RESUMO
Cells sense elevated temperatures and mount an adaptive heat shock response that involves changes in gene expression, but the underlying mechanisms, particularly on the level of translation, remain unknown. Here we report that, in budding yeast, the essential translation initiation factor Ded1p undergoes heat-induced phase separation into gel-like condensates. Using ribosome profiling and an in vitro translation assay, we reveal that condensate formation inactivates Ded1p and represses translation of housekeeping mRNAs while promoting translation of stress mRNAs. Testing a variant of Ded1p with altered phase behavior as well as Ded1p homologs from diverse species, we demonstrate that Ded1p condensation is adaptive and fine-tuned to the maximum growth temperature of the respective organism. We conclude that Ded1p condensation is an integral part of an extended heat shock response that selectively represses translation of housekeeping mRNAs to promote survival under conditions of severe heat stress.
Assuntos
RNA Helicases DEAD-box/metabolismo , Regulação Fúngica da Expressão Gênica/genética , Biossíntese de Proteínas/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , RNA Helicases DEAD-box/fisiologia , Expressão Gênica/genética , Genes Essenciais/genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , RNA Mensageiro/metabolismo , Ribossomos/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/fisiologiaRESUMO
Cellular homeostasis is constantly challenged by a myriad of extrinsic and intrinsic stressors. To mitigate the stress-induced damage, cells activate transient survival programs. The heat shock response (HSR) is an evolutionarily well-conserved survival program that is activated in response to proteotoxic stress. The HSR encompasses a dual regulation of transcription, characterized by rapid activation of genes encoding molecular chaperones and concomitant global attenuation of non-chaperone genes. Recent genome-wide approaches have delineated the molecular depth of stress-induced transcriptional reprogramming. The dramatic rewiring of gene and enhancer networks is driven by key transcription factors, including heat shock factors (HSFs), that together with chromatin-modifying enzymes remodel the 3D chromatin architecture, determining the selection of either gene activation or repression. Here, we highlight the current advancements of molecular mechanisms driving transcriptional reprogramming during acute heat stress. We also discuss the emerging implications of HSF-mediated stress signaling in the context of physiological and pathological conditions.
Assuntos
Proteostase , Fatores de Transcrição , Proteostase/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta ao Choque Térmico/genética , Chaperonas Moleculares/genética , Cromatina/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismoRESUMO
RNA polymerase II (RNA Pol II)-mediated transcription is a critical, highly regulated process aided by protein complexes at distinct steps. Here, to investigate RNA Pol II and transcription-factor-binding and dissociation dynamics, we generated endogenous photoactivatable-GFP (PA-GFP) and HaloTag knockins using CRISPR-Cas9, allowing us to track a population of molecules at the induced Hsp70 loci in Drosophila melanogaster polytene chromosomes. We found that early in the heat-shock response, little RNA Pol II and DRB sensitivity-inducing factor (DSIF) are reused for iterative rounds of transcription. Surprisingly, although PAF1 and Spt6 are found throughout the gene body by chromatin immunoprecipitation (ChIP) assays, they show markedly different binding behaviors. Additionally, we found that PAF1 and Spt6 are only recruited after positive transcription elongation factor (P-TEFb)-mediated phosphorylation and RNA Pol II promoter-proximal pause escape. Finally, we observed that PAF1 may be expendable for transcription of highly expressed genes where nucleosome density is low. Thus, our live-cell imaging data provide key constraints to mechanistic models of transcription regulation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , RNA Polimerase II , Transcrição Gênica , Fatores de Elongação da Transcrição , RNA Polimerase II/metabolismo , RNA Polimerase II/genética , Animais , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Fatores de Elongação da Transcrição/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Fator B de Elongação Transcricional Positiva/genética , Regiões Promotoras Genéticas , Sistemas CRISPR-Cas , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genética , Cromossomos Politênicos/genética , Cromossomos Politênicos/metabolismo , Regulação da Expressão Gênica , Fosforilação , Ligação Proteica , Resposta ao Choque Térmico/genética , Proteínas Nucleares/metabolismo , Proteínas Nucleares/genética , Nucleossomos/metabolismo , Nucleossomos/genéticaRESUMO
Heat-shocked cells prioritize the translation of heat shock (HS) mRNAs, but the underlying mechanism is unclear. We report that HS in budding yeast induces the disassembly of the eIF4F complex, where eIF4G and eIF4E assemble into translationally arrested mRNA ribonucleoprotein particles (mRNPs) and HS granules (HSGs), whereas eIF4A promotes HS translation. Using in vitro reconstitution biochemistry, we show that a conformational rearrangement of the thermo-sensing eIF4A-binding domain of eIF4G dissociates eIF4A and promotes the assembly with mRNA into HS-mRNPs, which recruit additional translation factors, including Pab1p and eIF4E, to form multi-component condensates. Using extracts and cellular experiments, we demonstrate that HS-mRNPs and condensates repress the translation of associated mRNA and deplete translation factors that are required for housekeeping translation, whereas HS mRNAs can be efficiently translated by eIF4A. We conclude that the eIF4F complex is a thermo-sensing node that regulates translation during HS.
