Project description:Since the discovery that cytosine deoxynucleotides could be methylated (dC5m), not much is known in higher eukaryotes about modifications affecting other deoxynucleotides. Here, we now report that we detected N-6-methyl-deoxyadenosine (dA6m) not only in frog DNA, but also in other species including mouse and human. Our methylome analysis revealed that dA6m is widely distributed across the eukaryotic genome, is present in different cell types, but commonly depleted from gene exons. Since dA6m is a direct modification of DNA, it has the potential to generate a significant impact on different biological areas. Ultimately, our work shows that deoxycytidine modifications might not be the only ones in higher eukaryotes, suggesting that such direct DNA modifications might be more widespread than previously thought.
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
Project description:This data set was downloaded from MetaboLights (http://www.ebi.ac.uk/metabolights/) accession number MTBLS276 Abstract:"Since the discovery that cytosine deoxynucleotides could be methylated (dC5m), not much is known in higher eukaryotes about modifications affecting other deoxynucleotides. Here, we now report that we detected N-6-methyl-deoxyadenosine (dA6m) not only in frog DNA, but also in other species including mouse and human. Our methylome analysis revealed that dA6m is widely distributed across the eukaryotic genome, is present in different cell types, but commonly depleted from gene exons. Since dA6m is a direct modification of DNA, it has the potential to generate a significant impact on different biological areas. Ultimately, our work shows that deoxycytidine modifications might not be the only ones in higher eukaryotes, suggesting that such direct DNA modifications might be more widespread than previously thought."
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.
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.
Project description:DNA methylation plays a crucial role for gene regulation among eukaryotes, but its regulatory function is less understood in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803 five DNA methyltransferases have been identified. Among them, M.Ssp6803II is responsible for the specific methylation of the first cytosine in the frequently occurring motif GGCC, leading to N4-methylcytosine (GGm4CC). The mutation of the corresponding gene sll0729 led to a strong phenotype, including a lowered chlorophyll/phycocyanin ratio, impaired growth, and alterations of the expression of two genes encoding hypothetical proteins. However, prolonged cultivation revealed instability of the initially obtained phenotype, with colonies showing normal pigmentation and wild-type-like growth appearing regularly and in high frequencies on agar plates. These colonies represent suppressor mutants, since the sll0729 gene was still completely inactivated and the GGCC sites remained un-methylated. Two suppressor clones were further characterized and showed a smaller cell size and lowered DNA content per cell. Moreover, they exhibited a decreased tolerance against UV stress compared to wild type. Proteomics revealed the reduction of DNA topoisomerase in suppressor mutant cells. Collectively, these results indicate that GGm4CC methylation is involved in the regulation of gene expression, in the fine-tuning of DNA replication, and DNA repair mechanisms.
Project description:DNA methylation plays a crucial role for gene regulation among eukaryotes, but its regulatory function is less understood in bacteria. In the cyanobacterium Synechocystis sp. PCC 6803 five DNA methyltransferases have been identified. Among them, M.Ssp6803II is responsible for the specific methylation of the first cytosine in the frequently occurring motif GGCC, leading to N4-methylcytosine (GGm4CC). The mutation of the corresponding gene sll0729 led to a strong phenotype, including a lowered chlorophyll/phycocyanin ratio, impaired growth, and alterations of the expression of two genes encoding hypothetical proteins. However, prolonged cultivation revealed instability of the initially obtained phenotype, with colonies showing normal pigmentation and wild-type-like growth appearing regularly and in high frequencies on agar plates. These colonies represent suppressor mutants, since the sll0729 gene was still completely inactivated and the GGCC sites remained un-methylated. Two suppressor clones were further characterized and showed a smaller cell size and lowered DNA content per cell. Moreover, they exhibited a decreased tolerance against UV stress compared to wild type. Proteomics revealed the reduction of DNA topoisomerase in suppressor mutant cells. Collectively, these results indicate that GGm4CC methylation is involved in the regulation of gene expression, in the fine-tuning of DNA replication, and DNA repair mechanisms.