Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Tet1 and hydroxymethylcytosine in transcription and DNA methylation fidelity (ChIP/DIP-Seq data)

ABSTRACT: We analyzed the genome-wide binding of Tet1 in control (shScr) and Tet1 knockdown (shTet1) mouse ES cells using two different Tet1 antibodies (Tet1-C and Tet1-N). Furthermore, we generated genome-wide mapping of hydroxymethyl cytosine (hmC) and methyl cytosine (mC). We find that hmC, in contrast to mC, is also found at transcription start sites (TSSs), and that there is a significant overlap between Tet1 binding and hmC positive regions. Surprisingly, our results also suggest, that Tet1 has a role in transcriptional repression. We showed that Tet1 associates with Sin3A co-repressor complex, and by performing ChIP-sequencing of Sin3A, we find co-localisation of Tet1 and Sin3a throughout the genome Examination of Tet1 and Sin3A binding as well as hmC and mC localization in mouse ES cells

ORGANISM(S): Mus musculus

SUBMITTER: Kristine Williams

PROVIDER: E-GEOD-24841 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity.

Williams Kristine K Christensen Jesper J Pedersen Marianne Terndrup MT Johansen Jens V JV Cloos Paul A C PA Rappsilber Juri J Helin Kristian K

Nature 20110413 7347

Enzymes catalysing the methylation of the 5-position of cytosine (mC) have essential roles in regulating gene expression and maintaining cellular identity. Recently, TET1 was found to hydroxylate the methyl group of mC, converting it to 5-hydroxymethyl cytosine (hmC). Here we show that TET1 binds throughout the genome of embryonic stem cells, with the majority of binding sites located at transcription start sites (TSSs) of CpG-rich promoters and within genes. The hmC modification is found in gen ...[more]

PMID: 21490601

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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.

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, 200 M-NM-<g/ml) for 72 hours. RNA was harvested from biological triplicates for each condition and hybridized to Affymetrix microarrays. 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).

Dataset Information

Tet1 and hydroxymethylcytosine in transcription and DNA methylation fidelity (ChIP/DIP-Seq data)

Publications

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity.

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