Project description:As a central component during Okazaki fragment maturation, flap endonuclease 1 (FEN1) removes the 5â flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. Arabidopsis fen1-1 mutant is hypersensitive to MMS and shows reduced telomere length. Interestingly, genome-wide ChIP-seq and RNA-seq results demonstrate that FEN1 mutation leads to a decrease in the H3K27me3 level and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS besides maintaining genome stability in Arabidopsis. To characterized the role of FEN1 in epigenetic silencing, we examine histone modification and RNA expression changes by whole-genome RNA sequencing; H3K27me3-, H3K4me3-, H3K9me2-, H3-ChIP-seq in A. thaliana transgenic wild type (TWT) and fen1 mutant
Project description:As a central component during Okazaki fragment maturation, flap endonuclease 1 (FEN1) removes the 5’ flap and maintains genomic stability. Here, FEN1 was cloned as a suppressor of transcriptional gene silencing (TGS) from a forward genetic screen. FEN1 is abundant in the root and shoot apical meristems and FEN1-GFP shows a nucleolus-localized signal in tobacco cells. Arabidopsis fen1-1 mutant is hypersensitive to MMS and shows reduced telomere length. Interestingly, genome-wide ChIP-seq and RNA-seq results demonstrate that FEN1 mutation leads to a decrease in the H3K27me3 level and an increase in the expression of a subset of genes marked with H3K27me3. Overall, these results uncover a role for FEN1 in mediating TGS besides maintaining genome stability in Arabidopsis.
Project description:We developed an artificial genome evolution system, which we termed ‘TAQing’, by introducing multiple genomic DNA double-strand breaks using a heat-activatable endonuclease in Arabidopsis plant. The heat-activated endonuclease, TaqI, induced random DSBs, which resulted in diverse types of chromosomal rearrangements including translocations. To evaluate the potential of TAQing in multicellular organisms, we tested it in diploid and tetraploid Arabidopsis plants. In 9 out of 96 TQ4 plants, we detected 22 large copy number variations (CNVs) events compared witn wild type plant genome, whereas no CNV was found in the 16 control tetraploid plants, and 12 TQ2 plants. The combination of artificially introduced DSBs with whole-genome duplication (WGD) in plants enabled more complex genome reorganization.
Project description:Oxygen is a key signalling component of plant biology, and whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana, key features of the associated PCO N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance, and are essential for root development and survival under oxygen limiting conditions in the soil. We determine the combined effects of ERFVIIs in controlling genome expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine sulphonic acid oxidation level of ERFVIIs was observed, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.
Project description:DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, implying that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis, as in cancer, or meiosis, leading to epiallelic variation. Here, we demonstrate the existence of an efficient and faithful mechanism that protects against transgenerational loss of DNA methylation in the plant Arabidopsis. This process is specific to the subset of heavily methylated genomic repeats that are targeted by the RNAi machinery, and does not spread into flanking regions. Remethylation is often progressive over two to four sexual generations. This differential and incremental correction of epigenetic defects may preserve genome stability while increasing adaptive opportunities.
Project description:Eukaryotic genomes are heavily regulated by epigenetic marks that often act to modulate the transcriptional control of genetic elements. In Arabidopsis thaliana the ATXR5 and ATXR6 histone methyltransferases, and their cognate H3K27 monomethylation mark, act in transcriptional silencing while also maintaining genome stability by preventing generation of excess DNA corresponding to pericentromeric heterochromatin. In this study we characterize the atxr5 atxr6 transcriptome and its relationship to the DNA damage response which suggests that the atxr5 atxr6 transcriptional defects may be epistatic to the genome instability defects in the mutants. In addition we isolate several factors that modulate both the transcriptional and genomic instability phenotypes of atxr5 atxr6 mutants, which suggest a mechanism for atxr5 atxr6-induced extra DNA involving conflicts between the replicative and transcriptional processes in the cell.
Project description:Oxygen is a key signalling component of plant biology and, whilst an oxygen-sensing mechanism was previously described in Arabidopsis thaliana. However, key features of the associated PCO N-degron pathway and Group VII ETHYLENE RESPONSE FACTOR (ERFVII) transcription factor substrates remain unknown. We demonstrate that ERFVIIs show non-autonomous activation of root hypoxia tolerance, and are essential for root development and survival under oxygen limiting conditions in the soil. We determine the combined effects of ERFVIIs in controlling genome expression and define genetic and environmental components required for proteasome-dependent oxygen-regulated stability of ERFVIIs through the PCO N-degron pathway. Using a plant extract, unexpected amino-terminal cysteine oxidation to sulphonic acid oxidation level of ERFVIIs was defined, suggesting a requirement for additional enzymatic activity within the pathway. Our results provide a holistic understanding of the properties, functions and readouts of this oxygen-sensing mechanism defined through its role in modulating ERFVII stability.
Project description:DNA methylation is essential for silencing transposable elements and some genes in higher eukaryotes, implying that this modification must be tightly controlled. However, accidental changes in DNA methylation can be transmitted through mitosis, as in cancer, or meiosis, leading to epiallelic variation. Here, we demonstrate the existence of an efficient and faithful mechanism that protects against transgenerational loss of DNA methylation in the plant Arabidopsis. This process is specific to the subset of heavily methylated genomic repeats that are targeted by the RNAi machinery, and does not spread into flanking regions. Remethylation is often progressive over two to four sexual generations. This differential and incremental correction of epigenetic defects may preserve genome stability while increasing adaptive opportunities. 2 samples examined: wild type, and ddm1 mutant.