small number of chromatin modifiers have been studied in HR regulation so far. Here, we investigated the function of CURLY LEAF (CLF), a Polycomb‐ group (PcG) gene responsible for histone3 lysine 27 trimethylation (H3K27me3), in somatic and meiotic HR in Arabidopsis thaliana. Although fluorescent protein reporter assays in pollen and seeds showed that the frequency of meiotic cross‐over in the loss‐of‐function mutant clf‐29 was not significantly different from that in wild type, there was a lower frequency of HR in clf‐ 29 than in wild type under normal conditions and under bleomycin treatment. The DNA damage levels were comparable between clf‐29 and wild type, even though several DNA damage repair genes (e.g. ATM, BRCA2a, RAD50, RAD51, RAD54,
and PARP2) were expressed at lower levels in clf‐29. Under bleomycin treatment, the expression levels of DNA repair genes were similar in clf‐29 and wild type, thus CLF may also regulate HR via other mechanisms. These findings expand the current knowledge of PcG function and contribute to general interests of epigenetic regulation in genome stability regulation.
INTRODUCTION
tion of the histone H2A variant H2AX (Sharma et al. 2012). Studies in yeast and mammalian cells have shown that phosphorylation of a serine residue at the C terminus of H2AX occurs rapidly after DNA breakage. This phosphorylation helps to recruit the key protein RAD51, histone modifiers such as NuA4 histone acetyltransferase, chromatin remodelers like INO80, and cohesion, the sister chromatid‐pairing protein (van Attikum and Gasser 2005; Sharma et al. 2012). Recently, other histone modifications, including H2B phosphorylation and ubiquitylation, H4 phosphorylation, H3 and H4 acetylations, and even H3 and H4 methylations, which are considered to be the relatively stable chromatin markers, have also been implicated in the DNA damage response (van Attikum and Gasser 2005; Faucher and Wellinger 2010). Studies on mammalian cells and yeast suggested that the methylation of lysine 79 on histone H3 (H3K79) and lysine 20 on histone H4 (H4K20) can control the recruitment of the DNA damage response mediator 53BP1 and its yeast ortholog Crb2, respectively, to DNA damage sites (Huyen et al. 2004; Sanders et al. 2004). The trimethylation of H3K4 (H3K4me3), which is known as a transcription activation marker, is also important for the proper response of budding yeast cells to DNA damaging agents (Faucher and Wellinger 2010). Studies on yeast and mouse cells also revealed the involvement of H3K4me3 in double‐strand break (DSB) formation during the initiation of meiotic recombination (Borde et al. 2009; Buard et al. 2009).
Homologous recombination (HR) is a powerful mechanism in biological processes (Schuermann et al. 2005). During meiosis, HR produces new combinations of DNA sequences by chromosome pairing and exchange, hence generating genetic variation in offspring (Wijeratne and Ma 2007). In somatic cells, HR maintains genome integrity and stability through the accurate repair of DNA lesions, thus contributing to the normal growth and development of organisms (Krejci et al. 2012). Multiple molecular mechanisms have evolved to regulate HR to ensure it occurs at the right time, in the proper place, and in an appropriate manner. One of the central players in HR is the RAD51 protein, which binds the ends of DNA breaks to form RAD51‐DNA nucleoprotein filaments, thereby mediating homologous pairing and strand exchange (Krejci et al. 2012). Other factors, such as BRCA1 and RAD54, are also involved in promoting the assembly and stabilization of nucleoprotein complexes and are crucial for HR (Heyer et al. 2010; Krejci et al. 2012). Chromatin is a highly ordered structure that packages genomic DNA, histones, and other molecules. Modifications to chromatin such as DNA methylation and histone modifications are important for all DNA‐related processes, including transcription, replication, recombination, and repair of DNA damage. In recent years, an increasing number of studies have focused on HR regulation in the context of chromatin. One crucial histone modification involved in HR is the phosphorylaJune 2014 | Volume 56 | Issue 6 | 550–558
