Vitamin C induces Tet-dependent DNA demethylation in ES cells to promote a blastocyst-like methylome [Affymetrix]
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ABSTRACT: 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-
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.
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.
Project description: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
Project description:Covalent modification of DNA distinguishes cellular identities and is crucial for regulating the pluripotency and differentiation of embryonic stem (ES) cells. The recent demonstration that 5-methylcytosine (5-mC) may be further modified to 5-hydroxymethylcytosine (5-hmC) in ES cells has revealed a novel regulatory paradigm to modulate the epigenetic landscape of pluripotency. To understand the role of 5-hmC in the epigenomic landscape of pluripotent cells, here we profile the genome-wide 5-hmC distribution and correlate it with the genomic profiles of 11 diverse histone modifications and six transcription factors in human ES cells. By integrating genomic 5-hmC signals with maps of histone enrichment, we link particular pluripotency-associated chromatin contexts with 5-hmC. Intriguingly, through additional correlations with defined chromatin signatures at promoter and enhancer subtypes, we show distinct enrichment of 5-hmC at enhancers marked with H3K4me1 and H3K27ac. These results suggest potential role(s) for 5-hmC in the regulation of specific promoters and enhancers. In addition, our results provide a detailed epigenomic map of 5-hmC from which to pursue future functional studies on the diverse regulatory roles associated with 5-hmC. Genome wide enrichment profile of 5-hmC in H1 human embryonic stem cells
Project description:Tet1 is a hydroxylase known for its role in the conversion of 5-methylcytosines (5mC) to 5-hydroxymethylcytosines (5hmC) involved in the possible active demethylation process and gene expression regulation1-5.M-BM- As somatic cell reprogramming involves the re-activation of pluripotency genes and the silencing of somatic ones6, it remains unclear whether Tet1 plays a positive or negative role in the reprogramming process. Here we show that Tet1 deficiency enhances reprogramming and its overexpression impairs reprogramming. Mechanistically, we demonstrated that Tet1 represses the early obligatory process of mesenchymal to epithelial transition (MET) during reprogramming7,8. Thus, our findings not only define a negative role for Tet1 in somatic cell reprogramming, but also suggest that the Tet enzymes regulate cell fate through distinctive mechanisms. Examination of genome DNA hmC modifications in 2 conditions: individually overexpressed Tet1CD or Tet2CD during MEF reprogramming; Examination of mRNA levels in five different conditions: individually overexpressed DR or Tet1CD or Tet1CDmut or Tet2CD or Tet2CDmut, during MEF reprogrammig.
Project description: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.
Project description:5-hydroxymethylcytosine (5hmC) is the first oxidative product of the TET-mediated 5-methylcytosine (5mC) demethylation pathway. It is a key intermediate in cytosine demethylation, and have potential regulatory functions with emerging importance in mammalian biology. In this work, we used a chemical capture-based technique that coupled with next-generation sequencing to investigate the global 5hmC methylation in five brain subregions (cerebellum, cortex, hippocampus, hypothalamus and thalamus) and liver tissues from female and male adult mice. We also performed total RNA sequencing to study the association between 5hmC and gene expression. The enriched 5-hmC library was sequenced on a HiSeq2500 by paired-end sequencing with 100 bp read length.
Project description:DNA methylation at the 5-position of cytosine (5-mC) is a key epigenetic mark critical for varius biological and pathological processes. 5-mC can be converted to 5-hydroxymethylcytosine (5-hmC) by the Ten-Eleven Translocation (TET) family of DNA hydroxylases. Here we report that "loss of 5-hmC" is an epigenetic hallmark of melanoma with diagonostic and prognostic implications. Genome-wide mapping of 5-hmC in nevi and melanomas for the first time revealed loss of 5-hmC landscape in the melanoma epigenome. Downregulation of Isocitrate Dehydrogenase 2 (IDH2) and TET family enzymes proved to be one of the mechanisms underlying the loss of 5-hmC during melanoma development, and rebuilding the 5-hmC landscape in the melanoma epigenome by reintroducing active TET2 or IDH2 suppressed melanoma growth and increased tumor-free survival. Thus, our study establishes that "loss of 5-hmC" is a new epigenetic hallmark of melanoma and links IDH and TET family enzymes-mediated 5-hmC putative tumor suppressor pathway to the suppression of melanoma progression. Determine the genome-wide distribution of 5mC and 5hmC in benign nevus tissue, melanoma tissue, A2058 MOCK cells (overexpressing empty vector), A2058 TET2 cells (overexpressing wild type human TET), and A2058 TET2M cells (overexpressing mutant human TET).
Project description:Histone methylation patterns regulate gene expression and are highly dynamic during development. The erasure of histone methylation is carried out by histone demethylase enzymes. We had previously shown that vitamin C enhances the activity of Tet enzymes in embryonic stem (ES) cells, leading to DNA demethylation and activation of germline genes. We report here that vitamin C induces a remarkably specific demethylation of histone H3 lysine 9 dimethylation (H3K9me2) in ES cells. Vitamin C treatment reduces global levels of H3K9me2, but not other histone methylation marks analyzed, as measured by Western blot, immunofluorescence and mass spectrometry. Vitamin C leads to widespread loss of H3K9me2 at large chromosomal domains as well as gene promoters and repeat elements. Vitamin C-induced loss of H3K9me2 occurs rapidly within 24 hours and is reversible. Importantly, we found that the histone demethylases Kdm3a and Kdm3b are required for vitamin C-induced demethylation of H3K9me2. Moreover, we show that vitamin C-induced Kdm3a/b-mediated H3K9me2 demethylation and Tet-mediated DNA demethylation are independent processes. Lastly, we document Kdm3a/b are partially required for the up-regulation of germline genes by vitamin C. These results reveal a specific role for vitamin C in histone demethylation in ES cells, and document that DNA methylation and H3K9me2 cooperate to silence germline genes in pluripotent cells.