p53 pulse modulation differentially regulates target gene promoters to regulate cell fate decisions.

The p53 tumor suppressor regulates distinct responses to cellular stresses. Although different stresses generate different p53 dynamics, the mechanisms by which cells decode p53 dynamics to differentially regulate target genes are not well understood. Here, we determined in individual cells how canonical p53 target gene promoters vary in responsiveness to features of p53 dynamics. Employing a chemical perturbation approach, we independently modulated p53 pulse amplitude, duration, or frequency, and we then monitored p53 levels and target promoter activation in individual cells. We identified distinct signal processing features-thresholding in response to amplitude modulation, a refractory period in response to duration modulation, and dynamic filtering in response to frequency modulation. We then showed that the signal processing features not only affect p53 target promoter activation, they also affect p53 regulation and downstream cellular functions. Our study shows how different promoters can differentially decode features of p53 dynamics to generate distinct responses, providing insight into how perturbing p53 dynamics can be used to generate distinct cell fates.

Determining the Limitations and Benefits of Noise in Gene Regulation and Signal Transduction through Single Cell, Microscopy-Based Analysis.

Stochastic fluctuations, termed "noise," in the level of biological molecules can greatly impact cellular functions. While biological noise can sometimes be detrimental, recent studies have provided an increasing number of examples in which biological noise can be functionally beneficial. Rather than provide an exhaustive review of the growing literature in this field, in this review, we focus on single-cell studies based on quantitative microscopy that have generated a deeper understanding of the sources, characteristics, limitations, and benefits of biological noise. Specifically, we highlight studies showing how noise can help coordinate the expression of multiple downstream target genes, impact the channel capacity of signaling networks, and interact synergistically with oscillatory dynamics to enhance the sensitivity of signal processing. We conclude with a discussion of current challenges and future opportunities.

Transcriptional regulation improves the throughput of three-hybrid counter selections in Saccharomyces cerevisiae.

The yeast three-hybrid (Y3H) assay expands the fields of drug discovery and protein engineering by enabling the search of large variant libraries for targets that do not inherently produce a distinct, measurable phenotype. The Y3H assay links the DNA-binding and activation domains of a transcription factor via a chemically synthesized heterodimeric small molecule, thereby activating a downstream reporter gene. Although the Y3H assay has been successfully applied as a positive selection to discover novel drug targets and to evolve proteins with improved functions, its expansion into applications requiring a high-throughput, versatile selection against transcriptional activation has been hindered by its limited dynamic range as a counter selection. Here, we describe the development of a second-generation Y3H counter selection that uses the dual tetracycline (Tet) system to tighten transcriptional regulation of the reporter gene. The Tet Y3H counter selection has an improved dynamic range and provides enrichment from mock libraries of up to 106, a 104-fold improvement over our original Y3H counter selection. This enhanced dynamic range brings the Y3H counter selection to a standard that is suitable for real-world protein engineering applications.