Unknown,Transcriptomics,Genomics,Proteomics

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DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis.

ABSTRACT: The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutation of the H3K27 methyltransferase genes, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs). Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs. DNA-seq: One gram of mature rosette leaves were collected from 3-4-week-old plants, chopped in 0.5 ml of filtered Galbraith buffer, and stained with propidium iodide. A BD FACS Aria II in the UCLA Jonsson Comprehensive Cancer Center (JCCC) Flow Cytometry Core Facility was used to sort the nuclei. For sequencing, 7,000-9,000 8C nuclei of each sample were collected, and purified DNA with Picopure purification kit (Arcturus) following manufacturer instructions. RNA-seq: RNA-seq experiments were performed in two biological replicates for each genotype. 0.1g of tissue was ground in Trizol. Total RNA were treated with DNaseI (Roche), and cleaned up with phenol-chlorophorm and precipitated with ethanol. Libraries were generated and sequenced following manufacturer instructions (Illumina). BS-seq: 0.5-1g of mature rosette leaves were collected, and genomic DNA was extracted using Plant DNeasey mini purification kit (Qiagen). Libraries were generated as previously described (Feng et al., Methods Mol Biol. 2011;733:223-38.).

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Hume Stroud

PROVIDER: E-GEOD-38286 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis.

Stroud Hume H Hale Christopher J CJ Feng Suhua S Caro Elena E Jacob Yannick Y Michaels Scott D SD Jacobsen Steven E SE

PLoS genetics 20120705 7

The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, Arabidopsis trithorax-related protein5 (ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochroma ...[more]

PMID: 22792077

Publication: 1/2

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Project description:DNA methylation is an epigenetic modification that plays critical roles in gene silencing, development, and the maintenance of genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and is targeted by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis and Pol-V, which functions in the downstream DNA methyltransferase targeting phase of the RdDM pathway to generate scaffold transcripts that recruit downstream RdDM factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV interacting protein3. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methyl H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals1,5, a further understanding of this early targeting step may aid in our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy. For wild type plants (ecotype Columbia) and RdDM mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. The Col and nrpe1 BS-seq libraries were previously reported (GSE39247) and so are not part of this submission. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of nrpd1 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPD1. Whole-genome bisulfite sequencing and small RNA sequencing was also performed on shh1 mutant plants transformed with the wild-type SHH1 protein-coding construct as well as multiple constructs containing point mutations. For these complementation libraries a separate shh1 mutant and Col control line were sequenced (“complementation replicates”).

Dataset Information

DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis.

Publications

DNA methyltransferases are required to induce heterochromatic re-replication in Arabidopsis.

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