Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax.

ABSTRACT: We report the existence of both methylcytosine and hydroxymethylcytosine in the genomic DNA of the ciliate Oxytricha trifallax during its genome rearrangement process. These modifications are dynamically added, de novo, to sequences targeted for elimination and are not present after the rearrangement process (in vegetative cells). We performed methyl-DNA immunoprecipitation-sequencing (meDIP-seq) with antibodies against methylcytosine and hydroxymethylcytosine. We performed methyl-DNA immunoprecipitation-sequencing (meDIP-seq) with antibodies against methylcytosine and hydroxymethylcytosine, and used an IgG control for nonspecific immunoprecpitation. Immunoprecipitation was performed on both vegetative (negative control) and 46h cells (with methylation). The data were normalized to RPKM, then the number of vegetative reads was subtracted from the number of 46h reads, giving excess reads at 46h, which we denote as the methylation/hydroxymethylation "signal". Those data are reported in the tab-deliminated data files included with this dataset.

ORGANISM(S): Oxytricha trifallax

SUBMITTER: John Bracht

PROVIDER: E-GEOD-41060 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax.

Bracht John R JR Perlman David H DH Landweber Laura F LF

Genome biology 20121017 10

<h4>Background</h4>Cytosine methylation of DNA is conserved across eukaryotes and plays important functional roles regulating gene expression during differentiation and development in animals, plants and fungi. Hydroxymethylation was recently identified as another epigenetic modification marking genes important for pluripotency in embryonic stem cells.<h4>Results</h4>Here we describe de novo cytosine methylation and hydroxymethylation in the ciliate Oxytricha trifallax. These DNA modifications o ...[more]

PMID: 23075511

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Project description:DNA methylation is a heritable epigenetic modification involved in gene silencing, imprinting, and the suppression of retrotransposons. Global DNA demethylation occurs in the early embryo and the germline and may be mediated by Tet (ten-eleven-translocation) enzymes, which convert 5-methylcytosine (mC) to 5-hydroxymethylcytosine (hmC). Tet enzymes have been extensively studied in mouse embryonic stem (ES) cells, which are generally cultured in the absence of Vitamin C, a potential co-factor for Fe(II) 2-oxoglutarate dioxygenase enzymes like Tets. Here we report that addition of Vitamin C to ES cells promotes Tet activity leading to a rapid and global increase in hmC. This is followed by DNA demethylation of numerous gene promoters and up-regulation of demethylated germline genes. Tet1 binding is enriched near the transcription start site (TSS) of genes affected by Vitamin C treatment. Importantly, Vitamin C, but not other antioxidants, enhances the activity of recombinant human Tet1 in a biochemical assay and the Vitamin C-induced changes in hmC and mC are entirely suppressed in Tet1/2 double knockout (Tet DKO) ES cells. Vitamin C has the strongest effects on regions that gain methylation in cultured ES cells compared to blastocysts and in vivo are methylated only after implantation. In contrast, imprinted regions and intracisternal A-particle (IAP) elements, which are resistant to demethylation in the early embryo, are resistant to Vitamin C-induced DNA demethylation. Collectively, this study establishes that Vitamin C is a direct regulator of Tet activity and DNA methylation fidelity in ES cells. Oct4-GiP mouse embryonic stem (ES) cells were cultured in the presence or absence of Vitamin C (L-ascorbic acid 2-phosphate, 100 M-NM-<g/ml) for 12 or 72 hours. Cells were maintained in N2B27 medium supplemented with LIF (1000 U/ml), MEK inhibitor PD0325901 (1 M-NM-<M), and GSK3M-NM-2 inhibitor CHIR99021 (3 M-NM-<M). Genomic DNA was used for DNA immunoprecipitation with antibodies against 5-hydroxymethylcytosine (hmC) or 5-methylcytosine (mC). Immunoprecipitated DNA was adaptor-ligated for paired-end sequencing on an Illumina HiSeq and sequence reads were aligned to the mm9 mouse reference genome for analysis.

Dataset Information

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax.

Publications

Cytosine methylation and hydroxymethylation mark DNA for elimination in Oxytricha trifallax.

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