Assuntos
Fator de Iniciação 4F em Eucariotos , Fator de Iniciação Eucariótico 4G , Resposta ao Choque Térmico , Proteínas de Ligação a Poli(A) , Biossíntese de Proteínas , RNA Mensageiro , Ribonucleoproteínas , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Resposta ao Choque Térmico/genética , Fator de Iniciação 4F em Eucariotos/metabolismo , Fator de Iniciação 4F em Eucariotos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fator de Iniciação Eucariótico 4G/metabolismo , Fator de Iniciação Eucariótico 4G/genética , Ribonucleoproteínas/metabolismo , Ribonucleoproteínas/genética , Fator de Iniciação 4E em Eucariotos/metabolismo , Fator de Iniciação 4E em Eucariotos/genética , Fator de Iniciação 4A em Eucariotos/metabolismo , Fator de Iniciação 4A em Eucariotos/genética , Regulação Fúngica da Expressão Gênica , Ligação Proteica , RNA Fúngico/metabolismo , RNA Fúngico/genéticaRESUMO
Eusocial insect reproductive females show strikingly longer life spans than nonreproductive female workers despite high genetic similarity. In the ant Harpegnathos saltator (Hsal), workers can transition to reproductive "gamergates," acquiring a fivefold prolonged life span by mechanisms that are poorly understood. We found that gamergates have elevated expression of heat shock response (HSR) genes in the absence of heat stress and enhanced survival with heat stress. This HSR gene elevation is driven in part by gamergate-specific up-regulation of the gene encoding a truncated form of a heat shock factor most similar to mammalian HSF2 (hsalHSF2). In workers, hsalHSF2 was bound to DNA only upon heat stress. In gamergates, hsalHSF2 bound to DNA even in the absence of heat stress and was localized to gamergate-biased HSR genes. Expression of hsalHSF2 in Drosophila melanogaster led to enhanced heat shock survival and extended life span in the absence of heat stress. Molecular characterization illuminated multiple parallels between long-lived flies and gamergates, underscoring the centrality of hsalHSF2 to extended ant life span. Hence, ant caste-specific heat stress resilience and extended longevity can be transferred to flies via hsalHSF2. These findings reinforce the critical role of proteostasis in health and aging and reveal novel mechanisms underlying facultative life span extension in ants.
Assuntos
Formigas , Longevidade , Animais , Feminino , Longevidade/genética , Formigas/genética , Drosophila melanogaster/genética , Envelhecimento , Resposta ao Choque Térmico/genética , MamíferosRESUMO
The heat shock transcription factors (HSFs) were discovered over 30 years ago as direct transcriptional activators of genes regulated by thermal stress, encoding heat shock proteins. The accepted paradigm posited that HSFs exclusively activate the expression of protein chaperones in response to conditions that cause protein misfolding by recognizing a simple promoter binding site referred to as a heat shock element. However, we now realize that the mammalian family of HSFs comprises proteins that independently or in concert drive combinatorial gene regulation events that activate or repress transcription in different contexts. Advances in our understanding of HSF structure, post-translational modifications and the breadth of HSF-regulated target genes have revealed exciting new mechanisms that modulate HSFs and shed new light on their roles in physiology and pathology. For example, the ability of HSF1 to protect cells from proteotoxicity and cell death is impaired in neurodegenerative diseases but can be exploited by cancer cells to support their growth, survival and metastasis. These new insights into HSF structure, function and regulation should facilitate the development tof new disease therapeutics to manipulate this transcription factor family.
