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Plant, Cell and Environment (2014) 37, 2128–2138
doi: 10.1111/pce.12299
Original Article
Histone chaperone ASF1 is involved in gene transcription activation in response to heat stress in Arabidopsis thaliana Minjie Weng1,†, Yue Yang1,†, Haiyang Feng1, Zongde Pan1, Wen-Hui Shen1,2, Yan Zhu1 & Aiwu Dong1 1
State Key Laboratory of Genetic Engineering, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 20043, China and 2Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg Cédex, France
ABSTRACT ANTI-SILENCING FUNCTION 1 (ASF1) is an evolutionarily conserved histone chaperone involved in diverse chromatin-based processes in eukaryotes. Yet, its role in transcription and the underlying molecular mechanisms remain largely elusive, particularly in plants. Here, we show that the Arabidopsis thaliana ASF1 homologous genes, AtASF1A and AtASF1B, are involved in gene transcription activation in response to heat stress. The Atasf1ab mutant displays defective basal as well as acquired thermotolerance phenotypes. Heat-induced expression of several key genes, including the HEAT SHOCK PROTEIN (HSP) genes Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, and the HEAT SHOCK FACTOR (HSF) gene HsfA2 but not HsfB1 is drastically impaired in Atasf1ab as compared with that in wild type. We found that AtASF1A/B proteins are recruited onto chromatin, and their enrichment is correlated with nucleosome removal and RNA polymerase II accumulation at the promoter and coding regions of HsfA2 and Hsa32 but not HsfB1. Moreover, AtASF1A/B facilitate H3K56 acetylation (H3K56ac), which is associated with HsfA2 and Hsa32 activation. Taken together, our study unravels an important function of AtASF1A/B in plant heat stress response and suggests that AtASF1A/B participate in transcription activation of some but not all HSF and HSP genes via nucleosome removal and H3K56ac stimulation. Key-words: ANTI-SILENCING FUNCTION 1; transcription regulation.
INTRODUCTION Nucleosome, the basic unit of chromatin, is composed of roughly 147 base pairs (bp) of DNA wrapped around the globular histone octamer comprising one centre-positioned (H3-H4)2 tetramer and two side-positioned H2A-H2B dimers. Histone chaperones facilitate histone–DNA interactions during nucleosome assembly and/or disassembly, and Correspondence: Y. Zhu. Fax: +86 21 55665673; e-mail: zhu_yan @fudan.edu.cn; A. Dong. Fax: +86 21 55665673; e-mail: aiwudong @fudan.edu.cn †
These authors contributed equally to this work.
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play crucial roles in chromatin dynamics relevant to DNA replication, repair, recombination and transcription (Avvakumov et al. 2011). ANTI-SILENCING FUNCTION 1 (ASF1) is a highly conserved histone chaperone specific for H3-H4, and its monomeric state is detected in the form of ASF1-H3-H4 ternary complex, which is mutually exclusive from that of (H3-H4)2 tetramer (English et al. 2006; Natsume et al. 2007). This suggests that loading of ASF1 likely has an (H3-H4)2 tetramer-disrupting activity in the context of nucleosomes. ASF1 also interacts with other chromatin factors, such as CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) and HISTONE REGULATORY HOMOLOG A (HIRA), which mediate replication-dependent and replication-independent H3 incorporation into chromatin, respectively (Tagami et al. 2004). Several studies have also revealed critical importance of ASF1 in establishment of H3K56 acetylation (H3K56ac; reviewed in Avvakumov et al. 2011). K56 is located within N-terminal α-helix of H3, and its acetylation requires ASF1 for catalysis by histone acetyltransferase Rtt109 and CBP/ p300 in yeast and animal cells, respectively (Schneider et al. 2006; Das et al. 2009; Avvakumov et al. 2011). Adding acetyl interrupts histone–DNA contact and increases rate of nucleosomal DNA ends unwrapping from histone octamer (Neumann et al. 2009), and promotes nucleosome disassembly during transcriptional activation in yeast (Rufiange et al. 2007; Williams et al. 2008; Lin & Schultz 2011). The Arabidopsis thaliana genome comprises two ASF1 homologous genes, AtASF1A and AtASF1B, which play redundant roles and participate in S-phase replicationdependent chromatin assembly (Zhu et al. 2011). The loss-offunction Atasf1ab double mutant displays chromatin instability and DNA damage as well as activation of expression of cell cycle checkpoint and DNA repair genes (Zhu et al. 2011). More recently, it was shown that AtASF1A and AtASF1B are involved in UV-induced DNA damage repair, likely through interaction with the TIP60-like histone acetyltransferases HAM1 and HAM2 (Lario et al. 2013). In the present study, we report a crucial function of AtASF1A and AtASF1B in plant heat stress tolerance. Heat stress is expecting to occur more frequently as a consequence of warming climate, which seriously challenges ecological plant species demography and agricultural crop © 2014 John Wiley & Sons Ltd
AtASF1 in heat stress response yield (Saidi et al. 2011; Qu et al. 2013). During evolution, plants have evolved various molecular and physiological mechanisms to cope with heat stress. Among others, HEAT SHOCK FACTOR (HSF) and HEAT SHOCK PROTEIN (HSP) play a central role in the onset of cellular thermotolerance (Saidi et al. 2011; Qu et al. 2013). The Arabidopsis HSF family comprises over 20 members, which can be grouped into A, B and C classes based on protein structural domain organization (Nover et al. 2001). The A class members contain a C-terminal AHA domain and most act as transcription activators. Although HsfA1a/b are not heat induced and function as immediate activators in heat response (Lohmann et al. 2004; Busch et al. 2005), HsfA2 is the most potently heat-induced gene and is responsible for amplification and long-term maintenance of heat response (Charng et al. 2007). The B class members, for example, HsfB1 and HsfB2b, possess a tetrapeptide LFGV motif and generally function as transcriptional repressors (Ikeda et al. 2011). Function of the unique C class member HsfC1 remains unknown so far. At downstream of HSFs, HSPs function as molecular chaperones involved in maintenance and/or restoration of protein homeostasis under heat stress, and are grouped into Hsp100, Hsp90, Hsp70, Hsp60 and small Hsp subgroups (Swindell et al. 2007). Intriguingly, some Hsps (e.g. Hsp90, Hsp70) also participate in the feedback control of subcellular compartmentalization and activity of plant HSFs (reviewed in Scharf et al. 2012). Detailed genetic and physiological analyses have implicated HSFs and HSPs in the regulation of plant basal thermotolerance upon direct exposure to a heat stress. Meanwhile, exposure of plants to a moderate level of high temperature (e.g. 37 °C for Arabidopsis) can efficiently activate the HSF-HSP gene networks, which significantly improves plant thermotolerance to a second more severe heat stress (e.g. 45 °C for Arabidopsis; reviewed in Saidi et al. 2011; Mittler et al. 2012). This process is generally referred as acclimation or ‘priming’, and the enhanced plant ability is referred as acquired thermotolerance. Chromatin remodelling is believed to be advantageous in regulation of a large set of genes and in gene priming in plant response to stresses (Berr et al. 2012; Zhu et al. 2012). The histone H2A variant, H2A.Z, was proposed as a thermosensor and was shown to be evicted from nucleosomes at Hsp70 and other genes following exposure of Arabidopsis plants to a moderately warm temperature (27 °C; Kumar & Wigge 2010). The arp6 mutant defective in H2A.Z incorporation showed constitutive up-regulation of Hsp70 and a genome-wide expression pattern mimicking wild-type (WT) plants grown at warm temperatures (Kumar & Wigge 2010). In Brachypodium distachyon, ARP6mediated H2A.Z incorporation was reported to play an important function in thermal stress response on grain yield (Boden et al. 2013). Long heat stress, 15–30 h at 37 °C or 48 h at 42 °C, was shown to release silencing of a reporter transgene as well as some endogenous heterochromatin loci in Arabidopsis (Lang-Mladek et al. 2010; Pecinka et al. 2010; Tittel-Elmer et al. 2010). The silencing release was not associated with any detectable changes of DNA methylation or histone methylation and mutants of several chromatin
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regulators, for example, CMT3, DDM1, DRM2, MOM1 and KYP, did not show any detectable perturbation in the heat stress response (Pecinka et al. 2010; Tittel-Elmer et al. 2010). In this study, we investigate the function of AtASF1A and AtASF1B in heat stress response. We show that the loss-offunction Atasf1ab double mutant exhibits both basal and acquired thermotolerance defects associated with impaired transcription activation of some HSF and HSP genes. We provide evidence highlighting a crucial role of AtASF1A/B in heat stress gene activation via nucleosome removal and H3K56ac stimulation.
MATERIALS AND METHODS Plant material and growth conditions Atasf1a, Atasf1b and Atasf1ab mutants have been described previously (Zhu et al. 2011). Seeds were produced in growth chamber at 22 °C in a 16 h light/8 h dark photoperiod.
Transcriptome analysis Twelve-day-old seedlings of Atasf1a, Atasf1b, Atasf1ab and WT grown on agar-solidified MS medium M0255 (Duchefa; http://www.duchefa.com) at 22 °C under a light intensity of about 100 μEm−2 s−1 in a 16 h light/8 h dark photoperiod were used in transcriptome analysis. RNA was extracted using the TRIzol kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Two biological replicates were analysed for gene expression using Agilent Arabidopsis4*44K (Palo Alto, CA, USA) oligonucleotide array containing 43603 probes (Shanghai Biotechnology Corporation, Pudong, Shanghai, China). The raw data of microarray have been deposited in public database NCBIGEO (GSE48854). Data analysis was performed by the online information analysis platform based on the statistic package R (http://sas.ebioservice.com). Genes with expression changed more than twofold between WT, and the mutants are considered as differentially expressed. Gene ontology (GO) analysis was performed as previously described (Liu et al. 2009b).
