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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. Copyright © 2007 John Wiley & Sons, Ltd.