Assuntos
Regulação da Expressão Gênica/genética , Fatores de Transcrição de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Transcrição Gênica/genética , Animais , Resposta ao Choque Térmico/genética , Humanos , Processamento de Proteína Pós-Traducional/genéticaRESUMO
The heat shock (HS) response involves rapid induction of HS genes, whereas transcriptional repression is established more slowly at most other genes. Previous data suggested that such repression results from inhibition of RNA polymerase II (RNAPII) pause release, but here, we show that HS strongly affects other phases of the transcription cycle. Intriguingly, while elongation rates increase upon HS, processivity markedly decreases, so that RNAPII frequently fails to reach the end of genes. Indeed, HS results in widespread premature transcript termination at cryptic, intronic polyadenylation (IPA) sites near gene 5'-ends, likely via inhibition of U1 telescripting. This results in dramatic reconfiguration of the human transcriptome with production of new, previously unannotated, short mRNAs that accumulate in the nucleus. Together, these results shed new light on the basic transcription mechanisms induced by growth at elevated temperature and show that a genome-wide shift toward usage of IPA sites can occur under physiological conditions.
Assuntos
Poliadenilação , Transcriptoma , Resposta ao Choque Térmico/genética , Humanos , RNA Polimerase II/genética , RNA Polimerase II/metabolismo , RNA Mensageiro/genéticaRESUMO
Mammalian developmental and disease-associated genes concentrate large quantities of the transcriptional machinery by forming membrane-less compartments known as transcriptional condensates. However, it is unknown whether these structures are evolutionarily conserved or involved in 3D genome reorganization. Here, we identify inducible transcriptional condensates in the yeast heat shock response (HSR). HSR condensates are biophysically dynamic spatiotemporal clusters of the sequence-specific transcription factor heat shock factor 1 (Hsf1) with Mediator and RNA Pol II. Uniquely, HSR condensates drive the coalescence of multiple Hsf1 target genes, even those located on different chromosomes. Binding of the chaperone Hsp70 to a site on Hsf1 represses clustering, whereas an intrinsically disordered region on Hsf1 promotes condensate formation and intergenic interactions. Mutation of both Hsf1 determinants reprograms HSR condensates to become constitutively active without intergenic coalescence, which comes at a fitness cost. These results suggest that transcriptional condensates are ancient and flexible compartments of eukaryotic gene control.
Assuntos
Resposta ao Choque Térmico , Corpos Nucleares , Animais , Resposta ao Choque Térmico/genética , Proteínas de Choque Térmico HSP70/genética , Mamíferos , RNA Polimerase II/genética , Saccharomyces cerevisiae/genética , GenomaRESUMO
Heat shock instantly reprograms transcription. Whether gene and enhancer transcription fully recover from stress and whether stress establishes a memory by provoking transcription regulation that persists through mitosis remained unknown. Here, we measured nascent transcription and chromatin accessibility in unconditioned cells and in the daughters of stress-exposed cells. Tracking transcription genome-wide at nucleotide-resolution revealed that cells precisely restored RNA polymerase II (Pol II) distribution at gene bodies and enhancers upon recovery from stress. However, a single heat exposure in embryonic fibroblasts primed a faster gene induction in their daughter cells by increasing promoter-proximal Pol II pausing and by accelerating the pause release. In K562 erythroleukemia cells, repeated stress refined basal and heat-induced transcription over mitotic division and decelerated termination-coupled pre-mRNA processing. The slower termination retained transcripts on the chromatin and reduced recycling of Pol II. These results demonstrate that heat-induced transcriptional memory acts through promoter-proximal pause release and pre-mRNA processing at transcription termination.
