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• W2985361230 abstract "Full text Figures and data Side by side Abstract eLife digest Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract Plants as sessile organisms can adapt to environmental stress to mitigate its adverse effects. As part of such adaptation they maintain an active memory of heat stress for several days that promotes a more efficient response to recurring stress. We show that this heat stress memory requires the activity of the FORGETTER1 (FGT1) locus, with fgt1 mutants displaying reduced maintenance of heat-induced gene expression. FGT1 encodes the Arabidopsis thaliana orthologue of Strawberry notch (Sno), and the protein globally associates with the promoter regions of actively expressed genes in a heat-dependent fashion. FGT1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Genomic targets of the BRM remodeler overlap significantly with FGT1 targets. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in fgt1. Together, our results suggest that by modulating nucleosome occupancy, FGT1 mediates stress-induced chromatin memory. https://doi.org/10.7554/eLife.17061.001 eLife digest In nature, plant growth is often limited by unfavourable conditions or disease. Plants have thus evolved sophisticated mechanisms to adapt to such stresses. In fact, brief exposure to stress can prime plants to be better prepared for a future stress following a period without stress. However, the molecular basis of this memory-like phenomenon is poorly understood. Now, Brzezinka, Altmann et al. have used priming by heat stress as a model to dissect the memory of environmental stresses in thale cress, Arabidopsis thaliana. First, a library of mutant plants were tested to identify a gene that is specifically required for heat stress memory but not for the initial responses to heat. Brzezinka, Altmann et al. identified one such gene and termed it FORGETTER1 (or FGT1 for short). Further experiments then revealed that the FGT1 protein binds directly to a specific class of heat-inducible genes that are relevant for heat stress memory. Brzezinka, Altmann et al. propose that the FGT1 protein makes sure that the heat-inducible genes are always accessible and active by modifying the way the DNA containing these genes is packaged. DNA is wrapped around protein complexes called nucleosomes and depending on how tightly the DNA of a gene is wrapped makes it more or less easy to activate the gene. In agreement with this model, FGT1 does interact with proteins that can reposition nucleosomes and leave the DNA more loosely packaged. Also, the fact that plants that lack a working FGT1 gene repackage the DNA of memory-related genes too early after experiencing heat stress provides further support for the model. Together these findings could lead to new approaches for breeding programmes to improve stress tolerance in crop plants. One future challenge will be to find out whether memories involving nucleosomes are also made in response to other stressful conditions, such as attack by pests and disease. https://doi.org/10.7554/eLife.17061.002 Introduction Abiotic stress is a major threat to global crop yields and this problem is likely to be exacerbated in the future. A large body of research has focused on the immediate stress responses. However, in nature, stress is frequently chronic or recurring, suggesting that temporal dynamics are an important, but under-researched, component of plant stress responses. Indeed, plants can be primed by a stress exposure such that they respond more efficiently to another stress incident that occurs after a stressless period (Hilker et al., 2015). Priming has been described in response to pathogen attack, heat stress (HS), drought, and salt stress (Charng et al., 2006; Conrath, 2011; Jaskiewicz et al., 2011; Ding et al., 2012; Sani et al., 2013). Such stress priming and memory may be particularly beneficial to plants due to their sessile life style. Chromatin structure can be modulated by nucleosome positioning, histone variants and posttranslational histone modifications that together control the access of sequence-specific transcription factors