Double and single stranded detection of 5-methylcytosine and 5-hydroxymethylcytosine with nanopore sequencing
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE279860 | GEO | 2024/10/23
REPOSITORIES: GEO
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Project description:Cytosine DNA bases can be methylated by DNA methyltransferases and subsequently oxidized by TET proteins. The resulting 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC), and 5-carboxylcytosine (5caC) are considered demethylation intermediates as well as stable epigenetic marks. To dissect the contribution of these cytosine modifying enzymes, we generated combinations of Tet knockout (KO) embryonic stem cells (ESCs) and systematically measured protein and DNA modification levels at the transition from naive to primed pluripotency. Whereas the increase of genomic 5-methylcytosine (5mC) levels during exit from pluripotency correlated with an upregulation of the de novo DNA methyltransferases DNMT3A and DNMT3B, the subsequent oxidation steps turned out to be far more complex. The strong increase of oxidized cytosine bases (5hmC, 5fC, and 5caC) was accompanied by a drop in TET2 levels, yet the analysis of KO cells suggested that TET2 is responsible for most 5fC formation. The comparison of modified cytosine and enzyme levels in Tet KO cells revealed distinct and differentiation-dependent contributions of TET1 and TET2 to 5hmC and 5fC formation arguing against a processive mechanism of 5mC oxidation. The apparent independent steps of 5hmC and 5fC formation suggest yet to be identified mechanisms regulating TET activity and may constitute another layer of epigenetic regulation.
2020-07-24 | PXD019514 | Pride
Project description:DNA methylation (5mC) plays important roles in epigenetic regulation of genome function, and recently the TET1-3 hydroxylases have been found to oxidize 5mC to hydroxymethylcytosine (5hmC), formylcytosine (5fC), and carboxylcytosine (5caC) in DNA. These derivatives have a role in demethylation of DNA but in addition may have epigenetic signaling functions in their own right. A recent study identified proteins with preferential binding to 5-methylcytosine (5mC) and its oxidized forms where readers for 5mC and 5hmC (5-hydroxymethylcytosine) showed little overlap while further oxidation forms enriched for repair proteins and transcription regulators. We extend this study by using promoter sequences as baits and compare protein binding patterns to unmodified or modified cytosine containing DNA using mouse embryonic stem cell (mESCs) extracts. The dataset contains 3 biological replicates each of mouse ES cell nuclear proteins binding to Pax6 and FGF15 promoter sequences containing different modified forms of cytosine. Data analysis: Mass spectrometric data were processed using Proteome Discoverer v1.3 and searched against a mammalian entries in Uniprot 2011.09 using Mascot v2.3 with the following parameters: Enzyme - trypsin; max 1 missed cleavage; Precursor Mass Tolerance - 10 ppm; Fragment Mass Tolerance - 0.6 Da; Dynamic Modification - Oxidation (M); Static Modification - Carbamidomethyl at C.
2013-10-21 | PXD000524 | Pride
Project description:Methylation at the 5-position of cytosine is a well-studied epigenetic pathway. In addition to 5-methylcytosine (5mC), substantial amounts of 5-hydroxymethylcytosine (5hmC) also referred to as the 6th DNA base, have been detected in certain tissues, most notably the brain. However, the genomic distribution of this cytosine modification is unknown. Here, we have developed an immunoprecipitation technique (5hmC-IP) to examine the occurrence of 5hmC in human DNA from brain frontal lobe tissue. The distribution of 5hmC was compared to that of 5mC. We show that 5hmC is more selectively targeted to genes than is 5mC. 5hmC is found at promoters and is particularly enriched in intragenic regions (gene bodies) but is largely absent from non-gene regions. 5hmC peaks at transcription start sites did not correlate with gene expression levels or H3K4me3 peaks. However, presence of 5hmC in gene bodies was more positively correlated with gene expression levels than was presence of 5mC. Promoters of testis-specific genes showed strong 5mC peaks in brain DNA but were almost completely devoid of 5hmC. Our data provide a first overview of the genomic distribution of 5hmC in human brain and will set the stage for further functional characterization of this novel DNA modification. Comparison between 5hmC and 5mC profiles
2011-03-01 | E-GEOD-27051 | biostudies-arrayexpress
