Author: Bo Zhou

Publisher:

Published: 2014

Total Pages:

ISBN-13:

One of the central aspects of the biological program that guide the development of an organism is embedded in the regulated and sequential expression of genes as development progresses. A large part of this regulation is achieved through the temporal activation and repression of transcriptional initiation by the selective binding of regulatory proteins, such as transcription factors, to promoters during specific stages of development. Thus, being able to globally and precisely identify these processes are important steps in gaining a systems-level insight and understanding of the developmental program. The cell cycle of Caulobacter crescentus, an alpha-proteobacteria that undergoes cell differentiation and asymmetric cell division, has been used extensively as a model organism to study bacterial development. A cyclical and integrated genetic circuit involving five master regulatory proteins, including DnaA, GcrA, CtrA, and SciP, and the DNA methyl-transferase CcrM, whose presence and activities oscillate in space and time, orchestrate the many facets of the Caulobacter cell cycle including DNA replication, DNA methylation, organelle biogenesis, and cytokinesis. This genetic circuit is at the core of the Caulobacter developmental program. While microarrays have shown 19% of mRNAs undergo changes in RNA level during the cell cycle and development, it is unclear exactly which regulatory factors of the core circuit drive the changes in transcription at each specific locus, and how these regulatory factors act combinatorially to effect transcriptional outcomes has not been systematically dissected. In order to achieve these goals and to further define the transcriptional regulatory landscape that guides the cell cycle, a thorough and global analysis of Caulobacter transcription as a function of the cell cycle and developmental progression is needed. In this thesis, I devised a novel protocol combining 5' rapid amplification of cDNA ends (5' RACE) and high-throughput sequencing to globally map the precise locations of transcriptional start sites (TSSs) in the Caulobacter genome, measured their transcription levels at multiple times in the cell cycle, and identified their transcription factor binding sites. Using the TSSs identified and a RNA sequencing dataset, I made a functional annotation of operons and other transcriptional units in the genome. A large number of antisense transcripts were identified, and many of them are within essential cell cycle-regulated genes, including two master regulators, a previous unknown feature of the core cell cycle control circuit. Many critical genes and operons have multiple promoters, and these promoters are often independently regulated. Furthermore, approximately 25% of the cell cycle-regulated promoters are co-regulated by two or more master regulatory proteins of the core genetic circuit. These results revealed surprising transcriptional complexity and uncovered multiple new layers of transcriptional control mediating the bacterial cell cycle and development and represent the first in-depth analysis of TSS control in as a function bacterial cell cycle and developmental progression.