and the general transcription machinery to gene loci (Struhl and Segal, 2013; Zentner and Henikoff, 2013). Chromatin mediates long-term stability of environmentally and developmentally-induced gene expression states (Gendrel and Heard, 2014; Steffen and Ringrose, 2014; Berry and Dean, 2015). Hence, the modification of chromatin structure has been suggested to mediate the priming and memory of stress-induced gene expression. Indeed, the above mentioned cases of plant stress priming are associated with lasting histone H3 methylation (Conrath, 2011; Jaskiewicz et al., 2011; Ding et al., 2012; Sani et al., 2013; Lämke et al., 2016). However, the underlying mechanism and the contribution of other determinants of chromatin structure such as nucleosome positioning and occupancy remain unknown. Moderate 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 phase is genetically separable from the acquisition phase (Charng et al., 2006, 2007; Meiri and Breiman, 2009; Stief et al., 2014). We refer to this maintenance phase as HS memory. The immediate HS response (acquisition phase) involves the activation of heat shock transcription factors (HSFs) that induce heat shock proteins (HSPs). Their chaperone activity ensures protein homeostasis (Scharf et al., 2012). The HS response is conserved in animals, plants and fungi (Richter et al., 2010). Among the 21 HSFs in Arabidopsis thaliana (Scharf et al., 2012), only HSFA2 is specifically required for HS memory (Charng et al., 2007). It activates HEAT-STRESS-ASSOCIATED32 (HSA32), a gene with chaperone-like activity, although no homology to known chaperone families (Wu et al., 2013). Like HSFA2, HSA32 is critically required for HS memory (Charng et al., 2006). HSA32 is induced by HS and this induction is sustained for at least three days. A set of HS memory-related genes was identified based on their similar expression pattern, which is in contrast to that of canonical HS-inducible genes (HSP70, HSP101) that show upregulation after HS, but not sustained induction (Stief et al., 2014). Among the HS memory-related genes are small HSPs (such as HSP21, HSP22.0, HSP18.2). A subset of these loci show transcriptional memory in the sense that recurring stress causes a more efficient re-activation compared to the first stress incident, even though active transcription has subsided before the second stress (Lämke et al., 2016). Sustained induction and transcriptional memory of these genes is associated with hyper-methylation of H3K4 (H3K4me2 and H3K4me3) and requires HSFA2, which binds directly to these genes (Lämke et al., 2016). Interestingly, HSFA2 dissociates from these loci before its requirement becomes apparent at the physiological and gene expression levels, thus implicating the existence of additional factors. Here, we report the identification of FGT1 from an unbiased screen for factors that are required for the sustained induction of HSA32. FGT1 is required for HS memory at the physiological and gene expression levels. FGT1 is the single A. thaliana orthologue of metazoan Strawberry notch, a highly conserved co-activator of the developmental regulator Notch. We show that FGT1 associates with memory genes in a HS-dependent way. Moreover, FGT1 is widely associated with the transcriptional start site of expressed genes. We further show that FGT1 interacts with highly conserved chromatin remodeling complexes and is required for proper nucleosome dynamics at HS-memory genes. Thus, FGT1 maintains its target loci in an open and transcription-competent state by interacting with remodeler complexes around the transcriptional start site. Results FGT1 is required for HS memory and sustained induction of HSA32 and other memory genes In order to identify regulators of HS memory we generated a transgenic HSA32::HSA32-LUCIFERASE (HSA32::HSA32-LUC) reporter line. LUC expression in this line was induced by HS and expression remained high for at least 3 d (Figure 1A), thus mimicking expression of the endogenous HSA32 (Charng et al., 2006). We mutagenized the HSA32::HSA32-LUC line with ethyl methanesulfonate and screened M2 families for mutants with modified maintenance of LUC activity after HS. To this