DNA methylation affects gene expression but not global chromatin structure in Escherichia coli

Abstract

The activity of DNA adenine methyltransferase (Dam) and DNA cytosine methyltransferase (Dcm) together account for nearly all methylated nucleotides in the Escherichia coli K-12 MG1655 genome. Previous studies have shown that perturbation of DNA methylation alters E. coli global gene expression, but it is unclear whether the methylation state of Dam or Dcm target sites regulates local transcription. In recent genome-wide experiments, we observed an underrepresentation of Dam sites in transcriptionally silent extended protein occupancy domains (EPODs), prompting us to hypothesize that EPOD formation is caused partially by low Dam site density. We thus hypothesized that a methylation-deficient version of MG1655 would show large-scale aberrations in chromatin structure. To test our hypothesis, we cloned methyltransferase deletion strains and performed global protein occupancy profiling using high resolution in vivo protein occupancy display (IPOD-HR), chromatin immunoprecipitation for RNA polymerase (RNAP-ChIP), and transcriptome abundance profiling using RNA-Seq. Our results indicate that loss of DNA methylation does not result in large-scale changes in genomic protein occupancy such as the formation of EPODs, indicating that the previously observed depletion of Dam sites in EPODs is correlative, rather than causal, in nature. However, loci with dense clustering of Dam methylation sites show methylation-dependent changes in local RNA polymerase and total protein occupancy, but local transcription is unaffected. Our transcriptome profiling data indicates that deletion of dam and/or dcm results in significant expression changes within some functional gene categories including SOS response, flagellar synthesis, and translation, but these expression changes appear to result from indirect regulatory consequences of methyltransferase deletion. In agreement with the downregulation of genes involved in flagellar synthesis, dam deletion is characterized by a swimming motility-deficient phenotype. We conclude that DNA methylation does not control the overall protein occupancy landscape of the E. coli genome, and that observable changes in gene regulation are generally not resulting from regulatory consequences of local methylation state. IMPORTANCEPrevious studies of E. coli chromatin structure revealed a statistical association between the presence of silenced, highly protein occupied regions of the genome and depletion of modification sites for Dam methyltransferase. Here, we show that loss of DNA methylation does not substantively affect global chromatin structure in E. coli, thus demonstrating that the previously observed correlation was not causal. However, we observed specific methylation-dependent changes in gene expression, particularly affecting the SOS response, flagellar synthesis, and translation. These effects appear to be indirect regulatory consequences of methyltransferase deletion. Our work clarifies the role of methylation in chromatin structure and regulation, providing new insights into the mechanistic basis of gene expression and chromatin structure in E. coli.