Doxycycline- and tetracycline-regulated transcriptional silencer enhance the expression level and transactivating performance of rtTA.
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.
Lai
Jen-Feng
JF
Department of Internal Medicine, National Taiwan University Hospital and National Taiwan University College of Medicine, No. 7 Chung-Shan S. Road, Taipei, 100 Taiwan.
Cheng
Hsin-Yuan
HY
Cheng
Tzu-Ling
TL
Lin
Yu-Yu
YY
Chen
Li-Chieh
LC
Lin
Mau-Ting
MT
Jou
Tzuu-Shuh
TS
D000818:Animals; D000900:Anti-Bacterial Agents / Q000494:pharmacology*; D015153:Blotting, Western; D004285:Dogs; D004318:Doxycycline / Q000494:pharmacology*; D020869:Gene Expression Profiling; D005786:Gene Expression Regulation / Q000187:drug effects*; D020868:Gene Silencing; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors; D015151:Immunoblotting; D007668:Kidney / Q000166:cytology; D008156:Luciferases / Q000032:analysis / Q000096:biosynthesis; D010957:Plasmids; D011401:Promoter Regions, Genetic; D046988:Proteasome Endopeptidase Complex; D037941:Silencer Elements, Transcriptional; D013752:Tetracycline / Q000494:pharmacology*; D015534:Trans-Activators; D025801:Ubiquitin / Q000494:pharmacology
D000900:Anti-Bacterial Agents; D015534:Trans-Activators; D025801:Ubiquitin; D008156:Luciferases; D046988:Proteasome Endopeptidase Complex; D013752:Tetracycline; D004318:Doxycycline