end, 4 d-old plate-grown seedlings were treated with an acclimatizing heat treatment (ACC, see Materials and methods). LUC-derived bioluminescence was monitored 1, 2 and 3 d later, and putative mutants were isolated that had normal LUC activity 1 d after ACC and reduced activity 3 d after ACC. Among the recovered mutants with such a LUC expression profile was forgetter1 (fgt1-1), on which we focused further analyses. LUC expression in fgt1-1 was induced normally, however, it declined precociously, which was most apparent 3 d after ACC. Figure 1 with 3 supplements see all Download asset Open asset FGT1 is required for HS memory and sustained induction of memory genes in A. thaliana. (A) fgt1-1 displays normal induction but reduced maintenance of pHSA32::HSA32-LUC expression. Bioluminescence of fgt1-1 or the parent assayed 1, 2, or 3 d after an acclimatizing HS (ACC). The color scale of relative LUC activity is shown. (B) fgt1-1 is impaired in HS memory at the physiological level. Seedlings of the indicated genotypes (cf. fgt1-1#2 in C–D) were acclimatized 4 d after germination and treated with a tester HS 2 or 3 d later. Pictures were taken after 14 d of recovery. (C–D) Quantification of the data shown in (B). The fgt1-1 lines represent independent backcrosses. Data are averaged over at least two independent assays (n>36). Fisher’s exact test, *p<0.05; **p<0.001. (E) Transcript levels of HS memory genes after ACC decline prematurely in fgt1-1. Expression values were normalized to the reference At4g26410 and the corresponding no-HS control (NHS). Data are averages and SE of two biological replicates. *p<0.05; **p<0.01 (Student’s t test). https://doi.org/10.7554/eLife.17061.003 We next investigated whether this correlated with modified HS memory at the physiological level by applying a tester HS 2 or 3 d after ACC. The tester HS is lethal to a naïve plant or a mutant with loss of HS memory. Indeed, fgt1-1 mutants displayed reduced growth and survival under these conditions (Figure 1B–D). To check whether fgt1-1 mutants had a generally impaired HS response, we also tested fgt1-1 seedlings for acquisition of thermotolerance and for basal thermotolerance, i. e. the amount of heat that can be tolerated without prior acclimation. fgt1-1 mutants behaved very similar to the parental line in these assays (Figure 1—figure supplements 1,2), indicating that the immediate responses to HS were not affected in fgt1-1. Thus, fgt1-1 is specifically impaired in HS memory. Notably, fgt1-1 did not have any obvious morphological alterations under standard growth conditions. We next examined whether the premature decline of LUC expression mimicked that of the endogenous HSA32 gene by quantitative RT-PCR (qRT-PCR) specific for the endogenous HSA32. HSA32endo and LUC transcripts were induced normally in fgt1-1 during the first day after ACC, but declined faster thereafter (Figure 1E, Figure 1—figure supplement 3). A similar defect was observed for the HS memory-related genes HSP21, HSP22.0 and HSP18.2, but not the HS-inducible non-memory genes HSP101 and HSP70. For intron-containing genes, we measured unspliced transcripts as a proxy for transcriptional activity. Unspliced HSA32 transcripts were induced in fgt1-1 similarly as in the parent up to 21 h, but declined faster thereafter. Notably, unspliced transcript levels in the parent were still elevated 20-fold relative to the non-HS control (NHS) at 69 h after ACC, indicating continued transcription throughout the memory phase. Similar results were obtained for HSP21, but not for the non-memory genes. This is in accordance with what was observed previously (Lämke et al., 2016). Thus, FGT1 is required to facilitate sustained transcription of HS memory genes after HS. FGT1 encodes the orthologue of Drosophila Strawberry notch, a protein involved in induction of Notch and EGFR target genes To identify the molecular lesion underlying the fgt1-1 mutant phenotype, we combined recombination breakpoint mapping with Illumina sequencing. We identified a 0.77 MB interval at the bottom of chromosome 1, containing a splice acceptor site mutation in At1g79350 (Figure 2A). This mutation caused retention of