| Summary: | Thesis (Ph. D.)--Massachusetts Institute of Technology, Dept. of Chemical Engineering, 2013. === This electronic version was submitted by the student author. The certified thesis is available in the Institute Archives and Special Collections. === Cataloged from student-submitted PDF version of thesis. === Includes bibliographical references. === The relationship between regulatory trans-factors binding a gene's cis-regulatory sequence elements and the transcriptional output of that gene is fundamental to even the most complex network behaviors such as metabolism and differentiation. In eukaryotes, chromatin dynamics on gene promoter sequences is an integral part of regulation, and nucleosome remodeling is often required for transcription activation. Though the transient response of these regulated genes is often important in biological contexts, the role of promoter chromatin architecture in activation kinetics is still unclear. We sought to investigate this relationship as well as possible links to the cell cycle, over which chromatin state experiences dramatic changes. To study the activation kinetics of individual promoters, we develop a method to infer real-time transcription rates from protein expression in single Saccharomyces cerevisiae cells using time-lapse fluorescence microscopy. Comparison between the instantaneous transcription rate and cell-cycle phase in each cell demonstrates the majority of transcriptional variability is due to cell cycle-dependent effects with noisy expression restricted to S/G2/M. This is in stark contrast to current stochastic models of gene expression, most of which do not account for extrinsic effects, and reveals a permissive activation period beginning each S-phase. We then employ a switchable transactivator system to probe transient response kinetics as a function of promoter chromatin architecture at the PHO5 promoter, a well-established model system for chromatin-regulated expression. While we show transactivator binding site affinity and location relative to nucleosomes influences promoter response kinetics, the effect is primarily through architecture-dependent reliance on a dominant, permissive activation period in S/G2. Together with similar observations at synthetic promoters using a chimerical, switchable transactivator, these results suggest the cell cycle has a general role in transcription activation. Based on the timing of the permissive period, DNA replication may play a direct role in transactivation. Thus, in network topologies involving noisy genes and positive feedback, the cell cycle-dependent transcription would lead to distinct predictions between frequently- and non-dividing cells. This work reveals an unappreciated yet dominant role for the cell cycle as a general regulator of transcription in eukaryotes with direct implications in better modeling and design of biological networks. === by Christopher J. Zopf. === Ph.D.
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