Heat and oxidative stress treatments For basal thermotolerance assay, 30 WT and 30 Atasf1ab seeds were germinated on a same plate containing agarsolidified MS medium. Each determination was performed with three replicate plates. The plates were incubated at 22 °C for 30 h, placed at 45 °C for 2 h and returned to 22 °C for 10 d before examination of heat effects. For acquired thermotolerance assay, the seed plates were incubated at 22 °C for 10 d, treated at 37 °C for 1.5 h, returned to 22 °C for 2 h (2H) or 2 d (2D) for recovery, treated at 45 °C for 2 h and finally returned to 22 °C for 6 d before examination of heat effects. Heat effects on H2O2 production was detected in the roots (from 30 plants per sample) by fluorescent dye 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA) staining and visualization under fluorescence microscopy as described
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2130 M. Weng et al. previously (Kim et al. 2012). For oxidative stress treatment, over 200 12-day-old seedlings per sample were transferred to the liquid MS medium M0255 without (control) or with 20 μm Rose Bengal (RB) (R3877; Sigma, St Louis, MO, USA), and grown at 22 °C under 100 μEm−2 s−1 light. Samples were collected at described time points and analysed for gene expression.
Chlorophylls and ion leakage measurements Total chlorophylls were measured by non-maceration methods. Seedlings (100 mg per sample) were incubated in 5 mL dimethyl sulfoxide (DMSO) at 65 °C for 4 h in the dark. Absorbance was recorded at 645, 663, 470 nm in a SmartSpecTM Plus Spectrophotometer (170–2525; Bio-Rad, Hercules, CA, USA). Chlorophyll and carotenoid contents were calculated as described previously (Chauhan et al. 2012). Ten-day-old seedlings (15 plants per sample) were used for the ion leakage measurement after different heat treatments according to Hong et al. (2003). Briefly, seedlings were incubated in deionized water for 1 d, and the resulting solution was measured for conductivity. Values were normalized to the total conductivity measured on dead seedlings after autoclaving, and the resulted percentage represents the relative ion leakage activity of living seedlings.
RT-PCR and Western blot analysis RNA isolation and RT-PCR were performed as described previously (Liu et al. 2009a), and the primers for RT-PCR were listed in the Supporting Information Table S1. Total proteins were extracted from the 12-day-old seedlings before and after heat treatment at 37 °C for 1.5 h and analysed by Western blot using anti-Hsp101 (ab80123; Abcam, Cambridge, UK) and anti-Hsp17.6 (ab80183; Abcam) antibodies. Histones were prepared as described previously (Gao et al. 2012) and analysed in Western blot using anti-H3 (ab1791; Abcam) and anti-H3K56ac (07–677; Millipore, Billerica, MA, USA) antibodies.
Chromatin immunoprecipitation (ChIP) analysis About 2 g per sample of 12-day-old seedlings untreated or treated by incubation at 37 °C for 1.5 h was used for ChIP analysis. ChIP experiments were performed as described previously (Johnson et al. 2002). The anti-AtASF1 rabbit polyclonal antiserum was generated against AtASF1A peptide (N-terminal 159 aa) through a commercial service by Abmart, Inc (http://www.abmart.cn). Other antibodies used in ChIP are anti-H3 (ab1791; Abcam), anti-H3K56ac (07– 677; Millipore) and anti-Pol II (sc-900; Santa Cruz Biotechnology, Santa Cruz, CA, USA). UBC28 was used as an internal reference gene (Pecinka et al. 2009). The genespecific primers used in ChIP are listed in the Supporting Information Table S1.
In vitro histone acetyltransferase (HAT) assay The open reading frame (ORF) of yeast Rtt109 was cloned into the expression vector pGEX-4T1 (Amersham,
Piscataway, NJ, USA) using BamHI and SalI sites. The ORF of AtASF1A was cloned into the expression vector pET-14b (Novagen, Madison, WI, USA) using BglII and XhoI sites. Recombinant proteins were expressed in Escherichia coli and purified according to a previously described procedure (Dong et al. 2005). In vitro HAT assay was performed as described previously (Driscoll et al. 2007) using mixture histones (223565; Roche, Indianapolis, IN, USA) as substrates and anti-H3K56ac antibody (07–677; Millipore) in Western blot detection.
RESULTS Transcriptome analysis reveals a large number of misregulated genes in the Atasf1ab mutant To investigate AtASF1A/B function in genome transcription regulation, we performed transcriptome analysis using Agilent Arabidopsis 44K oligonucleotide microarray on 12-day-old seedlings of WT and the double-mutant Atasf1ab as well as the single mutants Atasf1a and Atasf1b (Zhu et al. 2011). Compared with WT, Atasf1ab showed 695 upregulated genes and 1204 down-regulated genes whereas Atasf1a and Atasf1b only showed small numbers of perturbed genes (Fig. 1a; Supporting Information Tables S2–S7), which is in agreement with the redundant function of AtASF1A and AtASF1B. As expected, AtASF1A and AtASF1B were among the down-regulated genes found in the respective mutants (Supporting Information Tables S5– S7). The perturbed genes in Atasf1ab are distributed along all the five Arabidopsis chromosomes without any obvious preferential regions (Supporting Information Fig. S1). GO analysis revealed that genes involved in response to various stimuli, including heat and oxidative stress, are overrepresented in the down-regulation category of genes in Atasf1ab (Fig. 1b). RT-PCR analysis confirmed that several heat stress genes, for example, HsfA2, Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, are down-regulated in Atasf1ab (Supporting Information Fig. S2). Hereinafter, we focus on plant heat stress response to reveal AtASF1A/B functions.
Atasf1ab mutant plants show defects in both basal and acquired thermotolerances Thermotolerances of Atasf1ab mutant were assessed via well-established heat stress phenotypic assays (Yeh et al. 2012). We germinated the mutant and WT seeds on the same plate at standard growth chamber temperature (22 °C) for 30 h, heat treated at 45 °C for 2 h and then incubated at 22 °C for 10 d. As shown in Fig. 2a, about 100% of WT seedlings survived from the heat treatment whereas only about 40% of Atasf1ab seedlings could be recovered from the same heat treatment (n > 200). To assess Atasf1ab in acquired thermotolerance, 10-day-old mutant and WT seedlings were acclimated by incubation at 37 °C for 1.5 h followed by a recovery at 22 °C for 2 h (R-2H) or 2 d (R-2D), and then treated by heat stress at 45 °C for 2 h followed by additional 6 d of growth. We found that both WT and Atasf1ab plants
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Remarkably, acclimation greatly ameliorated plant tolerance and lower values of ion leakage were detected after the severe heat stress treatment. The R-2H recovery condition was superior over the R-2D condition in ion leakage reduction (Fig. 2d), which is consistent with the observed better recovery plant growth phenotype (Fig. 2b). At both R-2H and R-2D conditions, Atasf1ab showed higher ion leakage than WT did (Fig. 2d). Collectively, our plant growth and physiological analysis data clearly establish that both basal and acquired thermotolerances are impaired in the Atasf1ab mutant.
Activation of heat-induced genes is impaired in Atasf1ab mutant plants
Figure 1. Comparison of the differentially expressed genes in the Arabidopsis Atasf1a, Atasf1b and Atasf1ab mutants. (a) Venn diagrams show the number and overlap of significantly up- and down-regulated genes in the Atasf1a, Atasf1b and Atasf1ab mutants compared with wild type. (b) BiNGO analysis on down-regulated genes in the Atasf1ab mutant. As indicated by colour scale bar, the yellow to orange colour of the circles corresponds to the level of significance of over-represented gene ontology category according to a multiple t-test with false discovery rate-corrected P-value under 0.05. Circle size is proportional to number of genes in the category.
grow better with the R-2H than with the R-2D recovery treatment condition (Fig. 2b). Nonetheless, Atasf1ab compared with WT showed more severe growth inhibition at either R-2H or R-2D recovery condition, and the mutant but not WT seedlings displayed an obvious chlorotic phenotype (Fig. 2b). Chlorophyll content analysis confirmed that chlorophyll a/b and carotenoid levels are drastically reduced in Atasf1ab compared with WT (Fig. 2c). It is known that heat stress can change cell membrane permeability by affecting membrane transporter activities and membrane integrity (Hong et al. 2003). We investigated cell membrane permeability by ion leakage measurement using 10-day-old seedlings. Untreated (control) or acclimation temperature-treated (37 °C, 1.5 h) seedlings showed low values of ion leakage. Nevertheless, a statistically significant increase of ion leakage was detected in Atasf1ab compared with WT (Fig. 2d). The severe heat stress treatment (45 °C, 2 h) drastically induced ion leakage, which likely exceeds the normal membrane function capacity and in this case no difference between Atasf1ab and WT could be detected.
Most of HSF and HSP genes are expressed at relatively low levels in plants grown at 22 °C. Perturbed expression of HSF and HSP genes in Atasf1ab identified by our transcriptome analysis using plants grown in the standard growth chamber may not be necessarily temperature related. To test whether AtASF1A/B have a role in heat-related gene expression, we analysed expression levels of HSF and HSP genes in Atasf1ab and WT during a time course of 120 min incubation of plants at 37 °C (Fig. 3).We found that expression of HsfA1a/b remained largely unchanged, whereas HsfB1 expression was induced up to 75 folds,peaked at 60 and 90 min after the heat treatment.For these three genes, no significant differences of expression could be detected between Atasf1ab and WT. In contrast, the other examined genes, for example, HsfA2, Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, showed varied greater folds (several hundred to more than 10 000 at peak values) of heat induction, and their expression levels were overall drastically lower in Atasf1ab than in WT following the heat treatment (Fig. 3).The expression level of AtASF1A/B was not affected by heat treatment in WT (Supporting Information Fig. S3). Our Western blot analyses further confirmed that the Hsp101 and Hsp17.6 protein levels are drastically induced by the heat treatment and that they were lower in Atasf1ab than in WT (Supporting Information Fig. S4). Taken together, these data indicate that AtASF1A/B are required for a maximal heatinduced expression of HsfA2,Hsp101,Hsp70,Hsa32,Hsp17.6A and Hsp17.6B-CI, but not HsfB1.
