HSFA2 and HSFA3 binding after heat acclimation [ChIP-seq]
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ABSTRACT: Transcriptional regulation is a key aspect of environmental stress responses. Heat stress (HS) induces transcriptional memory that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crops is an important breeding goal. However, not all HS-inducible genes show sustained induction/transcriptional memory and it is unclear what distinguishes memory and non-memory genes. To address this issue and understand the (epi-) genome architecture in transcriptional memory after HS, we investigated genome-wide target genes of the two key memory heat shock transcription factors, HSFA2 and HSFA3. We determined the binding kinetics of these factors to their target genes and asked whether genes that show sustained induction of transcription carry specific features that allow prediction and potentially engineering of memory gene behaviour. HSFA2 and HSFA3 show near identical binding patterns. In vitro binding strength as determined by DAP-seq analysis correlates strongly with in vivo binding strength, confirming the importance of sequence features. However, no single distinctive sequence motif appears to be required for memory behaviour. Instead, HS memory genes are characterized by a combination of features: low expression levels in the absence of HS, chromatin environment and an enrichment of H3K4 methylation after HS. Our findings are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. In summary, our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants. They will contribute to the prediction and engineering of genes with transcriptional memory.
Project description:Transcriptional regulation is a key aspect of environmental stress responses. Heat stress (HS) induces transcriptional memory that allows plants to respond more efficiently to a recurrent HS. In light of more frequent temperature extremes due to climate change, improving heat tolerance in crops is an important breeding goal. However, not all HS-inducible genes show sustained induction/transcriptional memory and it is unclear what distinguishes memory and non-memory genes. To address this issue and understand the (epi-) genome architecture in transcriptional memory after HS, we investigated genome-wide target genes of the two key memory heat shock transcription factors, HSFA2 and HSFA3. We determined the binding kinetics of these factors to their target genes and asked whether genes that show sustained induction of transcription carry specific features that allow prediction and potentially engineering of memory gene behaviour. HSFA2 and HSFA3 show near identical binding patterns. In vitro binding strength as determined by DAP-seq analysis correlates strongly with in vivo binding strength, confirming the importance of sequence features. However, no single distinctive sequence motif appears to be required for memory behaviour. Instead, HS memory genes are characterized by a combination of features: low expression levels in the absence of HS, chromatin environment and an enrichment of H3K4 methylation after HS. Our findings are confirmed by an orthogonal transcriptomic data set using both de novo clustering and an established definition of memory genes. In summary, our findings provide an integrated view of HSF-dependent transcriptional memory and shed light on its sequence and chromatin determinants. They will contribute to the prediction and engineering of genes with transcriptional memory.
Project description:Adaptive plasticity in stress responses is a key element of plant survival strategies. For instance, moderate heat stress (HS) primes a plant to acquire thermotolerance, which allows subsequent survival of more severe HS conditions. Acquired thermotolerance is actively maintained over several days (HS memory) and involves the sustained induction of memory-related genes. We find FORGETTER3/ HEAT SHOCK TRANSCRIPTION FACTOR A3 (FGT3/HSFA3) to be specifically required for physiological HS memory and maintaining high memory-gene expression during the days following a HS exposure. HSFA3 mediates HS memory by direct transcriptional activation of memory-related genes after return to normal growth temperatures. HSFA3 binds HSFA2, and in vivo both proteins form heteromeric complexes with additional HSFs. Our results indicate that only complexes containing both HSFA2 and HSFA3 efficiently promote transcriptional memory by promoting histone H3 lysine 4 (H3K4) hyper-methylation. In summary, our work defines the major HSF complex controlling transcriptional memory and elucidates the in vivo dynamics of HSF complexes during somatic stress memory.
