N6-Methyladenine: A Conserved and Dynamic DNA Mark

O’Brown, Zach Klapholz; Greer, Eric Lieberman

doi:10.1007/978-3-319-43624-1_10

Cited by 105 publications

(64 citation statements)

References 215 publications

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“…1a). 5mC (+ 3mC) levels ranged from between 1.7-7% in all of the more recently evolved eukaryotes examined, but was not detected in three yeast species, S. pombe, S. cerevisiae, and S. japonicus, as previously reported [3] ( Fig. 2b).…”

Section: Resultssupporting

confidence: 82%

“…Directed DNA methylation by specific methyltransferase enzymes occurs in both prokaryotes and eukaryotes. These modifications can mark regions of the genome for control of a variety of processes, including base pairing, duplex stability, replication, repair, transcription, nucleosome positioning, X-chromosome inactivation, imprinting, and epigenetic memory [1][2][3]. The most well studied and abundant DNA methylation in eukaryotes is 5mC, a mark typically associated with repressed chromatin that occurs on~3-8% of cytosines in mammals [4].…”

Section: Introductionmentioning

confidence: 99%

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Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

et al. 2019

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Background: Directed DNA methylation on N6-adenine (6mA), N4-cytosine (4mC), and C5-cytosine (5mC) can potentially increase DNA coding capacity and regulate a variety of biological functions. These modifications are relatively abundant in bacteria, occurring in about a percent of all bases of most bacteria. Until recently, 5mC and its oxidized derivatives were thought to be the only directed DNA methylation events in metazoa. New and more sensitive detection techniques (ultra-high performance liquid chromatography coupled with mass spectrometry (UHPLC-ms/ms) and single molecule real-time sequencing (SMRTseq)) have suggested that 6mA and 4mC modifications could be present in a variety of metazoa. Results: Here, we find that both of these techniques are prone to inaccuracies, which overestimate DNA methylation concentrations in metazoan genomic DNA. Artifacts can arise from methylated bacterial DNA contamination of enzyme preparations used to digest DNA and contaminating bacterial DNA in eukaryotic DNA preparations. Moreover, DNA sonication introduces a novel modified base from 5mC that has a retention time near 4mC that can be confused with 4mC. Our analyses also suggest that SMRTseq systematically overestimates 4mC in prokaryotic and eukaryotic DNA and 6mA in DNA samples in which it is rare. Using UHPLC-ms/ms designed to minimize and subtract artifacts, we find low to undetectable levels of 4mC and 6mA in genomes of representative worms, insects, amphibians, birds, rodents and primates under normal growth conditions. We also find that mammalian cells incorporate exogenous methylated nucleosides into their genome, suggesting that a portion of 6mA modifications could derive from incorporation of nucleosides from bacteria in food or microbiota. However, gDNA samples from gnotobiotic mouse tissues found rare (0.9-3.7 ppm) 6mA modifications above background. Conclusions: Altogether these data demonstrate that 6mA and 4mC are rarer in metazoa than previously reported, and highlight the importance of careful sample preparation and measurement, and need for more accurate sequencing techniques.

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Section: Resultssupporting

confidence: 82%

Section: Introductionmentioning

confidence: 99%

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

et al. 2019

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“…Apart from 5mC, the most common types of enzyme-catalysed DNA modifications include N4-methylcytosine (4mC) and N6-methyladenine (6mA), which are widespread across bacteria. Notably, 6mA is also found in varying amounts in eukaryotes 30 and has recently been implicated in TE regulation in Drosophila melanogaster and mice 31,32 , although the presence and functional relevance of 6mA in higher eukaryotes remains controversial [33][34][35] . Moreover, 5mC can be oxidized by teneleven translocation (TET) enzymes to 5-hydroxymethylcytosine (5hmC), 5-formylcytosine (5fC) and 5carboxylcytosine (5caC), as part of a replication-independent pathway to DNA demethylation ( Figure 3).…”

