Nan Hao (l) and Erin O’Shea
Living organisms respond to external cues and initiate appropriate physiological responses through activation of transcription factors, which regulate expression of target genes. In many cases, such as p53 and NFkappaB, multiple signals are transmitted via a common transcription factor, which displays distinct activation dynamics in response to different signals. However, it remain unclear how transcription factors process upstream signaling inputs to generate diverse dynamic responses.
In a recent paper published in Science, the O’Shea lab attempted to address this problem using a simple model system, Msn2, a general stress responsive transcription factor in the budding yeast S. cerevisiae. In the absence of stress, Msn2 is phosphorylated by Protein Kinase A (PKA) and localized to the cytoplasm; in response to stress, Msn2 is dephosphorylated and translocates to the nucleus where it induces gene expression. Natural stresses elicit variable dynamics of Msn2 nuclear translocation, driven by stochastic oscillations in PKA signaling.
To systematically study how Msn2 processes oscillatory PKA inputs, we developed a method to artificially control the dynamics of PKA signaling. We engineered a yeast strain carrying Msn2-YFP and mutations in all three PKA isoforms that enables selective inhibition of PKA activity by a cell-permeable inhibitor, 1-NM-PP1. We then used this synthetic system and a microfluidics platform mounted on a microscope to produce oscillatory inputs of PKA inhibition and monitored translocation of Msn2 to the nucleus.
With this approach and a simple computational model, we found that the transcription factor Msn2 is a multifunctional signal processor, which can track, filter or integrate PKA signaling inputs in an amplitude-dependent manner. This versatile signal processing capability is crucial for a single transcription factor to generate diverse dynamic responses to different stresses and originates from dual regulation of nuclear import and export by phosphorylation, as Msn2 phosphorylation site mutants with only one form of regulation display only one signal processing behavior. These findings reveal how cells integrate sophisticated signal processing functions into a single transcription factor molecule by assembling independent functional modules and provide a guide for the design of transcription factors with “programmable” signal processing functions.