Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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A comparative analysis of 5-azacytidine and zebularine induced DNA demethylation using whole-genome bisulfite sequencing

ABSTRACT: The non-methylable cytosine analogs, 5-azacytidine and zebularine, are widely used to disrupt DNA methyltransferase activity and reduce genomic DNA methylation. In this study, whole-genome bisulfite sequencing is used to construct maps of DNA methylation with single base pair resolution in Arabidopsis thaliana seedlings treated with each demethylating agent. We find that 5-azacytidine and zebularine-treated seedlings have nearly indistinguishable patterns of DNA methylation genome-wide and that 5-azacytidine, despite being more unstable in aqueous solution, has a slightly greater demethylating effect at higher concentrations across the genome. Transcriptome analyses revealed a substantial number of up-regulated genes and transposable element genes, particularly CACTA-like elements, demonstrating that chemical demethylating agents have a disproportionately large effect on loci that are silenced by DNA methylation. Bisulfite-Seq and RNA-Seq

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Robert Schmitz

PROVIDER: E-GEOD-80300 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:During development histone modifying enzymes are required for cell identity and lineage commitment, however little is known about the regulatory origins of the epigenome during embryonic development. Here we generate a comprehensive set of embryonic epigenome reference maps, which we use to determine the extent to which maternal factors shape chromatin state in Xenopus embryos. Using Î±-amanitin to inhibit zygotic transcription, we find that the majority of H3K4me3 and H3K27me3-enriched regions form a maternally defined epigenetic regulatory space with an underlying logic of hypomethylated islands. This maternal regulatory space extends to a substantial proportion of neurula stage-activated promoters. In contrast, p300-recruitment to distal regulatory regions requires embryonic transcription at most loci. The results show that H3K4me3 and H3K27me3 are part of a regulatory space that exerts an extended maternal control well into post-gastrulation development, and highlight the combinatorial action of maternal and zygotic factors through proximal and distal regulatory sequences. We have performed ChIP-sequencing of eight histone modifications, RNA polymerase II (RNAPII) and the enhancer protein p300 at five stages of development: blastula (st. 9), gastrula (st. 10.5, 12.5), neurula (st. 16) and tailbud (st. 30). These experiments allow identification of enhancers (H3K4me1, p300), promoters (H3K4me3, H3K9ac), transcribed regions (H3K36me3, RNAPII) and repressed and heterochromatic domains (H3K27me3, H3K9me2, H3K9me3, H4K20me3). In addition we generated pre-MBT (st. 8) maps for three histone modifications (H3K4me3, H3K9ac, H3K27me3) and single-base resolution DNA methylome maps using whole genome bisulfite sequencing of blastula and gastrula (st. 9 and 10.5) embryos. To determine the maternal and zygotic contributions to chromatin state, we used alpha-amanitin to block embryonic transcription. Fertilised eggs were injected with 2.3 nl of 2.67 ng/ul alpha-amanitin and developed until the control embryos reached mid-gastrulation. Alpha-amanitin and control embryos were used for RNA-seq and ChIP-seq of RNAPII, H3K4me3, H3K27me3 and p300. For all ChIP-seq samples of the epigenome reference maps and RNAPII ChIP-seq samples of the Î±-amanitin experiments three biological replicates of different chromatin isolations of 45 embryos were pooled. Two biological replicates for H3K4me3 (Î±-amanitin injected: resp. 90 and 56 embryo equivalents (eeq); control: resp. 45 and 67 eeq), H3K27me3 (Î±-amanitin injected: resp. 90 and 180 eeq; control: resp. 45 and 202 eeq) and p300 (Î±-amanitin injected: resp. 112 and 56 eeq; control: resp. 112 and 67 eeq) ChIP-seq samples of the Î±-amanitin experiments were generated. For RNA-seq samples of the Î±-amanitin experiments RNA from 5 embryos from one biological replicate was isolated and depleted of ribosomal RNA

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A comparative analysis of 5-azacytidine and zebularine induced DNA demethylation using whole-genome bisulfite sequencing

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