title: TetR hybrid transcription factors report cell signaling and are inhibited by doxycycline.
abstract: We developed a bigenic reporter system composed of a hybrid transcription factor that combines the regulatory and activation domains of either Elk-1 or cyclic AMP-responsive element binding protein (CREB) with the DNA binding, dimerization, and regulatory domains from a synthetic variant of the bacterial Tet repressor (TetR). The novel hybrid transcription factor TetR-Elk-1 was regulated by MAPK ERK kinase 1 (MEK-1) overexpression, and TetR-CREB was regulated by protein kinase A (PKA) overexpression or elevation of cyclic AMP levels. These hybrid transcription factors could be useful reporters of cell signaling pathways because, unlike previous GAL4 hybrid reporters, TetR hybrid transcription factors are inhibited by the administration of doxycycline. We validated this system in cell culture transfection experiments utilizing luciferase assays to monitor reporter gene expression and Western blot analysis to monitor transcription factor expression and phosphorylation levels. This system may be useful in creating temporally restricted windows of response to cell signaling and may be of value in the advancement of methods used to study signal transduction.
authors.affiliation: University of Pennsylvania, Philadelphia, PA 19104, USA.
authors.forename: Michael B
authors.initials: MB
authors.lastname: Keeley
authors.forename: Johanna
authors.initials: J
authors.lastname: Busch
authors.forename: Rajesh
authors.initials: R
authors.lastname: Singh
authors.forename: Ted
authors.initials: T
authors.lastname: Abel
mesh_terms: D000900:Anti-Bacterial Agents / Q000494:pharmacology; D001709:Biotechnology / Q000295:instrumentation / Q000379:methods*; D015153:Blotting, Western; D002460:Cell Line; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000378:metabolism; D004247:DNA / Q000737:chemistry; D019281:Dimerization; D004318:Doxycycline / Q000494:pharmacology*; D017930:Genes, Reporter; D006801:Humans; D008156:Luciferases / Q000378:metabolism; D020935:MAP Kinase Signaling System; D010766:Phosphorylation; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics*; D015398:Signal Transduction; D014157:Transcription Factors / Q000378:metabolism; D014162:Transfection
chemical_list: D000900:Anti-Bacterial Agents; D012333:RNA, Messenger; D012097:Repressor Proteins; D014157:Transcription Factors; C034811:tetracycline resistance-encoding transposon repressor protein; D004247:DNA; D000242:Cyclic AMP; D008156:Luciferases; D017868:Cyclic AMP-Dependent Protein Kinases; D004318:Doxycycline