intron 19, resulting in a premature stop codon. At1g79350 was previously tentatively identified as EMB1135 with a reported embryo-defective phenotype (Meinke et al., 2008). However, it was not confirmed that the disruption of this gene in the emb1135 allele indeed causes the phenotype and neither fgt1-1, nor any of several putative loss-of-function T-DNA insertion lines showed any obvious morphological phenotype. Three independent lines of evidence show that FGT1 is At1g79350. First, we complemented the LUC expression and physiological memory phenotypes by expressing a genomic FGT1 fragment (SA13) in the fgt1-1 background (Figure 2B–D). Second, similar results were obtained for a FGT1-YFP fusion protein that was driven by the constitutive 35S CaMV promoter (Figure 2—figure supplement 1A,B). Finally, fgt1-2 and fgt1-3, two putative loss-of-function T-DNA alleles displayed reduced HS memory (Figure 2—figure supplement 1C–E). Figure 2 with 3 supplements see all Download asset Open asset FGT1 encodes the A. thaliana orthologue of Drosophila Sno and binds histones. (A) Gene model of FGT1 (At1g79350) with domains and location of mutations; exons (grey and colored bars); black line, intron. fgt1-1 has a C to T mutation at the splice acceptor site of intron 19/exon 20. (B–D) Complementation of fgt1-1 by a genomic FGT1 fragment (SA13). (B) pHSA32::HSA32-LUC-derived bioluminescence of indicated genotypes assayed 1, 2, or 3 d after ACC. (C,D) Seedlings of the indicated genotypes were acclimatized 5 d after germination and received a tester HS 3 d later. (C) Quantification; n = 48–49, Fisher’s exact test, **p<0.01. (D) Representative picture taken after 14 d recovery. (E) FGT1 transcript levels increase transiently after ACC. Relative FGT1 transcript levels were determined by qRT-PCR and normalized to At4g26410. Errors are SE of two biological replicates. (F) FGT1 is localized to the nucleus in 3 d-old seedling roots. 35S::FGT1-YFP transgenic seedlings were imaged for YFP fluorescence. Left, overlay; middle, bright field; right, YFP fluorescence. Scale bar, 40 µm. (G) FGT1 binds histone H3 in vivo. Nuclear protein extracts of transgenic 35S::FGT1-YFP, 35S::YFP and non-transgenic Col-0 seedlings harvested 28 h after the indicated treatments were immuno-precipitated with anti-GFP antibody. Co-purification of histone H3 was assessed by immunoblotting. https://doi.org/10.7554/eLife.17061.007 FGT1 contains an ATP-binding DExD/H-like helicase domain, a Helicase C-like domain, and a PHD finger (Figure 2A). FGT1 is a single copy gene in A. thaliana. Interestingly, it is highly homologous over the whole length of the protein with Sno from Drosophila melanogaster, human SBNO1 and SBNO2, and Caenorhabditis elegans let-765 (Figure 2—figure supplement 2) (Majumdar et al., 1997; Simms and Baillie, 2010; Grill et al., 2015). Sno genes are required for the expression of Notch and EGFR target genes and it has been hypothesized that they interact with co-activator proteins to spatio-temporally regulate transcription (Tsuda et al., 2002), yet no molecular mode of action has been demonstrated. Although DExD helicases have been ascribed a role in RNA processing and translation, roles in gene expression and transcription have been suggested (Fuller-Pace, 2006). FGT1 is expressed throughout the plant (Winter et al., 2007) and was slightly induced (1.7 fold) at 4 h after HS, but not thereafter (Figure 2E). We next tested the subcellular localization of the complementing FGT1-YFP fusion protein in roots of 3 d-old stably transformed seedlings. FGT1-YFP was localized to the nucleus (Figure 2F). PHD domains have the potential to bind to methylated histone H3 tails (Musselman and Kutateladze, 2011). We thus tested whether FGT1PHD-GST was precipitated by H3 histone tail peptides that were either unmethylated or mono-, di-, or trimethylated, respectively (Figure 2—figure supplement 3). We observed comparable binding to H3 aa 1–20, H3 aa 21–44 or H3 aa 1–20 methylated at K4 or K9. In contrast, the PHD domain of ING1 (Lee et al., 2009) bound under the same conditions only to the H3K4me3 peptide (Figure 2—figure supplement 3). This suggests that FGT1-PHD binds