Improved single-chain transactivators of the Tet-On gene expression system

Abstract

Background: The Tet-Off (tTA) and Tet-On (rtTA) regulatory systems are widely applied to control gene expression in eukaryotes. Both systems are based on the Tet repressor (TetR) from transposon Tn10, a dimeric DNA-binding protein that binds to specific operator sequences (tetO). To allow the independent regulation of multiple genes, novel Tet systems are being developed that respond to different effectors and bind to different tetO sites. To prevent heterodimerization when multiple Tet systems are expressed in the same cell, single-chain variants of the transactivators have been constructed. Unfortunately, the activity of the single-chain rtTA (sc-rtTA) is reduced when compared with the regular rtTA, which might limit its application.

Results: We recently identified amino acid substitutions in rtTA that greatly improved the transcriptional activity and doxycycline-sensitivity of the protein. To test whether we can similarly improve other TetR-based gene regulation systems, we introduced these mutations into tTA and sc-rtTA. Whereas none of the tested mutations improved tTA activity, they did significantly enhance sc-rtTA activity. We thus generated a novel sc-rtTA variant that is almost as active and dox-sensitive as the regular dimeric rtTA. This variant was also less sensitive to interference by co-expressed TetR-based tTS repressor protein and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used.

Conclusion: We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.

Figure 1

TetR-based transactivators. (A) tTA contains the N-terminal transposon Tn10-derived Tet repressor and the C-terminal herpes simplex virus VP16-derived activation domain (AD). The TetR part can be subdivided in a DNA-binding domain (BD) and a regulatory core domain (core). rtTA (rtTA2^S-S2 in ref. [6]) is a tTA variant with four amino acid substitutions in the TetR domain (shown above the protein). The mutations that enhance rtTA activity are also located in the TetR domain (shown under the protein). sc-rtTA is a single-chain version of rtTA that contains two TetR moieties connected head to tail by a peptide linker and a single activation domain at the C-terminal end. (B) Both tTA and rtTA are active as homodimers, whereas sc-rtTA folds intramolecularly.

Figure 2

Mutations that enhance rtTA activity do not improve tTA activity. The transcriptional activity of tTA variants was measured in HeLa X1/6 cells [28] containing chromosomally integrated copies of the CMV-7tetO luciferase reporter construct. Cells were transfected with the tTA expression plasmids indicated or pBluescript (-) as a negative control and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured in the presence of different dox concentrations (0–20 ng/ml). The ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the tTA activity. All values were related to the original (wild-type) tTA activity in the absence of dox, which was arbitrarily set at 100%. Average values of two transfections are shown with the error bars indicating the standard deviations.

Figure 3

Mutations observed in rtTA can improve sc-rtTA activity. The transcriptional activity of rtTA and sc-rtTA was measured in HeLa X1/6 cells, see Fig. 2 for details. Cells were cultured in the presence of different dox concentrations (0–1000 ng/ml). All values were related to the original (wild-type) sc-rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%. Average values of two transfections are plotted with the error bars indicating the standard deviations.

Figure 4

Transcriptional activity and dox-sensitivity of the sc-rtTA variants. Calculation is based on the data of Fig. 3. Transcriptional activity observed at 1000 ng/ml dox is shown as average value of two transfections with error bars indicating the standard deviations (wild-type sc-rtTA activity set at 100%). Dox-sensitivity is compared with the wild-type sc-rtTA (set at 1). For each variant, the dox concentration (ng/ml) that results in an activity comparable to that of the wild-type sc-rtTA activity at 1000 ng/ml dox is indicated between brackets.

Figure 5

Combinatorial regulation of gene expression by sc-rtTA and tTS. Transcription from the CMV-7tetO luciferase reporter construct was measured upon transfection of C33A cells with the indicated rtTA, sc-rtTA (wild-type or with mutations in both TetR domains) and tTS expression plasmids or pBluescript (-) as a negative control, and a plasmid constitutively expressing Renilla luciferase to correct for differences in transfection efficiency. Cells were cultured with (1000 ng/ml) or without dox. The ratio of the firefly and Renilla luciferase activities measured two days after transfection reflects the rtTA activity. All values were related to the original (wild-type) sc-rtTA activity at 1000 ng/ml dox, which was arbitrarily set at 100%. Average values of three transfections are plotted with the error bars indicating the standard deviations. The ratio between the induced (+ dox) and uninduced (no dox) promoter activity (fold induction) is indicated on top of the bars.