AtASF1A/B are also required for maximal activation of HSF and HSP genes in response to oxidative stress In agreement with previous reports (Volkov et al. 2006; Konigshofer et al. 2008), heat treatment induced hydrogen peroxide (H2O2) production (Fig. 4a), which was visualized by fluorescent dye H2DCFDA staining of roots (Kim et al. 2012). In this assay, we did not notice any significant difference between Atasf1ab and WT. We next asked the question whether Atasf1ab affects HSF and HSP gene expression in response to oxidative stress.We tested this by treating 12-dayold WT and Atasf1ab plants grown under light with the chemical RB, a photosensitizer-producing oxidative stress (Fischer et al. 2004). We found that RB treatments efficiently induced
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Figure 2. Comparison of basal and acquired thermotolerance between Arabidopsis wild-type (WT) and mutant Atasf1ab plants. (a) Comparison of plant basal thermotolerance. For heat treatment, 30-hour-old seedlings were incubated at 45 °C for 2 h, recovered and grown at 22 °C for 10 d before photographing. Untreated plants grown at 22 °C were shown as controls. (b) Comparison of plant acquired thermotolerance. For heat treatment, 10-day-old seedlings were first treated at 37 °C for 1.5 h and recovered at 22 °C for 2 h (R-2H) or 2 d (R-2D), and then treated at 45 °C for 2 h, recovered and grown at 22 °C for 6 d before photographing. Untreated plants grown at 22 °C were shown as controls. (c) Heat bleaching effect as measured by reduction levels of three major chlorophylls, chl a, chl b and carotenoids. Extracts from heat acclimated (R-2H) and untreated control seedlings described in (b) were analysed. The percentage of treated compared with untreated samples was shown as relative levels. The mean values and standard deviations from three independent experiments are shown. Significant difference at P < 0.05 between WT and Atasf1ab mutant is indicated by asterisk (*). (d) Heat effects measured by ion leakage. The mean value and standard deviation represent the results of three independent experiments. Significant difference at P < 0.05 between WT and Atasf1ab mutant is indicated by asterisk (*).
the expression of Hsp17.6A, HsfA2, Hsp17.6B-CI, Hsp101, Hsp70 and to a less extent Hsa32 (Fig. 4b). Compared with WT, Atasf1ab showed much reduced induction of these genes at most of the examined time points following the RB treatment (Fig. 4b). These data suggest that heat may induce HSF and HSP gene expression partly through signalling of oxidative stress and that AtASF1A/B are involved at the signal transduction terminal transcription.
AtASF1 binding and nucleosome occupancy at HSF and HSP genes To investigate molecular basis of AtASF1A/B function in transcription, we first asked the question whether
AtASF1A/B proteins bind chromatin at HSF and HSP genes. We generated polyclonal antibodies against the N-terminal 159 amino acids peptide of AtASF1A. In Western blot assay, the antibody specifically recognized the YFP-fused AtASF1A (Zhu et al. 2011) but not NRP1, another histone chaperone (Zhu et al. 2006), from total protein extracts of transgenic plants (Supporting Information Fig. S5). Because of highly conserved protein sequences between AtASF1A and AtASF1B (Zhu et al. 2011), this antibody likely can also recognize the AtASF1B protein. By ChIP assay using this antibody, we analysed AtASF1A/B protein enrichment at diverse regions of HsfA2, Hsa32 and HsfB1 (Fig. 5a). These three genes are all necessary for acclimation-induced acquired thermotolerance (Charng et al. 2006, 2007; Ikeda
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nucleosome removal is associated with heat-induced expression of HSF and HSP genes, and that AtASF1A/B positively regulate nucleosome dissociation at some but not all of these heat gene loci. To verify that AtASF1A/B regulated heat induction of HSF and HSP gene expression is indeed located at transcriptional level, we analysed RNA polymerase II (Pol II) enrichment at HsfA2, Hsa32 and HsfB1 genes in WT and Atasf1ab plants.As shown in Fig. 5d, Pol II levels at HsfA2 and Hsa32 were greatly increased in heat treated as compared with untreated plants of WT and to much less extents of Atasf1ab. At HsfB1, some moderate increases of Pol II levels were also observed in heat treated as compared with untreated plants, but the increase pattern is similar in WT and Atasf1ab. These data are in agreement with the expression profiles of these HSF and HSP gene (Fig. 3) and further corroborate AtASF1A/B binding (Fig. 5b) and H3 enrichment (Fig. 5c) data, together suggesting that AtASF1A/B proteins activate transcription of some heat-responsive genes (e.g. HsfA2 and Hsa32) by targeting and facilitating nucleosome dissociation at these gene loci.
AtASF1A/B are required for H3K56ac during HSF and HSP activation Figure 3. Comparison of heat-induced gene expression in Arabidopsis wild-type (WT) and mutant Atasf1ab plants. Quantitative RT-PCR analysis was performed on samples collected at indicated time point from 12-day-old seedlings incubated at 37 °C (over 200 seedlings per sample). The expression levels of the indicated genes were normalized to UBC28. The relative values were referenced to that of untreated WT, which is set as 1. Mean values and standard deviations are shown from three independent experiments.
et al. 2011). We reasoned that values obtained in the Atasf1ab mutant would represent non-specific backgrounds, which were subsequently set as 1 to normalize other values to facilitate comparisons between untreated and treated and between WT and mutant samples. From untreated WT plants, we observed low levels of AtASF1A/B binding at HsfA2, Hsa32 and HsfB1 chromatin (Fig. 5b). In heat-treated (90 min at 37 °C) WT plants, however, AtASF1A/B levels were greatly increased at chromatin regions throughout the HsfA2 and Hsa32 genes but not the HsfB1 gene, which is consistent with the observation that heat-induced expression of HsfA2 and Hsa32 but not HsfB1 is AtASF1A/B dependent (Fig. 3). As expected, in heat-treated Atasf1ab mutant only non-specific background levels were detected (Fig. 5b). Next we asked the question whether AtASF1A/B affect nucleosome occupancy at HsfA2, Hsa32 and HsfB1 genes. By ChIP assay using a commercially available histone H3-specific antibody, we detected broad reductions of H3 levels at almost all analysed regions of the HsfA2, Hsa32 and HsfB1 genes in heat treated as compared with untreated WT plants (Fig. 5c). In Atasf1ab, heat treatment caused H3 level reductions to much less extents at HsfA2 and Hsa32, whereas the reductions were similarly important at HsfB1 as compared with those in WT (Fig. 5c). These data suggest that
Studies in yeast indicate that ASF1 stimulates H3K56ac to facilitate nucleosome dissociation for induced gene transcription (Williams et al. 2008; Minard et al. 2011). To investigate whether or not heat-induced nucleosome dissociation is associated with H3K56ac, we first examined evolutionary conservation of ASF1 function in H3K56ac deposition. We expressed and purified recombinant GST-tagged Rtt109 and His-tagged AtASF1A proteins and tested their activity to acetylate core histones in vitro. Strong H3K56ac signal was detected only in the presence of both Rtt109 and AtASF1A in the reaction, indicating that AtASF1A has similar function to the yeast ASF1 in stimulation of Rtt109 in catalysing H3K56ac (Fig. 6a). We next examined by ChIP assay the H3K56ac levels at HSF and HSP genes. As shown in Fig. 6b, H3K56ac levels drastically increased at HsfA2 and Hsa32 gene loci in heat treated compared with untreated WT plants. Such increases were much impaired in Atasf1ab.Weak increases of H3K56ac were also detected at HsfB1, but in this case no difference could be noted between WT and Atasf1ab (Fig. 6b). These data are in agreement with the idea that H3K56ac facilitates nucleosome dissociation and indicate that AtASF1A/B are required for H3K56ac deposition, nucleosome dissociation and transcriptional activation of some heat-induced genes, for example, HsfA2 and Hsa32. The idea that AtASF1A/B regulate gene loci specific but not global H3K56ac levels was further supported by Western blot analysis, showing that the global H3K56ac level was roughly similar in heat-treated and untreated plants of WT and Atasf1ab (Supporting Information Fig. S6).
DISCUSSION As sessile organisms, plants cannot move and are thus constantly exposed to daily and seasonal temperature changes.
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Figure 4. Comparison of oxidative stress response of Arabidopsis wild-type (WT) and mutant Atasf1ab plants. (a) Comparison of H2O2 production. Roots of 5-day-old WT (indicated by red arrowhead) and Atasf1ab mutant (indicated by yellow arrowhead) seedlings before (control) and after heat treated at 45 °C (heat) for indicated time were stained with fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and imaged. (b) Comparison of expression levels of heat stress genes. Quantitative RT-PCR analysis was performed on samples collected at indicated time point from 12-day-old seedlings treated with the photosensitizer Rose Bengal (RB). The expression levels of the indicated genes were normalized to UBC28. The relative values were referenced to that of the mock-treated WT control, which is set as 1. Mean values and standard deviations are shown from three independent experiments.
