Project description: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:Arabidopsis telomeric repeat binding factors (TRBs) can bind telomeric DNA sequences to protect telomeres from degradation. TRBs can also recruit Polycomb Repressive Complex 2 (PRC2) to deposit tri-methylation of H3 lysine 27 (H3K27me3) over certain target loci. Here, we demonstrate that TRBs also associate and colocalize with JUMONJI14 (JMJ14) and trigger H3K4me3 demethylation at some loci. The trb1/2/3 triple mutant and the jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their target genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger (TRB-ZF) successfully triggers target gene silencing, as well as H3K27me3 deposition, and H3K4me3 removal. Interestingly, JMJ14 is predominantly recruited to ZF off-target sites with low levels of H3K4me3, which is accompanied with TRB-ZFs triggered H3K4me3 removal at these loci. These results suggest that TRB proteins coordinate PRC2 and JMJ14 activities to repress target genes via H3K27me3 deposition and H3K4me3 removal.
Project description:Histone marks H3K27me3 and H3K4me3 are mutual exclusive over plant genome, however, the underlying mechanism is not fully understood. Arabidopsis telomeric repeat binding factors (TRBs) are required for the deposition of H3K27me3 by recruiting Polycomb repressive complexes (PRCs). Here, we demonstrate that TRBs associate and colocalize with JUMONJI 14 (JMJ14) over gene body regions and trigger H3K4me3 demethylation. The trb1/2/3 triple mutant and jmj14-1 mutant show an increased level of H3K4me3 over TRB and JMJ14 binding sites, resulting in up-regulation of their targeting genes. Furthermore, tethering TRBs to the promoter region of genes with an artificial zinc finger successfully triggers target gene silencing. These results suggest that TRB proteins cooperate with PRC2 and JMJ14 complex to repress target gene by H3K4me3 demethylation and H3K27me3 deposition.
Project description:Background. Post-translational modifications of histones play important roles in regulating transcription by modulating the structural properties of the chromatin. In plants, methylation of histone H3 lysine4 (H3K4me) is associated with genes and required for normal plant development. Results. We have characterized the genome-wide distribution patterns of mono-, di- and trimethylation of H3K4 (H3K4me1, H3K4me2 and H3K4me3, respectively) in Arabidopsis thaliana using chromatin immunoprecipitation and high-resolution whole-genome tiling microarrays (ChIP-chip). All three types of H3K4me are found to be almost exclusively genic, and two thirds of Arabidopsis genes contain at least one type of H3K4me in seedlings. H3K4me2 and H3K4me3 accumulate predominantly in promoters and 5’ genic regions, whereas H3K4me1 is distributed within transcribed regions. In addition, H3K4me3-containing genes are highly expressed with low levels of tissue specificity, but H3K4me1 or H3K4me2 may not be directly involved in transcriptional activation. Furthermore, a genome-wide preferential co-localization of H3K4me3 and H3K27me3 found in mammals does not appear to exist in plants, but H3K4me2 and H3K27me3 co-localize at a higher-than-expected frequency. Finally, the relationship between H3K4me and DNA methylation was explored by comparing the genome-wide distribution patterns of H3K4me1, H3K4me2 and H3K4me3 in wild type plants and the met1 DNA methyltransferase mutant. Conclusions. H3K4me plays widespread roles in regulating gene expression in plants. Although many aspects of the mechanisms and functions of H3K4me appear to be conserved among all three kingdoms, we observed significant differences in the relationship between H3K4me and transcription or other epigenetic pathways in plants and mammals.