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• W2191069891 abstract "Article9 December 2015free access A hit-and-run heat shock factor governs sustained histone methylation and transcriptional stress memory Jörn Lämke Jörn Lämke Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Krzysztof Brzezinka Krzysztof Brzezinka Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Simone Altmann Simone Altmann Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Isabel Bäurle Corresponding Author Isabel Bäurle Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Jörn Lämke Jörn Lämke Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Krzysztof Brzezinka Krzysztof Brzezinka Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Simone Altmann Simone Altmann Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Isabel Bäurle Corresponding Author Isabel Bäurle Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany Search for more papers by this author Author Information Jörn Lämke1, Krzysztof Brzezinka1, Simone Altmann1,2 and Isabel Bäurle 1 1Institute for Biochemistry and Biology, University of Potsdam, Potsdam, Germany 2Present address: Institute of Biology, Free University Berlin, Berlin, Germany *Corresponding author. Tel: +49 331 9772647; E-mail: [email protected] The EMBO Journal (2016)35:162-175https://doi.org/10.15252/embj.201592593 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In nature, plants often encounter chronic or recurring stressful conditions. Recent results indicate that plants can remember a past exposure to stress to be better prepared for a future stress incident. However, the molecular basis of this is poorly understood. Here, we report the involvement of chromatin modifications in the maintenance of acquired thermotolerance (heat stress [HS] memory). HS memory is associated with the accumulation of histone H3 lysine 4 di- and trimethylation at memory-related loci. This accumulation outlasts their transcriptional activity and marks them as recently transcriptionally active. High accumulation of H3K4 methylation is associated with hyper-induction of gene expression upon a recurring HS. This transcriptional memory and the sustained accumulation of H3K4 methylation depend on HSFA2, a transcription factor that is required for HS memory, but not initial heat responses. Interestingly, HSFA2 associates with memory-related loci transiently during the early stages following HS. In summary, we show that transcriptional memory after HS is associated with sustained H3K4 hyper-methylation and depends on a hit-and-run transcription factor, thus providing a molecular framework for HS memory. Synopsis Stress exposure can prime for an enhanced response upon re-exposure. In Arabidopsis, heat stress-mediated priming is associated with stable histone H3 lysine 4 di- and trimethylation and requires transcription factor HSFA2 that only transiently binds to the primed gene loci. Heat stress primes genes for sustained activation and/or enhanced induction upon recurring stress. This transcriptional memory is linked to induction of histone H3 lysine 4 di- and trimethylation. Transcription factor HSFA2 is required for transcriptional memory. HSFA2 only transiently associates with the gene loci, suggesting it acts in a hit-and-run mode. Introduction Plants are sessile organisms that gauge and react to stressful conditions in order to ensure survival and reproductive success. Such stressful conditions include extreme temperatures, drought, salinity as well as pathogen and herbivore attacks. In nature, these are often chronic or recurring. Thus, plants have evolved strategies to remember a past exposure to stress to be better prepared for the next incident (Jaskiewicz et al, 2011; Ding et al, 2012a; Sani et al, 2013; Stief et al, 2014a). This area of research has only recently received increasing attention (Bruce et al, 2007; Conrath, 2011; Avramova, 2015; Hilker et al, 2015; Kim et al, 2015; Vriet et al, 2015), and the molecular basis of plant stress memory is still largely unknown. In this context, the term memory refers to the phenomenon where a signal of limited duration is perceived, stored and later retrieved, as evidenced by a modified response (Stief et al, 2014b). A related phenomenon in animals is hormesis, where a low-level stress treatment enhances resistance against the same and other stresses and increases growth, fecundity, and longevity (Gems & Partridge, 2008). Mechanistically, stress memory may be regulated at different levels ranging from metabolites to chromatin structure. In cases where stress memory involves modified gene expression patterns, a plausible hypothesis is that modifications of chromatin structure mediate such a memory. Chromatin structure is an important determinant of the regulation of gene expression. Chromatin structure is modified through nucleosome positioning, histone variants, and posttranslational modification of histones (Struhl & Segal, 2013; Zentner & Henikoff, 2013). Depending on their nature and position, these modifications can promote or repress transcription by altering chromatin accessibility or interaction with specific protein complexes. For example, histone acetylation is associated with active transcription and correlates closely with the rate of transcription (Zentner & Henikoff, 2013). Histone H3 lysine 4 (H3K4) can be mono-, di-, or trimethylated, and its functions are largely conserved between yeast, animals, and plants (Santos-Rosa et al, 2002; Zhang et al, 2009; Shilatifard, 2012). In plants and animals, H3K4 trimethylation (H3K4me3) is highly correlated with active transcription (Guenther et al, 2007; Zhang et al, 2009) and is thought to be required for efficient RNA polymerase II elongation (Ding et al, 2012b; Kwak & Lis, 2013). Similar to H3K4me3, H3K4 dimethylation (H3K4me2) is associated with the 5′-region of genes, but it does not correlate with active transcription (Guenther et al, 2007; Zhang et al, 2009). H3K4 monomethylation (H3K4me1) is not correlated with gene expression and accumulates within transcribed regions (Zhang et al, 2009). In mammals, H3K4me1 is enriched at promoters and enhancers (Cheng et al, 2014). It has been hypothesized that recent transcriptional activity of a locus may be marked to mediate a modified response following a second stimulus. H3K4me3 and H3K4me2 have been discussed as such chromatin marks. In yeast, H3K4me3 hyper-methylation was proposed to act as a memory of recent transcriptional activity (Ng et al, 2003). It was suggested that elevated H3K4me3 is important for genes to be rapidly switched on and off by environmental stimuli and that it acts to prevent the associated genes from being silenced (Ng et al, 2003). In mammals, H3K4me3 marks genes that are poised for expression (Guenther et al, 2007). Transcriptional stress memory in plants has been described for recurring drought stress (Ding et al, 2012a), hyper-osmotic stress (Sani et al, 2013), and defense priming (Conrath, 2011; Jaskiewicz et al, 2011). Priming refers to the ability for quicker and more effective activation of specific cellular defenses upon a previous exposure to stress (Hilker et al, 2015). Originally used mainly for biotic stress responses, it is now also used to describe abiotic stress responses. In the above-mentioned cases of priming, molecular responses were associated with lasting changes in chromatin modifications. Transcriptional memory following drought stress was correlated with elevated H3K4me3 and stalled RNA polymerase II that is phosphorylated at Ser5 (Ding et al, 2012a). Whether chromatin modifications are involved in priming-like phenomena in response to other abiotic stresses such as HS is unknown. What triggers and maintains the deposition of these plant memory marks remains unclear in all reported cases. In particular, the interaction of such chromatin modifications with the transcription factors that govern the induction or sustained activation of the corresponding loci remains unclear. Moderate heat stress (HS) allows a plant to acquire thermotolerance and subsequently withstand high temperatures that are lethal to a plant in the naïve state (Mittler et al, 2012). After returning to non-stress temperatures, acquired thermotolerance is maintained over several days, and this maintenance is genetically separable from the acquisition itself (Charng et al, 2006, 2007; Meiri & Breiman, 2009; Yeh et al, 