Engineered methionine adenosyltransferase cascades for metabolic labeling of individual DNA methylomes in live cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE267304 | GEO | 2025/05/01
REPOSITORIES: GEO
Similar Datasets
Project description:Enzymatic methylation of cytosine to 5-methylcytosine in DNA is a fundamental epigenetic mechanism involved in mammalian development and disease. DNA methylation is brought about by collective action of three AdoMet-dependent DNA methyltransferases (DNMTs), whose catalytic interactions and temporal interplay are poorly understood. We used structure-guided engineering of the Dnmt1 methyltransferase to enable catalytic transfer of azide tags onto DNA from a synthetic cofactor analog, Ado-6-azide, in vitro . We then CRISPR-edited the Dnmt1 locus in mouse embryonic stem cells to install the engineered codon, which, following pulse- internalization of the Ado-6-azide cofactor by electroporation, permitted selective azide-tagging of Dnmt1-specific genomic targets in cellula . The deposited covalent tags were exploited as ‘click’ handles for reading adjoining sequences and precise genomic mapping of the methylation sites. The proposed approach, Dnmt-TOP-seq, enables high-resolution temporal tracking of the Dnmt1 catalysis in mammalian cells paving the way to selective studies of other methylation pathways in eukaryotic systems.
2022-02-04 | GSE182445 | GEO
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.
2012-10-04 | E-GEOD-40133 | biostudies-arrayexpress
Project description:DNA methylation is an important regulator of genome function in the eukaryotes, but it is currently unclear if the same is true in prokaryotes. While regulatory functions have been demonstrated for a small number of bacteria, there have been no large-scale studies of prokaryotic methylomes and the full repertoire of targets and biological functions of DNA methylation remains unclear. Here we applied single-molecule, real-time sequencing to directly study the methylomes of 232 phylogenetically diverse prokaryotes. Collectively, we identified 834 methylated motifs, enabling the specific annotation of 415 DNA methyltransferases (MTases), and adding substantially to existing databases of MTase specificities. While the majority of MTases function as components of restriction-modification systems, 139 MTases have no cognate restriction enzyme in the genome, suggesting some other functional role. Several of these âorphanâ MTases are conserved across species and exhibit patterns of DNA methylation consistent with known regulatory MTases. Based on these patterns of methylation, we identify candidate novel regulators of gene expression in several phyla of bacteria, and candidate regulators of DNA replication in Haloarchaea. Together these data substantially advance our knowledge of DNA restriction-modification systems, and hint at a wider role for methylation in prokaryotic genome regulation. Single-molecule, real-time sequencing of DNA modifications across 232 diverse prokaryotic genomes.
2016-01-20 | E-GEOD-69872 | biostudies-arrayexpress
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences.
2012-10-04 | GSE40133 | GEO
Project description:Protein methylation is one of the major post-translational modifications (PTMs) in the cell. In Saccharomyces cerevisiae, over twenty protein methyltransferases (MTases) and their respective substrates have been identified. However, the way in which these MTases are modified, and potentially subject to regulation, remains poorly understood. Here, we investigated six S. cerevisiae protein MTases (Rkm1, Rkm4, Efm4, Efm7, Set5 and Hmt1) to identify PTMs of potential functional relevance. We identified 51 PTM sites across the six MTases, including phosphorylation, acetylation and methylation. 45 sites are novel. We contextualised the PTM sites in structural models of the MTases and revealed that many fell in catalytic pockets or enzyme-substrate interfaces. These may regulate MTase activity. Finally, we compared PTMs on Hmt1 with those on its human homologs PRMT1, PRMT3, CARM1, PRMT6 and PRMT8. This revealed that several PTMs are conserved from yeast to human, whereas others are only found in Hmt1.
2017-12-18 | PXD006767 | Pride
Project description:Cytosine methylation is a conserved base modification, but explanations for its interspecific variation remain elusive. Only through taxonomic sampling of disparate groups can unifying explanations for interspecific variation be thoroughly tested. Here we leverage phylogenetic resolution of cytosine DNA methyltransferases (DNA MTases) and genome evolution to better understand widespread interspecific variation across 40 diverse fungal species. DNA MTase genotypes have diversified from the ancestral DNMT1+DNMT5 genotype through numerous loss events, and duplications, whereas, DIM-2 and RID-1 are more recently derived in fungi. Methylation is typically enriched at intergenic regions, which includes repeats and transposons. Unlike certain Insecta and Angiosperm species, Fungi lack canonical gene body methylation. Some fungi species possess large clusters of contiguous methylation encompassing many genes, repetitive DNA and transposons, and are not ancient in origin. Broadly, methylation is partially explained by DNA MTase genotype and repetitive DNA content. Basidiomycota on average have the highest level of methylation, and repeat content, compared to other phyla. However, exceptions exist across Fungi. Other traits, including DNA repair mechanisms, might contribute to interspecific methylation variation within Fungi. Our results show mechanism and genome evolution are unifying explanations for interspecific methylation variation across Fungi.