Keywords: Arabidopsis; CLF; DNA repair; homologous recombination; meiosis Citation: Chen N, Zhou WB, Wang YX, Dong AW, Yu Y (2014) Polycomb‐ group histone methyltransferase CLF is required for proper somatic recombination in Arabidopsis. J Integr Plant Biol 56: 550–558. doi: 10.1111/jipb.12157 Edited by: John J. Harada, University of California, Davis, USA Received Sept. 9, 2013; Accepted Dec. 30, 2013 Available online on Jan. 7, 2014 at www.wileyonlinelibrary.com/ journal/jipb © 2014 Institute of Botany, Chinese Academy of Sciences
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Role of CLF in homologous recombination Methylations of H3K9 and H3K27 on the N‐terminal tail of histone H3 are well‐known epigenetic markers associated with repressive chromatin. A study on histone markers of transcriptional silencing after ionizing irradiation showed that H3K27me3, but not H3K9me2, accumulated in chromatin regions labeled by g‐H2AX (phosphorylation of H2AX at Ser 139) in mammalian cells (Seiler et al. 2011). The mammalian Polycomb‐ group (PcG) protein, H3K27 methyltransferase EZH2, was found to be recruited to sites of DNA damage (Chou et al. 2010). In Arabidopsis thaliana, genome‐wide analysis showed that H3K27me3 is highly enriched in a large number of genes within euchromatin (Zhang et al. 2007). Three EZH2 homologs, MEDEA (MEA), CURLY LEAF (CLF), and SWINGER (SWN), are known to be responsible for catalyzing H3K27 methylation in Arabidopsis (Yu et al. 2009). MEDEA functions specifically in the female gametophyte and during seed development (Grossniklaus et al. 1998). CLF is widely expressed, and controls leaf and flower morphology, as well as flowering time, via repressing floral homeotic gene AGAMOUS (AG) and the Class I KNOX gene SHOOT MERISTEMLESS (STM) by H3K27me3 (Goodrich et al. 1997; Schubert et al. 2006). CLF is also required for WUSCHEL (WUS) repression, which is important in controlling the fate of the floral meristem (Liu et al. 2011), and to maintain root meristem activity (Aichinger et al. 2011). SWN has a partially redundant function with CLF in regulating both vegetative and reproductive development; the clf swm double mutant showed a loss‐of‐cell differentiation after germination, causing callus formation on seedlings (Chanvivattana et al. 2004). In this study, we used previously reported HR reporter constructs (Melamed‐Bessudo et al. 2005; Francis et al. 2007;
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Schuermann et al. 2009) to investigate the function of CLF in both somatic HR and meiotic HR. Although the ratio of meiotic cross‐over (CO) in clf‐29 was not significantly different from that in wild type, somatic HR was impaired in the clf‐29 mutants under normal growth conditions as well as under bleomycin treatment. We analyzed DNA damage levels and the expression levels of DNA repair genes in clf‐29 and wild type. Our results provide insights into the function of CLF in somatic HR, and therefore, increase the current state of knowledge about the biological functions of PcGs.
RESULTS Somatic intra‐ and inter‐molecular HR frequencies are reduced in clf‐29 First, we investigated the role of CLF in somatic HR using both the intramolecular HR reporter line 1406 and the intermolecular reporter line IC9C (Schuermann et al. 2009). This substrate for recombination consists of two parts of the b‐glucuronidase (GUS) gene, which partially overlap. The two parts can recombine to form a functional GUS gene after a HR event (Figure 1A, D). As shown in Figure 1B, the restored GUS activity can be visualized as a blue spot after histochemical staining. We introduced the reporter lines 1,406 and IC9C into the clf‐ 29 mutant background by genetic crosses, and the corresponding homozygous lines were analyzed to detect GUS activity. Wild‐type plants segregating from the crosses were used as the controls. For each genotype, we analyzed 50 plants to determine the proportion showing substrate recombination.
