Genome wide analysis of the chromatin state and gene expression in Wild-type, tor-es mutant, and fie mutants
Ontology highlight
ABSTRACT: Using Western blot, we found the level of H3K27me3, but not H3K4me3, H3K9me2 and H3K36me3, was specifically reduced in the tor-es mutant. To gain a genome-wide view of the effects of TOR activity on H3K27me3 distribution, we performed quantitative chromatin immunoprecipitation with an exogenous reference genome (ChIP-Rx) followed by deep-sequencing. We find a global reduction of H3K27me3 occupancy in tor-es, whereas the H3K9me2 level was largely unaffected. These results suggest that TOR may be a specific and direct regulator of global deposition of H3K27me3. To investigate the function of TOR phosphorylation of FIE, we complemented the heterozygous fie/+ plants with GFP-FIE or the phosphorylation site mutant (SSTS/AAAA) under the control of the FIE promoter. To provide a parallel comparison with SSTS/AAAA/fie, we generated estradiol-inducible fie-amiR-es transgenic lines, that eliminated FIE protein. Quantitative ChIP-seq analyses revealed greatly reduced H3K27me3 levels across the genome in SSTS/AAAA and fie-amiR-es mutants. And transcriptome profiling by RNA-Seq was conducted to globally identify thousands of genes coordinately dysregulated in the shoots of SSTS/AAAA and fie-amiR-es plants. Furthermore, gene Ontology analysis of 986 TOR-FIE-PRC2 target genes revealed significant enrichment for transcription factors/regulators controlling a broad spectrum of developmental programs.
Project description:The aim was to identify genes associated to the down-regulation TARGET OF RAPAMYCIN (TOR) pathway in Arabidopsis. For this purpose, three independent amiR-tor lines (amiR-tor9, amiR-tor17 and amiR-tor 20) and EV lines 3 and 6 days after EST- or non-induction were used for expression profile analysis using Affymetrix microarray. Two weeks-old transgenic seedlings were transferred to MS plate with 20µM estradiol to induce the overexpression of amiR-tor (consequently, repression of AtTOR levels) under the control of an estradiol-(EST) inducible promoter. Identically treated wild-type Col-0 and pER8 empty-vector (EV) transformed seedlings were served as controls. Seedlings were harvested after 3 and 6 (amiR-tor lines) days. genetic modification
Project description:The aim was to identify genes associated to the down-regulation TARGET OF RAPAMYCIN (TOR) pathway in Arabidopsis. For this purpose, three independent amiR-tor lines (amiR-tor9, amiR-tor17 and amiR-tor 20) and EV lines 3 and 6 days after EST- or non-induction were used for expression profile analysis using Affymetrix microarray. Two weeks-old transgenic seedlings were transferred to MS plate with 20µM estradiol to induce the overexpression of amiR-tor (consequently, repression of AtTOR levels) under the control of an estradiol-(EST) inducible promoter. Identically treated wild-type Col-0 and pER8 empty-vector (EV) transformed seedlings were served as controls. Seedlings were harvested after 3 and 6 (amiR-tor lines) days.
Project description:To get a genome wide picture of the effects of TOR knockdown during the early phase of lateral root formation, we compared by RNA-seq the transcriptomes of roots 6h after the synchronous induction of lateral formation by auxin treatment (Himanen et al, 2002 - http://dx.doi.org/10.1105/tpc.004960) in the inducible UB10pro>>amiR-TOR background.