2012; Stief et al, 2014a). We refer to this maintenance of acquired thermotolerance as HS memory. While the molecular events that lead to the acquisition of thermotolerance are relatively well understood, little is known about the mechanism of HS memory. The acquisition of thermotolerance involves the activation of heat shock transcription factors (HSFs) that induce the expression of heat shock proteins (HSPs), which protect cellular proteins from denaturation (Scharf et al, 2012). This HS response is conserved in plants, animals, and fungi (Richter et al, 2010). Beyond its function in the HS response, mammalian HSF1 has important roles in aging and pathologies (Vihervaara & Sistonen, 2014). Yeast and animals have only one or a few copies of HSF genes, yet many plants contain more than 20 copies with specialized functions. Among the 21 HSFs in Arabidopsis thaliana, so far eight have been shown to act in the responses to HS (Charng et al, 2007; Schramm et al, 2008; Ikeda et al, 2011; Liu et al, 2011; Scharf et al, 2012). Of these, HSFA2 has received special attention, as its expression is highly induced by heat (Nishizawa et al, 2006; Schramm et al, 2006). Interestingly, HSFA2 is not required for the acquisition of thermotolerance, but specifically for its maintenance (Charng et al, 2007). Similar effects have been described for HSA32 (Charng et al, 2006), and miR156 (Stief et al, 2014a). Micro-array analyses have identified a number of HS memory-related genes that were classified based on their sustained induction after HS, which lasts for at least 3 days (Stief et al, 2014a). They comprise many small HSPs (such as HSP21, HSP22.0, and HSP18.2), but also ASCORBATE PEROXIDASE 2 (APX2). Their expression pattern is in strong contrast to that of HS-inducible non-memory genes such as HSP70 and HSP101, whose expression peaks soon after HS and declines relatively quickly. HSFA2 was reported to be required for the maintenance of high expression levels of several HS memory-related genes, but not for their induction, suggesting they could be direct targets of HSFA2 (Nishizawa et al, 2006; Charng et al, 2007). This idea was confirmed by in vitro binding studies (Schramm et al, 2006), but so far not in planta. How HS induction is maintained on some HSPs for several days but not on others remains an open question. Also, it is unknown how the molecular responses to a recurring HS during the memory period differ from the responses to the initial HS, that is, whether there is a transcriptional memory in the classical sense. In general, the term “transcriptional memory” has been used to describe at least two different phenomena (D'Urso & Brickner, 2014). The first phenomenon is where transcriptional response upon a stimulus (such as induction) is modified if the gene was recently active compared to a copy of the gene that was not active before (type 1). An example is transcriptional memory of INO1 in yeast (Light et al, 2010). The second type of transcriptional memory refers to a phenomenon during which gene expression levels are lastingly modified after a transient signal (type 2). A classic example here is the epigenetic silencing of FLC in response to cold by vernalization (Berry & Dean, 2015). During HS memory, a modified re-induction after a second HS would indicate type 1, whereas the sustained induction of HS memory-related genes indicates type 2. For the sake of distinction and clarity, we here refer to type 1 transcriptional memory as such, whereas type 2 transcriptional memory will be referred to as sustained induction. This study investigates a possible involvement of histone modifications during HS memory and their interaction with HSFA2. We show that HSFA2 is required for the maintenance of induction of HS memory-associated genes and directly binds to these loci in planta. Interestingly, this binding occurs transiently during the first few hours after HS. In addition, we identify sustained H3K4me3 and H3K4me2 as chromatin marks that discriminate HS memory-related genes from other HS-inducible genes. Elevated H3K4me3 and H3K4me2 levels persist after HSFA2 association with the locus has declined. Loci that maintain very high levels of H3K4 methylation