Understanding and Engineering Dynamics and Variability in Gene Regulation and Beyond

I will discuss three examples describing the utility of understanding and/or exploiting both epigenetic and genetic variability in populations of yeast cells. First, I describe an unappreciated and dominant role for cell-cycle phase on transcriptional variability and dynamics. We show that for a model “noisy” gene in S. cerevisiae, the common view that large variability observed in mRNA numbers is due to a transcriptional bursting, where a promoter undergoes random and intermittent periods of active transcription, is incomplete and possibly incorrect. Rather, variable mRNA distributions are largely driven by differences in transcriptional activity between G1 and S/G2/M phases of the cell cycle. The cell-cycle phase is also paramount when probing variability in the kinetics of gene activation, with early S/G2 appearing to be far more permissive for activation. This global cell-cycle dependence may be essential to consider when using stochastic models to predict the behavior of both natural and synthetically engineered gene networks, especially in conditions when growth rate changes.

Second, I describe how variable numbers of tandemly repeated “decoy” transcription factor (TF) binding sites that bind a cognate transcriptional activator can reduce expression at the activator’s target genes, qualitatively converting the dose-response from a linear to steeper sigmoidal-like threshold response. The results imply that 1) transcription factor (TF) / promoter binding may be weaker than expected in the context of gene expression and 2) there may be a previously unappreciated negative cooperativity in TF binding to clustered sites. Therefore, even small quantitative changes in the highly variable length of repetitive DNA containing TF binding sites found in eukaryotic genomes can have qualitative effects on gene expression, and perhaps ultimately phenotype.

Third, I describe a technique we have developed for the repeated and targeted in vivo mutagenesis of multiple genes in S. cerevisiae. We show selective damage of DNA and subsequent repair by error-prone homologous recombination pathways can lead to selective >800-fold increase in mutation rate in a user-defined 20 kb target region. We discuss applications of this method for the directed evolution of multigenic phenotypes. Deployment is simple and our constructs and protocols are available to interested researchers.