Assuntos
Mitose/genética , Regiões Promotoras Genéticas/genética , Estresse Fisiológico/genética , Transcrição Gênica/genética , Linhagem Celular Tumoral , Cromatina/genética , Fibroblastos/fisiologia , Regulação da Expressão Gênica/genética , Genoma/genética , Resposta ao Choque Térmico/genética , Humanos , Células K562 , RNA Polimerase II/genética , RNA Mensageiro/genéticaRESUMO
In response to stress, human cells coordinately downregulate transcription and translation of housekeeping genes. To downregulate transcription, the negative elongation factor (NELF) is recruited to gene promoters impairing RNA polymerase II elongation. Here we report that NELF rapidly forms nuclear condensates upon stress in human cells. Condensate formation requires NELF dephosphorylation and SUMOylation induced by stress. The intrinsically disordered region (IDR) in NELFA is necessary for nuclear NELF condensation and can be functionally replaced by the IDR of FUS or EWSR1 protein. We find that biomolecular condensation facilitates enhanced recruitment of NELF to promoters upon stress to drive transcriptional downregulation. Importantly, NELF condensation is required for cellular viability under stressful conditions. We propose that stress-induced NELF condensates reported here are nuclear counterparts of cytosolic stress granules. These two stress-inducible condensates may drive the coordinated downregulation of transcription and translation, likely forming a critical node of the stress survival strategy.
Assuntos
Resposta ao Choque Térmico/genética , Proteínas Intrinsicamente Desordenadas/genética , Processamento de Proteína Pós-Traducional , RNA Polimerase II/genética , Transcrição Gênica , Fatores de Elongação da Transcrição/genética , Aminoaciltransferases/genética , Aminoaciltransferases/metabolismo , Cromatina/química , Cromatina/metabolismo , Células Clonais , Quinase 9 Dependente de Ciclina/genética , Quinase 9 Dependente de Ciclina/metabolismo , Genes Reporter , Células HEK293 , Células HeLa , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Fosforilação , Fator B de Elongação Transcricional Positiva/genética , Fator B de Elongação Transcricional Positiva/metabolismo , Regiões Promotoras Genéticas , RNA Polimerase II/metabolismo , Transdução de Sinais , Estresse Fisiológico , Sumoilação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Fatores de Elongação da Transcrição/química , Fatores de Elongação da Transcrição/metabolismo , Proteína Vermelha FluorescenteRESUMO
Plants are often exposed to recurring adverse environmental conditions in the wild. Acclimation to high temperatures entails transcriptional responses, which prime plants to better withstand subsequent stress events. Heat stress (HS)-induced transcriptional memory results in more efficient re-induction of transcription upon recurrence of heat stress. Here, we identified CDK8 and MED12, two subunits of the kinase module of the transcription co-regulator complex, Mediator, as promoters of heat stress memory and associated histone modifications in Arabidopsis. CDK8 is recruited to heat-stress memory genes by HEAT SHOCK TRANSCRIPTION FACTOR A2 (HSFA2). Like HSFA2, CDK8 is largely dispensable for the initial gene induction upon HS, and its function in transcriptional memory is thus independent of primary gene activation. In addition to the promoter and transcriptional start region of target genes, CDK8 also binds their 3'-region, where it may promote elongation, termination, or rapid re-initiation of RNA polymerase II (Pol II) complexes during transcriptional memory bursts. Our work presents a complex role for the Mediator kinase module during transcriptional memory in multicellular eukaryotes, through interactions with transcription factors, chromatin modifications, and promotion of Pol II efficiency.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Resposta ao Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Ativação Transcricional , Nucleotidiltransferases/metabolismo , Complexo Mediador/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Quinase 8 Dependente de Ciclina/genética , Quinase 8 Dependente de Ciclina/metabolismoRESUMO
RNA polymerase II (RNA Pol II) is generally paused at promoter-proximal regions in most metazoans, and based on in vitro studies, this function has been attributed to the negative elongation factor (NELF). Here, we show that upon rapid depletion of NELF, RNA Pol II fails to be released into gene bodies, stopping instead around the +1 nucleosomal dyad-associated region. The transition to the 2nd pause region is independent of positive transcription elongation factor P-TEFb. During the heat shock response, RNA Pol II is rapidly released from pausing at heat shock-induced genes, while most genes are paused and transcriptionally downregulated. Both of these aspects of the heat shock response remain intact upon NELF loss. We find that NELF depletion results in global loss of cap-binding complex from chromatin without global reduction of nascent transcript 5' cap stability. Thus, our studies implicate NELF functioning in early elongation complexes distinct from RNA Pol II pause-release.
