DOI: 10.5061/dryad.zpc866tdg
title: Data for: Cooperative assembly confers regulatory specificity and long-term genetic circuit stability

description: A ubiquitous feature of eukaryotic transcriptional regulation is
 cooperative self-assembly between transcription factors (TFs) and
 DNA cis-regulatory motifs. It is thought that this strategy
 enables specific regulatory connections to be formed in gene networks
 between otherwise weakly-interacting, low-specificity molecular
 components. Here, using synthetic gene circuits constructed in
 yeast, we find that high regulatory specificity can emerge from
 cooperative, multivalent interactions among artificial zinc finger-based
 TFs. We show that circuits ‘wired’ using the strategy of
 cooperative TF assembly are effectively insulated from aberrant
 misregulation of the host cell genome. As we demonstrate
 in experiments and mathematical models, this mechanism is
 sufficient to rescue circuit-driven fitness defects, resulting in genetic
 and functional stability of circuits in long-termcontinuous culture. Our
 naturally-inspired approach offers a simple, generalizable
 means for building high-fidelity, evolutionarily robust gene
 circuits that can be scaled to a wide range of host organisms and
 applications.
description: These data were generated to support the characterization of a
 toolkit used to construct cooperatively binding synthetic transcription
 factors (synTFs) in yeast to both build stable genetic circuits and better
 understand eukaryotic transcriptional regulation. Our inducible synthetic
 zinc finger-derived transcription factors harbor an activation domain and
 bind to 9 bp cis regulatory elements that we integrate into the yeast
 genome. We use these transcriptional activators to build insulated genetic
 circuits, characterizing their performance, and the fitness of yeast cells
 expressing the activators, in our published work. We demonstrate circuit
 stability in competition experiments as well as long-term evolution
 experiments and describe a circuit with memory that functions via positive
 feedback following a short induction period. Our
 datasets, comprised of data gathered using flow cytometry from which we
 calculate activation, as well as cellular fitness measurements and growth
 rate measurements, support the seven major figures of our
 manuscript: Introduction of a cellular
 fitness cost from nonspecific binding of transcription factors in
 endogenous and synthetic regulatory networks. A
 thermodynamic model indicates that increased TF cooperativity improves
 binding fidelity. Characterization of synTF concentration, affinity, and
 multi-synTF complex size from a synthetic TF cooperativity toolkit in
 yeast. Determining a synTF activation expression tradeoff with cellular
 fitness for nine candidate library synTFs.
 RNA-sequencing analysis of differential gene expression in yeast
 strains containing either cooperatively binding or non-cooperative
 synTFs. Chromatin immunoprecipitation-sequencing
 analysis of differential gene expression in yeast strains containing
 either cooperatively binding or non-cooperative synTFs. Differential gene
 expression at ten endogenous loci indicated as likely synTF binding
 events. Long-term culturing experiments determine the
 cellular fitness cost and stability of synthetic circuits expressing
 cooperatively binding or non-cooperative synTFs.
 Long-term culturing experiments indicate stable activation
 memory via a positive feedback loop in cells expressing cooperatively
 binding synTFs following a short inducer pulse.
 synTF-cooperativity mediated long-term circuit stability is a
 promising strategy for industrial applications.  
 The arranged datasets for each figure are included in .xlsx
 files, named “Fig#_arranged,” and the processed data are presented in
 .pzfx files, named “Fig#_processed.” They are accompanied by the relevant
 raw dataset files. Please refer to the README file for
 a more detailed description of the data and file structure.
description: The .fcs raw datasets require FlowJo to open, and the .pzfx
 processed data files require GraphPad Prism.