Heat stress can affect nucleosome composition and stability, and greatly impacts plant genome function (Kumar & Wigge 2010; Lang-Mladek et al. 2010; Pecinka et al. 2010; TittelElmer et al. 2010; Boden et al. 2013). Therefore, identification of chromatin regulators involved in heat stress response has crucial importance in understanding plant adaptation to environments. In this study, we found that the histone chaperone AtASF1 plays important roles in plant thermotolerance, in HSF and HSP gene transcription, and in nucleosome removal and H3K56ac enrichment at some HSF and HSP genes. Our phenotypic and physiological analyses clearly showed that the Atasf1ab mutant plants have reduced basal and acquired thermotolerance. This differs from the absence of heat stress growth phenotype of the mutants deprived of regulators involved in DNA methylation (CMT3, DRM2) or chromatin remodelling (DDM1, MOM1) or histone methylation (KYP, SUVH2) (Pecinka et al. 2010; Tittel-Elmer et al. 2010; Popova et al. 2013). Reduced basal thermotolerance was reported in the more recent study for several mutants related to RNA-directed DNA methylation (RdDM) pathway, for example, nrpd2, hda6, ago4, dcl3 and rdr2 (Popova et al. 2013). Although its thermotolerance to high temperature has not yet been analysed, the H2A.Z incorporation defective mutant arp6 exhibits warm temperature (27 °C), responsive growth and developmental phenotypes, including plant architecture and flowering time (Kumar & Wigge 2010). Our study together with these previous studies indicates that multiple
epigenetic pathways are involved in various aspects of plant growth response to heat. Yet future studies are required to determine whether some of these epigenetic factors might work together to regulate same aspect of plant heat response. In nature heat is frequently accompanied by strong sunlight. AtASF1 has been reported as required for repair of DNA damage induced by UV-B (Lario et al. 2013) as well as by other types of genotoxic stress (Zhu et al. 2011). Heat induces H2O2 accumulation, which is a potent agent causing damage to DNA, proteins and other molecules (Gill & Tuteja 2010). Our study shows that the H2O2 production is normal, but oxidative response genes are down-regulated in Atasf1ab. Down-regulation of heat and oxidative stress response genes contrasts the unchanged expression or up-regulation of DNA damage repair genes previously reported in Atasf1ab (Zhu et al. 2011; Lario et al. 2013). Our focused study on several genes, including HsfA2, Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, revealed that AtASF1A/B positively regulate basal as well as heat and oxidative stress-responsive expression of these genes. The strong heat-induced expression provides an attractive system to address mechanisms of AtASF1A/B function in gene expression control. Our gene expression and ChIP data show that heat induces HsfA2 and Hsa32 transcription, which is associated with Pol II and AtASF1A/B accumulation at these gene loci. Positioning of nucleosome in the path of transcriptionally engaged Pol II is generally detrimental (Petesch & Lis 2012). For transcription initiation and elongation to proceed, Pol
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Figure 5. Analysis of AtASF1, H3 and polymerase II (Pol II) protein levels at gene-specific chromatin regions in Arabidopsis wild-type (WT) and mutant Atasf1ab plants and in response to heat stress. (a) A schematic representation of gene structure indicating the regions examined by chromatin immunoprecipitation (ChIP). Black boxes represent exons; white boxes represent untranslated region; lines represent promoter and introns; number-labelled bars represent regions amplified by corresponding primer pairs. (b–d) ChIP analysis for the relative occupancy of AtASF1 (b), H3 (c) and Pol II (d) at the indicated gene chromatin regions. WT and Atasf1ab plants untreated or treated by incubation at 37 °C for 1.5 h were analysed. Values were referenced to those of untreated Atasf1ab, which were set as 1. Mean values and standard deviations are shown from two independent experiments. The symbols * and # indicate statistically significant differences (P < 0.05) observed for heat effect in either WT or Atasf1ab and for Atasf1ab effect on the heat induction compared with WT, respectively.
II-containing transcriptional machinery needs to overcome the polar barrier of nucleosomes at the transcription start site and throughout the gene body. Our data reveal that HsfA2 and Hsa32 activation is associated with AtASF1A/Bdependent nucleosome removal, suggesting a positive role of AtASF1A/B in nucleosome dissociation. In line with this latter proposal, AtASF1A/B specifically bind H3 (Zhu et al. 2011) and the yeast and human ASF1-H3-H4 ternary complex is mutually exclusive from the nucleosomal core (H3-H4)2 tetramer (English et al. 2006; Natsume et al. 2007). The heat response is a universally conserved reaction, and nucleosome loss is also associated with yeast and animal HSF and HSP genes (Erkina et al. 2010; Teves & Henikoff 2011).
Therefore, it is reasonable to speculate that our newly discovered role of ASF1 may extend to heat response in other organisms. Yeast ASF1 stimulates Rtt109 in catalysing H3K56ac (Schneider et al. 2006; Avvakumov et al. 2011). Our in vitro assay revealed that AtASF1A can also stimulate H3K56ac production by Rtt109. Nevertheless, Western blot analysis revealed roughly similar levels of H3K56ac in WT and Atasf1ab, indicating that AtASF1A/B are not major determining factors of global H3K56ac in Arabidopsis. In animals, both CBP/p300 and GCN5 acetyltransferases are involved in catalysing H3K56ac (Das et al. 2009; Kong et al. 2011). Arabidopsis possesses five CBP/p300-type acetyltransferases
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Figure 6. Analysis of H3K56ac levels at gene-specific chromatin regions in Arabidopsis wild-type (WT) and mutant Atasf1ab plants and in response to heat stress. (a) In vitro assay for H3K56 acetylation. Recombinant proteins GST-Rtt109 (indicated by black arrowhead) and His-AtASF1A (indicated by black arrow) were produced and tested in the enzyme activity assay. The histone mixture (indicated by open arrowhead) was used as substrates. The upper panel shows protein gel staining by Coomassie Brilliant Blue (CBB) for loading control, and the lower panel shows Western blot (WB) detection by anti-H3K56ac antibody. (b) Relative H3K56ac levels at indicated gene chromatin regions. WT and Atasf1ab plants untreated or treated by incubation at 37 °C for 1.5 h were analysed using anti-H3 and anti-H3K56ac. H3K56ac-to-H3 ratio values were referenced to those of untreated Atasf1ab, which were set as 1. Mean values and standard deviations are shown from two independent experiments. The symbols * and # indicate statistically significant differences (P < 0.05) observed for heat effect in either WT or Atasf1ab and for Atasf1ab effect on the heat induction compared with WT, respectively.
(AtHAC1/2/4/5/12; Pandey et al. 2002), and AtHAC1/5/12 have been shown to possess broad substrate specificities in vitro, including H3K9/14 and H4K5/8/12/16 sites (Earley et al. 2007). The Arabidopsis ortholog AtGCN5 can also acetylate H3K14 in vitro (Earley et al. 2007), and its deprivation affects H3K9/27 acetylation levels in planta (Benhamed et al. 2006). It is yet unknown whether AtHAC1/2/4/5/12 and AtGCN5 are also involved in H3K56ac deposition. Our ChIP analysis has uncovered a substantial role of AtASF1A/B in H3K56ac deposition at HsfA2 and Hsa32 loci. We found that AtASF1A/B are required for maximal levels of H3K56ac enrichment, which occurs at HsfA2 and Hsa32 loci in plant response to heat treatment. It has been reported that AtASF1A/B bind HAM1/2 (Lario et al. 2013), and that HAM1/2 are required for H4K5ac deposition at Arabidopsisflowering regulatory genes FLC and MAF3/4 (Xiao et al. 2013). It is currently unclear whether HAM1/2 have H3K56accatalysing activity or whether another (yet unknown identity) enzyme is responsible for H3K56ac deposition at HsfA2 and Hsa32. In any case, our observed H3K56ac increase in response to heat is positively correlated with nucleosome removal in HsfA2 and Hsa32 activation. This is in agreement with the function of H3K56ac in facilitating nucleosome dissociation. A recent study in yeast found that H3K56ac promotes H2A.Z removal from the nucleosome through changing the chromatin remodelling factor SWR-C substrate specificity, providing a link between H3K56ac and H2A.Z in the regulation of nucleosome turnover (Watanabe et al. 2013).
Warm temperature (27 °C) treatment of Arabidopsis plants affects H2A.Z/H2A exchange activity at many genes including HSP70, and the arp6 mutant defective in H2A.Z incorporation displays a constitutive heat response phenotype (Kumar & Wigge 2010). Future characterization of Arabidopsis enzymes involved in H3K56ac deposition and chromatin remodelling factors involved in H2A.Z exchange in relation with AtASF1 function will shed further insight into mechanisms of nucleosome dynamics in plant response to heat stress.
ACKNOWLEDGMENTS This work was supported by the Chinese Ministry of Science and Technology (grant no. 2012CB910500 and 2011CB944600) and the National Natural Science Foundation of China (31271374 and 30971443). The research was conducted within the context of the International Associated Laboratory Plant Epigenome Research, LIA PER.