Assuntos
Fator B de Elongação Transcricional Positiva/genética , RNA Polimerase II/genética , Fatores de Transcrição/genética , Transcrição Gênica , Animais , Resposta ao Choque Térmico/genética , Humanos , Camundongos , Nucleossomos/genética , Regiões Promotoras GenéticasRESUMO
Plants often experience recurrent stressful events, for example, during heat waves. They can be primed by heat stress (HS) to improve the survival of more severe heat stress conditions. At certain genes, sustained expression is induced for several days beyond the initial heat stress. This transcriptional memory is associated with hyper-methylation of histone H3 lysine 4 (H3K4me3), but it is unclear how this is maintained for extended periods. Here, we determined histone turnover by measuring the chromatin association of HS-induced histone H3.3. Genome-wide histone turnover was not homogenous; in particular, H3.3 was retained longer at heat stress memory genes compared to HS-induced non-memory genes during the memory phase. While low nucleosome turnover retained H3K4 methylation, methylation loss did not affect turnover, suggesting that low nucleosome turnover sustains H3K4 methylation, but not vice versa. Together, our results unveil the modulation of histone turnover as a mechanism to retain environmentally mediated epigenetic modifications.
Assuntos
Histonas , Nucleossomos , Histonas/genética , Histonas/metabolismo , Nucleossomos/genética , Cromatina/genética , Resposta ao Choque Térmico/genética , Epigênese GenéticaRESUMO
Temperature shapes the geographical distribution and behavior of plants. Understanding the regulatory mechanisms underlying the plant heat stress response is important for developing climate-resilient crops, including maize (Zea mays). To identify transcription factors (TFs) that may contribute to the maize heat stress response, we generated a dataset of short- and long-term transcriptome changes following a heat treatment time course in the inbred line B73. Co-expression network analysis highlighted several TFs, including the class B2a heat shock factor (HSF) ZmHSF20. Zmhsf20 mutant seedlings exhibited enhanced tolerance to heat stress. Furthermore, DNA affinity purification sequencing and Cleavage Under Targets and Tagmentation assays demonstrated that ZmHSF20 binds to the promoters of Cellulose synthase A2 (ZmCesA2) and three class A Hsf genes, including ZmHsf4, repressing their transcription. We showed that ZmCesA2 and ZmHSF4 promote the heat stress response, with ZmHSF4 directly activating ZmCesA2 transcription. In agreement with the transcriptome analysis, ZmHSF20 inhibited cellulose accumulation and repressed the expression of cell wall-related genes. Importantly, the Zmhsf20 Zmhsf4 double mutant exhibited decreased thermotolerance, placing ZmHsf4 downstream of ZmHsf20. We proposed an expanded model of the heat stress response in maize, whereby ZmHSF20 lowers seedling heat tolerance by repressing ZmHsf4 and ZmCesA2, thus balancing seedling growth and defense.
