title: Light-controlled gene switches in mammalian cells.
abstract: Remote control of cells is a desirable feature in synthetic biology. We established a light-switchable interfering peptide (iPEP) which controls gene expression by modulating the activity of a transcription factor. For photo-switching, the iPEP is cross-linked with a cis-trans isomerizable cross-linker in such a way that the light-activated cis form enables inhibitor folding rendering it active, whereas the dark-adapted trans form forces the inhibitor into an inactive form. Switching can be repeated in both directions. The iPEP acts as dominant-negative inhibitor targeting c-Jun and c-Fos of the transcription factor activator protein-1 (AP-1). Light-activated peptides exhibited much stronger inhibition of AP-1:DNA complexes and interference with gene transcription than their nonactivated counter parts. In this chapter, we provide protocols for cross-linking, peptide purification, observation of structural changes upon photo-switching, DNA binding analyses as well as gene expression studies in mammalian cells.
authors.affiliation: BIOSS Centre for Biological Signalling Studies, University of Freiburg, Freiburg, Germany.
authors.forename: Fuzhong
authors.initials: F
authors.lastname: Zhang
authors.forename: Kristian M
authors.initials: KM
authors.lastname: Müller
authors.forename: G Andrew
authors.initials: GA
authors.lastname: Woolley
authors.forename: Katja M
authors.initials: KM
authors.lastname: Arndt
mesh_terms: D050976:Basic-Leucine Zipper Transcription Factors / Q000737:chemistry / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression / Q000528:radiation effects; D017930:Genes, Reporter / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D007536:Isomerism; D008027:Light; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D009154:Mutation; D009690:Nucleic Acid Conformation; D010446:Peptide Fragments / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D055668:Photochemical Processes; D017433:Protein Structure, Secondary; D058615:Synthetic Biology / Q000379:methods*; D018808:Transcription Factor AP-1 / Q000378:metabolism; D014158:Transcription, Genetic / Q000528:radiation effects; D014162:Transfection; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
chemical_list: D050976:Basic-Leucine Zipper Transcription Factors; D010446:Peptide Fragments; D018808:Transcription Factor AP-1; D004247:DNA; D008156:Luciferases; D001616:beta-Galactosidase