show an increased response after a recurring HS 2 days after the primary HS, consistent with the definition of transcriptional memory. Thus, HS memory consists of two components, first, the sustained induction of HS memory-associated genes, and second, differential response upon recurring HS. Both components are associated with elevated levels of H3K4me3 and H3K4me2 and depend on functional HSFA2. In summary, our study identifies H3K4 methylation as a HS memory mark and reveals its dependency on a transiently binding transcription factor. Results Prolonged expression of HS memory-related genes is associated with changes in chromatin modifications We hypothesized that sustained activation of HS memory-related genes may be associated with changes in chromatin modifications and that such modifications may prime the plant for recurring HS. To test this hypothesis, we investigated H3K4me3, H3K4me2, and H3K9 acetylation (H3K9ac) at the HSP22.0 and HSP70 loci as representatives of the HS memory-related and non-memory HS-inducible genes, respectively. We analyzed histone modifications in the A. thaliana accession Col-0 by chromatin immunoprecipitation followed by quantitative real-time PCR (ChIP-qPCR) 4, 28 and 52 h after an acclimatizing HS (ACC), consisting of 60 min at 37°C, 90 min recovery at 23°C, 45 min at 44°C (Stief et al, 2014a). A temperature of 44°C is commonly used in A. thaliana HS studies (Yeh et al, 2012). In nature, full insolation causes leaf temperatures to rise much higher than air temperatures (Salisbury & Spomer, 1964). During the 2 days following ACC, HSP22.0 transcript levels remain elevated while HSP70 transcript levels are elevated only during the first 28 h (Stief et al, 2014a). For both genes, we analyzed one genic region at the 5′-end of the gene and one flanking region about three kb away in an intergenic region. For H3K9ac, we observed strong enrichment at 4 h that was specific for the genic region at both loci (Fig 1). This enrichment declined rapidly in HSP70, where it was no longer significantly enriched at 28 h and undetectable at 52 h relative to no-HS (NHS) control conditions (Fig 1B). In contrast, H3K9ac at HSP22.0 declined more slowly and was still significantly enriched at 52 h (Fig 1A). We next investigated H3K4me3 levels at HSP70. A moderate enrichment (threefold) was observed 4 h after ACC that declined over the next 2 days and returned to baseline levels by 52 h. In contrast, HSP22.0 showed strongly elevated H3K4me3 levels relative to NHS (up to 75-fold enrichment). These remained very highly elevated over the course of the experiment. For H3K4me2, no HS-dependent changes were observed at the HSP70 locus (Fig 1B). However, at HSP22.0, enrichment of H3K4me2 was observed following HS. Interestingly, this modification accumulated only at the later time points (28, 52 h), while at 4 h no significant enrichment compared to the NHS control was found. Together, these results indicate that H3K4me3 and H3K4me2 accumulated on HSP22.0 especially toward the later phases during HS memory, when expression and acetylation levels have declined (Figs 1A and 2A). Thus, given their sustained enrichment in comparison with H3K9ac, H3K4me3 and H3K4me2 at HSP22.0 may store information of recent transcriptional activity. Interestingly, the genic regions of HSP22.0 and HSP70 displayed a slight enrichment of H3K4me2 and H3K4me3 relative to their respective intergenic control regions also in the absence of any HS (compare regions 1 and 2 in Fig 1, see Fig EV1 for no antibody control). This suggests that HSP22.0 and HSP70 may both be constitutively poised for rapid activation in response to elevated temperatures. Notably, our results for HSP70 confirm the notion that there is no close correlation between high expression levels and H3K4me2 accumulation. At the physiological level, HS memory is detectable for at least 3 days after ACC (Stief et al, 2014a). Thus, the duration of the physiological memory phase is in good accordance with elevated H3K4me3 and H3K4me2 at HSP22.0. Figure 1. HS induces sustained H3K4me3 and H3K4me2 methylation at HSP22.0, but not at HSP70 A, B. Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 wild type at HSP22.0 (A) and HSP70 (B). Seedlings were subjected to ACC or a control treatment (NHS) 4 days after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with antibodies against H3K9ac, H3K4me3, H3K4me2, and H3. Schematics show positions of regions analyzed. Amplicon positions relative to TSS are HSP22.0: 1, −2,570 bp; 2, +235 bp. HSP70: 1, 4,192 bp downstream of the 3′UTR; 2, +47 bp. Data shown are averages over three biological replicates. Amplification values were normalized to input, H3 and 4 h NHS region 2. The bottom panel shows the H3 signal normalized to input and 4 h NHS region 2. Squares and triangles within bars mark significant differences (P < 0.01 and P < 0.05, respectively, Student′s t-test) between ACC and NHS samples of the same time point. Error bars indicate SE. Download figure Download PowerPoint Figure 2. Sustained induction of HS memory-related gene expression after an acclimatizing HS depends on HSFA2 Transcript levels of the indicated HS-inducible genes after ACC in Col-0 (blue bars) and hsfa2 (orange bars) as determined by quantitative reverse transcription PCR (qRT–PCR). Transcript levels were normalized to TUB6 and the respective NHS harvested at the same time point ([GENE OF INTERESTACC x h/TUB6ACC x h]/[GENE OF INTERESTNHS x h/TUB6NHS x h]). Note that the y-axis is on a log10 scale. Error bars show SD of at least three biological replicates. Asterisks show significant differences between the genotypes at the same time point based on Student′s t-test (*P < 0.05; **P < 0.01). Immunoblotting of HSP21 and HSP101 in Col-0 (C) and hsfa2 (h) at the indicated time points after ACC or NHS. α-tubulin was used as a loading control. M, marker. Download figure Download PowerPoint Click here to expand this figure. Figure EV1. Comparison of enrichment of DNA after ChIP with either anti-H3K4me3 antibody or no antibody at the HSP22.0 locus Dynamics of histone modifications after an acclimatizing HS (ACC) or no HS (NHS) in Col-0 at HSP22.0 as detected by ChIP-qPCR. Seedlings were subjected to ACC or a control treatment (NHS) 4 days after germination. At the indicated time points after the treatments, ChIP-qPCR was performed with an antibody against H3K4me3 or no antibody (no Ab). Schematic shows positions of regions analyzed. Amplicon positions are as in Fig 3. H3K4me3 data correspond to those shown in Fig 3. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and Col-0 4 h NHS region 2. Error bars indicate SE. Schematic (gray bars, UTR; black bar, exons) on top right shows positions of regions analyzed as black boxes below. Results as shown in (A) but with a blow-up of the y-axis to show low enrichment of no Ab samples. Download figure Download PowerPoint Sustained activation of HS memory-related genes in response to ACC depends on HSFA2 HSFA2 was previously shown to be required for the maintenance of acquired thermotolerance (HS memory), but not acquisition per se (Charng et al, 2007; see also Stief et al, 2014a). We next investigated whether the expression profiles of HS memory-related and non-memory HS-inducible genes depended on HSFA2 during a time course of 3 days (76 h) following ACC (Fig 2A). To put further analyses on a broader basis, we investigated four memory genes and two HS-inducible non-memory genes. For the memory-related genes HSP18.2, HSP22.0, and HSP21, we observed unchanged induction 4 h after ACC in hsfa2 mutants compared to wild type. This induction declined faster in hsfa2 mutants over the next 3 days. The dependency on HSFA2 was most pronounced in HSP18.2 and somewhat weaker in HSP21 and HSP22.0. For the memory gene APX2, transcript levels in hsfa2 mutants were already lower at 4 h and declined strongly thereafter. These results indicate that for HSP18.2, HSP21, and HSP22.0, HSFA2 is not required for the initial induction, but rather for the maintenance of high expression levels. For APX2, HSFA2 was required for all time points analyzed. However, it is important to keep in mind that the first time point analyzed was already 4 h after ACC. In contrast, transcript levels of the non-memory genes HSP70 and HSP101 declined much faster. Moreover, HSP70 and HSP101 transcript levels were not dependent on hsfa2. These