Section: [H1] Introductionmentioning

confidence: 99%

Regulation of transposable elements by DNA modifications

2019

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Maintenance of genome stability requires control over the expression of transposable elements (TEs), whose activity can have substantial deleterious effects on the host. Chemical modification of DNA is a commonly used strategy to achieve this, and it has long been argued that the emergence of 5-methylcytosine (5mC) in many species was driven by the requirement to silence TEs. Potential roles in TE regulation have also been suggested for other DNA modifications, such as N6-methyladenine and oxidation derivatives of 5mC, although the underlying mechanistic relationships are poorly understood. Here, we discuss current evidence implicating DNA modifications and DNA modifying enzymes in TE regulation across different species. 100 LINE-1 elements account for virtually all of the transposition activity observed today 18 , including retrotransposition of non-autonomous short interspersed nuclear elements (SINEs), which depend on proteins encoded by LINE-1 elements 19. Although many TEs are neutral in their effect on the host, some insertions can disrupt gene function or lead to harmful chromosomal rearrangements, as demonstrated by over 120 disease-causing TE insertions in humans 20. Additionally, exacerbated TE expression in the germline can lead to sterility in mice and fruit flies 21,22. The resulting selective pressure has driven the evolution of numerous transcriptional and posttranscriptional host defence mechanisms that repress TE expression (Figure 2), which have been recently reviewed by Molaro and Malik 22. Small RNAs, including PIWI-interacting RNAs [G] (piRNAs), are the

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“…To address this, we exploited N6-methyladenine (m6A). In contrast to cytosine methylation, which is abundant in animals and typically acts to repress genes (Bernstein et al, 2007), m6A is rarely found in metazoan genomes, and its existence and potential function remain unclear in human cells (Heyn and Esteller, 2015;O'Brown and Greer, 2016). The orthogonal properties of DNA adenine methylation were previously harnessed to develop technology for mapping chromatin-associated proteins in eukaryotic genomes (Kind et al, 2013;van Steensel and Henikoff, 2000).…”

Section: Introductionmentioning

confidence: 99%

Engineering Epigenetic Regulation Using Synthetic Read-Write Modules

Park

Patel

Keung

et al. 2019

Cell

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Graphical Abstract HIGHLIGHTS d A synthetic epigenetic regulatory system in human cells using m6A DNA modification d Engineered writers and readers of m6A enable construction of regulatory circuits d Read-write circuits drive spatial propagation and hallmarks of chromatin spreading d Read-write circuits enable epigenetic memory of transcriptional states A synthetic, modular, and programmable read-write system allows isolated and orthogonal epigenetic control in mammalian cells. SUMMARYChemical modifications to DNA and histone proteins are involved in epigenetic programs underlying cellular differentiation and development. Regulatory networks involving molecular writers and readers of chromatin marks are thought to control these programs. Guided by this common principle, we established an orthogonal epigenetic regulatory system in mammalian cells using N6-methyladenine (m6A), a DNA modification not commonly found in metazoan epigenomes. Our system utilizes synthetic factors that write and read m6A and consequently recruit transcriptional regulators to control reporter loci. Inspired by models of chromatin spreading and epigenetic inheritance, we used our system and mathematical models to construct regulatory circuits that induce m6A-dependent transcriptional states, promote their spatial propagation, and maintain epigenetic memory of the states. These minimal circuits were able to program epigenetic functions de novo, conceptually validating ''read-write'' architectures. This work provides a toolkit for investigating models of epigenetic regulation and encoding additional layers of epigenetic information in cells.

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N6-Methyladenine: A Conserved and Dynamic DNA Mark

Cited by 105 publications

References 215 publications

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

Sources of artifact in measurements of 6mA and 4mC abundance in eukaryotic genomic DNA

Regulation of transposable elements by DNA modifications

Engineering Epigenetic Regulation Using Synthetic Read-Write Modules

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