title: Synthetic mammalian trigger-controlled bipartite transcription factors.
abstract: Synthetic biology has significantly advanced the design of synthetic control devices, gene circuits and networks that can reprogram mammalian cells in a trigger-inducible manner. Prokaryotic helix-turn-helix motifs have become the standard resource to design synthetic mammalian transcription factors that tune chimeric promoters in a small molecule-responsive manner. We have identified a family of Actinomycetes transcriptional repressor proteins showing a tandem TetR-family signature and have used a synthetic biology-inspired approach to reveal the potential control dynamics of these bi-partite regulators. Daisy-chain assembly of well-characterized prokaryotic repressor proteins such as TetR, ScbR, TtgR or VanR and fusion to either the Herpes simplex transactivation domain VP16 or the Krueppel-associated box domain (KRAB) of the human kox-1 gene resulted in synthetic bi- and even tri-partite mammalian transcription factors that could reversibly program their individual chimeric or hybrid promoters for trigger-adjustable transgene expression using tetracycline (TET), γ-butyrolactones, phloretin and vanillic acid. Detailed characterization of the bi-partite ScbR-TetR-VP16 (ST-TA) transcription factor revealed independent control of TET- and γ-butyrolactone-responsive promoters at high and double-pole double-throw (DPDT) relay switch qualities at low intracellular concentrations. Similar to electromagnetically operated mechanical DPDT relay switches that control two electric circuits by a fully isolated low-power signal, TET programs ST-TA to progressively switch from TetR-specific promoter-driven expression of transgene one to ScbR-specific promoter-driven transcription of transgene two while ST-TA flips back to exclusive transgene 1 expression in the absence of the trigger antibiotic. We suggest that natural repressors and activators with tandem TetR-family signatures may also provide independent as well as DPDT-mediated control of two sets of transgenes in bacteria, and that their synthetic transcription-factor analogs may enable the design of compact therapeutic gene circuits for gene and cell-based therapies.
authors.affiliation: Department of Biosystems Science and Engineering, ETH Zurich, Mattenstrasse 26, CH-4058 Basel, Switzerland.
authors.forename: Marc
authors.initials: M
authors.lastname: Folcher
authors.forename: Mingqi
authors.initials: M
authors.lastname: Xie
authors.forename: Andrea
authors.initials: A
authors.lastname: Spinnler
authors.forename: Martin
authors.initials: M
authors.lastname: Fussenegger
mesh_terms: D039903:Actinobacteria / Q000235:genetics; D020816:Amino Acid Motifs; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D002478:Cells, Cultured; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000235:genetics; D011401:Promoter Regions, Genetic; D015202:Protein Engineering / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000737:chemistry; D012097:Repressor Proteins / Q000737:chemistry* / Q000145:classification / Q000235:genetics / Q000378:metabolism; D058615:Synthetic Biology / Q000379:methods; D015534:Trans-Activators / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019076:Transgenes
chemical_list: D001426:Bacterial Proteins; D004268:DNA-Binding Proteins; D017405:Herpes Simplex Virus Protein Vmw65; D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; D012097:Repressor Proteins; C446304:ScbR protein, Streptomyces coelicolor; D015534:Trans-Activators; C109484:ZNF10 protein, human; C034811:tetracycline resistance-encoding transposon repressor protein