findings confirm and extend previous work (Charng et al, 2007). To estimate whether transcript levels represented ongoing transcriptional activity, we sought to analyze unspliced transcript levels as a proxy for ongoing transcriptional activity (Bäurle et al, 2007; Kabelitz et al, 2014). Unspliced APX2 transcripts were significantly induced in wild type between 4 and 52 h, indicating that active transcription continues for at least 2 days after ACC (Fig EV2A). In hsfa2, we observed a significantly lower induction of unspliced APX2 transcripts at 4 h that was undetectable at later points. The same was true for the HS memory gene HSP21 (Fig EV2B). In contrast, unspliced HSP70 transcripts in wild type and hsfa2 were strongly induced at 4 h, but quickly declined thereafter (Fig EV2C). Neither HSP18.2 nor HSP22.0 contains an intron, thus precluding corresponding analyses for these genes. In summary, the overall dynamics of unspliced transcripts closely mimicked that of the spliced transcripts, indicating that the sustained accumulation of APX2 and HSP21 transcripts reflects ongoing transcriptional activity for at least 52 h after ACC. In contrast, unspliced HSP70 transcripts were not induced at 28 h and thereafter, corroborating the distinction between the two classes. Thus, our results indicate that HSFA2 regulates APX2 and HSP21 at the level of transcription. Click here to expand this figure. Figure EV2. Unspliced transcript levels of APX2 and HSP21, but not HSP70, are increased for at least 2 days after an acclimatizing HS A–C. Quantification of unspliced transcript levels of APX2 (A), HSP21 (B), and HSP70 (C) by qRT–PCR in Col-0 (blue bars) and hsfa2 (orange bars) at the indicated times after an acclimatizing HS (ACC) or control treatment (NHS). NHS controls were harvested at the same time points as the corresponding ACC samples. Transcript levels were normalized to TUB6 (GENE OF INTERESTACC x h/TUB6ACC x h) and (GENE OF INTERESTNHS x h/TUB6NHS x h), respectively. Small parentheses indicate comparisons between the two genotypes at the same treatment and time point; larger parentheses compare the same genotype between heat-treated and NHS control samples at the same time point (p Col-0/p hsfa2). *P < 0.05; **P < 0.01; Student's t-test. Error bars are SE of three biological replicates. Download figure Download PowerPoint We next sought to confirm our findings at the protein level. Unfortunately, antibodies were only available for HSP21 and HSP101. We analyzed protein levels of HSP21 and HSP101 in wild type and hsfa2 after ACC using commercially available antibodies. HSP21 protein levels peaked only at 28–52 h after ACC and remained highly elevated until 76 h (Fig 2B). In contrast, HSP21 levels in hsfa2 were much reduced between 28 and 76 h. HSP101 was induced by ACC and peaked at 4 h. Elevated protein levels were still observed at 76 h. There was no difference in hsfa2, indicating that HSP101 expression did not depend on HSFA2. Thus, HSP101 protein levels are still elevated at 76 h despite the earlier decline in transcript levels, suggesting that the HSP101 protein may have a longer half life than the HSP21 protein. In summary, our protein analysis correlates well with the corresponding transcript analysis and indicates that proteins may be more stable than transcripts. HSFA2 is required for sustained accumulation of H3K4me3 and H3K4me2 We next asked whether HSFA2 is also required for the accumulation of H3K4me3 and H3K4me2 at the memory-associated loci. To this end, we compared the accumulation of H3K4me2, H3K4me3, and H3K9ac in response to ACC in wild-type and hsfa2 mutants by ChIP-qPCR. We analyzed HSP18.2, HSP22.0, and APX2 as HS memory-related loci, and HSP70 as a HS-inducible non-memory locus whose expression is independent of HSFA2 (see Fig 2A). In wild type, H3K4me3 was strongly induced at the 5′-region of HSP18.2, HSP22.0, and APX2 throughout the analyzed time course (4–52 h after ACC, compared to NHS controls, Figs 1A and 3A). There was no enrichment in intergenic regions flanking the loci. For HSP22.0 and APX2, H3K4me3 remained very high at 52 h after ACC, and for HSP18.2, the levels were still elevated, but lower than at 4 h (Fig 