RNA-seq of med14-3 and uvh6-3 mutants in heat stress (37°C) and control stress (23°C), with WT controls. RNA-seq of double mutants: med14-3 or uvh6-3 combined with either ddm1-2 or mom1-2 mutants. Bisulfite-seq of WT samples subjected to control or heat stress. Bisulfite-seq of med14-3 and WT samples in control stress.
ABSTRACT: Heterochromatin transcription is functionally important, for instance to establish heterochromatin silencing or condensation, but the factors allowing transcription in a repressive chromatin environment are little known. To gain insight in this process, we triggered heterochromatin transcription using heat stress or mutations for the silencing factors DDM1 or MOM1 and generated mRNA profiles of med14 and uvh6 mutants in these different contexts. We used rosette leaves incubated 24h in dH2O at 23°C for control stress and 37°C for heat stress. Our data and analysis suggest that UVH6/XPD/RAD3 is involved in a heat stress specific transcriptional process where it promotes transcription of most but not all genes. MED14 promotes transcription at a more reduced number of loci during heat stress, and preferentially targets heterochromatic loci. In addition, MED14 is required for heterochromatin transcription without heat stress, but this function is lost when heterochromatin properties are disrupted, suggesting it is specifically involved in transcription of heterochromatic sequences. In addition, bisulfite-seq of med14 mutants indicates that MED14 participates in RNA-directed DNA methylation, suggesting that MED14 is required both for heterochromatin transcription and formation.
Project description:DNA replication requires the faithful propagation of both genetic and epigenetic information. There is evidence that DNA polymerases play a role in transcriptional silencing, but the extent of their contribution and how it relates to heterochromatin maintenance is unclear. Analyzing a new hypomorphic pol2a mutant allele, we find that POL2A, the catalytic subunit of the DNA polymerase epsilon, maintains heterochromatin silencing and nuclear organization. Here, we profiled the DNA methylation landscape using BS-seq in various pol2a mutant alleles in Arabidopsis, in combination or not with other silencing and methyltransferase mutants. We also generated methylomes of wild-type and pol2a mutants exposed to hydroxyurea, to explore the consequences of replicative stress onto DNA methylation maintenance.
Project description:Arabidopsis 5’-3’ exoribonuclease, AtXRN4, a homolog of yeast Xrn1p, functions in degradation of uncapped RNAs after de-capping step. While Xrn1p-dependent on plant XRN4’s targets for degradation is still limited. For understanding biological function of AtXRN4, we tested survivability of atxrn4 mutants under heat stress. Our results showed that atxrn4 mutants increased survival rate under short-term degradation is a main mRNA decay in yeast, knowledge heat stress compared with WT plants. Our microarray and mRNA decay assay showed that loss of AtXRN4 function caused reduction of mRNA degradation of heat shock factor A2 (HSFA2) and ethylene response factor 1 (ERF1). HSFA2 has been known as a key regulator in heat acclimation, was found as a target for AtXRN4 for degradation at non-stress condition. Heat stress applied on atxrn4-3 hsfa2 double mutant severely lacked heat tolerance phenotype of atxrn4 mutant. These results suggest that AtXRN4-mediated mRNA degradation linked to suppress heat acclimation. In the study here, 2 week-old WT and atxrn4-3 mutant plants were exposure to non-stress (22oC) and heat-stress (37oC, 1 h). Custom microarray was applied to acquire expression profile of 32788 Arabidopsis genes. 3 biological repeats of WT (non-stress), WT(heat stress), atxrn4-3 (non-stress) and atxrn4-3 (heat stress) were used for microarray analysis
Project description:Using whole genome bisulfite sequencing to provide single-base resulution of DNA methylation status in rdm16ros1, ros1, nrpd1ros1 mutants and examine the effect of RDM16 on DNA methylation 4 samples examined: C24 wild type with RD29A-LUC transgene, rdm16ros1 double mutant, ros1 mutant, nrpd1ros1 mutant (all in C24 background with RD29A-LUC transgene)
Project description:The yeast protein PBP1 has been implicated in diverse pathways such as polyadenylation, translation, RNA-DNA hybrid formation, stress granule homeostasis, mitochondrial dysfunction, and TORC1 sequestration. Intriguingly, its deletion mitigates the toxicity of human neurodegeneration factors, but the molecular mechanisms of these effects are poorly understood. Here we performed label-free quantitative global proteomics to identify crucial downstream factors, comparing two PBP1 deletion strains (DB and SM) and two cell stress conditions (heat and NaN3). In all four analyses, downregulations of key bioenergetics enzymes (CIT1, SDH1, MLS1), cell wall mannoproteins (HSP150, PST1) and the prion protein RNQ1 as well as upregulations of the leucine biosynthesis enzyme LEU1 and the transcription factor TAF6 were documented. Consistently for both unstressed PBP1-deleted strains, over 2-fold dysregulations were documented for potential PBP1 interactors such as MKT1 or RPL39 and the stress granule component NRP1. Upregulation of the ribosomal biogenesis factor NOP10 was observed as in the mouse mutant. Consistently for both PBP1 deletion strains, heat stress triggered changes of the stress granule component GIS2 and several of its interactors.
