Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Tet1 and hydroxymethylcytosine in transcription and DNA methylation fidelity (Affymetrix gene expression data)

ABSTRACT: Enzymes catalyzing the methylation of the 5-position of cytosine (mC) have essential roles in regulating gene expression, genome stability, and maintaining cellular identity. Recently Tet1, which is highly expressed in embryonic stem (ES) cells, was found to oxidize the methyl group of mC converting it to 5-hydroxymethyl cytosine (hmC)3. Here, we present the genome-wide mapping of Tet1 and hmC in mouse ES cells. We show that Tet1 binds throughout the genome with the majority of binding sites located at transcription start sites (TSSs) and within genes. Similar to Tet1 and mC, also hmC is found throughout the genome and in particular in gene bodies. However, in contrast to mC, hmC is enriched at TSSs. Tet1 and hmC are associated with genes critical for the control of development and differentiation, which become methylated during differentiation. Surprisingly our results also suggest that Tet1 has a role in transcriptional repression. We show that Tet1 binds to a significant proportion of target genes that are positive for the Polycomb repressive histone mark H3K27me3, and that downregulation of Tet1 also leads to increased expression of a group of Tet1 target genes. In agreement with a potential repressive function, we show that Tet1 associates with the Sin3A co-repressor complex, which also co-localises with Tet1 throughout the genome. We propose that Tet1 fulfils dual functions in transcriptional regulation, where it fine-tunes DNA methylation and associates with the Sin3A co-repressor complex to prevent transcriptional activation. [GSM611209-GSM611217] Control (shScr) or two different Tet1 knockdown (shTet1#4 or shTet1#5) mouse ES cells were used. Each experiment was performed in triplicates. [GSM675884-GSM675889] Control (shScr) or Sin3A knockdown (shSin3A) mouse ES cells were used.Each experiment was performed in triplicates.

ORGANISM(S): Mus musculus

SUBMITTER: Kristine Williams

PROVIDER: E-GEOD-24842 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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).

Project description:Epigenetic modification of the mammalian genome by DNA methylation (5-methylcytosine) has a profound impact on chromatin structure, gene expression and maintenance of cellular identity. Recent demonstration that members of the Ten-eleven translocation (Tet) family proteins can convert 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) raised the possibility that Tet proteins are capable of establishing a distinct epigenetic state. We have recently demonstrated that Tet1 is specifically expressed in murine embryonic stem (ES) cells and is required for ES cell self-renewal and maintenance. Using chromatin immunoprecipitation coupled with high-throughput DNA sequencing (ChIP-seq), here we show that Tet1 is preferentially bound to CpG-rich sequences at promoters of both transcriptionally active and Polycomb-repressed genes. Despite a general increase in levels of DNA methylation at Tet1 binding-sites, Tet1 depletion does not lead to down-regulation of all the Tet1 targets. Interestingly, while Tet1-mediated promoter hypomethylation is required for maintaining the expression of a group of transcriptionally active genes, it is also required for repression of Polycomb-targeted developmental regulators. Tet1 contributes to silencing of this group of genes by facilitating recruitment of PRC2 to CpG-rich gene promoters. Thus, our study not only establishes a role for Tet1 in modulating DNA methylation levels at CpG-rich promoters, but also reveals a dual function of Tet1 in promoting transcription of pluripotency factors as well as participating in the repression of Polycomb-targeted developmental regulators. Mouse ES cells infected with control knockdown (KD) or Tet1 KD lentiviruses were FACS-sorted for RNA extraction and hybridization on Affymetrix microarrays. We also investigated the effect of Nanog overexpression (OE) in Tet1 KD mouse ES cells on dys-regulated Tet1 targets. We have collected four biologically independent replicates for each treatment.

Dataset Information

Tet1 and hydroxymethylcytosine in transcription and DNA methylation fidelity (Affymetrix gene expression data)

Publications

TET1 and hydroxymethylcytosine in transcription and DNA methylation fidelity.

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