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.
Highlights
Epigenetic signals, such as methylation of DNA, play an important role in the regulation of gene expression in eukaryotes
Methylation-dependent DNA binding of GcrA orthologs To explore if GcrA-controlled functions are conserved across the Alphaproteobacteria, we introduced the GcrA ortholog [20] from Brucella melitensis biovar abortus 2308 (BAB1_0329) and Sinorhizobium meliloti Rm1021 (SMc02139) under the control of an xyloseinducible promoter on a low-copy plasmid [30] in C. crescentus, harbouring a temperature sensitive allele of gcrA with a ThrRPro mutation at position 10 and evaluated their ability to support growth at the restrictive temperature [16]
With the identification and genetic/biochemical characterizations of the m6A-marked promoters and the transcriptional effector(s) hemi-methylated state at a specific time in the cell-cycle that is dictated by the relative distance of the promoter from the origin of replication
Summary
Epigenetic signals, such as methylation of DNA, play an important role in the regulation of gene expression in eukaryotes. The cell cycle role of CcrM was originally described in C. crescentus [5,7]. Upon nutrient availability the swarmer cell differentiates in a stalked cell, resembling the eukaryotic G1RS transition. In this cyclical progression, a crucial role is played by CtrA, an essential transcriptional regulator that targets many cell cycle genes [10]. In G1, CtrA,P inhibits DNA replication by repression of the origin of replication [11] and only upon CtrA proteolysis or dephosphorylation, DnaA-mediated chromosome replication initiation occurs [12] committing cells to the S phase. While in Caulobacter GcrA accumulates in early S phase and is confined to
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