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

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.

[1]  David Botstein,et al.  Combinatorial control of diverse metabolic and physiological functions by transcriptional regulators of the yeast sulfur assimilation pathway , 2012, Molecular biology of the cell.

[2]  David Botstein,et al.  Perturbation-based analysis and modeling of combinatorial regulation in the yeast sulfur assimilation pathway , 2012, Molecular biology of the cell.

[3]  Ahmad S. Khalil,et al.  A Synthetic Biology Framework for Programming Eukaryotic Transcription Functions , 2012, Cell.

[4]  Pamela A. Silver,et al.  Engineering synthetic TAL effectors with orthogonal target sites , 2012, Nucleic acids research.

[5]  Edith D. Wong,et al.  Saccharomyces Genome Database: the genomics resource of budding yeast , 2011, Nucleic Acids Res..

[6]  M. Noyes.  Analysis of specific protein-DNA interactions by bacterial one-hybrid assay. , 2012, Methods in molecular biology.

[7]  Megan N. McClean,et al.  Fast-acting and Nearly Gratuitous Induction of Gene Expression and Protein Depletion in Saccharomyces Cerevisiae Graduate Program in Quantitative and Computational Biology, And , 2022 .

[8]  D. Botstein,et al.  Coordinated regulation of sulfur and phospholipid metabolism reflects the importance of methylation in the growth of yeast , 2011, Molecular biology of the cell.

[9]  Jared E. Toettcher,et al.  Light-based feedback for controlling intracellular signaling dynamics , 2011, Nature Methods.

[10]  D. Botstein,et al.  Survival of starving yeast is correlated with oxidative stress response and nonrespiratory mitochondrial function , 2011, Proceedings of the National Academy of Sciences.

[11]  Ross D. Alexander,et al.  RiboSys, a high-resolution, quantitative approach to measure the in vivo kinetics of pre-mRNA splicing and 3'-end processing in Saccharomyces cerevisiae. , 2010, RNA: A publication of the RNA Society.

[12]  U. Bonas,et al.  Xanthomonas AvrBs3 family-type III effectors: discovery and function. , 2010, Annual review of phytopathology.

[13]  Aaron Klug,et al.  The discovery of zinc fingers and their development for practical applications in gene regulation and genome manipulation , 2010, Quarterly Reviews of Biophysics.

[14]  Jens Boch,et al.  Breaking the Code of DNA Binding Specificity of TAL-Type III Effectors , 2009, Science.

[15]  Matthew J. Moscou,et al.  A Simple Cipher Governs DNA Recognition by TAL Effectors , 2009, Science.

[16]  Tatsuo Fukagawa,et al.  An auxin-based degron system for the rapid depletion of proteins in nonplant cells , 2009, Nature Methods.

[17]  Ronnie J Winfrey,et al.  Rapid "open-source" engineering of customized zinc-finger nucleases for highly efficient gene modification. , 2008, Molecular cell.

[18]  Megan N. McClean,et al.  Signal processing by the HOG MAP kinase pathway , 2008, Proceedings of the National Academy of Sciences.

[19]  Toni Cathomen,et al.  Unexpected failure rates for modular assembly of engineered zinc fingers , 2008, Nature Methods.

[20]  M. Noyes,et al.  A systematic characterization of factors that regulate Drosophila segmentation via a bacterial one-hybrid system , 2008, Nucleic acids research.

[21]  Matthew J. Brauer,et al.  Coordination of growth rate, cell cycle, stress response, and metabolic activity in yeast. , 2008, Molecular biology of the cell.

[22]  Luigi Naldini,et al.  Gene editing in human stem cells using zinc finger nucleases and integrase-defective lentiviral vector delivery , 2007, Nature Biotechnology.

[23]  N. Slonim,et al.  A universal framework for regulatory element discovery across all genomes and data types. , 2007, Molecular cell.

[24]  L. Pease,et al.  Gene splicing and mutagenesis by PCR-driven overlap extension , 2007, Nature Protocols.

[25]  W. Liang,et al.  TM4 microarray software suite. , 2006, Methods in enzymology.

[26]  W. Liang,et al.  9) TM4 Microarray Software Suite , 2006 .

[27]  Ting Wang,et al.  An improved map of conserved regulatory sites for Saccharomyces cerevisiae , 2006, BMC Bioinformatics.

[28]  S Miyano,et al.  Open source clustering software. , 2004, Bioinformatics.

[29]  Mark Isalan,et al.  Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[30]  C. Pabo,et al.  Rearrangement of side-chains in a Zif268 mutant highlights the complexities of zinc finger-DNA recognition. , 2001, Journal of molecular biology.

[31]  Ka Yee Yeung,et al.  Validating clustering for gene expression data , 2001, Bioinform..

[32]  O. Nyanguile,et al.  A versatile framework for the design of ligand-dependent, transgene-specific transcription factors. , 2001, Molecular therapy : the journal of the American Society of Gene Therapy.

[33]  U. Schopfer,et al.  Chemically Regulated Zinc Finger Transcription Factors* , 2000, The Journal of Biological Chemistry.

[34]  J. Joung,et al.  A bacterial two-hybrid selection system for studying protein-DNA and protein-protein interactions. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[35]  Jie Rao,et al.  Control of estrogen receptor ligand binding by Hsp90 , 2000, The Journal of Steroid Biochemistry and Molecular Biology.

[36]  C. Pabo,et al.  DNA recognition by Cys2His2 zinc finger proteins. , 2000, Annual review of biophysics and biomolecular structure.

[37]  D J Segal,et al.  Toward controlling gene expression at will: selection and design of zinc finger domains recognizing each of the 5'-GNN-3' DNA target sequences. , 1999, Proceedings of the National Academy of Sciences of the United States of America.

[38]  C. Pabo,et al.  Analysis of zinc fingers optimized via phage display: evaluating the utility of a recognition code. , 1999, Journal of molecular biology.

[39]  D. Thiele,et al.  Copper ion inducible and repressible promoter systems in yeast. , 1999, Methods in enzymology.

[40]  D. Botstein,et al.  Cluster analysis and display of genome-wide expression patterns. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[41]  G. Bellí,et al.  An activator/repressor dual system allows tight tetracycline-regulated gene expression in budding yeast. , 1998, Nucleic acids research.

[42]  R. Müller,et al.  Use of conditional promoters for expression of heterologous proteins in Saccharomyces cerevisiae. , 1997, Methods in enzymology.

[43]  C. Pabo,et al.  Zinc finger phage: affinity selection of fingers with new DNA-binding specificities. , 1994, Science.

[44]  D. Picard,et al.  Fusion of GAL4-VP16 to a steroid-binding domain provides a tool for gratuitous induction of galactose-responsive genes in yeast. , 1993, Gene.

[45]  C. Scorer,et al.  Foreign gene expression in yeast: a review , 1992, Yeast.

[46]  Robert C. Wolpert,et al.  A Review of the , 1985 .

[47]  G. Fink,et al.  Positive regulation in the general amino acid control of Saccharomyces cerevisiae. , 1983, Proceedings of the National Academy of Sciences of the United States of America.