Genome wide analysis of the chromatin state and gene expression in Wild-type, tor-es mutant, and fie mutants

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. Overall design: ChIP-seq analysis of H3K2me2 and H3K27me3 in Wild-type, tor-es mutant, and fie mutants. RNA-seq analysis of FIE amiRNA mutant and FIE phosphorylation site mutant