Abstract
DNA methylation is the most common epigenetic modification observed in the genomic DNA (gDNA) of prokaryotes and eukaryotes. Methylated nucleobases, N6-methyl-adenine (m6A), N4-methyl-cytosine (m4C), and 5-methyl-cytosine (m5C), detected on gDNA represent the discrimination mark between self and non-self DNA when they are part of restriction-modification systems in prokaryotes (Bacteria and Archaea). In addition, m5C in Eukaryotes and m6A in Bacteria play an important role in the regulation of key cellular processes. Although archaeal genomes present modified bases as in the two other domains of life, the significance of DNA methylations as regulatory mechanisms remains largely uncharacterized in Archaea. Here, we began by investigating the DNA methylome of Sulfolobus acidocaldarius. The strategy behind this initial study entailed the use of combined digestion assays, dot blots, and genome resequencing, which utilizes specific restriction enzymes, antibodies specifically raised against m6A and m5C and single-molecule real-time (SMRT) sequencing, respectively, to identify DNA methylations occurring in exponentially growing cells. The previously identified restriction-modification system, specific of S. acidocaldarius, was confirmed by digestion assay and SMRT sequencing while, the presence of m6A was revealed by dot blot and identified on the characteristic Dam motif by SMRT sequencing. No m5C was detected by dot blot under the conditions tested. Furthermore, by comparing the distribution of both detected methylations along the genome and, by analyzing DNA methylation profiles in synchronized cells, we investigated in which cellular pathways, in particular the cell cycle, this m6A methylation could be a key player. The analysis of sequencing data rejected a role for m6A methylation in another defense system and also raised new questions about a potential involvement of this modification in the regulation of other biological functions in S. acidocaldarius.
Highlights
DNA methylations are one of the most well-known epigenetic modifications
The nucleic acid sequence of the latter substrate was identical to the genomic DNA (gDNA) but epigenetic marks were absent and, comparing digestion profiles of the sample and the negative control (WGA) by BsuRI allows us to check for the presence/absence of methylated cytosine (m4C) on the 5′-GGCC-3′ recognition sequence
This experiment clearly showed that the 5′-GGCC-3′ recognition sequences present on the gDNA were modified and these specific modifications protected it from digestion
Summary
DNA methylations are one of the most well-known epigenetic modifications. The addition of a methyl group on the exogenous nitrogen of the adenine or cytosine forming N6-methyl-adenine (m6A) or N4-methyl-cytosine (m4C) modifications, respectively, or directly on the endogenous carbon leading to 5-methyl-cytosine (m5C) modification is carried out by DNA methyltransferases (DNA MTases) (Jeltsch, 2002). The development of new sequencing technologies such as the single-molecule real-time (SMRT) sequencing, as an example, allows for identification of and direct mapping of the methylated motifs along the genome of an organism This technology, based on the speed of incorporation of each nucleotides with kinetic signatures specific for each nucleobase modification (Flusberg et al, 2010; Clark et al, 2012), has identified the genomic distribution pattern of m6A in bacteria (Fang et al, 2012; Murray et al, 2012; Kozdon et al, 2013) and, more recently, in multicellular eukaryotes (Greer et al, 2015; Zhang et al, 2015) as well as in one archaeal strain (Ouellette et al, 2015). These covalent modifications of nucleobases are widespread in the three domains of life and participate in different cellular processes
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