Figure 1. Somatic homologous recombination (HR) is reduced in the clf‐29 mutant compared to wild type (A) Recombination event in intramolecular HR line 1,406. Constructs consist of two fragments of the GUS gene, which partially overlap and can recombine to form a functional GUS gene after a HR event. (B) Arabidopsis leaf with blue spots, which represent a functional GUS gene (A). Arrows indicate independent HR events. (C) HR capacity in clf‐29 and wild type (WT) in intramolecular HR line 1,406, under normal conditions or bleomycin treatment. (D) Recombination event in intermolecular HR line IC9C. Recombination requires intermolecular interaction to restore a functional GUS gene via an HR. (E) HR capacity in clf‐29 and WT in intermolecular HR line IC9C, under normal conditions or bleomycin treatment. www.jipb.net
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Using the intramolecular HR line 1,406, we found that the HR capacity in the clf‐29 mutant was reduced to approximately half of that in wild type under normal growth conditions (Figure 1C). When the strand‐break generating agent bleomycin was applied at various concentrations, the HR capacity was drastically induced in both the mutant and wild type (Figure 1C). Upon bleomycin treatment, there was also an obvious reduction in the HR frequency in the mutant when compared with that in the wild type. Because the clf‐29 plants were slightly smaller than wild‐type plants, the observed HR reduction could be explained by a possible reduced number of cells in the mutant. To compare HR on an equal cell number basis, we measured the quantity of genomic DNA and determined the relative HR frequencies after normalization to the amount of genomic DNA (Figure S1). In this case, the HR reduction in clf‐29 compared with that in wild type was still significant in both untreated and bleomycin‐treated plants (Figure 1C). In the analysis using the intermolecular reporter line IC9C, clf‐29 also showed a dramatically lower HR capacity than that of wild type under normal growth conditions and under bleomycin treatment (Figure 1E). Therefore, both the intra‐ and the inter‐molecular HR assays showed that there was a lower HR frequency in the clf‐29 mutant than in wild type, thus indicating a role of CLF in somatic HR in plants. DNA damage repair genes are downregulated in clf‐29 We examined GUS reporter gene expression in different genotypes. There were similar levels of GUS expression driven by the 35S promoter in clf‐29 and wild‐type lines (Figure S2), thus excluding the possibility that differences were due to reporter gene suppression. To explore the reason for the decreased frequencies of somatic HR in clf‐29, we performed a comet assay to directly monitor the extent of DNA damage in wild‐type and clf‐29 seedlings. The comet assays were carried out using plants grown under normal conditions and those treated with bleomycin. The typical nuclei comets observed in each genotype are shown in Figure 2A. The percentage of DNA in comet tails was not significantly different between clf‐29 and wild type under both normal and bleomycin‐treated conditions. This result suggested that in somatic HR, the function of CLF is likely to be downstream of DNA damage. Next, we analyzed the expression levels of several key genes involved in HR, including RAD50, RAD51, RAD54, BRCA2a, and PARP2. We included Ataxia Telangiectasia Mutated (ATM), which encodes a signaling kinase expressed in response to DNA damage (Ismail et al. 2005), in this analysis. Consistent with the decreased HR frequencies in clf‐29, all of the tested genes were downregulated in clf‐29 compared with their respective expression levels in wild type under normal growth conditions (Figure 2C). Upon bleomycin treatment, HR genes and ATM were drastically induced in clf‐29 and wild type (Figure 2C), but their expression levels did not significantly differ between clf‐ 29 and wild type. Given that the HR reduction in clf‐29 was still significant after bleomycin treatment, we propose that CLF also may be involved in other aspects of the recombination machinery after the induction of HR. Meiotic CO frequencies are not significantly affected in clf‐29 In addition, we investigated the role of CLF in meiotic recombination using the previously developed Fluorescent June 2014 | Volume 56 | Issue 6 | 550–558
