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. 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

通过蛋白质印迹法（Western blot）实验，我们发现tor-es突变体中H3K27me3的水平发生特异性降低，而H3K4me3、H3K9me2及H3K36me3的水平无显著变化。为从全基因组层面解析TOR活性对H3K27me3分布的调控效应，我们开展了带有外源参考基因组的定量染色质免疫沉淀（ChIP-Rx）联合深度测序实验。结果显示，tor-es突变体中H3K27me3的基因组占据水平呈整体下降趋势，而H3K9me2的水平基本不受影响。上述结果表明，TOR可能是H3K27me3全局沉积的特异性直接调控因子。为探究TOR对FIE的磷酸化调控功能，我们利用FIE启动子驱动GFP-FIE或其磷酸化位点突变体（SSTS/AAAA），对杂合子fie/+植株进行遗传互补。为与SSTS/AAAA/fie材料开展平行对照实验，我们构建了可通过雌二醇诱导敲除FIE蛋白的fie-amiR-es转基因株系。定量染色质免疫沉淀测序（ChIP-seq）分析显示，SSTS/AAAA与fie-amiR-es突变体的全基因组H3K27me3水平显著降低。此外，我们通过RNA测序（RNA-seq）开展转录组分析，在SSTS/AAAA与fie-amiR-es植株的地上部分中，全局鉴定出数千个协同失调的基因。进一步对986个TOR-FIE-PRC2靶基因开展基因本体论（Gene Ontology，GO）分析发现，其在调控各类发育程序的转录因子/调控因子中显著富集。本研究还完成了野生型、tor-es突变体及fie突变体中H3K2me2与H3K27me3的ChIP-seq分析，以及FIE人工microRNA突变体与FIE磷酸化位点突变体的RNA-seq分析。