Generation and characterization of tTS-H4: a novel transcriptional repressor that is compatible with the reverse tetracycline-controlled TET-ON system.
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.
Bockamp
Ernesto
E
Institute of Toxicology/Mouse Genetics, Johannes Gutenberg University, D-55131 Mainz, Germany. bockamp@mail.uni-mainz.de
Christel
Cerstin
C
Hameyer
Dorothe
D
Khobta
Andriy
A
Maringer
Marko
M
Reis
Marco
M
Heck
Rosario
R
Cabezas-Wallscheid
Nina
N
Epe
Bernd
B
Oesch-Bartlomowicz
Barbara
B
Kaina
Bernd
B
Schmitt
Steffen
S
Eshkind
Leonid
L
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
D006657:Histones; D012097:Repressor Proteins; C400300:HDAC4 protein, human; D006655:Histone Deacetylases; D013752:Tetracycline