Project description:In nature, plants are often exposed to recurring adverse environmental conditions. Acclimation to high temperature stress entails transcriptional responses that are mediated by heat-shock transcription factors (HSFs), and they are primed to better withstand subsequent stress events. This heat stress (HS)-induced transcriptional memory results in more efficient re-induction of transcription upon recurring HS. However, the mechanisms by which HSFs recruit and enact the transcriptional machinery remain unclear. Here, we identified two subunits of the kinase module of the Mediator transcriptional co-regulator complex, CDK8 and MED12, as regulators of HS memory in Arabidopsis thaliana. Enhanced re-induction of gene expression after recurrent HS and physiological HS memory, as well as H3K4 methylation are compromised in cdk8 and med12 mutants. HSFA2 interacts with CDK8 during and after HS and recruits it to memory gene loci, where CDK8 binds in the promoter but also the gene body, together with core Mediator and RNA polymerase II (Pol II). Our data suggest that CDK8 resolves stalled Pol II complexes or promotes efficient recycling for subsequent cycles of transcription. As HSFA2, CDK8 is largely dispensable for the initial induction of gene expression after HS and thus promotes transcriptional memory independently of HS-dependent primary gene induction. Our findings provide a model for the complex role of the Mediator kinase module during transcriptional memory in multicellular eukaryotes through interaction with transcription factors, chromatin modifications and promotion of Pol II productivity.
Project description:In nature, plants are constantly exposed to many transient, but recurring, stresses. Thus, to complete their life cycles they require a dynamic balance between capacities to recover following cessation of stress and maintenance of stress memory. Recently, we uncovered a new functional role of autophagy in regulating recovery from heat stress (HS) and resetting cellular memory of HS in Arabidopsis thaliana. Here, we demonstrate that NBR1 (Next-to-BRCA1) plays a crucial role as an adaptor receptor for selective autophagy during recovery from HS. Immunoblot analysis and confocal microscopy revealed that levels of NBR1 protein, NBR1-labeled puncta and NBR1 activities were all higher during the HS recovery phase than before and after this phase. Co-immunoprecipitation analysis of proteins interacting with NBR1 and comparative proteomic analysis of a nbr1 knockout mutant and wild-type plants identified 58 proteins as potential novel targets of NBR1. Cellular, biochemical and functional genetic studies confirmed that NBR1 targets Heat Shock Protein 90 (HSP90) and ROF1, a member of the FKBP family, and mediates their degradation by autophagy, which represses the expression of HSPs regulated by HsfA2 transcription factor and the response to HS. Accordingly, loss-of-function mutation of NBR1 resulted in a stronger HS memory phenotype. Together our results provide new insights into the mechanistic principles by which autophagy regulates plant response to recurrent HS.
Project description:Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by heat stress transcription factors (Hsfs). The role of Hsfs and Hsps in HS-response in tomato was initially examined by transcriptome analysis using the Massive Analysis of cDNA Ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points toward two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato. 2 samples
Project description:Heat shock proteins (Hsps) are molecular chaperones primarily involved in maintenance of protein homeostasis. Their function has been best characterized in heat stress (HS) response during which Hsps are transcriptionally controlled by heat stress transcription factors (Hsfs). The role of Hsfs and Hsps in HS-response in tomato was initially examined by transcriptome analysis using the Massive Analysis of cDNA Ends (MACE) method. Approximately 9.6% of all genes expressed in leaves are enhanced in response to HS, including a subset of Hsfs and Hsps. The underlying Hsp-Hsf networks with potential functions in stress responses or developmental processes were further explored by meta-analysis of existing microarray datasets. We identified clusters with differential transcript profiles with respect to abiotic stresses, plant organs and developmental stages. The composition of two clusters points toward two major chaperone networks. One cluster consisted of constitutively expressed plastidial chaperones and other genes involved in chloroplast protein homeostasis. The second cluster represents genes strongly induced by heat, drought and salinity stress, including HsfA2 and many stress-inducible chaperones, but also potential targets of HsfA2 not related to protein homeostasis. This observation attributes a central regulatory role to HsfA2 in controlling different aspects of abiotic stress response and tolerance in tomato.
Project description:Plants often experience recurrent stressful events, for example during heat waves. They can adapt to such recurrent heat stress (HS), allowing subsequent survival of more severe HS conditions. At certain genes HS induces sustained expression for several days beyond the actual HS. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), however, how this is maintained for extended periods of time is unclear. Here, we determined histone turnover by measuring chromatin association of a HS-induced histone H3.3. Genome-wide Histone turnover was not homogenous, in particular, H3.3 was retained longer at HS memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, its loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation (and not vice versa). Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally-mediated epigenetic modifications.