Tagged Line (FTL) tetrad analysis system in Arabidopsis (Francis et al. 2007). The FTL tetrad analysis system is a visual assay based on the expressions of transgenes encoding fluorescent proteins. The transgenes are expressed in the pollen of the quartet mutant (qrt1‐2), and the pollen grains resulting from a meiosis event remain physically attached as a tetrad. We crossed the mutant clf‐29 into the qrt1‐2 background, which contains three genetically linked reporter transgenes on chromosome 5. The reporter genes result in different colors of fluorescence that can be observed in the pollen grains (Figure 3A). As a control, we used wild‐type plants segregating from the cross. We monitored meiotic recombination in the F2 progeny from these crosses that were homozygous for clf‐29 (or homozygotes for CLF in the wild‐ type control) and heterozygous for fluorescent markers. Examples of the fluorescence patterns in the pollen tetrads are shown in Figure 3B. We observed no CO (NCO), single COs (SCOI1 and SCOI2), and one type of double CO (DCO) occurring between two strands. Approximately 10 flower buds from 10 plants were analyzed for each sample in either the clf‐29 or wild‐type background. The ratio of CO frequencies was 8.88% in clf‐29 and 8.90% in wild type (Figure 3C). These results indicate that in our tetrad assay, the meiotic CO frequency ratio in clf‐29 was not significantly different from that in wild type. As an alternative approach to measure meiotic recombination, we used a seed‐based assay (Melamed‐Bessudo et al. 2005) in which green and red fluorescent proteins (GFP and RFP, respectively) were expressed under the control of a seed‐specific promoter (Figure 4A, B). The marker line was introgressed into the clf‐29 mutant. Wild‐type plants segregating from the cross served as the control. For each genotype, thousands of seeds were examined under a fluorescence microscope (Figure 4C). The recombination rate, as measured by the ratio of the number of recombinant seeds showing red RFP or green GFP, was 16.20% in clf‐29 and 16.28% in wild type (Figure 4D). Thus, consistent with the tetrad assay, the seed‐ based assay showed that the meiotic CO frequency was not significantly different between clf‐29 and wild type.
DISCUSSION Chromatin structure has crucial functions in HR. To date, only a few chromatin modifiers have been studied in terms of their roles in regulating HR in plants, including the ATP‐dependent chromatin‐remodeling factor INO80, the H2A/H2B‐type histone chaperone NUCLEOSOME ASSEMBLY PROTEIN1 (NAP1) group proteins, and the H3/H4‐type histone chaperone CHROMATIN ASSEMBLY FACTOR‐1 (CAF‐1) (Fritsch et al. 2004; Endo et al. 2006; Kirik et al. 2006; Gao et al. 2012). Although previous studies showed that the repressive PcG complex and H3K27 methylation accumulate on sites of DNA damage, a specific role for H3K27 methyltransferase in HR has not been uncovered in plants so far. Here, we showed that the PcG histone methyltransferase CLF is required for somatic recombination in Arabidopsis. Using the GUS reporter system, we found that HR occurred at significantly lower frequencies in clf‐29 plants than in wild‐type plants under normal conditions and under bleomycin treatment. The levels of DNA lesions were not significantly different between clf‐29 and wild type under www.jipb.net
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Figure 2. Effects of clf‐29 on DNA damage and expression of DNA repair genes (A) Typical nuclei comets of wild type and clf‐29. (B) DNA damage levels in wild type and clf‐29 as measured by percentage of DNA nuclei tails in the comet assay. More than 100 individual nuclei were analyzed for each line. Error bars show SD. (C) Expression levels of DNA repair genes as measured by quantitative reverse transcription polymerase chain reaction. Values are relative to those in untreated wild type (set to 1). Error bars show SD. Three biological repeats were analyzed.
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Figure 3. Meiotic recombination in wild type and clf‐29 mutant as determined by the Fluorescent Tagged Line (FTL) tetrad analysis system (A) Genomic positions of FTL markers along chromosome 5 of Arabidopsis. Red, yellow, and cyan circles show FTL markers dsRED2, eYFP, and CFP, respectively. (B) Examples of tetrad fluorescent patterns with no cross‐over (NCO), two types of single cross‐overs (SCOI1 and SCOI2), and one type of double cross‐over (DCO) occurring in two strands. Schematic representation of corresponding CO events is shown at left of each tetrad class. (C) Number of tetrads observed in wild type and clf‐29; CO frequencies were calculated as follows: ((SCOI1 þ SCOI2 þ DCO 2) / total tetrads) 100%.