2019-02-18 | GSE112636 | GEO
Project description:Many cellular proteins are post-translationally modified by methylation of lysine residues. This has been most intensively studied in the case of histone proteins, where lysine methylations in the flexible tails are determinants of chromatin state and gene expression. Lysine methylations on non-histone proteins are also frequent, but in most cases the functional significance of the methylation event, as well as the identity of the responsible lysine (K) specific methyltransferase (KMT), remain unknown. While the majority of identified human KMTs belong to the SET (Su(var)3-9, Enhancer-of-zeste, Trithorax)-domain class of methyltransferases (MTases), several recently discovered KMTs belong to a different MTase class, the seven-beta-strand (7BS) MTases. Here, we have investigated an uncharacterized human 7BS MTase currently annotated as being part of the endothelin converting enzyme 2 (ECE2). However, transcriptomics data indicate that this MTase is not part of ECE2, and should be considered a separate protein. Combining in vitro enzymology and analyses of knockout cells, we demonstrate that this MTase efficiently methylates K36 in eukaryotic translation elongation factor 1 alpha (eEF1A) in vitro and in vivo. We suggest that this novel KMT is named eEF1A-KMT4 (gene name EEF1AKMT4), in agreement with the recently established nomenclature. Furthermore, by ribosome profiling we show that the absence of K36 methylation affects translation dynamics and changes translation speed of distinct codons. Finally, we demonstrate that eEF1A-KMT4 is part of a novel family of human KMTs, defined by a shared sequence motif in the active site, and we show the importance of this motif for catalytic activity.
2017-05-23 | GSE97140 | GEO
Project description:DNA methylation is an important regulator of genome function in the eukaryotes, but it is currently unclear if the same is true in prokaryotes. While regulatory functions have been demonstrated for a small number of bacteria, there have been no large-scale studies of prokaryotic methylomes and the full repertoire of targets and biological functions of DNA methylation remains unclear. Here we applied single-molecule, real-time sequencing to directly study the methylomes of 232 phylogenetically diverse prokaryotes. Collectively, we identified 834 methylated motifs, enabling the specific annotation of 415 DNA methyltransferases (MTases), and adding substantially to existing databases of MTase specificities. While the majority of MTases function as components of restriction-modification systems, 139 MTases have no cognate restriction enzyme in the genome, suggesting some other functional role. Several of these ‘orphan’ MTases are conserved across species and exhibit patterns of DNA methylation consistent with known regulatory MTases. Based on these patterns of methylation, we identify candidate novel regulators of gene expression in several phyla of bacteria, and candidate regulators of DNA replication in Haloarchaea. Together these data substantially advance our knowledge of DNA restriction-modification systems, and hint at a wider role for methylation in prokaryotic genome regulation.
2016-01-20 | GSE69872 | GEO
Project description:We performed repeated endoscopic colon biopsies of C57BL/6J mice weekly and found that the mesenteric adipose tissue (MAT) was enlarged and it wrapped around the colon at wound sites after 4 cycles of biopsy. On histological view, fibrous bands extended from the intestine into the adjacent MAT, mimicking the fibrosis feature in Crohn’s disease. To investigate the gene signature in the MAT, laser capture microdissection was performed to collect the MAT and associated fibrosis in the repeated biopsy mice and controls. Agilent Microarray was used for the transcriptomic analysis.
2021-07-16 | E-MTAB-8566 | biostudies-arrayexpress
Project description:RNA methylations are essential both for RNA structure and function, and are introduced by a number of distinct methyltransferases (MTases). In recent years, N6-methyladenosine (m6A) modification of eukaryotic mRNA has been subject to intense studies, and it has been demonstrated that m6A is a reversible modification that regulates several aspects of mRNA function. However, m6A is also found in other RNAs, such as mammalian 18S and 28S ribosomal RNAs (rRNAs), but the responsible MTases have remained elusive. 28S rRNA carries a single m6A modification, found at position A4220 (alternatively referred to as A4190) within a stem-loop structure, and here we show that the MTase ZCCHC4 is the enzyme responsible for introducing this modification. Accordingly, we found that ZCCHC4 localises to nucleoli, the site of ribosome assembly, and that proteins involved in RNA metabolism are overrepresented in the ZCCHC4 interactome. Interestingly, the absence of m6A4220 perturbs codon-specific translation dynamics and shifts gene expression at the translational level. In summary, we establish ZCCHC4 as the enzyme responsible for m6A modification of human 28S rRNA, and demonstrate its functional significance in mRNA translation.
2019-11-25 | GSE128994 | GEO