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Doxycycline‐ and tetracycline‐regulated transcriptional silencer enhance the expression level and transactivating performance of rtTA
Author(s) -
Lai JenFeng,
Cheng HsinYuan,
Cheng TzuLing,
Lin YuYu,
Chen LiChieh,
Lin MauTing,
Jou TzuuShuh
Publication year - 2004
Publication title -
the journal of gene medicine
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.689
H-Index - 91
eISSN - 1521-2254
pISSN - 1099-498X
DOI - 10.1002/jgm.614
Subject(s) - doxycycline , transactivation , silencer , biology , microbiology and biotechnology , transcription (linguistics) , gene expression , regulation of gene expression , repressor , tetracycline , ubiquitin , gene , genetics , antibiotics , mechanical engineering , linguistics , philosophy , engineering , inlet
Background The tetracycline‐regulated transcriptional silencer (tTS) has been demonstrated to mitigate leaky expression of the tetracycline‐inducible promoter under uninduced condition, and, when conjugated with reverse‐type tetracycline‐controlled transactivator (rtTA), shows great promise for gene therapy. This effect was attributed to the effectiveness of tTS as a repressor of transcription at the tetracycline‐regulated promoter. However, we observed an unexpected increase in transactivational activity by rtTA in the presence of tTS under inducible condition. Methods To explore the nature of this co‐activational effect of tTS on rtTA, we examined the expression patterns of rtTA by Western blotting analysis of total cellular lysates or an enriched ubiquitinated pool of proteins under various conditions, including the one when proteasomal degradation is inhibited. Results We demonstrate tTS, in addition to its established role as a transcriptional silencer, can enhance rtTA expression level by salvaging rtTA from the ubiquitin‐dependent proteasomal degradation pathway. Along with this finding, we also demonstrate that doxycycline, a commonly used tetracycline analogue, inhibits the susceptibility of rtTA to ubiquitin/proteasome‐mediated degradation and enhances the expression level of rtTA. Conclusions Taken together, our data establish an unappreciated role of doxycycline and tTS in tetracycline‐regulated gene expression and the functionality of rtTA, and should shed light on the design of gene therapy vectors based on tetracycline‐controlled transcriptional regulation systems. Copyright © 2004 John Wiley & Sons, Ltd.