Synthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast

Abstract

A general method for the dynamic control of single gene expression in eukaryotes, with no off-target effects, is a long-sought tool for molecular and systems biologists. We engineered two artificial transcription factors (ATFs) that contain Cys2His2 zinc-finger DNA-binding domains of either the mouse transcription factor Zif268 (9 bp of specificity) or a rationally designed array of four zinc fingers (12 bp of specificity). These domains were expressed as fusions to the human estrogen receptor and VP16 activation domain. The ATFs can rapidly induce a single gene driven by a synthetic promoter in response to introduction of an otherwise inert hormone with no detectable off-target effects. In the absence of inducer, the synthetic promoter is inactive and the regulated gene product is not detected. Following addition of inducer, transcripts are induced >50-fold within 15 min. We present a quantitative characterization of these ATFs and provide constructs for making their implementation straightforward. These new tools allow for the elucidation of regulatory network elements dynamically, which we demonstrate with a major metabolic regulator, Gcn4p.

Identifying genetic modulators of the connectivity between transcription factors and their transcriptional targets

“…To test susceptibilities for selected TFs, we used a time course of genome-wide mRNA levels after synthetic induction of GCN4 using a hormone-controlled artificial transcription factor (34). For Ste12p, we used the pheromone response pathway induction expression data (35).…”

“…To further assess the quality of the susceptibility signature derived for Gcn4p, we took genome-wide data on mRNA abundance obtained over a time course after synthetic induction of the TF (34). For Ste12p, we used mRNA expression data generated from cells treated with α-pheromone (35).…”

Tuning gene expression variability and multi-gene regulation by dynamic transcription factor control

“…18 . The promoter was constructed by exchanging the UAS-GAL region (containing Gal4p binding sites) of the GAL1 promoter with the 5xBS sequence from pcDNA-C120-mCherry 6 .…”

Towards combinatorial transcriptional engineering

“…The knowledge of such polymorphisms may be used to rationally design tunable effectors with desired target specificities. The engineering of three and four part ZNFs that show limited off-target activity in the host is another illustration of the power and need for rational engineering approaches (Khalil et al, 2012;McIsaac et al, 2013). Computational tools may aid in increasing the specificity of sTF technologies, as recently illustrated by Didovyk et al, (2016).…”

“…Synthetic ZNF TFs enabled the detection of multiple genes that were coexpressed or repressed upon induction of the native GCN4 networks in S. cerevisiae (McIsaac et al, 2013). The study revealed 327 genes that were up-regulated by >2 fold, and 255 genes that were repressed >2 fold upon GCN4 expression.…”