normal conditions and under bleomycin treatment; this suggested that CLF functions downstream of DNA damage in somatic HR. Consistent with the HR reduction in clf‐29, we found that expressions of all of the tested DNA repair genes were repressed in clf‐29 under normal conditions. It is noteworthy that clf‐29 showed a markedly decreased expression level of ATM, which encodes an essential kinase that specifically responds to DSB DNA damage (Ismail et al. 2005). ATM is a key player in the initiation of the DNA damage June 2014 | Volume 56 | Issue 6 | 550–558
response, and the reduced levels of ATM suggests that CLF may be involved in ATM‐coordinated DNA damage response. The lower recombination frequencies in clf‐29 under normal growth conditions may be strongly correlated with the deficiency of ATM expression. Under bleomycin treatment, the expression levels of the DNA repair genes and the sensitivity to the DNA damaging agent (Figure S3) were comparable between clf‐29 and wild type, but the HR reduction in clf‐29 was still significant. This result suggested that as well www.jipb.net
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Figure 4. Meiotic recombination in wild type and clf‐29 mutant as determined by seed reporter system (A) Distributions of fluorescence markers along chromosome 3 of Arabidopsis. (B) Schematic representation of cross‐over (CO) events. (C) Visualization of Arabidopsis seeds under visible light (left panel), or ultraviolet (UV) light with two different filters, one for green fluorescent protein (GFP; middle panel), and one for red fluorescent protein (RFP; right panel). R, RFP expression only; G, GFP expression only; B, expression of both RFP and GFP; N, no fluorescence markers expressed. (D) Number of corresponding seeds observed in wild type and clf‐29. Recombination rates between GFP and RFP markers are shown as CO frequencies, calculated as follows: (G þ R) / total seeds 100%.
as its role in modulating HR‐related gene expression, CLF may also play roles in other aspects of the recombination machinery after HR induction. Our analyses using the GUS reporter system showed that somatic HR was impaired in the clf‐29 mutant. However, the meiotic CO frequencies in clf‐29 were not significantly different from those in wild type, as revealed in our assays based on fluorescent markers specifically expressed in pollen and seeds. This is possibly because of the differences in sensitivity between GUS and the fluorescent protein reporter assays. Because GUS activity can be monitored with a histochemical assay, subtle changes in signal intensity can be detected in plant tissues. The www.jipb.net
differences in cell cycle and type of damage between meiotic and somatic recombination may also cause the differences of CLF function in the execution of each. Meiotic recombination happens in prophase of meiosis I and is initiated by the programmed DSBs, whereas the scope and diversity of somatic recombination are broader, which happens throughout the cell cycle and is initiated by DSBs including simple breaks, gaps, and ends with damaged based (Andersen and Sekelsky 2010). In fact, by cytological analysis, we found that the chromosome behavior in the clf‐29 mutant was almost the same as that in the wild type through all phases of meiosis (only that in the mutant is shown in Figure S4). However, we cannot exclude the possibility that CLF June 2014 | Volume 56 | Issue 6 | 550–558
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affected meiotic recombination, given that the CO frequency is highly variable along eukaryotic chromosomes. For example, there are fewer COs in centromeric regions and more abundant COs in gene‐dense regions (Drouaud et al. 2007; Giraut et al. 2011). A recent study on the meiotic CO frequency in DNA methyltransferase mutants of Arabidopsis showed that the loss‐of‐function of METHYLTRANSFERASE1 (MET1) resulted in epigenetic remodeling of CO frequencies. The met1 mutant showed increased CO frequencies in the centromeric regions, although the total numbers of COs were similar between the mutant and wild type (Yelina et al. 2012). Therefore, to test the effect of CLF on meiotic recombination, it will be interesting to investigate CO frequencies using more markers with wider chromosome coverage. Our results indicated a role of the PcG H3K27 methyltransferase CLF in somatic recombination in Arabidopsis. In other studies, it was shown that the loss‐of‐Polycomb components such as EZH2 and EZH1 resulted in increased radiation sensitivity of mammalian cells and Caenorhabditis elegans (Chou et al. 2010; Gieni et al. 2011). In contrast to the repressive H3K27me3 modification, methylated H3K4, catalyzed by Trithorax‐group (TrxG) proteins, is associated with active transcription. PcG and TrxG proteins, which catalyze methylation of histone H3K27 and H3K4, respectively, were shown to play antagonistic roles in the maintenance of cell fate in Drosophila and mammals (Simon and Tamkun 2002; Schuettengruber et al. 2009). There are several lines of evidence for the possible antagonistic functions of H3K4 and H3K27 methylation in the control of flowering time and floral identity in Arabidopsis (Alvarez‐Venegas et al. 2003; Saleh et al. 2007). SET DOMAIN GROUP 2 (SDG2) is responsible for the global genome‐wide H3K4me3 deposition (Berr et al. 2010; Guo et al. 2010). Very interestingly, a recent study showed that loss of SDG2 leads to increased DNA damage and activated expression levels of DNA repair genes such as RAD51, RAD54, and PARP1 (Yao et al. 2013). Given that the H3K27 methyltransferase CLF represses gene expression and the H3K4 methyltransferase SDG2 activates gene expression, the decreased expression levels of DNA repair genes in clf‐29 and the oppositely activated levels in sdg2 suggested that these DNA repair genes may not be direct targets of CLF and SDG2. Therefore, the functions of CLF and SDG2 likely differ in terms of their roles in regulating genome integrity and modulating chromatin function, and ultimately, their effects on HR after DNA damage. More detailed analyses should be carried out to explore the molecular mechanisms of these histone modifiers.