to the N-terminal region of H3, albeit not in a methylation-specific manner (at least with respect to methylated K4 and K9). In addition, histone H3 was co-immunoprecipitated with FGT1-YFP, but not YFP alone, from extracts of transgenic A. thaliana seedlings (Figure 2G). In summary, nuclear FGT1 is the A. thaliana orthologue of the DExD helicase Sno and associates with H3 in vivo, consistent with a function as a co-activator. FGT1 associates with HSA32 and other memory genes Given its potential function as a co-activator, we next asked whether FGT1 binds directly to its putative target genes during HS memory. To test this, we performed chromatin immunoprecipitation (ChIP) followed by qPCR analysis on 35S::FGT1-YFP plants (Figure 3). FGT1 bound to a broad region around the transcriptional start sites (TSS) of HSA32, HSP18.2, and HSP22.0. The enrichment of FGT1 in the heat-treated samples was highest 4 h and 28 h after ACC compared to the NHS, and was still present at 52 h. Such heat-dependent enrichment was not observed at the ACTIN7 (ACT7) and AtMu1 control loci. The enrichment at the active ACT7 gene was comparable to that of HSA32 before HS, suggesting that FGT1 binds HSA32 and ACT7 already pre-HS. The ChIP-signal in FGT1-YFP plants at bound loci was strongly enhanced compared to non-transgenic control samples (Figure 3—figure supplement 1A). A comparable but overall weaker binding pattern was observed for a FGT1-YFP driven by the endogenous promoter (Figure 3—figure supplement 1B,C), indicating that the observed binding pattern does not result from FGT1 overexpression. Thus, FGT1 binds to memory genes in a region encompassing the TSS and proximal promoter, where it may mediate their sustained expression after HS. Figure 3 with 1 supplement see all Download asset Open asset FGT1 binds memory-associated genes in a HS-dependent manner. FGT1 binds to HSA32, HSP18.2 and HSP22.0. ChIP-qPCR on 35S::FGT1-YFP seedlings was performed 4, 28 or 52 h after an acclimatizing HS (ACC) or no HS (NHS). As controls, the active gene ACT7 or the inactive transposon AtMu1 were used. Schematics show regions analyzed relative to TSS. HSA32 1–4: −175, −75, +57, +194 bp. HSP18.2 1–3: −1068, −367, +32 bp. HSP22.0 1–2: −3000, −60 bp; ACT7: +55 bp. AtMu1: -175 bp relative to ATG. Amplification values were normalized to input and region 2HSA32 at 28 h NHS (HSA32, AtMu1 and ACT7), region 2HSP18.2 at 28 h NHS or region 3HSP22.0 at 28 h NHS, respectively. Data are averages of at least three biological replicates. Error bars indicate SE. https://doi.org/10.7554/eLife.17061.011 FGT1 binds widely to the proximal promoter of expressed genes Because of the highly conserved nature of FGT1 and the binding to ACT7, we suspected that FGT1 may have genomic targets beyond the tested candidates. To obtain a global view, we performed ChIP-seq on 35S::FGT1-YFP plants 28 h after ACC or NHS. Peak calling identified 942 (60) genes with FGT1 enrichment after ACC (NHS), and no binding in the corresponding non-transgenic control samples. While the NHS peaks remained associated with FGT1 after ACC, the ACC peaks were overall less strongly enriched under NHS conditions (Figure 4—figure supplement 1). Coverage profiling of the FGT1-associated genes indicated that FGT1 bound primarily to the proximal promoter just upstream of the TSS and somewhat more weakly to the region downstream of the transcription termination site (TTS). In contrast, the signal was very low in the transcribed region. For both conditions (ACC and NHS), FGT1-associated genes showed a higher expression in seedlings under normal growth conditions compared to non-target genes (Figure 4A), suggesting that FGT1 binding is positively correlated with transcription. Figure 4 with 1 supplement see all Download asset Open asset FGT1 globally binds expressed genes upstream of the TSS. (A) FGT1-associated genes (ChIP-seq peaks) are more highly expressed than other genes. Violin plot indicates expression levels of FGT1-bound genes compared to all other genes (Figure 4—figure supplement 1). Expression data were taken from (Gan et al., 2011). (B) Normalized global read coverages of ACC or NHS FGT1-YFP or wild-type