Cellular generation of 5-Hydroxymethylcytosine by redox-active chemicals via an unprecedent non-enzymatic mechanism
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE44457 | GEO | 2013/12/05
SECONDARY ACCESSION(S): PRJNA190013
REPOSITORIES: GEO
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Project description:We proposed that besides TET family dioxygenase oxidizing 5mC to 5hmC, there is a non-enzyme pathway which is due to hydroxyl radica l(OH) or OH-like species also involvement in demethylation of 5mC forming 5hmC. This pathway includes classical fenton reagents such as H2O2 and Fe2+, and more important redox-activity quinoid compounds, especially, tetrachloro-1,4-benzoquinone (TCBQ), which was reported producing hydroxyl radicals independent of transition metal Examination of 5hmC and transcriptome levels with TCBQ and DMSO in human MRC-5 cell lines
2013-12-05 | E-GEOD-44457 | biostudies-arrayexpress
Project description:We studied the transcriptomic response of S. pneumoniae to moxifloxacin, a fluoroquinolone that inhibits DNA gyrase. At 10 × MIC, 30 and 140 responsive genes were detected at 15 and 30 minutes, respectively. Several pathways leading to an increase in pyruvate showed up-regulation. These included 2 genes introducing P-sugars into the glycolysis and 3 out of 7 genes of the glycolysis pathway, which converts fructose-6P to pyruvate. In addition, an increase in acetyl-coA would be expected from the down-regulation of the genes coding for the acetyl-coA carboxylase, and in turn, to a further increase in pyruvate by the up-regulation of formate acetyltransferase. Since pyruvate is converted to hydrogen peroxide (H2O2) by pyruvate oxidase (SpxB), its increase would lead to an equivalent increase in the intracellular amount of H2O2, and in turn, in those of hydroxyl radicals resulting from the Fenton reaction, which damage DNA, lipids and proteins. We observed similar increases in the production of H2O2 and hydroxyl radicals under moxifloxacin treatment. These reactive oxygen species contributed to the lethality of the drug, as showed by the attenuation of its lethality in a strain lacking SpxB. These results support the production of redox alterations by fluoroquinolones and are in agreement with our previous findings showing that levofloxacin, an inhibitor of topoisomerase IV, triggers the transcriptional activation of iron transport genes. Both fluoroquinolones stimulate the Fenton reaction in their mechanism of action, by increasing the amounts of any of their two components: iron by levofloxacin or H2O2 by moxifloxacin.
2016-02-05 | GSE68947 | GEO
Project description:The emergence of drug resistance among tuberculosis (TB) patients is often associated with their non-compliance to the length of the chemotherapy, which can reach up to 2 years for the treatment of multi-drug-resistant (MDR) TB. Drugs that would kill TB faster and would not lead to the development of drug resistance could shorten chemotherapy significantly. In Escherichia coli, the common mechanism of cell death by bactericidal antibiotics is the generation of highly reactive hydroxyl radicals via the Fenton reaction. Since ascorbic acid (vitamin C) is known to drive the Fenton reaction, we tested whether the Fenton reaction could lead to a bactericidal event in Mycobacterium tuberculosis by treating M. tuberculosis cultures with vitamin C. Here, we report that the addition of vitamin C to drug-susceptible, MDR and extensively drug-resistant (XDR) M. tuberculosis strains results in sterilization of the cultures in vitro. We show that the sterilizing effect of vitamin C on M. tuberculosis was dependent on the production of high ferrous ion levels and reactive oxygen species. Although, this potent sterilizing activity of vitamin C against M. tuberculosis in vitro was not observed in mice, we believe this activity needs further investigation. Comparison of vitamin C treated Mycobacterium tuberculosis transcriptome relative to untreated; Three biological replicates, second is a dye flip
2013-05-24 | E-GEOD-42293 | biostudies-arrayexpress
Project description:The emergence of drug resistance among tuberculosis (TB) patients is often associated with their non-compliance to the length of the chemotherapy, which can reach up to 2 years for the treatment of multi-drug-resistant (MDR) TB. Drugs that would kill TB faster and would not lead to the development of drug resistance could shorten chemotherapy significantly. In Escherichia coli, the common mechanism of cell death by bactericidal antibiotics is the generation of highly reactive hydroxyl radicals via the Fenton reaction. Since ascorbic acid (vitamin C) is known to drive the Fenton reaction, we tested whether the Fenton reaction could lead to a bactericidal event in Mycobacterium tuberculosis by treating M. tuberculosis cultures with vitamin C. Here, we report that the addition of vitamin C to drug-susceptible, MDR and extensively drug-resistant (XDR) M. tuberculosis strains results in sterilization of the cultures in vitro. We show that the sterilizing effect of vitamin C on M. tuberculosis was dependent on the production of high ferrous ion levels and reactive oxygen species. Although, this potent sterilizing activity of vitamin C against M. tuberculosis in vitro was not observed in mice, we believe this activity needs further investigation.
2013-05-24 | GSE42293 | GEO
Project description:We describe the use of a novel DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5hmC at low occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follows the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the mono-methylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or de-methylation intermediate, while others may suggest a locally accessible chromosomal environment to the TET enzymatic apparatus. Mapping of genomic 5-hydroxymethylcytosine in mouse embryonic stem cell by enzymatic digistion of AbaSI coupled with high-throughput sequencing, in replicates.
2013-01-01 | E-GEOD-42898 | biostudies-arrayexpress
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:We describe the use of a novel DNA modification-dependent restriction endonuclease AbaSI coupled with sequencing (Aba-seq) to map high-resolution hydroxymethylome of mouse E14 embryonic stem cells. The specificity of AbaSI enables sensitive detection of 5hmC at low occupancy regions. Bioinformatic analysis suggests 5hmCs in genic regions closely follows the 5mC distribution. 5hmC is generally depleted in CpG islands and only enriched in a small set of repetitive elements. A regularly spaced and oscillating 5hmC pattern was observed at the binding sites of CTCF. 5hmC is enriched at the poised enhancers with the mono-methylated histone H3 lysine 4 (H3K4me1) marks, but not at the active enhancers with the acetylated histone H3 lysine 27 (H3K27Ac) marks. Non-CG hydroxymethylation appears to be prevalent in the mitochondrial genome. We propose that some amounts of transiently stable 5hmCs may indicate a poised epigenetic state or de-methylation intermediate, while others may suggest a locally accessible chromosomal environment to the TET enzymatic apparatus.
2013-01-01 | GSE42898 | GEO
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:Oxidative modification of 5-methylcytosine (5mC) by TET DNA dioxygenases generates 5-hydroxymethylcytosine (5hmC), the most abundant form of oxidized 5mC. Existing single-cell bisulfite sequencing methods cannot resolve 5mC and 5hmC, leaving the cell-type-specific regulatory mechanisms of TET and 5hmC largely unknown. Here we present Joint single-nucleus (hydroxy)methylcytosine sequencing (Joint-snhmC-seq), a scalable and quantitative approach that simultaneously profiles 5hmC and true 5mC in single cells by harnessing differential deaminase activity of APOBEC3A towards 5mC and chemically protected 5hmC. Joint-snhmC-seq profiling of single nuclei from the mouse brains reveals an unprecedented level of epigenetic heterogeneity of both 5hmC and true 5mC at single-cell resolution. We show that cell-type-specific profiles of 5hmC or true 5mC improve multi-modal single-cell data integration, enable accurate identification of neuronal subtypes, and uncover context-specific regulatory effects of cell-type-specific genes by TET enzymes.
2023-07-14 | GSE236789 | GEO