MATERIALS AND METHODS Plant materials and growth conditions All plant lines used in this study were derived from the Columbia ecotype (Arabidopsis thaliana L.). The mutant clf‐29 has been described previously (Xu and Shen 2008). Seeds were surface‐ sterilized (70% ethanol and 0.1% Tween 20 for 10 min) and plated on solid Murashige–Skoog (MS) medium M0255 (Duchefa, Haarlem, the Netherlands) supplemented with 0.9% sucrose and cultivated under a 16:8 h light : dark photoperiod at 21 °C. For bleomycin treatment, sterilized seeds were spread onto solid MS containing 2 mmol/L bleomycin. After 14 d, the effect of the DNA damaging agent on plant growth was evaluated. June 2014 | Volume 56 | Issue 6 | 550–558
Homologous recombination assays in somatic cells The intramolecular HR reporter line 1406 and the intermolecular reporter line IC9C (Schuermann et al. 2009) were each introduced into the clf‐29 background by genetic crosses. Wild‐ type plants segregating from the crosses were used as the control. The homozygous lines for each reporter were used for GUS histochemical staining and HR assessment as described previously (Gao et al. 2012). For bleomycin treatment, 14 d old plants were incubated in liquid MS supplemented with indicated concentrations of bleomycin for 1 d, and were then grown in bleomycin‐free MS medium for 3 d before evaluating HR events. The HR assays were repeated independently at least three times, and mean values and standard deviations were determined. Comet assay We incubated 14 d old seedlings in liquid MS or MS supplemented with indicated concentrations of bleomycin for 6 h, and then harvested the plants for the comet assay. The experimental procedures and evaluations were conducted as described elsewhere (Zhu et al. 2006). The seedlings were sliced and the nuclei suspension was mixed with the same volume of liquid 1% low melting point agarose and spread on a slide precoated with 1% normal melting point agarose. After lysis in high salt buffer (2.5 mol NaCl, 10 mmol/L Tris‐HCl, pH 7.5, 100 mmol/L ethylenediaminetetraacetic acid), the slide was placed on a horizontal gel electrophoresis unit and electrophoresis was carried out for 6 min at 40 V. After clearing, drying, and staining, images of nuclei were captured under an Imager A2 microscope (Zeiss, Jena, Germany). Signals were quantified using CometScore software (http://autocomet.com). Gene expression analysis Total RNA from untreated and bleomycin‐treated seedlings was prepared using the TRIzol kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). The RNA was then used for reverse transcription using Improm‐II reverse transcriptase (Promega, Madison, WI, USA). Gene expression was analyzed by quantitative real‐time reverse transcription polymerase chain reaction (RT‐PCR) as previously described (Sui et al. 2012). ACTIN2 was used as the reference gene. Gene‐specific primers for ATM, RAD50, RAD51, RAD54, BRCA2a, and PARP2 used in PCR are listed in Table S1. Measurement of CO frequencies by the FTL tetrad analysis system The clf‐29 mutant was crossed with lines carrying transgenic marker genes encoding fluorescent proteins (dsRED2, eYFP, and CFP), which are expressed specifically in pollen in the qrt1‐2 background; these lines were described previously (Berchowitz and Copenhaver 2008). F2 plants homozygous for clf‐29 and heterozygous for fluorescent markers were selected by monitoring patterns of pollen fluorescence in attached tetrads. We used wild‐type plants segregating from the cross as the control. To assess the three different colors of fluorescence, each tetrad was visualized using under an Imager A2 microscope (Zeiss) using each of three different fluorescent filters (red, yellow, and cyan). Pollen tetrads showing all three colors of fluorescence in two of the pollen grains were considered as NCO pollen tetrads. An SCO yielded a pollen tetrad in which one grain showed all three colors of www.jipb.net
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fluorescence, one grain showed two colors, one showed one color, and one showed no color. Double COs yielded pollen tetrads with two grains with two colors of fluorescence and two grains with one color. One recombinant pollen grain was considered to represent an SCO; two recombinant pollen grains were considered to represent a DCO. The CO frequencies were calculated using the following formula: ((SCOI1 þ SCOI2 þ DCO 2)/total tetrads) 100%.