Project description:NFATc2 is a sequence specific DNA binding protein in the Rel family that binds a 5-mer motif CGGAA better when both cytosines in the CG dinucleotide are methylated. Using protein binding microarrays (PBMs) we examined the DNA binding of NFATc2 to three additional types of DNA: (1) single-stranded DNA (ssDNA), (2) double stranded DNA (dsDNA) with 5-methylcytosine (5mC, M) in one strand and cytosine in the second strand (dsDNA(5mC|C)) and (3) DNA with 5-hydroxymethylcytosine (5hmC, H) in one strand and cytosine in the second strand (dsDNA(5hmC|C)). NFATc2 binding to ssDNA is strongest for the 7-mer ATTTCCA. While NFAT can bind ssDNA, the average binding to the 40,000 DNA features of the PBM was twice as high for ssDNA compared to dsDNA, with maximal binding observed to dsDNA, suggesting that NFAT binding to dsDNA is more specific. dsDNA sequences containing the 5-mer MGGAA with 5mC in one DNA strand are better bound than CGGAA, with methylation of a single cytosine being sufficient for the stronger binding seen when both cytosines in the CG dinucleotide are methylated. Several DNA sequences containing HGGAA are also better bound than those containing cytosine, however the effect of 5hmC is not as dramatic as that of 5mC. Analysis of available NFATc2:DNA complexes rationalizes our PBM data.
2018-12-31 | GSE104631 | GEO
Project description:DNA methylation (5mC) is an epigenetic modification classified by the addition of a methyl group (Ch3-) catalysed by DNA methyltransferases (DNMTs) and S-adenosyl methionine (SAM). Recently hydroxymethylation at the same carbon-5 residue of the cytosine base catalysed by the TET (Ten–Eleven Translocation) family of proteins has been shown occur in mouse embryonic stem cells and the brain. 5-hydroxymethylcytosine (5hmC) has been shown to be distributed at a lesser extent in most mammalian tissues with an increased occurrence in Purkinje cells of the mouse brain. Here we assess genome-wide 5hmC distribution relative to 5mC enrichment in the human cerebellum and peripheral blood lymphocytes (PBLs) using high-resolution sequencing by synthesis. We found a significant increase and change in 5hmC distribution in human cerebellum at loci highly expressed in the human brain and postsynaptic density (PSD) compared to PBLs. Minimal changes in 5mC distribution were observed at these same loci and genome-wide in both human cerebellum and PBLs. Using high-throughput RNA-Seq of total RNA from human brain, cerebellum and PBLs we quantified elevated expression of gene products implicated in neuronal activity and having significantly increased 5hmC distribution across their gene bodies only in brain tissue compared to PBLs. Furthermore we show elevated expression of gene loci in the cerebellum to co-localise with increased 5hmC in Purkinje neurons. Our data provide new evidence for 5hmC mediated regulation of gene loci highly expressed in the human brain and PSD and implicated in neuronal activity and development.
2015-09-01 | GSE35235 | GEO
Project description:DNA methylation (5mC) is an epigenetic modification classified by the addition of a methyl group (Ch3-) catalysed by DNA methyltransferases (DNMTs) and S-adenosyl methionine (SAM). Recently hydroxymethylation at the same carbon-5 residue of the cytosine base catalysed by the TET (Ten–Eleven Translocation) family of proteins has been shown occur in mouse embryonic stem cells and the brain. 5-hydroxymethylcytosine (5hmC) has been shown to be distributed at a lesser extent in most mammalian tissues with an increased occurrence in Purkinje cells of the mouse brain. Here we assess genome-wide 5hmC distribution relative to 5mC enrichment in the human cerebellum and peripheral blood lymphocytes (PBLs) using high-resolution sequencing by synthesis. We found a significant increase and change in 5hmC distribution in human cerebellum at loci highly expressed in the human brain and postsynaptic density (PSD) compared to PBLs. Minimal changes in 5mC distribution were observed at these same loci and genome-wide in both human cerebellum and PBLs. Using high-throughput RNA-Seq of total RNA from human brain, cerebellum and PBLs we quantified elevated expression of gene products implicated in neuronal activity and having significantly increased 5hmC distribution across their gene bodies only in brain tissue compared to PBLs. Furthermore we show elevated expression of gene loci in the cerebellum to co-localise with increased 5hmC in Purkinje neurons. Our data provide new evidence for 5hmC mediated regulation of gene loci highly expressed in the human brain and PSD and implicated in neuronal activity and development.