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Received 6 December 2013; received in revised form 29 January 2014; accepted for publication 29 January 2014
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Distribution of genes perturbed in Atasf1ab on the five Arabidopsis chromosomes. Figure S2. Relative expression levels of several key heat stress genes in Arabidopsis wild-type (WT) and Atasf1ab mutant under standard growth conditions. Figure S3. Relative expression levels of AtASF1A/B during heat treatment. Figure S4. Western blot analysis of Hsp101 and HSP17.6 protein levels in Arabidopsis wild-type (WT) and Atasf1ab mutant plants. Figure S5. Specificity control of anti-AtASF1A antibody on Western blot. Figure S6. Western blot analysis of H3K56ac levels in Arabidopsis wild-type (WT) and Atasf1ab mutant plants. Table S1. Primers used in this study. Table S2. List of up-regulated genes in Atasf1a from transcriptome analysis. Table S3. List of up-regulated genes in Atasf1b from transcriptome analysis. Table S4. List of up-regulated genes in Atasf1ab from transcriptome analysis. Table S5. List of down-regulated genes in Atasf1a from transcriptome analysis. Table S6. List of down-regulated genes in Atasf1b from transcriptome analysis. Table S7. List of down-regulated genes in Atasf1ab from transcriptome analysis.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2128–2138
Plant, Cell and Environment (2014) 37, 2128–2138
doi: 10.1111/pce.12299
Original Article
Histone chaperone ASF1 is involved in gene transcription activation in response to heat stress in Arabidopsis thaliana Minjie Weng1,†, Yue Yang1,†, Haiyang Feng1, Zongde Pan1, Wen-Hui Shen1,2, Yan Zhu1 & Aiwu Dong1 1
State Key Laboratory of Genetic Engineering, International Associated Laboratory of CNRS-Fudan-HUNAU on Plant Epigenome Research, Department of Biochemistry, Institute of Plant Biology, School of Life Sciences, Fudan University, Shanghai 20043, China and 2Institut de Biologie Moléculaire des Plantes du CNRS, Université de Strasbourg, 12 rue du Général Zimmer, 67084 Strasbourg Cédex, France
ABSTRACT ANTI-SILENCING FUNCTION 1 (ASF1) is an evolutionarily conserved histone chaperone involved in diverse chromatin-based processes in eukaryotes. Yet, its role in transcription and the underlying molecular mechanisms remain largely elusive, particularly in plants. Here, we show that the Arabidopsis thaliana ASF1 homologous genes, AtASF1A and AtASF1B, are involved in gene transcription activation in response to heat stress. The Atasf1ab mutant displays defective basal as well as acquired thermotolerance phenotypes. Heat-induced expression of several key genes, including the HEAT SHOCK PROTEIN (HSP) genes Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, and the HEAT SHOCK FACTOR (HSF) gene HsfA2 but not HsfB1 is drastically impaired in Atasf1ab as compared with that in wild type. We found that AtASF1A/B proteins are recruited onto chromatin, and their enrichment is correlated with nucleosome removal and RNA polymerase II accumulation at the promoter and coding regions of HsfA2 and Hsa32 but not HsfB1. Moreover, AtASF1A/B facilitate H3K56 acetylation (H3K56ac), which is associated with HsfA2 and Hsa32 activation. Taken together, our study unravels an important function of AtASF1A/B in plant heat stress response and suggests that AtASF1A/B participate in transcription activation of some but not all HSF and HSP genes via nucleosome removal and H3K56ac stimulation. Key-words: ANTI-SILENCING FUNCTION 1; transcription regulation.
INTRODUCTION Nucleosome, the basic unit of chromatin, is composed of roughly 147 base pairs (bp) of DNA wrapped around the globular histone octamer comprising one centre-positioned (H3-H4)2 tetramer and two side-positioned H2A-H2B dimers. Histone chaperones facilitate histone–DNA interactions during nucleosome assembly and/or disassembly, and Correspondence: Y. Zhu. Fax: +86 21 55665673; e-mail: zhu_yan @fudan.edu.cn; A. Dong. Fax: +86 21 55665673; e-mail: aiwudong @fudan.edu.cn †
These authors contributed equally to this work.
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play crucial roles in chromatin dynamics relevant to DNA replication, repair, recombination and transcription (Avvakumov et al. 2011). ANTI-SILENCING FUNCTION 1 (ASF1) is a highly conserved histone chaperone specific for H3-H4, and its monomeric state is detected in the form of ASF1-H3-H4 ternary complex, which is mutually exclusive from that of (H3-H4)2 tetramer (English et al. 2006; Natsume et al. 2007). This suggests that loading of ASF1 likely has an (H3-H4)2 tetramer-disrupting activity in the context of nucleosomes. ASF1 also interacts with other chromatin factors, such as CHROMATIN ASSEMBLY FACTOR-1 (CAF-1) and HISTONE REGULATORY HOMOLOG A (HIRA), which mediate replication-dependent and replication-independent H3 incorporation into chromatin, respectively (Tagami et al. 2004). Several studies have also revealed critical importance of ASF1 in establishment of H3K56 acetylation (H3K56ac; reviewed in Avvakumov et al. 2011). K56 is located within N-terminal α-helix of H3, and its acetylation requires ASF1 for catalysis by histone acetyltransferase Rtt109 and CBP/ p300 in yeast and animal cells, respectively (Schneider et al. 2006; Das et al. 2009; Avvakumov et al. 2011). Adding acetyl interrupts histone–DNA contact and increases rate of nucleosomal DNA ends unwrapping from histone octamer (Neumann et al. 2009), and promotes nucleosome disassembly during transcriptional activation in yeast (Rufiange et al. 2007; Williams et al. 2008; Lin & Schultz 2011). The Arabidopsis thaliana genome comprises two ASF1 homologous genes, AtASF1A and AtASF1B, which play redundant roles and participate in S-phase replicationdependent chromatin assembly (Zhu et al. 2011). The loss-offunction Atasf1ab double mutant displays chromatin instability and DNA damage as well as activation of expression of cell cycle checkpoint and DNA repair genes (Zhu et al. 2011). More recently, it was shown that AtASF1A and AtASF1B are involved in UV-induced DNA damage repair, likely through interaction with the TIP60-like histone acetyltransferases HAM1 and HAM2 (Lario et al. 2013). In the present study, we report a crucial function of AtASF1A and AtASF1B in plant heat stress tolerance. Heat stress is expecting to occur more frequently as a consequence of warming climate, which seriously challenges ecological plant species demography and agricultural crop © 2014 John Wiley & Sons Ltd
AtASF1 in heat stress response yield (Saidi et al. 2011; Qu et al. 2013). During evolution, plants have evolved various molecular and physiological mechanisms to cope with heat stress. Among others, HEAT SHOCK FACTOR (HSF) and HEAT SHOCK PROTEIN (HSP) play a central role in the onset of cellular thermotolerance (Saidi et al. 2011; Qu et al. 2013). The Arabidopsis HSF family comprises over 20 members, which can be grouped into A, B and C classes based on protein structural domain organization (Nover et al. 2001). The A class members contain a C-terminal AHA domain and most act as transcription activators. Although HsfA1a/b are not heat induced and function as immediate activators in heat response (Lohmann et al. 2004; Busch et al. 2005), HsfA2 is the most potently heat-induced gene and is responsible for amplification and long-term maintenance of heat response (Charng et al. 2007). The B class members, for example, HsfB1 and HsfB2b, possess a tetrapeptide LFGV motif and generally function as transcriptional repressors (Ikeda et al. 2011). Function of the unique C class member HsfC1 remains unknown so far. At downstream of HSFs, HSPs function as molecular chaperones involved in maintenance and/or restoration of protein homeostasis under heat stress, and are grouped into Hsp100, Hsp90, Hsp70, Hsp60 and small Hsp subgroups (Swindell et al. 2007). Intriguingly, some Hsps (e.g. Hsp90, Hsp70) also participate in the feedback control of subcellular compartmentalization and activity of plant HSFs (reviewed in Scharf et al. 2012). Detailed genetic and physiological analyses have implicated HSFs and HSPs in the regulation of plant basal thermotolerance upon direct exposure to a heat stress. Meanwhile, exposure of plants to a moderate level of high temperature (e.g. 37 °C for Arabidopsis) can efficiently activate the HSF-HSP gene networks, which significantly improves plant thermotolerance to a second more severe heat stress (e.g. 45 °C for Arabidopsis; reviewed in Saidi et al. 2011; Mittler et al. 2012). This process is generally referred as acclimation or ‘priming’, and the enhanced plant ability is referred as acquired thermotolerance. Chromatin remodelling is believed to be advantageous in regulation of a large set of genes and in gene priming in plant response to stresses (Berr et al. 2012; Zhu et al. 2012). The histone H2A variant, H2A.Z, was proposed as a thermosensor and was shown to be evicted from nucleosomes at Hsp70 and other genes following exposure of Arabidopsis plants to a moderately warm temperature (27 °C; Kumar & Wigge 2010). The arp6 mutant defective in H2A.Z incorporation showed constitutive up-regulation of Hsp70 and a genome-wide expression pattern mimicking wild-type (WT) plants grown at warm temperatures (Kumar & Wigge 2010). In Brachypodium distachyon, ARP6mediated H2A.Z incorporation was reported to play an important function in thermal stress response on grain yield (Boden et al. 2013). Long heat stress, 15–30 h at 37 °C or 48 h at 42 °C, was shown to release silencing of a reporter transgene as well as some endogenous heterochromatin loci in Arabidopsis (Lang-Mladek et al. 2010; Pecinka et al. 2010; Tittel-Elmer et al. 2010). The silencing release was not associated with any detectable changes of DNA methylation or histone methylation and mutants of several chromatin
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regulators, for example, CMT3, DDM1, DRM2, MOM1 and KYP, did not show any detectable perturbation in the heat stress response (Pecinka et al. 2010; Tittel-Elmer et al. 2010). In this study, we investigate the function of AtASF1A and AtASF1B in heat stress response. We show that the loss-offunction Atasf1ab double mutant exhibits both basal and acquired thermotolerance defects associated with impaired transcription activation of some HSF and HSP genes. We provide evidence highlighting a crucial role of AtASF1A/B in heat stress gene activation via nucleosome removal and H3K56ac stimulation.
MATERIALS AND METHODS Plant material and growth conditions Atasf1a, Atasf1b and Atasf1ab mutants have been described previously (Zhu et al. 2011). Seeds were produced in growth chamber at 22 °C in a 16 h light/8 h dark photoperiod.
Transcriptome analysis Twelve-day-old seedlings of Atasf1a, Atasf1b, Atasf1ab and WT grown on agar-solidified MS medium M0255 (Duchefa; http://www.duchefa.com) at 22 °C under a light intensity of about 100 μEm−2 s−1 in a 16 h light/8 h dark photoperiod were used in transcriptome analysis. RNA was extracted using the TRIzol kit according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). Two biological replicates were analysed for gene expression using Agilent Arabidopsis4*44K (Palo Alto, CA, USA) oligonucleotide array containing 43603 probes (Shanghai Biotechnology Corporation, Pudong, Shanghai, China). The raw data of microarray have been deposited in public database NCBIGEO (GSE48854). Data analysis was performed by the online information analysis platform based on the statistic package R (http://sas.ebioservice.com). Genes with expression changed more than twofold between WT, and the mutants are considered as differentially expressed. Gene ontology (GO) analysis was performed as previously described (Liu et al. 2009b).
