Gene expression analysis of 35S::JMJ30-HA overexpression transgenic lines grown at 22°C under long day conditions
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ABSTRACT: Analysis of overexpression of JMJ30 in Arabidopsis thaliana (Col). JMJ30 encode the histone demethylases that contain the catalytic JmjC domain. The trimethylation of Histone H3 lysine 27 (H3K27me3) plays a key role in gene repression and developmental regulation. It is actively and dynamically deposited and removed in plants. However, while the H3K27 methyltransferases had been extensively studied, findings on the H3K27 demethylase were few. Here, we show that JMJ30 encodes a H3K27me3 demethylase, and genes up-regulated in 35S::JMJ30-HA transgenic lines are enriched for H3K27me3 targets. Columbia wild-type (Col WT) and 35S::JMJ30-HA transgenic plants were grown at 22°C under long day conditions, and seedling samples were collected at 13 day-after-germination (DAG). Two independent sets of WT and 35S::JMJ30-HA seedling mRNA samples were used for this array.
Project description:Analysis of loss-of-function mutants of JUMONJI30 (JMJ30) and JMJ32 in Arabidopsis thaliana (Col). JMJ30 and JMJ32 encode the histone demethylases that contain the catalytic JmjC domain. The trimethylation of Histone H3 lysine 27 (H3K27me3) plays a key role in gene repression and developmental regulation. It is actively and dynamically deposited and removed in plants. However, while the H3K27 methyltransferases had been extensively studied, findings on the H3K27 demethylase were few. Here, we show that JMJ30 and JMJ32 encode H3K27me3 demethylases, and genes down-regulated in jmj30 jmj32 mutant are enriched for H3K27me3 targets. Columbia wild-type (Col WT) and jmj30 jmj32 mutant were grown at 29°C, and seedling samples were collected at 13 day-after-germination (DAG). Two independent sets of WT and jmj30 jmj32 seedling mRNA samples were used for this array.
Project description:Background. Polycomb Repressive Complex 2 (PRC2) is an essential regulator of gene expression that maintains genes in a repressed state by marking chromatin with trimethylated Histone H3 lysine 27 (H3K27me3). In Arabidopsis, loss of PRC2 function leads to pleiotropic effects on growth and development thought to be due to ectopic expression of seed and embryo-specific genes. While there is some understanding of the mechanisms by which specific genes are targeted by PRC2 in animal systems, it is still not clear how PRC2 is recruited to specific regions of plant genomes.Results. We used ChIP-seq to determine the genome-wide distribution of hemagglutinin (HA)-tagged FERTLIZATION INDEPENDENT ENDOSPERM (FIE-HA), the Extra Sex Combs homolog protein present in all Arabidopsis PRC2 complexes. We found that the FIE-HA binding sites co-locate with a subset of the H3K27me3 sites in the genome and the associated genes were more likely to be de-repressed in mutants of PRC2 components. The FIE-HA binding sites are enriched for three sequence motifs including a putative GAGA factor binding site that is also found in Drosophila Polycomb Response Elements (PREs).Conclusions. Our results suggest that PRC2 binding sites in plant genomes share some sequence features with Drosophila PREs. However, RNA was extracted from intermediate phenotype siFIE plants, clf-7 swn-28 and Col using Qiagen Plant RNeasy mini kit. For each sample, three pools of 10-12 plants were used. The siFIE plants were analysed for FIE mRNA by RT-qPCR; the maximum level of FIE mRNA was found to be 10% of wildtype.
Project description:Analysis of overexpression of JMJ30 in Arabidopsis thaliana (Col). JMJ30 encode the histone demethylases that contain the catalytic JmjC domain. The trimethylation of Histone H3 lysine 27 (H3K27me3) plays a key role in gene repression and developmental regulation. It is actively and dynamically deposited and removed in plants. However, while the H3K27 methyltransferases had been extensively studied, findings on the H3K27 demethylase were few. Here, we show that JMJ30 encodes a H3K27me3 demethylase, and genes up-regulated in 35S::JMJ30-HA transgenic lines are enriched for H3K27me3 targets.