Project description:Male reproductive tissues are more sensitive to heat stress compared to vegetative tissues, however the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection and recovery from heat stress. HsfA2 has been characterized as co-activator of HsfA1a in tomato and is considered as one of the major Hsfs accumulating in response to elevated temperatures. The role of HsfA2 in heat stress response of different tissues was examined by exploring the composition and structure of the tissue-specific regulatory networks in transgenic tomato plants with suppressed HsfA2 expression (A2AS). Transcriptome analysis revealed that HsfA2 acts in condition- and tissue-specific manner and that only a subset of heat stress induced genes require HsfA2 for higher expression. Remarkably, although HsfA2 is not essential for thermotolerance in seedlings and flowering plants, it is required for maintenance pollen viability under stress conditions. We show that the activation of Hsf networks is important for the developmentally regulated priming of heat stress response occurring at early stages of anther and pollen development. Thereby, HsfA2 is involved in pollen thermotolerance by directly regulating heat stress responsive genes but also by stimulating the synthesis of molecular chaperones under non-stress conditions.
Project description:Male reproductive tissues are more sensitive to heat stress compared to vegetative tissues, however the basis of this phenomenon is poorly understood. Heat stress transcription factors (Hsfs) regulate the transcriptional changes required for protection and recovery from heat stress. HsfA2 has been characterized as co-activator of HsfA1a in tomato and is considered as one of the major Hsfs accumulating in response to elevated temperatures. The role of HsfA2 in heat stress response of different tissues was examined by exploring the composition and structure of the tissue-specific regulatory networks in transgenic tomato plants with suppressed HsfA2 expression (A2AS). Transcriptome analysis revealed that HsfA2 acts in condition- and tissue-specific manner and that only a subset of heat stress induced genes require HsfA2 for higher expression. Remarkably, although HsfA2 is not essential for thermotolerance in seedlings and flowering plants, it is required for maintenance pollen viability under stress conditions. We show that the activation of Hsf networks is important for the developmentally regulated priming of heat stress response occurring at early stages of anther and pollen development. Thereby, HsfA2 is involved in pollen thermotolerance by directly regulating heat stress responsive genes but also by stimulating the synthesis of molecular chaperones under non-stress conditions. 8 samples
Project description:The expression of heat-shock proteins (Hsps) induced by a non-lethal heat treatment confers acquired thermotolerance (AT) to organisms against a subsequent challenge of otherwise lethal temperature. After stress signal lifted, AT gradually decayed with the decline of Hsps during recovery period. The duration of AT may be critical for sessile organisms, such as plants, to survive repeated heat stress in the environment. To identify heat-induced genes involved in duration of AT, we took a reverse-genetics approach by screening for Arabidopsis T-DNA insertion mutants that show decreased thermotolerance after a long recovery at non-stress condition following a conditioning treatment. Among the tested mutants corresponding to 47 genes, only the HsfA2 knockout mutant showed significant phenotype. The mutant plants were more sensitive to severe heat stress than the wild type after long but not short recovery following a pretreatment at 37oC, which can be complemented by introducing a wild-type copy of the gene. Quantitative hypocotyl elongation assay also revealed that AT decayed faster in the absence of HsfA2. Significant decline of the transcript levels of several highly heat-induced genes was observed in the HsfA2 knockout plants after a 4-h recovery or after 2 h of prolonged heat stress. Immunoblot anlysis showed that Hsa32 and class I small Hsp were lower in the mutant than in the wild type after a long recovery. Our results suggest that HsfA2 as a heat-induced transactivator sustains the post-stress expression of Hsp genes and extends the duration of AT in Arabidopsis. Experiment Overall Design: Total RNA was isolated from the seedlings of 5-d old wild-type and HsfA2 knockout mutant seedlings (a pool of about 100 plants per treatment in duplicates) harvested immediately after heat shock treatment. In this experiment, total 12 chips were used, 1 each for 2 biological replicates of the control and HS-treated samples for the wild type and mutant plants.