Heat and oxidative stress treatments For basal thermotolerance assay, 30 WT and 30 Atasf1ab seeds were germinated on a same plate containing agarsolidified MS medium. Each determination was performed with three replicate plates. The plates were incubated at 22 °C for 30 h, placed at 45 °C for 2 h and returned to 22 °C for 10 d before examination of heat effects. For acquired thermotolerance assay, the seed plates were incubated at 22 °C for 10 d, treated at 37 °C for 1.5 h, returned to 22 °C for 2 h (2H) or 2 d (2D) for recovery, treated at 45 °C for 2 h and finally returned to 22 °C for 6 d before examination of heat effects. Heat effects on H2O2 production was detected in the roots (from 30 plants per sample) by fluorescent dye 2′,7′dichlorodihydrofluorescein diacetate (H2DCFDA) staining and visualization under fluorescence microscopy as described
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2130 M. Weng et al. previously (Kim et al. 2012). For oxidative stress treatment, over 200 12-day-old seedlings per sample were transferred to the liquid MS medium M0255 without (control) or with 20 μm Rose Bengal (RB) (R3877; Sigma, St Louis, MO, USA), and grown at 22 °C under 100 μEm−2 s−1 light. Samples were collected at described time points and analysed for gene expression.
Chlorophylls and ion leakage measurements Total chlorophylls were measured by non-maceration methods. Seedlings (100 mg per sample) were incubated in 5 mL dimethyl sulfoxide (DMSO) at 65 °C for 4 h in the dark. Absorbance was recorded at 645, 663, 470 nm in a SmartSpecTM Plus Spectrophotometer (170–2525; Bio-Rad, Hercules, CA, USA). Chlorophyll and carotenoid contents were calculated as described previously (Chauhan et al. 2012). Ten-day-old seedlings (15 plants per sample) were used for the ion leakage measurement after different heat treatments according to Hong et al. (2003). Briefly, seedlings were incubated in deionized water for 1 d, and the resulting solution was measured for conductivity. Values were normalized to the total conductivity measured on dead seedlings after autoclaving, and the resulted percentage represents the relative ion leakage activity of living seedlings.
RT-PCR and Western blot analysis RNA isolation and RT-PCR were performed as described previously (Liu et al. 2009a), and the primers for RT-PCR were listed in the Supporting Information Table S1. Total proteins were extracted from the 12-day-old seedlings before and after heat treatment at 37 °C for 1.5 h and analysed by Western blot using anti-Hsp101 (ab80123; Abcam, Cambridge, UK) and anti-Hsp17.6 (ab80183; Abcam) antibodies. Histones were prepared as described previously (Gao et al. 2012) and analysed in Western blot using anti-H3 (ab1791; Abcam) and anti-H3K56ac (07–677; Millipore, Billerica, MA, USA) antibodies.
Chromatin immunoprecipitation (ChIP) analysis About 2 g per sample of 12-day-old seedlings untreated or treated by incubation at 37 °C for 1.5 h was used for ChIP analysis. ChIP experiments were performed as described previously (Johnson et al. 2002). The anti-AtASF1 rabbit polyclonal antiserum was generated against AtASF1A peptide (N-terminal 159 aa) through a commercial service by Abmart, Inc (http://www.abmart.cn). Other antibodies used in ChIP are anti-H3 (ab1791; Abcam), anti-H3K56ac (07– 677; Millipore) and anti-Pol II (sc-900; Santa Cruz Biotechnology, Santa Cruz, CA, USA). UBC28 was used as an internal reference gene (Pecinka et al. 2009). The genespecific primers used in ChIP are listed in the Supporting Information Table S1.
In vitro histone acetyltransferase (HAT) assay The open reading frame (ORF) of yeast Rtt109 was cloned into the expression vector pGEX-4T1 (Amersham,
Piscataway, NJ, USA) using BamHI and SalI sites. The ORF of AtASF1A was cloned into the expression vector pET-14b (Novagen, Madison, WI, USA) using BglII and XhoI sites. Recombinant proteins were expressed in Escherichia coli and purified according to a previously described procedure (Dong et al. 2005). In vitro HAT assay was performed as described previously (Driscoll et al. 2007) using mixture histones (223565; Roche, Indianapolis, IN, USA) as substrates and anti-H3K56ac antibody (07–677; Millipore) in Western blot detection.
RESULTS Transcriptome analysis reveals a large number of misregulated genes in the Atasf1ab mutant To investigate AtASF1A/B function in genome transcription regulation, we performed transcriptome analysis using Agilent Arabidopsis 44K oligonucleotide microarray on 12-day-old seedlings of WT and the double-mutant Atasf1ab as well as the single mutants Atasf1a and Atasf1b (Zhu et al. 2011). Compared with WT, Atasf1ab showed 695 upregulated genes and 1204 down-regulated genes whereas Atasf1a and Atasf1b only showed small numbers of perturbed genes (Fig. 1a; Supporting Information Tables S2–S7), which is in agreement with the redundant function of AtASF1A and AtASF1B. As expected, AtASF1A and AtASF1B were among the down-regulated genes found in the respective mutants (Supporting Information Tables S5– S7). The perturbed genes in Atasf1ab are distributed along all the five Arabidopsis chromosomes without any obvious preferential regions (Supporting Information Fig. S1). GO analysis revealed that genes involved in response to various stimuli, including heat and oxidative stress, are overrepresented in the down-regulation category of genes in Atasf1ab (Fig. 1b). RT-PCR analysis confirmed that several heat stress genes, for example, HsfA2, Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, are down-regulated in Atasf1ab (Supporting Information Fig. S2). Hereinafter, we focus on plant heat stress response to reveal AtASF1A/B functions.
Atasf1ab mutant plants show defects in both basal and acquired thermotolerances Thermotolerances of Atasf1ab mutant were assessed via well-established heat stress phenotypic assays (Yeh et al. 2012). We germinated the mutant and WT seeds on the same plate at standard growth chamber temperature (22 °C) for 30 h, heat treated at 45 °C for 2 h and then incubated at 22 °C for 10 d. As shown in Fig. 2a, about 100% of WT seedlings survived from the heat treatment whereas only about 40% of Atasf1ab seedlings could be recovered from the same heat treatment (n > 200). To assess Atasf1ab in acquired thermotolerance, 10-day-old mutant and WT seedlings were acclimated by incubation at 37 °C for 1.5 h followed by a recovery at 22 °C for 2 h (R-2H) or 2 d (R-2D), and then treated by heat stress at 45 °C for 2 h followed by additional 6 d of growth. We found that both WT and Atasf1ab plants
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Remarkably, acclimation greatly ameliorated plant tolerance and lower values of ion leakage were detected after the severe heat stress treatment. The R-2H recovery condition was superior over the R-2D condition in ion leakage reduction (Fig. 2d), which is consistent with the observed better recovery plant growth phenotype (Fig. 2b). At both R-2H and R-2D conditions, Atasf1ab showed higher ion leakage than WT did (Fig. 2d). Collectively, our plant growth and physiological analysis data clearly establish that both basal and acquired thermotolerances are impaired in the Atasf1ab mutant.
Activation of heat-induced genes is impaired in Atasf1ab mutant plants
Figure 1. Comparison of the differentially expressed genes in the Arabidopsis Atasf1a, Atasf1b and Atasf1ab mutants. (a) Venn diagrams show the number and overlap of significantly up- and down-regulated genes in the Atasf1a, Atasf1b and Atasf1ab mutants compared with wild type. (b) BiNGO analysis on down-regulated genes in the Atasf1ab mutant. As indicated by colour scale bar, the yellow to orange colour of the circles corresponds to the level of significance of over-represented gene ontology category according to a multiple t-test with false discovery rate-corrected P-value under 0.05. Circle size is proportional to number of genes in the category.
grow better with the R-2H than with the R-2D recovery treatment condition (Fig. 2b). Nonetheless, Atasf1ab compared with WT showed more severe growth inhibition at either R-2H or R-2D recovery condition, and the mutant but not WT seedlings displayed an obvious chlorotic phenotype (Fig. 2b). Chlorophyll content analysis confirmed that chlorophyll a/b and carotenoid levels are drastically reduced in Atasf1ab compared with WT (Fig. 2c). It is known that heat stress can change cell membrane permeability by affecting membrane transporter activities and membrane integrity (Hong et al. 2003). We investigated cell membrane permeability by ion leakage measurement using 10-day-old seedlings. Untreated (control) or acclimation temperature-treated (37 °C, 1.5 h) seedlings showed low values of ion leakage. Nevertheless, a statistically significant increase of ion leakage was detected in Atasf1ab compared with WT (Fig. 2d). The severe heat stress treatment (45 °C, 2 h) drastically induced ion leakage, which likely exceeds the normal membrane function capacity and in this case no difference between Atasf1ab and WT could be detected.
Most of HSF and HSP genes are expressed at relatively low levels in plants grown at 22 °C. Perturbed expression of HSF and HSP genes in Atasf1ab identified by our transcriptome analysis using plants grown in the standard growth chamber may not be necessarily temperature related. To test whether AtASF1A/B have a role in heat-related gene expression, we analysed expression levels of HSF and HSP genes in Atasf1ab and WT during a time course of 120 min incubation of plants at 37 °C (Fig. 3).We found that expression of HsfA1a/b remained largely unchanged, whereas HsfB1 expression was induced up to 75 folds,peaked at 60 and 90 min after the heat treatment.For these three genes, no significant differences of expression could be detected between Atasf1ab and WT. In contrast, the other examined genes, for example, HsfA2, Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, showed varied greater folds (several hundred to more than 10 000 at peak values) of heat induction, and their expression levels were overall drastically lower in Atasf1ab than in WT following the heat treatment (Fig. 3).The expression level of AtASF1A/B was not affected by heat treatment in WT (Supporting Information Fig. S3). Our Western blot analyses further confirmed that the Hsp101 and Hsp17.6 protein levels are drastically induced by the heat treatment and that they were lower in Atasf1ab than in WT (Supporting Information Fig. S4). Taken together, these data indicate that AtASF1A/B are required for a maximal heatinduced expression of HsfA2,Hsp101,Hsp70,Hsa32,Hsp17.6A and Hsp17.6B-CI, but not HsfB1.
