Transcription factor networks, synthetic gene expression switches, and chaotic cell-cycle oscillations

Abstract

A conserved aspect of cellular life is the need to respond and adapt to a dynamic environment. Complex interwoven regulatory networks process extracellular cues to coordinate appropriate adaptive responses. At the promoter level, these responses are driven by the presence or absence of transcription factor proteins, which facilitate or block expression of target genes. In the fungus (and model eukaryotic system) Saccharomyces cerevisiae, a relatively small number of transcription factors (~200) are involved in the regulation of ~5700 genes.

In this thesis, new synthetic tools that allow an experimenter to rapidly perturb the levels of single regulators dynamically are described. These tools are functional under physiologically diverse conditions and (other then the factor being perturbed) are nearly or completely inert with respect to the cellular physiology. By coupling these synthetic perturbation systems with the microarray technology platform, one can globally assess the strength and dynamics with which a perturbed factor affects (at the transcriptional level) every gene in the genome.

As a proof of principle of this approach, the role of five different transcription factors in the combinatorial regulation of the yeast sulfur metabolic network were dissected. These data were coupled with bioinformatic analyses and biophysical mod- eling to describe the dynamic response of downstream effectors. Given the success of this approach in elucidating the regulatory architecture within the sulfur metabolic network, a panel of 70 strains containing different inducible alleles was constructed. Perturbation data for 22 different transcription factors (some under multiple environ- ment conditions) and a total of 213 microarrays have been collected to date.

Finally, numerical simulations were employed to investigate a different type of cellular regulatory network: the embryonic cell-cycle control circuit. Following fer- tilization, embryos from many species undergo synchronous, clock-like cell divisions. Taking the equations previously developed to describe the cell cycle of the African tree frog Xenopus laevis and adding a spatial component, we discovered that these equations are susceptible to chaotic behavior, and establish a novel design principle whereby the widely conserved post-fertilization calcium wave initiates and ensures synchrony of cellular divisions.