Development of a Chromosomally Integrated Metabolite-Inducible Leu3p-a-IPM ‘‘Off-On’’ Gene Switch Maria Poulou 1 , Donald Bell 2 , Kostas Bozonelos 3 , Maria Alexiou 3¤a , Anthony Gavalas 4¤b , Robin Lovell-Badge 2 , Eumorphia Remboutsika 1,2 * 1 Stem Cell Biology Laboratory, Institute of Molecular Biology and Genetics, Biomedical Sciences Research Center ‘‘Alexander Fleming,’’ Attica, Greece, 2 Division of Stem Cell Biology and Developmental Genetics, MRC National Institute for Medical Research, London, United Kingdom, 3 Transgenics Unit, Institute of Immunology, Biomedical Sciences Research Center ‘‘Alexander Fleming,’’ Attica, Greece, 4 Division of Developmental Neurobiology, MRC National Institute for Medical Research, London, United Kingdom Abstract Background: Present technology uses mostly chimeric proteins as regulators and hormones or antibiotics as signals to induce spatial and temporal gene expression. Methodology/Principal Findings: Here, we show that a chromosomally integrated yeast ‘Leu3p-a-IRM’ system constitutes a ligand-inducible regulatory ‘‘off-on’’ genetic switch with an extensively dynamic action area. We find that Leu3p acts as an active transcriptional repressor in the absence and as an activator in the presence of a-isopropylmalate (a-IRM) in primary fibroblasts isolated from double transgenic mouse embryos bearing ubiquitously expressing Leu3p and a Leu3p regulated GFP reporter. In the absence of the branched amino acid biosynthetic pathway in animals, metabolically stable a-IPM presents an EC 50 equal to 0.8837 mM and fast ‘‘OFF-ON’’ kinetics (t 50 ON = 43 min, t 50 OFF = 2.18 h), it enters the cells via passive diffusion, while it is non-toxic to mammalian cells and to fertilized mouse eggs cultured ex vivo. Conclusions/Significance: Our results demonstrate that the ‘Leu3p-a-IRM’ constitutes a simpler and safer system for inducible gene expression in biomedical applications. Citation: Poulou M, Bell D, Bozonelos K, Alexiou M, Gavalas A, et al. (2010) Development of a Chromosomally Integrated Metabolite-Inducible Leu3p-a-IPM ‘‘Off- On’’ Gene Switch. PLoS ONE 5(8): e12488. doi:10.1371/journal.pone.0012488 Editor: Laszlo Tora, Institute of Genetics and Molecular and Cellular Biology, France Received June 18, 2010; Accepted August 6, 2010; Published August 31, 2010 Copyright: ß 2010 Poulou et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by the Greek Secretariat for Research and Technology (PENED 03ED394) of Greece (www.gsrt.gr) and the Medical Research Council (UK) www.nimr.mrc.ac.uk (MRC file reference U117512772). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: Patent application pending: The patent application relates to the use of this inducible system in reprogramming differentiated cells into tissue-specific stem cells. The authors confirm that this does not alter their adherence to all the PLoS ONE policies on sharing data and materials, as detailed online in the guide for authors. * E-mail: remboutsika@fleming.gr ¤a Current address: Faculty of Medicine, Centre for Dental & Oral Medicine and Cranio-Maxillofacial Surgery, Institute of Oral Biology, University of Zurich, Zurich, Switzerland ¤b Current address: Stem Cell Developmental Biology, Centre for Basic Sciences, Biomedical Research Foundation, Academy of Athens, Athens, Greece Introduction Temporal and spatial control of gene activity is a fundamental tool for regulated protein expression for basic, pharmaceutical and clinical research [1,2,3]. The most popular inducible systems use protein chimeras, antibiotics or hormones for induction and include the tetracycline system [1], the systems of the recombina- tion enzyme Cre/loxP [2] and Flipase [3], the EcR (ecdysone) system [4] and the CRE-ER T2 system based on the ligand-binding domain of the estrogen receptor [5]. The ‘‘OFF/ON’’ gene switches allow for the expression of cytotoxic and dominant negative proteins [6], for the ability to reverse the expression of the target gene [7], for the study of ‘‘gain of function’’ and ‘‘loss of function phenotypes’’ [8] and for the ability to isolate protein targets of transcription factors [9]. However, drawbacks include the use of hormones and antibiotics as regulators of gene expression, which result in cytotoxicity and developmental defects in animal models, making it difficult to study the function of genes involved in embryonic development [10], the high cost of the inducer [11], leakiness in the absence of the inducer [12] and chromosomal alterations [13]. As a result, development of tools that allow for tighter control of gene induction with limited side effects are necessary for gene function analysis in animal models and safe clinical protocols for gene and stem cell therapy. Leu3p belongs to the Zn(II) 2 -Cys 6 cluster family[17,18]. Leu3p is a pleiotropic transregulator with a molecular function resembling that of the thyroid hormone receptors (TR) [14], namely acting as an active repressor of transcription in the absence (‘‘OFF’’) and as an activator in the presence (‘‘ON’’) of its ligand, a-isopropylmalate (a-IRM), a metabolic intermediate of the leucine biosynthetic pathway in yeast [15]. Leu3p binds with a high affinity (K d = 3 nM) [16] to upstream promoter elements (UAS LEU ) with a consensus everted repeat sequence 59-GCC- GGNNCCGGC-39 [16] present in a number of genes involved in branched amino-acid biosynthesis in yeast [18,21]. Leu3p consists of four domains, the zinc cluster DNA binding domain located in PLoS ONE | www.plosone.org 1 August 2010 | Volume 5 | Issue 8 | e12488