AtASF1A/B are also required for maximal activation of HSF and HSP genes in response to oxidative stress In agreement with previous reports (Volkov et al. 2006; Konigshofer et al. 2008), heat treatment induced hydrogen peroxide (H2O2) production (Fig. 4a), which was visualized by fluorescent dye H2DCFDA staining of roots (Kim et al. 2012). In this assay, we did not notice any significant difference between Atasf1ab and WT. We next asked the question whether Atasf1ab affects HSF and HSP gene expression in response to oxidative stress.We tested this by treating 12-dayold WT and Atasf1ab plants grown under light with the chemical RB, a photosensitizer-producing oxidative stress (Fischer et al. 2004). We found that RB treatments efficiently induced
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Figure 2. Comparison of basal and acquired thermotolerance between Arabidopsis wild-type (WT) and mutant Atasf1ab plants. (a) Comparison of plant basal thermotolerance. For heat treatment, 30-hour-old seedlings were incubated at 45 °C for 2 h, recovered and grown at 22 °C for 10 d before photographing. Untreated plants grown at 22 °C were shown as controls. (b) Comparison of plant acquired thermotolerance. For heat treatment, 10-day-old seedlings were first treated at 37 °C for 1.5 h and recovered at 22 °C for 2 h (R-2H) or 2 d (R-2D), and then treated at 45 °C for 2 h, recovered and grown at 22 °C for 6 d before photographing. Untreated plants grown at 22 °C were shown as controls. (c) Heat bleaching effect as measured by reduction levels of three major chlorophylls, chl a, chl b and carotenoids. Extracts from heat acclimated (R-2H) and untreated control seedlings described in (b) were analysed. The percentage of treated compared with untreated samples was shown as relative levels. The mean values and standard deviations from three independent experiments are shown. Significant difference at P < 0.05 between WT and Atasf1ab mutant is indicated by asterisk (*). (d) Heat effects measured by ion leakage. The mean value and standard deviation represent the results of three independent experiments. Significant difference at P < 0.05 between WT and Atasf1ab mutant is indicated by asterisk (*).
the expression of Hsp17.6A, HsfA2, Hsp17.6B-CI, Hsp101, Hsp70 and to a less extent Hsa32 (Fig. 4b). Compared with WT, Atasf1ab showed much reduced induction of these genes at most of the examined time points following the RB treatment (Fig. 4b). These data suggest that heat may induce HSF and HSP gene expression partly through signalling of oxidative stress and that AtASF1A/B are involved at the signal transduction terminal transcription.
AtASF1 binding and nucleosome occupancy at HSF and HSP genes To investigate molecular basis of AtASF1A/B function in transcription, we first asked the question whether
AtASF1A/B proteins bind chromatin at HSF and HSP genes. We generated polyclonal antibodies against the N-terminal 159 amino acids peptide of AtASF1A. In Western blot assay, the antibody specifically recognized the YFP-fused AtASF1A (Zhu et al. 2011) but not NRP1, another histone chaperone (Zhu et al. 2006), from total protein extracts of transgenic plants (Supporting Information Fig. S5). Because of highly conserved protein sequences between AtASF1A and AtASF1B (Zhu et al. 2011), this antibody likely can also recognize the AtASF1B protein. By ChIP assay using this antibody, we analysed AtASF1A/B protein enrichment at diverse regions of HsfA2, Hsa32 and HsfB1 (Fig. 5a). These three genes are all necessary for acclimation-induced acquired thermotolerance (Charng et al. 2006, 2007; Ikeda
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nucleosome removal is associated with heat-induced expression of HSF and HSP genes, and that AtASF1A/B positively regulate nucleosome dissociation at some but not all of these heat gene loci. To verify that AtASF1A/B regulated heat induction of HSF and HSP gene expression is indeed located at transcriptional level, we analysed RNA polymerase II (Pol II) enrichment at HsfA2, Hsa32 and HsfB1 genes in WT and Atasf1ab plants.As shown in Fig. 5d, Pol II levels at HsfA2 and Hsa32 were greatly increased in heat treated as compared with untreated plants of WT and to much less extents of Atasf1ab. At HsfB1, some moderate increases of Pol II levels were also observed in heat treated as compared with untreated plants, but the increase pattern is similar in WT and Atasf1ab. These data are in agreement with the expression profiles of these HSF and HSP gene (Fig. 3) and further corroborate AtASF1A/B binding (Fig. 5b) and H3 enrichment (Fig. 5c) data, together suggesting that AtASF1A/B proteins activate transcription of some heat-responsive genes (e.g. HsfA2 and Hsa32) by targeting and facilitating nucleosome dissociation at these gene loci.
AtASF1A/B are required for H3K56ac during HSF and HSP activation Figure 3. Comparison of heat-induced gene expression in Arabidopsis wild-type (WT) and mutant Atasf1ab plants. Quantitative RT-PCR analysis was performed on samples collected at indicated time point from 12-day-old seedlings incubated at 37 °C (over 200 seedlings per sample). The expression levels of the indicated genes were normalized to UBC28. The relative values were referenced to that of untreated WT, which is set as 1. Mean values and standard deviations are shown from three independent experiments.
et al. 2011). We reasoned that values obtained in the Atasf1ab mutant would represent non-specific backgrounds, which were subsequently set as 1 to normalize other values to facilitate comparisons between untreated and treated and between WT and mutant samples. From untreated WT plants, we observed low levels of AtASF1A/B binding at HsfA2, Hsa32 and HsfB1 chromatin (Fig. 5b). In heat-treated (90 min at 37 °C) WT plants, however, AtASF1A/B levels were greatly increased at chromatin regions throughout the HsfA2 and Hsa32 genes but not the HsfB1 gene, which is consistent with the observation that heat-induced expression of HsfA2 and Hsa32 but not HsfB1 is AtASF1A/B dependent (Fig. 3). As expected, in heat-treated Atasf1ab mutant only non-specific background levels were detected (Fig. 5b). Next we asked the question whether AtASF1A/B affect nucleosome occupancy at HsfA2, Hsa32 and HsfB1 genes. By ChIP assay using a commercially available histone H3-specific antibody, we detected broad reductions of H3 levels at almost all analysed regions of the HsfA2, Hsa32 and HsfB1 genes in heat treated as compared with untreated WT plants (Fig. 5c). In Atasf1ab, heat treatment caused H3 level reductions to much less extents at HsfA2 and Hsa32, whereas the reductions were similarly important at HsfB1 as compared with those in WT (Fig. 5c). These data suggest that
Studies in yeast indicate that ASF1 stimulates H3K56ac to facilitate nucleosome dissociation for induced gene transcription (Williams et al. 2008; Minard et al. 2011). To investigate whether or not heat-induced nucleosome dissociation is associated with H3K56ac, we first examined evolutionary conservation of ASF1 function in H3K56ac deposition. We expressed and purified recombinant GST-tagged Rtt109 and His-tagged AtASF1A proteins and tested their activity to acetylate core histones in vitro. Strong H3K56ac signal was detected only in the presence of both Rtt109 and AtASF1A in the reaction, indicating that AtASF1A has similar function to the yeast ASF1 in stimulation of Rtt109 in catalysing H3K56ac (Fig. 6a). We next examined by ChIP assay the H3K56ac levels at HSF and HSP genes. As shown in Fig. 6b, H3K56ac levels drastically increased at HsfA2 and Hsa32 gene loci in heat treated compared with untreated WT plants. Such increases were much impaired in Atasf1ab.Weak increases of H3K56ac were also detected at HsfB1, but in this case no difference could be noted between WT and Atasf1ab (Fig. 6b). These data are in agreement with the idea that H3K56ac facilitates nucleosome dissociation and indicate that AtASF1A/B are required for H3K56ac deposition, nucleosome dissociation and transcriptional activation of some heat-induced genes, for example, HsfA2 and Hsa32. The idea that AtASF1A/B regulate gene loci specific but not global H3K56ac levels was further supported by Western blot analysis, showing that the global H3K56ac level was roughly similar in heat-treated and untreated plants of WT and Atasf1ab (Supporting Information Fig. S6).
DISCUSSION As sessile organisms, plants cannot move and are thus constantly exposed to daily and seasonal temperature changes.
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Figure 4. Comparison of oxidative stress response of Arabidopsis wild-type (WT) and mutant Atasf1ab plants. (a) Comparison of H2O2 production. Roots of 5-day-old WT (indicated by red arrowhead) and Atasf1ab mutant (indicated by yellow arrowhead) seedlings before (control) and after heat treated at 45 °C (heat) for indicated time were stained with fluorescent dye 2′,7′-dichlorodihydrofluorescein diacetate (H2DCFDA) and imaged. (b) Comparison of expression levels of heat stress genes. Quantitative RT-PCR analysis was performed on samples collected at indicated time point from 12-day-old seedlings treated with the photosensitizer Rose Bengal (RB). The expression levels of the indicated genes were normalized to UBC28. The relative values were referenced to that of the mock-treated WT control, which is set as 1. Mean values and standard deviations are shown from three independent experiments.