3A). In contrast, at the HSP70 locus, we observed only a moderate increase in H3K4me3 after ACC (Figs 1B and 3A). H3K4me3 accumulation at HSP18.2, HSP22.0, and APX2 was dependent on HSFA2 as HSP18.2 and APX2 had significantly lower H3K4me3 in hsfa2 at all time points, and HSP22.0 had a trend for lower H3K4me3 at 4 and 28 h and significantly lower levels at 52 h (Fig 3A). The slight increase in H3K4me3 at HSP70 at 4 and 28 h was largely independent of HSFA2, in line with the expression analysis, which suggested that HSP70 is not a target. In summary, we observed sustained H3K4me3 accumulation at HS memory-related loci (HSP18.2, HSP22.0, APX2) that lasted for several days after ACC; this sustained H3K4me3 accumulation required functional HSFA2. Figure 3. Sustained H3K4me3 and H3K4me2 at HS memory-related loci depends on HSFA2 A, B. H3K4me3 (A) or H3K4me2 (B) levels after an acclimatizing HS (ACC) in Col-0 and hsfa2 at HSP18.2, HSP22.0, HSP70, and APX2 as detected by ChIP-qPCR. Col-0 (blue bars) and hsfa2 (orange bars) seedlings were subjected to ACC or no treatment (NHS) 4 d after germination. At the indicated time points after the treatment, ChIP-qPCR was performed with antibodies against H3K4me3 (A), H3K4me2 (B) and H3 (for normalization). Schematics show positions of regions analyzed (gray bars, UTR; black bar, exons). Intergenic control region 1 is 3,123 bp (APX2) or 2,570 bp (HSP22.0) upstream of the TSS, or 5,311 bp (HSP18.2) or 6,725 bp (HSP70) downstream of the TSS, respectively. Data are averages over four biological replicates. Amplification values were normalized to input and H3 and the Col-0 4 h NHS region 2 (HSP18.2, HSP22.0, and HSP70) or region 3 (for APX2). *P < 0.05; **P < 0.01 for differences between genotypes at the same time point and treatment; squares and triangles within bars mark significant differences (P < 0.01 and P < 0.05, respectively) between ACC and NHS samples of the same time point and genotype, Student′s t-test. Error bars indicate SE. Download figure Download PowerPoint We next analyzed H3K4me2 accumulation (Fig 3B). Similar to what we observed previously for HSP22.0 (Fig 1A), H3K4me2 at the 5′-region of HSP18.2 and APX2 was clearly enriched after ACC in wild type (Fig 3B). Remarkably, this enrichment tended to increase during the course of the experiment and reached highest levels only at 28 or 52 h after ACC. For HSP70, H3K4me2 after ACC was not increased. At the APX2 locus, H3K4me2 enrichment was high and depended on HSFA2 at 28 h and 52 h. Similar results were obtained for HSP18.2 and HSP22.0. In contrast, H3K4me2 levels at HSP70 were not induced after ACC and this did not change in hsfa2. For all genes analyzed, no accumulation of H3K4me2 was observed at the intergenic region. In summary, H3K4me2 does not directly correlate with transcriptional activity. Instead, our findings support the notion that H3K4me2 acts as a mark of recent transcriptional activity in HS memory and that HSFA2 is required for the sustained accumulation of this mark. Lastly, we analyzed H3K9ac levels after ACC (Fig 4). At HSP70, we observed enrichment at 4 h but not thereafter. This enrichment was not affected in hsfa2. At HSP18.2 and APX2, H3K9ac was enriched strongly at 4 h and weaker thereafter and the enrichment depended on HSFA2. At HSP22.0, sustained accumulation of H3K9ac depended on HSFA2 during the later time points (Figs 1A and 4). Thus, H3K9ac levels during HS memory largely follow transcriptional activity, however, at most HS memory-related genes investigated, acetylation levels appear to decline earlier than transcription. In summary, especially for APX2 and HSP18.2, H3K4me3 and H3K4me2 persist longer than H3K9ac after ACC." @default.
• W2191069891 created "2016-06-24" @default.
• W2191069891 creator A5021738968 @default.
• W2191069891 creator A5050918621 @default.
• W2191069891 creator A5090829768 @default.
• W2191069891 creator A5091294517 @default.
• W2191069891 date "2015-12-09" @default.
• W2191069891 modified "2023-10-18" @default.
• W2191069891 title "A hit‐and‐run heat shock factor governs sustained histone methylation and transcriptional stress memory" @default.
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