DNA Binding of the Cell Cycle Transcriptional Regulator GcrA Depends on N6-Adenosine Methylation in Caulobacter crescentus and Other Alphaproteobacteria

Abstract

Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle.

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“…The cell cycle-regulated methyltransferase CcrM facilitates the binding of the global transcriptional regulator GcrA to its preferred target promoters (Fioravanti et al 2013) via N6 adenine methylation (m6A) of nearly all of the 4000 5 ′ -GANTC-3 ′ sites in the C. crescentus genome. Following replication, these sites are hemimethylated, and CcrM remethylates them once it accumulates in late S phase ( Fig.…”

“…GcrA, synthesized in early S phase, preferentially binds and activates target promoters carrying such m6A marks. Mutation of the methylation motif in these promoters or inactivation of CcrM impairs binding of GcrA to these promoters (Fioravanti et al 2013). Several important cell cycle proteins are directly regulated by GcrA/CcrM, such as the master cell cycle regulator CtrA, the cytokinetic tubulin FtsZ, the cell division positioning factor MipZ, the FtsN division protein, and the PodJ polarity determinant (Laub et al 2000;Viollier et al 2002;Thanbichler and Shapiro 2006;Fioravanti et al 2013;Gonzalez and Collier 2013).…”

“…Several important cell cycle proteins are directly regulated by GcrA/CcrM, such as the master cell cycle regulator CtrA, the cytokinetic tubulin FtsZ, the cell division positioning factor MipZ, the FtsN division protein, and the PodJ polarity determinant (Laub et al 2000;Viollier et al 2002;Thanbichler and Shapiro 2006;Fioravanti et al 2013;Gonzalez and Collier 2013). Reasoning that other cell cycle factors operating specifically during the S phase are also under GcrA/CcrM control, we analyzed several uncharacterized GcrA/CcrM targets identified in ChIP-seq (chromatin immunoprecipitation [ChIP] combined with "deep" sequencing) experiments using antibodies to GcrA and m6A (Fioravanti et al 2013). Bioinformatic analysis using a low-stringency cutoff identified 97 putative promoters as targets of GcrA/CcrM (Fig.…”

“…1B). Of these, 68 targets had upstream "GANTC" sites (Supplemental Table S1; Fioravanti et al 2013).…”

“…Also shown are the replication and sequestration of the newly replicated origin (yellow) and the presence of ParC (magenta) and ParE (blue) during the cell cycle in C. crescentus. (B) Network image showing putative promoters (gray dots) regulated by CcrM, detected using m6A (brown) and GcrA (green), as inferred from ChIP-seq (chromatin immunoprecipitation [ChIP] combined with "deep" sequencing) results of wild-type C. crescentus (Fioravanti et al 2013). The common targets between m6A and GcrA are shown in orange.…”