Project description:Dnmt2 genes are highly conserved tRNA methyltransferases with biological roles in cellular stress responses. Dnmt2 has recently been implicated in transposon silencing in Drosophila but the exact molecular mechanisms are unclear. Adult Dnmt2 mutants were heat shocked and RNA sequencing was performed on visible high-molecular weight RNAs to determine the identity of up-regulated transposons. Dnmt2 mutants accumulated almost all families of transposons after heat shock, indicating a general mis-regulation of transposon silencing in Dnmt2 mutants during the stress response.
Project description:Dnmt2 genes are highly conserved tRNA methyltransferases with biological roles in cellular stress responses. The absence of obvious mutant phenotypes under laboratory conditions suggested a function for Dnmt2 under non-physiological conditions. Indeed, Dnmt2 has recently been implicated in various aspects of the cellular stress response and the tRNA methyltransferase activity of Dnmt2 has been shown to interfere with stress-induced fragmentation of various tRNAs. We used adult animals and small RNA sequencing during a heat stress experiment to determine the tRNA fragment abundance and identities in wild-type and Dnmt2 mutant somatic tissues. Dnmt2 mutants produced tRNA fragments with different identities when compared to wild-type controls, indicating the accumulation of non-physiological tRNA-derived molecules in tissues without Dnmt2. 6 samples examined: heterozygous and Dnmt2 mutant under control, heat shock and recovery conditions
Project description:It has been claimed previously that loss of (cytosine-5) DNA methylation in Dnmt2 mutant embryos affected the establishment of silent chromatin at Invader4 LTRs (Phalke et al., 2009). The inability of Dnmt2 mutants to control TE expression after heat shock (GSE40432) raised the possibility that DNA methylation could be required for control of Invader 4 during the heat shock response. We used DNA bisulfite sequencing at Inv4 LTRs that were previously suggested to be methylated (Phalke et al., 2009).
Project description:To understand how microRNAs are involved in stress response, we examined their expression changes in C. elegans animals that were exposed to stress conditions, including heat shock, oxidation, hypoxia and starvation. Total RNAs were purified from young adult animals that were exposed to each stress, and used for cDNA library preparation for small RNAs. In this experiment, spe-9(hc88), a temperature sensitive sterile mutant, that were cultured at 23dC, was used in order to avoid the effect from developing embryos. Stress conditions we examined include: Heat shock (32M-BM-0C, 6 hrs), Recovery from heat shock (6 hrs recovery at 23M-BM-0C after heat shock treatment at 32M-BM-0C for 6 hrs), Hypoxia (0.01%, 6 hrs), Oxidation (Juglone 750 M-NM-<M, 6 hrs), Starvation (complete food deprivation, 12 hrs). In addition to these stress conditions, RNAs were prepared from normally cultured, untreated animals at three time points, 0 hr (baseline), 6 hrs (as controls for heat shock, hypoxia and oxidation) and 12 hrs (as controls for heat shock recovery and starvation) after starting stress exposure. These cDNA libraries established were sequenced with Illumina Genome Analyzer II.
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. Keywords: heat shock response
Project description:Post-embryonic plant development must be coordinated in response to and with environmental feedback. Development of above-ground organs is orchestrated from stem cells in the center of the shoot apical meristem (SAM). Heat can pose significant abiotic stress to plants and induce a rapid heat shock response, developmental alterations, chromatin decondensation, and activation of transposable elements (TEs). However, most plant heat-stress studies are conducted with seedlings, and we know very little about cell-type-specific responses. Here we use fluorescent-activated nuclear sorting to isolate and characterize stem cells of wild type and mutants defective in TE defense and chromatin compaction after heat shock and after a long recovery. Our results indicate that stem cells can suppress heat shock response pathways to maintain developmental programs. Furthermore, mutants defective in DNA methylation fail to recover efficiently from heat stress and persistently activate heat shock factors and heat-inducible TEs. Heat stress also induces DNA methylation epimutations, especially in the CHG context, and we find hundreds of DNA methylation changes three weeks after stress. Our results underline the importance of disentangling cell type-specific environmental responses for understanding plant development.
Project description:Post-embryonic plant development must be coordinated in response to and with environmental feedback. Development of above-ground organs is orchestrated from stem cells in the center of the shoot apical meristem (SAM). Heat can pose significant abiotic stress to plants and induce a rapid heat shock response, developmental alterations, chromatin decondensation, and activation of transposable elements (TEs). However, most plant heat-stress studies are conducted with seedlings, and we know very little about cell-type-specific responses. Here we use fluorescent-activated nuclear sorting to isolate and characterize stem cells of wild type and mutants defective in TE defense and chromatin compaction after heat shock and after a long recovery. Our results indicate that stem cells can suppress heat shock response pathways to maintain developmental programs. Furthermore, mutants defective in DNA methylation fail to recover efficiently from heat stress and persistently activate heat shock factors and heat-inducible TEs. Heat stress also induces DNA methylation epimutations, especially in the CHG context, and we find hundreds of DNA methylation changes three weeks after stress. Our results underline the importance of disentangling cell type-specific environmental responses for understanding plant development.
Project description:Dnmt2 genes are highly conserved tRNA methyltransferases with biological roles in cellular stress responses. The absence of obvious mutant phenotypes under laboratory conditions suggested a function for Dnmt2 under non-physiological conditions. Indeed, Dnmt2 has recently been implicated in various aspects of the cellular stress response and the tRNA methyltransferase activity of Dnmt2 has been shown to interfere with stress-induced fragmentation of various tRNAs. We used adult animals and small RNA sequencing during a heat stress experiment to determine the tRNA fragment abundance and identities in wild-type and Dnmt2 mutant somatic tissues. Dnmt2 mutants produced tRNA fragments with different identities when compared to wild-type controls, indicating the accumulation of non-physiological tRNA-derived molecules in tissues without Dnmt2.
Project description:FBXW7 modulates stress response by post-translational modification of HSF1 HSF1 orchestrates the heat-shock response upon exposure to heat stress and activates a transcriptional program vital for cancer cells. Genes positively regulated by HSF1 show increeased expression during heat shock while their expression is reduced during recovery. Genes negatively regulated by HSF1 show the opposite pattern. In this study we utilized the HCT116 FBXW7 KO colon cell line and its wild type counterpart to monitor gene expression changes during heat shock (42oC, 1 hour) and recovery (37oC for 2 hours post heat shock) using RNA sequencing. These results revealed that the heat-shock response pathway is prolonged in cells deficient for FBXW7. Whole RNA was extracted from 1 million HCT116 WT or FBXW7KO cells using the RNAeasy kit (Qiagen) according to the manufacturer’s protocol. Poly-A+ (magnetic oligodT-containing beads (Invitrogen)) or Ribominus RNA was used for library preparation. cDNA preparation and strand-specific library construction was performed using the dUTP method. Libraries were sequenced on the Illumina HiSeq 2000 using 50bp single-read method. Differential gene expression analysis was performed for each matched recovery versus heat-shock pairs, separately in each biological replicate and cell line (WT or KO). Two types of comparisons were tested: (a) WT recovery vs WT heat shock, (b) FBXW7 KO recovery vs heat shock.