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:NF-YC10 is a subunit of the NF-Y transcription factor complex in Arabidopsis thaliana. We identified NF-YC10 as an interactor of the transcription factor DREB2A, which binds to DRE and activate expression of target genes under heat and/or dehydration stress conditions. Transgenic Arabidopsis plants that overexpress NF-YC10 showed stronger expression of DREB2A target genes under heat stress and were more tolerant to heat stress tolerance than the vector control plants. We conducted a transcript profiling by microarray aiming to identify target genes of NF-YC10 under heat stress
Project description:Mycoplasma gallisepticum proteome reponse under heat stress was studied. Heat stress has been shown previously to induce the most widespread and at the same time the most intense response at transcription level among the panel of several stresses. Here the corresponding proteome response was characterized.
Project description:To ensure cell survival and growth during temperature increase, eukaryotic organisms respond with transcriptional activation that results in accumulation of proteins that protect against damage, and facilitate recovery. To define the global cellular adaptation response to heat stress, we performed a systematic genetic screen that yielded 277 yeast genes required for growth at high temperature. Of these, the Rpd3 histone deacetylase complex was enriched. Global gene expression analysis showed that Rpd3 partially regulated gene expression upon heat shock. The Hsf1 and Msn2/4 transcription factors are the main regulators of gene activation in response to heat stress. RPD3-deficient cells had impaired activation of Msn2/4-dependent genes, while activation of genes controlled by Hsf1 was deacetylase independent. Rpd3 bound to heat stress-dependent promoters through the Msn2/4 transcription factors, allowing entry of RNA Pol II and activation of transcription upon stress. Finally, we found that the large, but not the small Rpd3 complex regulated cell adaptation in response to heat stress. Three independent 200 ml cultures of wild-type and rpd3Δ mutant strains were grown to mid-log phase in YPD rich medium at 25ºC (control) or at 39 ºC for 20 min (heat stressed). Results were analyzed comparing thermo-responsive gene expression respect to the control in each individual strain.
Project description: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 mediates a more efficient response to recurring stress. We identified a mutant that is specifically affected in this heat stress memory. The forgetter1 (frg1) mutant is defective in the Arabidopsis orthologue of Strawberry notch and displays reduced maintenance of heat-induced gene expression. FRG1 globally associates with the promoter region of actively expressed genes in a heat-dependent fashion. FRG1 interacts with chromatin remodelers of the SWI/SNF and ISWI families, which also display reduced heat stress memory. Accordingly, nucleosome dynamics at loci with altered maintenance of heat-induced expression are affected in frg1. Thus, FRG1 is required to sustain a transcription-competent chromatin environment by nucleosome positioning. Our findings suggest a mechanism for the highly conserved Strawberry notch proteins in development and pathologies. Overall design: Chromatin binding of FRG1.
Project description:To ensure cell survival and growth during temperature increase, eukaryotic organisms respond with transcriptional activation that results in accumulation of proteins that protect against damage, and facilitate recovery. To define the global cellular adaptation response to heat stress, we performed a systematic genetic screen that yielded 277 yeast genes required for growth at high temperature. Of these, the Rpd3 histone deacetylase complex was enriched. Global gene expression analysis showed that Rpd3 partially regulated gene expression upon heat shock. The Hsf1 and Msn2/4 transcription factors are the main regulators of gene activation in response to heat stress. RPD3-deficient cells had impaired activation of Msn2/4-dependent genes, while activation of genes controlled by Hsf1 was deacetylase independent. Rpd3 bound to heat stress-dependent promoters through the Msn2/4 transcription factors, allowing entry of RNA Pol II and activation of transcription upon stress. Finally, we found that the large, but not the small Rpd3 complex regulated cell adaptation in response to heat stress. Overall design: Three independent 200 ml cultures of wild-type and rpd3Δ mutant strains were grown to mid-log phase in YPD rich medium at 25ºC (control) or at 39 ºC for 20 min (heat stressed). Results were analyzed comparing thermo-responsive gene expression respect to the control in each individual strain.