Assuntos
Regulação da Expressão Gênica de Plantas , Glucosiltransferases , Fatores de Transcrição de Choque Térmico , Resposta ao Choque Térmico , Proteínas de Plantas , Zea mays , Zea mays/genética , Zea mays/fisiologia , Zea mays/metabolismo , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Resposta ao Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismo , Termotolerância/genética , Celulose/metabolismo , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
Bacterial pathogens deliver effectors into host cells to suppress immunity. How host cells target these effectors is critical in pathogen-host interactions. SUMOylation, an important type of posttranslational modification in eukaryotic cells, plays a critical role in immunity, but its effect on bacterial effectors remains unclear in plant cells. In this study, using bioinformatic and biochemical approaches, we found that at least 16 effectors from the bacterial pathogen Pseudomonas syringae pv. tomato DC3000 are SUMOylated by the enzyme cascade from Arabidopsis thaliana. Mutation of SUMOylation sites on the effector HopB1 enhances its function in the induction of plant cell death via stability attenuation of a plant receptor kinase BRASSINOSTEROID INSENSITIVE 1 (BRI1)-ASSOCIATED RECEPTOR KINASE 1. By contrast, SUMOylation is essential for the function of another effector, HopG1, in the inhibition of mitochondria activity and jasmonic acid signaling. SUMOylation of both HopB1 and HopG1 is increased by heat treatment, and this modification modulates the functions of these 2 effectors in different ways in the regulation of plant survival rates, gene expression, and bacterial infection under high temperatures. Therefore, the current work on the SUMOylation of effectors in plant cells improves our understanding of the function of dynamic protein modifications in plant-pathogen interactions in response to environmental conditions.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Resposta ao Choque Térmico , Pseudomonas syringae , Sumoilação , Arabidopsis/microbiologia , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Bactérias/metabolismo , Proteínas de Bactérias/genética , Morte Celular , Ciclopentanos/metabolismo , Regulação da Expressão Gênica de Plantas , Resposta ao Choque Térmico/genética , Interações Hospedeiro-Patógeno , Temperatura Alta , Células Vegetais/metabolismo , Células Vegetais/microbiologia , Doenças das Plantas/microbiologia , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Pseudomonas syringae/patogenicidade , Pseudomonas syringae/fisiologia , Transdução de SinaisRESUMO
The number of meiotic crossovers is tightly controlled and most depend on pro-crossover ZMM proteins, such as the E3 ligase HEI10. Despite the importance of HEI10 dosage for crossover formation, how HEI10 transcription is controlled remains unexplored. In a forward genetic screen using a fluorescent crossover reporter in Arabidopsis thaliana, we identify heat shock factor binding protein (HSBP) as a repressor of HEI10 transcription and crossover numbers. Using genome-wide crossover mapping and cytogenetics, we show that hsbp mutations or meiotic HSBP knockdowns increase ZMM-dependent crossovers toward the telomeres, mirroring the effects of HEI10 overexpression. Through RNA sequencing, DNA methylome, and chromatin immunoprecipitation analysis, we reveal that HSBP is required to repress HEI10 transcription by binding with heat shock factors (HSFs) at the HEI10 promoter and maintaining DNA methylation over the HEI10 5' untranslated region. Our findings provide insights into how the temperature response regulator HSBP restricts meiotic HEI10 transcription and crossover number by attenuating HSF activity.
Assuntos
Proteínas de Arabidopsis , Arabidopsis , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas Cromossômicas não Histona/genética , Troca Genética , Proteínas de Choque Térmico/metabolismo , Resposta ao Choque Térmico/genética , Meiose/genética , Ubiquitina-Proteína Ligases/metabolismoRESUMO
Although protein aggregation can cause cytotoxicity, such aggregates can also form to mitigate cytotoxicity from misfolded proteins, although the nature of these contrasting aggregates remains unclear. We previously found that overproduction (op) of a three green fluorescent protein-linked protein (3×GFP) induces giant aggregates and is detrimental to growth. Here, we investigated the mechanism of growth inhibition by 3×GFP-op using non-aggregative 3×MOX-op as a control in Saccharomyces cerevisiae. The 3×GFP aggregates were induced by misfolding, and 3×GFP-op had higher cytotoxicity than 3×MOX-op because it perturbed the ubiquitin-proteasome system. Static aggregates formed by 3×GFP-op dynamically trapped Hsp70 family proteins (Ssa1 and Ssa2 in yeast), causing the heat-shock response. Systematic analysis of mutants deficient in the protein quality control suggested that 3×GFP-op did not cause a critical Hsp70 depletion and aggregation functioned in the direction of mitigating toxicity. Artificial trapping of essential cell cycle regulators into 3×GFP aggregates caused abnormalities in the cell cycle. In conclusion, the formation of the giant 3×GFP aggregates itself is not cytotoxic, as it does not entrap and deplete essential proteins. Rather, it is productive, inducing the heat-shock response while preventing an overload to the degradation system.