Heat stress can affect nucleosome composition and stability, and greatly impacts plant genome function (Kumar & Wigge 2010; Lang-Mladek et al. 2010; Pecinka et al. 2010; TittelElmer et al. 2010; Boden et al. 2013). Therefore, identification of chromatin regulators involved in heat stress response has crucial importance in understanding plant adaptation to environments. In this study, we found that the histone chaperone AtASF1 plays important roles in plant thermotolerance, in HSF and HSP gene transcription, and in nucleosome removal and H3K56ac enrichment at some HSF and HSP genes. Our phenotypic and physiological analyses clearly showed that the Atasf1ab mutant plants have reduced basal and acquired thermotolerance. This differs from the absence of heat stress growth phenotype of the mutants deprived of regulators involved in DNA methylation (CMT3, DRM2) or chromatin remodelling (DDM1, MOM1) or histone methylation (KYP, SUVH2) (Pecinka et al. 2010; Tittel-Elmer et al. 2010; Popova et al. 2013). Reduced basal thermotolerance was reported in the more recent study for several mutants related to RNA-directed DNA methylation (RdDM) pathway, for example, nrpd2, hda6, ago4, dcl3 and rdr2 (Popova et al. 2013). Although its thermotolerance to high temperature has not yet been analysed, the H2A.Z incorporation defective mutant arp6 exhibits warm temperature (27 °C), responsive growth and developmental phenotypes, including plant architecture and flowering time (Kumar & Wigge 2010). Our study together with these previous studies indicates that multiple
epigenetic pathways are involved in various aspects of plant growth response to heat. Yet future studies are required to determine whether some of these epigenetic factors might work together to regulate same aspect of plant heat response. In nature heat is frequently accompanied by strong sunlight. AtASF1 has been reported as required for repair of DNA damage induced by UV-B (Lario et al. 2013) as well as by other types of genotoxic stress (Zhu et al. 2011). Heat induces H2O2 accumulation, which is a potent agent causing damage to DNA, proteins and other molecules (Gill & Tuteja 2010). Our study shows that the H2O2 production is normal, but oxidative response genes are down-regulated in Atasf1ab. Down-regulation of heat and oxidative stress response genes contrasts the unchanged expression or up-regulation of DNA damage repair genes previously reported in Atasf1ab (Zhu et al. 2011; Lario et al. 2013). Our focused study on several genes, including HsfA2, Hsp101, Hsp70, Hsa32, Hsp17.6A and Hsp17.6B-CI, revealed that AtASF1A/B positively regulate basal as well as heat and oxidative stress-responsive expression of these genes. The strong heat-induced expression provides an attractive system to address mechanisms of AtASF1A/B function in gene expression control. Our gene expression and ChIP data show that heat induces HsfA2 and Hsa32 transcription, which is associated with Pol II and AtASF1A/B accumulation at these gene loci. Positioning of nucleosome in the path of transcriptionally engaged Pol II is generally detrimental (Petesch & Lis 2012). For transcription initiation and elongation to proceed, Pol
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Figure 5. Analysis of AtASF1, H3 and polymerase II (Pol II) protein levels at gene-specific chromatin regions in Arabidopsis wild-type (WT) and mutant Atasf1ab plants and in response to heat stress. (a) A schematic representation of gene structure indicating the regions examined by chromatin immunoprecipitation (ChIP). Black boxes represent exons; white boxes represent untranslated region; lines represent promoter and introns; number-labelled bars represent regions amplified by corresponding primer pairs. (b–d) ChIP analysis for the relative occupancy of AtASF1 (b), H3 (c) and Pol II (d) at the indicated gene chromatin regions. WT and Atasf1ab plants untreated or treated by incubation at 37 °C for 1.5 h were analysed. Values were referenced to those of untreated Atasf1ab, which were set as 1. Mean values and standard deviations are shown from two independent experiments. The symbols * and # indicate statistically significant differences (P < 0.05) observed for heat effect in either WT or Atasf1ab and for Atasf1ab effect on the heat induction compared with WT, respectively.
II-containing transcriptional machinery needs to overcome the polar barrier of nucleosomes at the transcription start site and throughout the gene body. Our data reveal that HsfA2 and Hsa32 activation is associated with AtASF1A/Bdependent nucleosome removal, suggesting a positive role of AtASF1A/B in nucleosome dissociation. In line with this latter proposal, AtASF1A/B specifically bind H3 (Zhu et al. 2011) and the yeast and human ASF1-H3-H4 ternary complex is mutually exclusive from the nucleosomal core (H3-H4)2 tetramer (English et al. 2006; Natsume et al. 2007). The heat response is a universally conserved reaction, and nucleosome loss is also associated with yeast and animal HSF and HSP genes (Erkina et al. 2010; Teves & Henikoff 2011).
Therefore, it is reasonable to speculate that our newly discovered role of ASF1 may extend to heat response in other organisms. Yeast ASF1 stimulates Rtt109 in catalysing H3K56ac (Schneider et al. 2006; Avvakumov et al. 2011). Our in vitro assay revealed that AtASF1A can also stimulate H3K56ac production by Rtt109. Nevertheless, Western blot analysis revealed roughly similar levels of H3K56ac in WT and Atasf1ab, indicating that AtASF1A/B are not major determining factors of global H3K56ac in Arabidopsis. In animals, both CBP/p300 and GCN5 acetyltransferases are involved in catalysing H3K56ac (Das et al. 2009; Kong et al. 2011). Arabidopsis possesses five CBP/p300-type acetyltransferases
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2128–2138
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Figure 6. Analysis of H3K56ac levels at gene-specific chromatin regions in Arabidopsis wild-type (WT) and mutant Atasf1ab plants and in response to heat stress. (a) In vitro assay for H3K56 acetylation. Recombinant proteins GST-Rtt109 (indicated by black arrowhead) and His-AtASF1A (indicated by black arrow) were produced and tested in the enzyme activity assay. The histone mixture (indicated by open arrowhead) was used as substrates. The upper panel shows protein gel staining by Coomassie Brilliant Blue (CBB) for loading control, and the lower panel shows Western blot (WB) detection by anti-H3K56ac antibody. (b) Relative H3K56ac levels at indicated gene chromatin regions. WT and Atasf1ab plants untreated or treated by incubation at 37 °C for 1.5 h were analysed using anti-H3 and anti-H3K56ac. H3K56ac-to-H3 ratio values were referenced to those of untreated Atasf1ab, which were set as 1. Mean values and standard deviations are shown from two independent experiments. The symbols * and # indicate statistically significant differences (P < 0.05) observed for heat effect in either WT or Atasf1ab and for Atasf1ab effect on the heat induction compared with WT, respectively.
(AtHAC1/2/4/5/12; Pandey et al. 2002), and AtHAC1/5/12 have been shown to possess broad substrate specificities in vitro, including H3K9/14 and H4K5/8/12/16 sites (Earley et al. 2007). The Arabidopsis ortholog AtGCN5 can also acetylate H3K14 in vitro (Earley et al. 2007), and its deprivation affects H3K9/27 acetylation levels in planta (Benhamed et al. 2006). It is yet unknown whether AtHAC1/2/4/5/12 and AtGCN5 are also involved in H3K56ac deposition. Our ChIP analysis has uncovered a substantial role of AtASF1A/B in H3K56ac deposition at HsfA2 and Hsa32 loci. We found that AtASF1A/B are required for maximal levels of H3K56ac enrichment, which occurs at HsfA2 and Hsa32 loci in plant response to heat treatment. It has been reported that AtASF1A/B bind HAM1/2 (Lario et al. 2013), and that HAM1/2 are required for H4K5ac deposition at Arabidopsisflowering regulatory genes FLC and MAF3/4 (Xiao et al. 2013). It is currently unclear whether HAM1/2 have H3K56accatalysing activity or whether another (yet unknown identity) enzyme is responsible for H3K56ac deposition at HsfA2 and Hsa32. In any case, our observed H3K56ac increase in response to heat is positively correlated with nucleosome removal in HsfA2 and Hsa32 activation. This is in agreement with the function of H3K56ac in facilitating nucleosome dissociation. A recent study in yeast found that H3K56ac promotes H2A.Z removal from the nucleosome through changing the chromatin remodelling factor SWR-C substrate specificity, providing a link between H3K56ac and H2A.Z in the regulation of nucleosome turnover (Watanabe et al. 2013).
Warm temperature (27 °C) treatment of Arabidopsis plants affects H2A.Z/H2A exchange activity at many genes including HSP70, and the arp6 mutant defective in H2A.Z incorporation displays a constitutive heat response phenotype (Kumar & Wigge 2010). Future characterization of Arabidopsis enzymes involved in H3K56ac deposition and chromatin remodelling factors involved in H2A.Z exchange in relation with AtASF1 function will shed further insight into mechanisms of nucleosome dynamics in plant response to heat stress.
ACKNOWLEDGMENTS This work was supported by the Chinese Ministry of Science and Technology (grant no. 2012CB910500 and 2011CB944600) and the National Natural Science Foundation of China (31271374 and 30971443). The research was conducted within the context of the International Associated Laboratory Plant Epigenome Research, LIA PER.
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Received 6 December 2013; received in revised form 29 January 2014; accepted for publication 29 January 2014
SUPPORTING INFORMATION Additional Supporting Information may be found in the online version of this article at the publisher’s web-site: Figure S1. Distribution of genes perturbed in Atasf1ab on the five Arabidopsis chromosomes. Figure S2. Relative expression levels of several key heat stress genes in Arabidopsis wild-type (WT) and Atasf1ab mutant under standard growth conditions. Figure S3. Relative expression levels of AtASF1A/B during heat treatment. Figure S4. Western blot analysis of Hsp101 and HSP17.6 protein levels in Arabidopsis wild-type (WT) and Atasf1ab mutant plants. Figure S5. Specificity control of anti-AtASF1A antibody on Western blot. Figure S6. Western blot analysis of H3K56ac levels in Arabidopsis wild-type (WT) and Atasf1ab mutant plants. Table S1. Primers used in this study. Table S2. List of up-regulated genes in Atasf1a from transcriptome analysis. Table S3. List of up-regulated genes in Atasf1b from transcriptome analysis. Table S4. List of up-regulated genes in Atasf1ab from transcriptome analysis. Table S5. List of down-regulated genes in Atasf1a from transcriptome analysis. Table S6. List of down-regulated genes in Atasf1b from transcriptome analysis. Table S7. List of down-regulated genes in Atasf1ab from transcriptome analysis.
© 2014 John Wiley & Sons Ltd, Plant, Cell and Environment, 37, 2128–2138
