title: Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system.
abstract: BACKGROUND
  Conditional gene regulatory systems ensuring tight and adjustable expression of therapeutic genes are central for developing future gene therapy strategies. Among various regulatory systems, tetracycline-controlled gene expression has emerged as a safe and reliable option. Moreover, the tightness of tetracycline-regulated gene switches can be substantially improved by complementing transcriptional activators with antagonizing repressors.
  
  
  METHODS
  To develop novel tetracycline-responsive transcriptional repressors, we fused various transcriptional silencing domains to the TetR (B/E) DNA-binding and dimerization domain of the Tn10-encoded tetracycline resistance operon (TetR (B/E)). The resulting fusion proteins were individually tested for their ability to repress transcription of the constitutively active hypoxanthine phosphoribosyltransferase (HPRT) promoter. In addition, compatibility with the commonly used reverse tetracycline-controlled transactivator system (rtTA-system) and responsiveness to the pharmacological effector doxycycline (DOX) were evaluated. Finally, inducibility, effector-dependent promoter activity and the modification of histone H3 and H4 of the active versus the repressed target promoter were determined.
  
  
  RESULTS
  Fusion of the human deacetylase 4 (HDAC4) carboxy-terminal silencing domain to TetR (B/E) resulted in a functional transcriptional repressor. This novel repressor, termed tTS-H4, efficiently reduced the activity of the murine HPRT promoter and a constitutively active human cytomegalovirus (hCMV) minimal promoter. Furthermore, combining tTS-H4 with the rtTA transcriptional activator allowed for grading, turning off and resuming target gene expression over several orders of magnitude without background.
  
  
  CONCLUSIONS
  The tTS-H4 repressor is compatible with the commonly used rtTA transcriptional activation system and is a versatile new tool for tightly and adjustably regulating conditional gene expression.
authors.affiliation: Institute of Toxicology/Mouse Genetics, Johannes Gutenberg University, D-55131 Mainz, Germany. bockamp@mail.uni-mainz.de
authors.forename: Ernesto
authors.initials: E
authors.lastname: Bockamp
authors.forename: Cerstin
authors.initials: C
authors.lastname: Christel
authors.forename: Dorothe
authors.initials: D
authors.lastname: Hameyer
authors.forename: Andriy
authors.initials: A
authors.lastname: Khobta
authors.forename: Marko
authors.initials: M
authors.lastname: Maringer
authors.forename: Marco
authors.initials: M
authors.lastname: Reis
authors.forename: Rosario
authors.initials: R
authors.lastname: Heck
authors.forename: Nina
authors.initials: N
authors.lastname: Cabezas-Wallscheid
authors.forename: Bernd
authors.initials: B
authors.lastname: Epe
authors.forename: Barbara
authors.initials: B
authors.lastname: Oesch-Bartlomowicz
authors.forename: Bernd
authors.initials: B
authors.lastname: Kaina
authors.forename: Steffen
authors.initials: S
authors.lastname: Schmitt
authors.forename: Leonid
authors.initials: L
authors.lastname: Eshkind
mesh_terms: D000818:Animals; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D015316:Genetic Therapy / Q000379:methods*; D006655:Histone Deacetylases / Q000235:genetics / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D051379:Mice; D041681:NIH 3T3 Cells; D011401:Promoter Regions, Genetic; D012097:Repressor Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D013752:Tetracycline / Q000737:chemistry / Q000378:metabolism*; D014158:Transcription, Genetic
chemical_list: D006657:Histones; D012097:Repressor Proteins; C400300:HDAC4 protein, human; D006655:Histone Deacetylases; D013752:Tetracycline