Epigenetic and transcriptional control of the Caulobacter cell cycle [electronic resource]

In order for a cell to propagate, numerous events must be coordinated spatially and temporally within the cell. Caulobacter crescentus, an [alpha]-proteobacterium that divides asymmetrically, employs many layers of control such as phospho-relay systems, protein localization, targeted proteolysis, epigenetic regulation, and transcriptional regulation to achieve this spatial and temporal coordination over its cell cycle. This work focuses on two of these layers: the epigenetic role of the CcrM DNA methyltransferase and the regulatory roles of the GcrA and SciP transcription factors. The timing of transcriptional activation of at least four essential Caulobacter genes is affected by the methylation state of GANTC sites in their promoters. To explore the global scope of this regulatory mechanism, we determined the methylation state of the entire genome over the cell cycle (Chapter 2). This led to the identification of four novel methylation motifs in addition to the previously characterized CcrM methyltransferase motif. Only the GANTC motif which is methylated by CcrM exhibited a dynamic methylation pattern, shifting from fully methylated to hemi-methylated concomitant with replication fork passage. Using these cell cycle-dependent methylation profiles of GANTC sites as well as gene expression data from synchronized cell populations, we identified candidate genes whose transcription might be affected by the methylation state of their promoters. In addition, 27 GANTC sites were found to be unmethylated throughout the cell cycle. In Chapter 3, we used DNA footprinting to show that the GcrA transcription factor contains an N-terminal helix-turn-helix domain that binds DNA between the -10 and -35 promoter elements of three genes: podJ, parE, and ctrA. Each of the three footprinted regions contains a GANTC methylation site. Furthermore, we demonstrated through ChIP-qPCR that GcrA binds these sites in vivo. Mutations in these GcrA-binding regions reduced promoter activity, indicating that GcrA binding at these sites is important for activity. Finally, in Chapter 4, we identified a novel set of signaling pathways in the cell cycle network established by the SciP transcription factor, and we show that SciP binds DNA and identified its binding motif. At least 58 genes are in the SciP regulon, including many flagellar and chemotaxis genes. The SciP transcription factor functions to repress ctrA and CtrA target genes, restricting the expression of these genes to a precise time in the cell cycle when CtrA is available but SciP is not, and thus enhances the robustness of the Caulobacter cell cycle. This work expands our understanding of the master regulatory network that is involved in controlling the Caulobacter cell cycle. In addition, it describes the global methylation state of each base in the chromosome as a function of the cell cycle which will facilitate the discovery of novel methylation-mediated regulatory mechanisms in Caulobacter.

Keywords: Chemistry.
Advisor: McAdams Harley primary advisor. Thesis advisor
Shapiro Lucy advisor. Thesis advisor
Zare Richard N. advisor. Thesis advisor