Identification of N6-adenine methylation in C. elegans DNA
Ontology highlight
ABSTRACT:
ORGANISM(S): Caenorhabditis elegans
PROVIDER: GSE66504 | GEO | 2015/05/05
SECONDARY ACCESSION(S): PRJNA278967
REPOSITORIES: GEO
Similar Datasets
Project description:In mammalian cells, DNA methylation on the 5th position of cytosine (5mC) plays an important role as an epigenetic mark. However, DNA methylation was considered to be absent in C. elegans because of the lack of detectable 5mC as well as homologs of the cytosine DNA methyltransferases. Here, using multiple approaches, we demonstrate the presence of adenine N6-methylation (6mA) in C. elegans DNA. We further demonstrate that this modification increases trans-generationally in a paradigm of epigenetic inheritance. Importantly, we identify a DNA demethylase, NMAD-1, and a potential DNA methyltransferase, DAMT-1, which regulate 6mA levels and crosstalk between methylation of histone H3K4me2 and 6mA, and control the epigenetic inheritance of phenotypes associated with the loss of the H3K4me2 demethylase spr-5. Together, these data identify a novel DNA modification in C. elegans and raise the exciting possibility that 6mA may be a carrier of heritable epigenetic information in eukaryotes. SMRT-sequencing for a mixed cell population of wildtype worms 6mA ChIP-Seq for a mixed cell population of wildtype worms
2015-05-05 | E-GEOD-66504 | biostudies-arrayexpress
Project description:DNA methylation is a key mechanism for regulation of DNA repair, DNA replication, and gene expression. In bacteria, DNA is modified by methylation on C5-cytosine (5mC), N6-adenine (6mA) and N4-cytosine (4mC). Metazoans were thought to only use 5mC to regulate gene expression until the recent discovery of N6-adenine methylation in DNA of diverse metazoan species, including mammals. Here we show by dot blot, immunoprecipitation with 4mC-specific antibodies, and ultra-high performance liquid chromatography coupled with triple-quadrupole mass spectrometry that 4mC also occurs in most eukaryotes. We further characterize 4mC sites on a near genome-wide scale by single molecule real time (SMRT) sequencing in the nematode C. elegans. In C. elegans, 4mC is found in all chromosomes but is relatively sparse on the X chromosome. 4mC is relatively enriched on introns, depleted on exons, and co-localizes with 6mA. In human cells, 4mC is anti-correlated with 5mC. Moreover, 4mC modifications are associated with increased gene expression. Transfection of human cells with a 4mC-modified luciferase reporter plasmid increases luciferase expression as compared to the unmethylated plasmid. These data suggest that 4mC promotes gene transcription, playing an opposing role to 5mC, which generally represses transcription. Thus 4mC modification of DNA is a previously unrecognized mechanism of gene regulation in eukaryotes, providing evidence for a complex epigenetic DNA code.
2018-09-01 | GSE86993 | 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:The DNA methylation program is at the bottom layer of the epigenetic regulatory cascade of vertebrate development. While the methylation at C-5 position of the cytosine (C) residues on the vertebrate genomes is achieved through the catalytic activities of the DNA methyltransferases (DNMTs), the conversion of the methylated cytosine (5mC) could be accomplished by the combined actions of the TET enzyme and DNA repair. Interestingly, it has been found recently that the mouse and human DNMTs also possess active DNA demethylation activity in vitro in a Ca2+- and redox condition-dependent manner. We report here the study of tracking down the fate of the methyl group removed from 5mC on DNA during in vitro demethylation reaction by mouse de novo DNMTs, i.e. DNMT3A and DNMT3B. Remarkably, the methyl group becomes covalently linked to the catalytic cysteines utilized by the two de novo DNMTs in their DNA methylation reactions. Thus, the forward and reverse reactions of DNA methylations by the DNMTs may utilize the same cysteine residue(s) as the active site despite of their distinctive pathways. Secondly, we demonstrate that active DNA demethylation of a heavily methylated GFP reporter plasmid by ectopically expressed DNMT3A or DNMT3B occurs in vivo in transfected human HEK 293 cells in culture. Furthermore, the extent of DNA demethylation by the DNMTs in this cell-based system is affected by Ca2+ homeostasis as well as by mutation of their putative active cysteines. These findings substantiate the roles of the vertebrate DNMTs as double-edged swords in DNA methylation-demethylation in vitro as well as in a cellular context.