Project description:Filamentous fungi produce a vast array of secondary metabolites (SMs) and some of them are applied in agriculture or pharmacology. Recent sequencing of the rice pathogen Fusarium fujikuroi revealed the presence of far more SM-encoding genes than known products. SM production is energy-consuming and thus tightly regulated, leaving the majority of SM gene clusters silent under laboratory conditions. It is now well established that one important regulatory layer in SM biosynthesis involves histone modifications that render the genes either silent or poised for transcription. In this study, we show that the majority of the putative SM gene clusters in F. fujikuroi are located within facultative heterochromatin marked by H3K27me3. In this study, we performed comparative transcriptomics of a knock-down mutant of the responsible methyltransferase Kmt6 involved in H3K27 methylation grown on either solid complete medium or solid synthetic ICI medium. Overall four so far cryptic and otherwise silent putative SM gene clusters were significantly induced in the KMT6kd strain accompanied by reduced H3K27me3 levels at the respective gene loci and accumulation of novel metabolites. One of the four putative SM gene clusters, the STC5 gene cluster, was analysed in detail and heterologous expression of the key enzyme allowed for the identification of the first pathway-specific intermediate (1R,4R,5S)-guaia-6,10(14)-diene. 2 strains were analysed in overall two conditions, and each with 3 biological replicates
Project description:Investigation of the effect of the knockdown of AT-hook motif DNA binding nuclear matrix protein TRANSPOSABLE ELEMENT KILLER (TEK) in the Arabidopsis Landsberg erecta (Ler) background Transposable elements (TEs) are silenced by epigenetic mechanisms of DNA and histone methylation. The repressive histone modification H3 lysine 9 dimethylation (H3K9me2) is TE-associated epigenetic hallmark, and is necessary for DNA methylation. However the mechanism to direct the repressive epigenetic modification in TEs has remained elusive. Here we show that knockdown of the AT-hook motif DNA binding nuclear matrix protein TRANSPOSABLE ELEMENT KILLER (TEK) in the Arabidopsis Landsberg erecta (Ler) background results in robust activation of various TEs, the repeat-containing floral repressor gene FWA and the TE-containing floral repressor FLOWERING LOCUS C (FLC). A four chip study using two separate wild-type seedling mRNA samples and two separate TEKi seedling mRNA samples
Project description:Constitutive expression of VvMYBPAR in Arabidopsis was found to accumulate proanthocyanidins when the plants were grown on sucrose-supplemented media to induce anthocyanins. To identify the putative targets of VvMYBPAR, the transcriptome analysis of the transgenic lines which highly express VvMYBPAR was carried out using NimbleGen microarray. Three transgenic lines in which VvMYBPAR were constitutively expressed under the control of 35S promoter vs. empty vector transformants were compared.
Project description:Transcriptional changes assayed with two bilogical replicates in wild type and RNAi mediated insulator knock-downs RNAi mediated insulator knock-downs cause changes in the H3K27me3 levels and spread of Topoisomerase II In order to understand the role of insulators in gene expression and regulation we used Drosophila Kc cells to knock-down single and multiple insulators in combination to assay for transcriptional changes. Two biologial replicates were prepared in independent experiments. Each cDNA samples were labeled with Cy3 dye and hybridized and scaned as per manufactures instructions at Florida State University Nimblegen facility.
Project description:Metaphase chromosome staining was used to provide a high level overview of the pattern of histone modifications (H3K27ac, H3K27me3 and H3K4me3) at a single cell level in human lymphoblastois cells. These epigenomic banding patterns were related to various genomic features including gene and CpG island density as well as gene expression. A 3 array study using RNA extracted from 3 separate cultures of human lymphoblastoid cells as biological replicates
Project description:This study describes the changes in epigenetic chromatin modifications during murine hematopoietic stem cell differentiation in vivo using a modified miniChIP-chip technology. We have addressed issues including bivalent (H3K4me3/H3K27me3) modifications, lineage priming hypothesis, and stem cell chromatin properties in our study described in Weishaupt et al., 2009 (Blood) Comparison of 4 murine hematopoietic stem, progenitor and mature cell types directly isolated from primary tissues using FACS. HSCs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150+, Flk2/Flt3- (LSKCD150+ cells). MPPs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1+, CD150-, Flk2/Flt3+ (LSKCD150- cells). PreMegEs are phenotypically identified in bone marrow as lineage-, cKit+, Sca1-, CD150+, CD105-, FcgRI/IIlo and CD41- cells as described in Pronk et al., 2007. Splenic-derived CD4+ T cells are phentypically identified as CD4+, CD8-, B220-, Nk1.1- cells as described in Rolf et al., 2008.
Project description:Investigation on expression levels of normal tissue from prostate cancer patients on locus 8q24. The region chr8:127640000-129120000 is tiled with isothermal probes (hg17) 7 chip study, using 7 independent samples.