2015-09-01 | GSE34612 | GEO
Project description:In mammalian cells, 5-methylcytosine (5mC) occurs in genomic double-stranded DNA (dsDNA) and is enzymatically oxidized to 5-hydroxymethylcytosine (5hmC), then to 5-formylcytosine (5fC), and finally to 5-carboxylcytosine (5caC). These cytosine modifications are enriched in regulatory regions of the genome. The effect of these oxidative products on five bZIP dimers (CREB1, ATF2, Zta, ATF3|cJun, and cFos|cJun) binding to five types of dsDNA was measured using protein binding microarrays. The five dsDNAs contain either cytosine in both DNA strands or cytosine in one strand and either 5mC, 5hmC, 5fC, or 5caC in the second strand. Some sequences containing the CEBP half-site GCAA are bound more strongly by all five bZIP domains when dsDNA contains 5mC, 5hmC, or 5fC. dsDNA containing 5caC in some TRE (AP-1)-like sequences, e.g., TGACTAA, is better bound by Zta, ATF3|cJun, and cFos|cJun.
2020-11-16 | GSE141050 | GEO
Project description:Cytosine base modifications 5-methylcytosine (5mC), 5-hydroxymethylcytosine (5hmC) and 5-formylcytosine (5fC) are present in mammalian DNA. Here, reduced bisulfite sequencing is developed for quantitatively sequencing 5fC at single-base resolution. This method is then applied with oxidative bisulfite sequencing to gain a map of 5mC, 5hmC and 5fC in mouse embryonic stem cells. 12 samples, reduced representation bisulphite treatment: 4 replicates each for bisulphite (BS), oxidative BS (oxBS) and reduced BS (redBS) for the detection of 5mC, 5hmC and 5fC. Mouse (strain B6C) embryonic stem cells.
2014-04-08 | E-GEOD-56572 | biostudies-arrayexpress
Project description:The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the binding of Zta to four kinds of double-stranded DNA: 1) DNA containing cytosine on both strands, 2) DNA with 5-methylcytosine (5mC) on one strand and cytosine on the second strand, 3) DNA with 5-hydroxymethylcytosine (5hmC) on one strand and cytosine on the second strand, and 4) DNA where both cytosines in all CG dinucleotides contain 5mC. We compared the resulting data to PBM data for three other B-ZIP proteins (CREB1 and CEBPB homodimers, and cFos-cJun heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M=5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide GCAA stronger when it contains 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, opening the potential for discovery of new viral and host regulatory programs controlled by EBV.
2017-10-26 | GSE100870 | GEO
Project description:The Epstein-Barr virus (EBV) B-ZIP transcription factor (TF) Zta binds to many DNA sequences containing methylated CG dinucleotides. Using protein binding microarrays (PBMs), we analyzed the binding of Zta to four kinds of double-stranded DNA: 1) DNA containing cytosine on both strands, 2) DNA with 5-methylcytosine (5mC) on one strand and cytosine on the second strand, 3) DNA with 5-hydroxymethylcytosine (5hmC) on one strand and cytosine on the second strand, and 4) DNA where both cytosines in all CG dinucleotides contain 5mC. We compared the resulting data to PBM data for three other B-ZIP proteins (CREB1 and CEBPB homodimers, and cFos-cJun heterodimers). With cytosine, Zta binds the TRE motif TGAC/GTCA as previously reported. With CG dinucleotides containing 5mC on both strands, many TRE motif variants containing a methylated CG dinucleotide at two positions in the motif, such as MGAGTCA and TGAGMGA (where M=5mC) were preferentially bound. 5mC inhibits Zta binding to both TRE motif half sites GTCA and CTCA. Like the CREB1 homodimer, the Zta homodimer and the cJun|cFos heterodimer bind the C/EBP half site tetranucleotide GCAA stronger when it contains 5mC. Our results identify new DNA sequences that are well-bound by the viral B-ZIP protein Zta only when they contain 5mC or 5hmC, opening the potential for discovery of new viral and host regulatory programs controlled by EBV.
2017-10-26 | GSE100869 | GEO