Abstract
The genomes of organisms from all three domains of life harbor endogenous base modifications in the form of DNA methylation. In bacterial genomes, methylation occurs on adenosine and cytidine residues to include N6-methyladenine (m6A), 5-methylcytosine (m5C), and N4-methylcytosine (m4C). Bacterial DNA methylation has been well characterized in the context of restriction-modification (RM) systems, where methylation regulates DNA incision by the cognate restriction endonuclease. Relative to RM systems less is known about how m6A contributes to the epigenetic regulation of cellular functions in Gram-positive bacteria. Here, we characterize site-specific m6A modifications in the non-palindromic sequence GACGmAG within the genomes of Bacillus subtilis strains. We demonstrate that the yeeA gene is a methyltransferase responsible for the presence of m6A modifications. We show that methylation from YeeA does not function to limit DNA uptake during natural transformation. Instead, we identify a subset of promoters that contain the methylation consensus sequence and show that loss of methylation within promoter regions causes a decrease in reporter expression. Further, we identify a transcriptional repressor that preferentially binds an unmethylated promoter used in the reporter assays. With these results we suggest that m6A modifications in B. subtilis function to promote gene expression.
Highlights
DNA methylation is pervasive across all three domains of life
5-methylcytosine (m5C) modifications have been shown to function in development and the regulation of gene expression, with aberrant methylation implicated in human health, including cancer, autoimmune diseases, and metabolic disorders [for review, [1,2]]. m5C in promoter regions has been linked to the repression of downstream gene transcription, whereas gene body methylation has been positively correlated with gene expression [for review [3]]
Pacific Biosciences (PacBio) SMRT sequencing was used to determine if DNA modifications were present in the genome of several B. subtilis strains with the results deposited on the publicly available web resource REBASE maintained by New England Biolabs
Summary
DNA methylation is pervasive across all three domains of life. In eukaryotes, 5-methylcytosine (m5C) modifications have been shown to function in development and the regulation of gene expression, with aberrant methylation implicated in human health, including cancer, autoimmune diseases, and metabolic disorders [for review, [1,2]]. m5C in promoter regions has been linked to the repression of downstream gene transcription, whereas gene body methylation has been positively correlated with gene expression [for review [3]]. 5-methylcytosine (m5C) modifications have been shown to function in development and the regulation of gene expression, with aberrant methylation implicated in human health, including cancer, autoimmune diseases, and metabolic disorders [for review, [1,2]]. M5C in promoter regions has been linked to the repression of downstream gene transcription, whereas gene body methylation has been positively correlated with gene expression [for review [3]]. Recent studies have identified m6A in the genomes of Chlamydomonas, Caenorhabditis elegans and Drosophila melanogaster [4,5,6]. In contrast to promoter m5C, m6A modifications appear to function in gene activation in the algae Chlamydomonas [4] and promoter m6A is important in early Drosophila development [5]. There is a growing recognition that m6A is critical for the regulation of gene expression in a broad range of eukaryotic organisms
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