control samples as determined by ChIP-seq. Genes were categorized into not expressed genes and equally sized groups of highly, moderately and lowly expressed genes (Gan et al., 2011). Coverage profiles include 2 kb up- and downstream of the TSS and TTS, respectively. Genic regions were normalized to a standard length. (C) Normalized read coverages of HS-responsive genes from ACC or NHS FGT1-YFP. The panels show global analysis of genes in the respective expression class (cf. B) according to their expression pattern 4 h after ACC (Stief et al., 2014) in wild type (up, down, others). (D) FGT1 is enriched in chromatin state 2 (Sequeira-Mendes et al., 2014). Relative enrichment of FGT1-bound sequences or WT control after ACC or NHS in different chromatin states indicated depletion of FGT1 in heterochromatin (states 8, 9) and enrichment in state 2 (promoter and intergenic regions, open chromatin). Lines denote average of replicates. https://doi.org/10.7554/eLife.17061.013 Given that the identified peaks were wide and flat, we hypothesized that the peak calling may have underestimated the number of targets. Thus, we investigated the global correlation of FGT1 binding and expression under NHS conditions by plotting global coverage profiles grouped according to the relative expression in non-stressed seedlings (Gan et al., 2011). This revealed that FGT1 is preferentially associated with expressed genes at a global scale (Figure 4B). FGT1 is most strongly associated with genes that have high or intermediate expression in a pattern similar to that observed for the peak genes (Figure 4—figure supplement 1). We next asked whether FGT1 shows differential association with genes that are HS-responsive (Stief et al., 2014). FGT1 associated most strongly with those expressed genes that are upregulated at 4 h after ACC, irrespective of their expression level before HS (Figure 4C). This is especially true for genes that are lowly expressed without HS; this category contains typical HS-responsive genes. Accordingly, FGT1 associated most strongly with genes that are upregulated at 4 h and/ or 52 h after ACC and this is more pronounced in the ACC samples (Figure 7C). Thus, HS increases binding of FGT1 to HS-responsive genes globally, and these genes are associated with low levels of FGT1 already before HS. The A. thaliana genome was categorized into nine chromatin states based on the differential presence of histone modifications, variants and DNA methylation (Sequeira-Mendes et al., 2014). Analyzing the overlap between FGT1-bound sequences with different chromatin states, we found that FGT1 was highly enriched in sequences annotated as chromatin state 2 (Figure 4D). This state is found in poised chromatin, mostly in promoters and intergenic regions (hence transcript levels are low). It is enriched in H3.3, H3K4me2, H3K4me3, H2A.Z, H2Bub, H3K27me3, AT-rich and relatively low in overall nucleosome abundance. Strikingly, state 2 peaks immediately before the TSS, and has a smaller peak just after the TTS, mimicking closely the global coverage profile of FGT1 (Sequeira-Mendes et al., 2014). In contrast, FGT1 was depleted from the heterochromatic states 8 and 9. Thus, FGT1 binding globally associates with the nucleosome-poor regions flanking the transcription units of expressed genes. FGT1 interacts with SWI/SNF and ISWI chromatin remodelers To elucidate the mechanism of how FGT1 promotes gene expression, we isolated FGT1-interacting proteins. To this end, we purified native FGT1-YFP complexes from 35S::FGT1-YFP seedlings 28 h after ACC or control (NHS) treatment. FGT1-YFP and associated proteins were then subjected to LC-MS/MS analysis. As controls, we performed purifications on 35S::YFP and Col-0 plants, respectively. Among the peptides identified specifically in the FGT1-YFP samples were both A. thaliana orthologues of the ISWI chromatin remodeler, CHR11 and CHR17, and the SWI/SNF chromatin remodeler BRAHMA (BRM), suggesting that FGT1 interacts with chromatin remodeling proteins (Table 1). Because of the high homology between CHR11 and CHR17, most of the identified peptides could not be assigned unequivocally to either of the two