Project description:Transcriptional gene silencing (TGS) can serve as an innate immunity against invading DNA viruses throughout Eukaryotes. Geminivirus code for TrAP protein to suppress the TGS pathway. Here we identified an Arabidopsis H3K9me2 histone methyltransferase, Su(var)3-9 homolog 4 (SUVH4/KYP), as a bona fide cellular target of TrAP. TrAP interacts with the catalytic domain of KYP and inhibits its activity in vitro. TrAP elicits developmental anomalies phenocopying several TGS mutants, reduces the repressive H3K9me2 mark and CHH DNA methylation, and reactivates numerous endogenous KYP-repressed loci in vivo. Moreover, KYP binds to the viral chromatin, and controls its methylation to combat virus infection. Notably, kyp mutants support systemic infection of TrAP-deficient Geminivirus. We conclude that TrAP attenuates the TGS of the viral chromatin by inhibiting KYP activity to evade host surveillance. These findings provide new insight on the molecular arms race between host antiviral defense and virus counter defense at an epigenetic level.
Project description:In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the epsilon-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation.
Project description:In Arabidopsis, CHG DNA methylation is controlled by the H3K9 methylation mark through a self-reinforcing loop between DNA methyltransferase CHROMOMETHYLASE3 (CMT3) and H3K9 histone methyltransferase KRYPTONITE/SUVH4 (KYP). We report on the structure of KYP in complex with methylated DNA, substrate H3 peptide and cofactor SAH, thereby defining the spatial positioning of the SRA domain relative to the SET domain. The methylated DNA is bound by the SRA domain with the 5mC flipped out of the DNA, while the H3(1-15) peptide substrate binds between the SET and post-SET domains, with the epsilon-ammonium of K9 positioned adjacent to bound SAH. These structural insights complemented by in vivo functional data on key mutants of residues lining the 5mC and H3K9-binding pockets within KYP, establish how methylated DNA recruits KYP to the histone substrate. Together, the structures of KYP and previously reported CMT3 complexes provide insights into molecular mechanisms linking DNA and histone methylation. Plants homozygous for null mutations in the KRYPTONITE H3K9 methyltransferase were stably transformed with transgenes encoding the wildtype KYP protein or transgenes carrying induced point mutations in the KYP active site. The resulting lines were assayed for DNA methylation by whole-genome bisulfite sequencing to learn the efficiency with which wildtype and mutant versions of the KYP protein could restore DNA methylation lost in a kyp mutant. Samples 7 and 8 were run as single Illumina lanes and as such were compared to a previous Col sample (GSM881756), this Col sample was realigned to the TAIR10 genome for this study and as such updated processed files are available with this submission. These samples were used to define kyp mutant CHG context DMRs that were complemented upon introduction of the wildtype KYP protein. Samples 1-6 were run as multiplexed samples and were used to assay the degree of complementation for various point mutants. All plants are in the Col ecotype background.
Project description:Investigation of genome-wide expression in the mutant of histone H3K9 methyltransferase KRYPTONITE (KYP) or DNA methyltransferase CHROMOMETHYLASE3 (CMT3) in Arabidopsis. These mutants showed decrease in H3K9 methylation and DNA methylation levels, and transcriptional activation at transposons and repeats. Using NimbleGen DNA microarray, global pattern of expression of genes and transposons were examined in these mutants.
Project description:Bacteria harbor diverse mechanisms to defend themselves against their viral predators, bacteriophages. In response, phages can evolve counter-defense systems, most of which remain poorly understood. In T4-like phages, the gene tifA prevents bacterial defense by the type III toxin-antitoxin (TA) system toxIN, but the mechanism by which TifA inhibits toxIN remains unclear. Here, we show that TifA directly binds both the endoribonuclease ToxN and RNA, leading to the formation of a high molecular weight ribonucleoprotein complex in which ToxN is inhibited. The RNA binding activity of TifA is necessary for its interaction with and inhibition of ToxN. Thus, we propose that TifA inhibits ToxN during phage infection by trapping ToxN on cellular RNA, particularly the abundant 16S rRNA, preventing cleavage of phage transcripts. Taken together, our results reveal a novel mechanism underlying inhibition of a phage-defensive RNase toxin by a small, phage-encoded protein.
Project description:DNA methylation occurs at preferred sites in eukaryotes, although the basis for preference is not known. We use a microarray-based profiling method to explore the involvement of Arabidopsis CMT3 and DRM DNA methyltransferases, a histone H3 lysine-9 methyltransferase (KYP) and an Argonaute-related RNA silencing component (AGO4) in methylating target loci. We find that KYP targets are also CMT3 targets, suggesting that histone methylation maintains CNG methylation genome-wide. CMT3 and KYP targets show similar proximal distributions that corresponds to the overall distribution of transposable elements of all types, whereas DRM targets are distributed more distally along the chromosome. We find an inverse relationship between element size and loss of methylation in ago4 and drm mutants. Our results suggest that RNA-directed DNA methylation is required to silence isolated elements that may be too small to be maintained in a silent state by a chromatin-based mechanism. Thus, parallel pathways would be needed to maintain silencing of transposable elements. Keywords: Methylation profiling using Msp I enzyme
Project description:Investigation of genome-wide expression in the mutant of histone H3K9 methyltransferase KRYPTONITE (KYP) or DNA methyltransferase CHROMOMETHYLASE3 (CMT3) in Arabidopsis. These mutants showed decrease in H3K9 methylation and DNA methylation levels, and transcriptional activation at transposons and repeats. Using NimbleGen DNA microarray, global pattern of expression of genes and transposons were examined in these mutants. Total RNA from leaves was isolated using the RNeasy Plant Mini kit (Qiagen) and was treated with DNase I (TAKARA). Double-stranded cDNA was synthesized using the SuperScript Double Stranded cDNA Synthesis kit (Invitrogen) and oligo (dT)20 primer. cDNA was labeled by Cy3, hybridized to 4 x 72k array, and scanned according to manufacture's instructions (www.nimblegen.com).
Project description:Purpose: The goals of this study are to compare NGS-derived transciptomes from engineered mouse tumors with activated BRAF (primary), repressed BRAF (simulating drug treatment- dormant) and tumors which escape dormant state (recurrent) Results: We found conserved expression changes in histone methyltransferase genes (HMT), including Enhancer Of Zeste 2 Polycomb Repressive Complex 2 Subunit (EZH2), Suppressor Of Variegation 3-9 Homolog 1(SUV39H1), Suppressor Of Variegation 3-9 Homolog 2 (SUV39H2), ASH2 Like, Histone Lysine Methyltransferase Complex Subunit (ASH2L), SET And MYND Domain Containing 2 (SYMD2), and Protein arginine methyltransferase 5 (PRMT5) and histone deacetylases (HDACs) HDACS 7, 9,10, and 11.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of KYP in 10 days old KYPpro::KYP:3xFLAG arabidopsis. By obtaining sequence from chromatin immunoprecipitated DNA, we mapped genome-wide binding levels of KYP:3xFLAG. ChIP was performed using anti-FLAG antibody (sigma M2), and ChIP DNA were analyzed by Illumina NovoSeq.