2022-02-24 | PXD003401 | Pride
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:Both RNAi-dependent and -independent mechanisms have been implicated in the establishment of heterochromatin domains, which may be stabilized by feedback loops involving chromatin proteins and modifications of histones and DNA. Neurospora crassa sports features of heterochromatin found in higher eukaryotes, namely cytosine methylation (5mC), methylation of histone H3 lysine9 (H3K9me) and HETEROCHROMATIN PROTEIN-1 (HP1), and provides a model to investigate heterochromatin establishment and maintenance. We mapped the distribution of HP1, 5mC, H3K9me3 and H3K4me2 at 100bp-resolution and explored their interplay. HP1, H3K9me3 and DNA methylation were extensively colocalized and defined 44 heterochromatic domains on linkage group VII, all relics of repeat-induced point mutation (RIP). Interestingly, the centromere was found in a striking ~350kb heterochromatic domain with no detectable H3K4me2. 5mC was not found in genes, in contrast to the situation in plants and animals. H3K9me3 is required for HP1 localization and DNA methylation. Here, we show that localization of H3K9me3 is independent of 5mC or HP1 at virtually all heterochromatin regions. In addition, we observed complete restoration of DNA methylation patterns after depletion and reintroduction of the H3K9 methylation machinery, indicating that the signals for de novo heterochromatin formation lie upstream of H3K9 methylation. These data show that A:T rich RIPâd DNA efficently directs methylation of H3K9, which in turn, directs methylation of associated cytosines. Immunoprecipitation experiments using antibodies to 5mC, H3K9me3, epitope-tagged HP1, and H3K4me2 were performed. The immunoprecipitate fraction was labeled with Cy5 and the total input was labeled with Cy3. Samples were hybridized to a N. crassa LGVII tiling path microarray.
2010-05-15 | E-GEOD-12690 | biostudies-arrayexpress
Project description:We report the application of 6mA IP-sequencing technology for high-throughput profiling in rice tissues of 12 days' leaf. By obtaining over 20 million high quality clean mapping sequenced reads from immunoprecipitated DNA, we generate genome-wide 6mA maps of rice 12 days' leaf. Using mass spectrometry and immunoprecipitation and validation with analysis of single-molecule real-time sequencing, we observed that about 0.2% of all adenines are 6mA-methylated in the rice genome. 6mA occurs most frequently at GAGG motifs and is mapped to about 20% of genes and 14% of transposable elements (TEs). In promoters, 6mA marks silent genes, but in bodies correlates with gene activity. 6mA overlaps with 5-methylated cytosine (5mC) at CG sites in gene bodies and is complementary to 5mC at CHH sites in TEs. We show that OsALKBH1 may be potentially involved in 6mA demethylation in rice. The results suggest that 6mA is complementary to 5mC as an epigenomic mark in rice and reinforces a distinct role for 6mA as a gene-expression associated epigenomic mark in eukaryotes.
2018-06-10 | GSE103145 | GEO
Project description:N6-methyldeoxyadenosine (6mA or m6dA), a well-known prokaryotic DNA modification has recently been shown to exist and play regulatory roles in eukaryotic genomic DNA. However, biological functions of 6mA in mammals remain to be explored largely due to its low abundance in most mammalian genomes. Here, we report a significant enrichment of 6mA in mammalian mitochondrial DNA (mtDNA), and identify METTL4 as a methyltransferase that installs 6mA. The level of 6mA in mtDNA is elevated up to over 0.02% of the total deoxyadenosines under hypoxia, representing ~7,00-fold enrichment over 6mA in the genomic DNA (gDNA) under normal growth conditions. The presence of 6mA negatively regulates mitochondrial transcription and balances mitochondrial function for cellular adaption to hypoxic stress. While DNA 5-methylcytosine (5mC) is a well-established, prevalent epigenetic mark in mammalian genomic DNA, our study reveals for the first time DNA 6mA methylation as a regulatory mark in mammalian mtDNA.
2020-03-24 | GSE102670 | GEO
Project description:DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and Human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom in place of O2 to form a dinitrosyliron complex (DNIC). In cancer cells treated with exogeneous NO, or cells endogenously synthesizing NO, there was a global increase in DNA 5mC, a substrate for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing mouse xenograft and patient derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC at gene-regulatory loci which correlated to the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO-signaling and represents a novel epigenetic regulatory mechanism of NO.
2024-05-01 | GSE248150 | GEO
Project description:DNA methylation at cytosine bases of eukaryotic DNA (5-methylcytosine, 5mC) is a heritable epigenetic mark that can regulate gene expression in health and disease. Enzymes that metabolize 5mC have been well-characterized yet the discovery of endogenously produced signaling molecules that regulate DNA methyl-modifying machinery have not been described. Herein, we report that the free radical signaling molecule nitric oxide (NO) can directly inhibit the Fe(II)/2-OG-dependent DNA demethylases ten-eleven translocation (TET) and Human AlkB homolog 2 (ALKBH2). Physiologic NO concentrations reversibly inhibited TET and ALKBH2 demethylase activity by binding to the mononuclear non-heme iron atom in place of O2 to form a dinitrosyliron complex (DNIC). In cancer cells treated with exogeneous NO, or cells endogenously synthesizing NO, there was a global increase in DNA 5mC, a substrate for TET, that could not be attributed to increased DNA methyltransferase activity. 5mC was also elevated in NO-producing mouse xenograft and patient derived xenograft tumors. Genome-wide DNA methylome analysis of cells chronically treated with NO (10 days) demonstrated enrichment of 5mC at gene-regulatory loci which correlated to the expression of NO-regulated tumor-associated genes. Regulation of DNA methylation is distinctly different from canonical NO-signaling and represents a novel epigenetic regulatory mechanism of NO.
2024-05-01 | GSE248149 | GEO