TitleSynthetic gene expression perturbation systems with rapid, tunable, single-gene specificity in yeast.
Publication TypeJournal Article
Year of Publication2013
AuthorsR McIsaac, S, Oakes, BL, Wang, X, Dummit, KA, Botstein, D, Noyes, MB
JournalNucleic Acids Res
Volume41
Issue4
Paginatione57
Date Published2013 Feb 1
KeywordsAnimals, Basic-Leucine Zipper Transcription Factors, Binding Sites, Cell Proliferation, Early Growth Response Protein 1, Estradiol, Gene Expression Regulation, Gene Regulatory Networks, Genetic Engineering, Genome, Fungal, Herpes Simplex Virus Protein Vmw65, Humans, Mice, Protein Structure, Tertiary, Receptors, Estrogen, Recombinant Fusion Proteins, Saccharomyces cerevisiae, Saccharomyces cerevisiae Proteins, Transcription, Genetic, Zinc Fingers
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 Cys(2)His(2) 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.

Alternate JournalNucleic Acids Res.