Chou DM, Adamson B, Dephoure NE, Tan X, Nottke AC, Hurov KE, Gygi SP, Colaiacovo MP, Elledge SJ (2010) A chromatin localization screen reveals poly (ADP ribose)‐regulated recruitment of the repressive Polycomb and NuRD complexes to sites of DNA damage. Proc Natl Acad Sci USA 107: 18475–18480
Seed reporter system for meiotic recombination The clf‐29 mutant was crossed with the previously described marker line Col3‐4/20, which contains GFP and RFP markers on chromosome 3, approximately 5 cM apart (Melamed‐Bessudo et al. 2005). Wild‐type plants segregating from the cross were used as the control. The plants homozygous for clf‐29 (or homozygous for CLF in the wild‐type control) and heterozygous for the fluorescent markers were identified under a fluorescence microscope. Recombination rates between GFP and RFP markers are expressed as CO frequencies, which were calculated as follows: (G þ R) / total seeds 100%.
Endo M, Ishikawa Y, Osakabe K, Nakayama S, Kaya H, Araki T, Shibahara K, Abe K, Ichikawa H, Valentine L, Hohn B, Toki S (2006) Increased frequency of homologous recombination and T‐DNA integration in Arabidopsis CAF‐1 mutants. EMBO J 25: 5579– 5590
ACKNOWLEDGEMENTS We thank Gregory P. Copenhaver, Avraham A. Levy, and Babara Hohn for generously providing us with the FTL tetrad line, seed reporter line, and HR reporter lines (1406 and IC9C), respectively. We thank Wen‐Hui Shen for critical reading of the manuscript and helpful discussion. This work was supported by the National Basic Research Program of China (973 Program, grants nos. 2012CB910500 and 2011CB944600) and the National Natural Science Foundation of China (31371304).
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SUPPORTING INFORMATION Additional supporting information can be found in the online version of this article: Figure S1. Amount of genomic DNA in wild type and clf‐29 mutant per plant Figure S2. Relative GUS expression levels in wild type and clf‐29 mutant lines under both normal and bleomycin‐treated conditions Values are relative to the untreated wild type (set to 1). Figure S3. Sensitivity of clf‐29 seedlings to bleomycin treatment The m123‐1 seedlings are as a positive control showing hypersensitivity to bleomycin treatment, whereas clf‐29 plants do not display a significant sensitivity when compared with the wild type. Figure S4. Chromosome behavior of clf‐29 mutant Chromosomes became condensed into visible thin lines at leptotene (A). Pairing of the homologous chromosomes began at zygotene (B) and fully synapsed into thick threads at pachytene (C). Further chromosome condensation occurred at diplotene (D) and diakinesis (E), and chiasmata corresponding to cross‐overs formed at pachytene became visible. At metaphase I, all five condensed bivalents were aligned along the equatorial plate (F), and after that, homologous chromosomes were separated and moved to the opposite direction sat anaphase I (G) and telophase I (H). From prophase II to telophase II, the sister chromatids of each chromosome segregated like in mitosis, resulting in the formation of four haploid set of chromatids (I–L). Table S1. Primers for RT‐PCR.
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