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.
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.
Authors: Weidong Xiong; Shyam Goverdhana; Sandra A Sciascia; Marianela Candolfi; Jeffrey M Zirger; Carlos Barcia; James F Curtin; Gwendalyn D King; Gabriela Jaita; Chunyan Liu; Kurt Kroeger; Hasmik Agadjanian; Lali Medina-Kauwe; Donna Palmer; Philip Ng; Pedro R Lowenstein; Maria G Castro Journal: J Virol Date: 2006-01 Impact factor: 5.103
Authors: James F Curtin; Marianela Candolfi; Weidong Xiong; Pedro R Lowenstein; Maria G Castro Journal: Mol Cancer Ther Date: 2008-03 Impact factor: 6.261
Authors: Marianela Candolfi; G Elizabeth Pluhar; Kurt Kroeger; Mariana Puntel; James Curtin; Carlos Barcia; A K M Ghulam Muhammad; Weidong Xiong; Chunyan Liu; Sonali Mondkar; William Kuoy; Terry Kang; Elizabeth A McNeil; Andrew B Freese; John R Ohlfest; Peter Moore; Donna Palmer; Phillip Ng; John D Young; Pedro R Lowenstein; Maria G Castro Journal: Neuro Oncol Date: 2007-05-23 Impact factor: 12.300