Assuntos
Proteínas de Fluorescência Verde , Proteínas de Choque Térmico HSP70 , Agregados Proteicos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Proteínas de Fluorescência Verde/metabolismo , Proteínas de Fluorescência Verde/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteólise , Complexo de Endopeptidases do Proteassoma/metabolismo , Resposta ao Choque Térmico/genética , Dobramento de Proteína , Ciclo Celular/genética , Adenosina TrifosfatasesRESUMO
Dissecting genetic components in crop plants associated with heat stress (HS) sensing and adaptation will facilitate the design of modern crop varieties with improved thermotolerance. However, the molecular mechanisms underlying the ON/OFF switch controlling HS responses (HSRs) in wheat (Triticum aestivum) remain largely unknown. In this study, we focused on the molecular action of TaHsfA1, a class A heat shock transcription factor, in sensing dynamically changing HS signals and regulating HSRs. We show that the TaHsfA1 protein is modified by small ubiquitin-related modifier (SUMO) and that this modification is essential for the full transcriptional activation activity of TaHsfA1 in triggering downstream gene expression. During sustained heat exposure, the SUMOylation of TaHsfA1 is suppressed, which partially reduces TaHsfA1 protein activity, thereby reducing the intensity of downstream HSRs. In addition, we demonstrate that TaHsfA1 interacts with the histone acetyltransferase TaHAG1 in a thermosensitive manner. Together, our findings emphasize the importance of TaHsfA1 in thermotolerance in wheat. In addition, they define a highly dynamic SUMOylation-dependent "ON/OFF" molecular switch that senses temperature signals and contributes to thermotolerance in crops.
Assuntos
Sumoilação , Triticum , Triticum/metabolismo , Regulação da Expressão Gênica de Plantas/genética , Resposta ao Choque Térmico/genética , Fatores de Transcrição de Choque Térmico/metabolismoRESUMO
The stem, consisting of nodes and internodes, is the shoot axis, which supports aboveground organs and connects them to roots. In contrast to other organs, developmental processes of the stem remain elusive, especially those initiating nodes and internodes. By introducing an intron into the Cre recombinase gene, we established a heat shock-inducible clonal analysis system in a single binary vector and applied it to the stem in the flag leaf phytomer of rice (Oryza sativa). With detailed characterizations of stem structure and development, we show that cell fate acquisition for each domain of the stem occurs stepwise. Cell fate for a single phytomer was established in the shoot apical meristem (SAM) by one plastochron before leaf initiation. Cells destined for the foot (nonelongating domain at the stem base) also started emerging before leaf initiation. Cell fate acquisition for the node began just before leaf initiation at the flank of the SAM, separating cell lineages for leaves and stems. Subsequently, cell fates for the axillary bud were established in early leaf primordia. Finally, cells committed to the internode emerged from, at most, a few cell tiers of the 12- to 25-cell stage stem epidermis. Thus, internode cell fate is established last during stem development. This study provides the groundwork to unveil underlying molecular mechanisms in stem development and a valuable tool for clonal analysis, which can be applied to various species.
Assuntos
Oryza , Oryza/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Diferenciação Celular , Meristema , Folhas de Planta/metabolismo , Resposta ao Choque Térmico/genética , Regulação da Expressão Gênica de Plantas/genéticaRESUMO
Heat stress (HS) adversely affects plant growth and productivity. The Class A1 HS transcription factors (HSFA1s) act as master regulators in the plant response to HS. However, how HSFA1-mediated transcriptional reprogramming is modulated during HS remains to be elucidated. Here, we report that a module formed by the microRNAs miR165 and miR166 and their target transcript, PHABULOSA (PHB), regulates HSFA1 at the transcriptional and translational levels to control plant HS responses. HS-triggered induction of MIR165/166 in Arabidopsis thaliana led to decreased expression of target genes including PHB. MIR165/166 overexpression lines and mutations in miR165/166 target genes enhanced HS tolerance, whereas miR165/166 knockdown lines and plants expressing a miR165/166-resistant form of PHB were sensitive to HS. PHB directly repressed the transcription of HSFA1s and globally modulated the expression of HS-responsive genes. PHB and HSFA1s share a common target gene, HSFA2, which is essential for activation of plant responses to HS. PHB physically interacted with HSFA1s and exerted an antagonistic effect on HSFA1 transcriptional activity. PHB and HSFA1s co-regulated transcriptome reprogramming upon HS. Together, these findings indicate that heat-triggered regulation of the miR165/166-PHB module controls HSFA1-mediated transcriptional reprogramming and plays a critical role during HS in Arabidopsis.