proteins, however, a few specific peptides were recovered demonstrating the presence of both ISWI proteins (Table 1). We also identified several known subunits (SWI3a, b, d, SWP73b) of the BRM complex. We did not observe differences between the ACC and NHS samples, suggesting that the mode of action of FGT1 is independent of HS. To confirm the interactions between FGT1 and the remodelers we used bimolecular fluorescent complementation in transiently transformed tobacco leaves (Walter et al., 2004). We thus confirmed the interaction of FGT1 with CHR11, CHR17 and BRM in the nucleus (Figure 5). In summary, FGT1 interacts with chromatin remodeling proteins of the ISWI and SWI/SNF classes. Table 1 FGT1 interacts with chromatin remodeling proteins in vivo. FGT1-interacting proteins identified by native co-immunoprecipitation followed by mass spectrometry (nHPLC-MS/MS) from 5 d-old 35S::FGT1-YFP seedlings subjected to ACC or NHS 28 h before sampling. Col-0 and 35S::YFP were used as controls. The data represent the number of unique peptides found in the indicated experiments. https://doi.org/10.7554/eLife.17061.015 BackgroundTreatmentExpNumber of peptidesFGT1CHR11/ CHR17Chr11Chr17BRMSWI3aSWI3bSWI3dSWP73b35S::FGT1-YFPACC15841------25612322311334311--2----NHS1332------1252114214--33514-------Col-0ACC1-3---------NHS1-3---------35S::YFPNHS1-3--------- Figure 5 Download asset Open asset FGT1 interacts in vivo with SWI/SNF (BRM) and ISWI (CHR11, CHR17) chromatin remodeling proteins. Bimolecular Fluorescence Complementation confirms the interaction of FGT1 and CHR11, CHR17 or BRM in the nucleus of tobacco leaf cells. The indicated constructs were co-transformed and analyzed 2 d later with an LSM710 confocal microscope. YFP, BiFC signal in the YFP spectrum; RFP, signal from co-expressed nuclear RFP-fusion protein. Size bar, 20 µm. https://doi.org/10.7554/eLife.17061.016 BRM and ISWI are required for HS memory To determine whether the interaction of FGT1 and the remodelers was functionally relevant during HS memory, we examined whether remodeler mutants displayed normal HS memory. As the loss of BRM causes sterility, we performed the assay on the progeny of a heterozygous brm-1/+ plant and genotyped individual seedlings after phenotyping. Indeed, brm-1 mutants displayed reduced HS memory (Figure 6A,B). The highly similar CHR11 and CHR17 proteins show functional redundancy and the double mutant displays severe developmental defects including dwarfism (Li et al., 2012). Thus, we performed the experiment on the progeny of a chr11/chr11 chr17/+ plant. As for brm-1, we genotyped individual seedlings after the phenotypic analysis was completed. We observed that chr11 single mutants and chr11/chr11 chr17/+ seedlings were defective in the physiological HS memory (Figure 6A,B). Due to their growth defects, we could not analyze chr11 chr17 double mutants. To test whether the remodeler mutants have a generally impaired HS response, we also tested their ability to acquire thermotolerance and their basal thermotolerance. Mutants in the remodelers behaved similar to the wild type or slightly better in these assays (Figure 6—figure supplements 1, 2), which indicates that the responses to acute HS were not compromised. Thus, the remodeler mutants under investigation displayed a specific impairment of HS memory and not a general defect in HS responses. We also tested the expression of HS-responsive genes after ACC in wild type, and brm-1/+ or chr11/chr11 chr17/+ -segregating lines, respectively. qRT-PCR analysis revealed that both mutant lines show a premature decline of expression of HSA32, HSP18.2, HSP21, HSP22 and HSP101 (Figure 6C). In many cases, transcript levels were already lower at the earliest time point measured (immediately after the end of ACC), suggesting that the remodelers were also necessary for full induction of these genes. Interestingly, this was not correlated with a reduced leve" @default.
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• W2985361230 title "Author response: Arabidopsis FORGETTER1 mediates stress-induced chromatin memory through nucleosome remodeling" @default.
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