Improved single-chain transactivators of the Tet-On gene expression system
© Zhou et al; licensee BioMed Central Ltd. 2007
Received: 28 August 2006
Accepted: 19 January 2007
Published: 19 January 2007
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.
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.
We developed an improved sc-rtTA variant that may replace regular rtTA in applications where multiple TetR-based regulatory systems are used.
Current research focuses on developing additional TetR-based transregulators that respond to specific Tc-derivatives and recognize distinct operator sequences [12–16]. Such novel Tet systems will allow the independent regulation of multiple (sets of) genes by different effectors [16, 17]. Again, the formation of heterodimers has to be prevented. Transfer of the mutations responsible for the altered phenotypes to other natural sequence variants represents one strategy, but such changes can lead to non-functional transregulators [17, 18]. As an alternative strategy, single-chain Tet transregulators have recently been developed in which two TetR domains are connected by a flexible peptide linker, and a single functional domain, like an activation, repression or oligomerization domain is fused to the C-terminus [18–20] (Fig. 1). These transregulators fold intramolecularly and do not dimerize with each other. Unfortunately, the single-chain version of rtTA (sc-rtTA) exhibits reduced activity when compared with the dimeric rtTA, and this may restrict its application .
We have previously incorporated the rtTA2S-S2 gene encoding a second-generation rtTA variant  and the tetO elements into the HIV-1 genome to control virus replication [21, 22]. During culturing of this dox-dependent virus, spontaneous evolution selected for improved virus variants in which the introduced Tet-On system was found to be optimized [23–27]. We have identified several amino acid substitutions that greatly enhance the transcriptional activity and dox-sensitivity of rtTA [24, 26]. To test whether these mutations can similarly improve other TetR-based transactivators, they were introduced into tTA and sc-rtTA. Whereas none of these mutations improve tTA activity, they all enhance sc-rtTA activity. The most active sc-rtTA variant that we generated is 30-fold more active than the original sc-rtTA, and almost as active as the regular rtTA.
Mutations that enhance rtTA activity do not improve tTA activity
Mutations observed in rtTA can improve sc-rtTA activity
Similar results were obtained upon introduction of the mutations in the C-terminal TetR part (Fig. 3B). However, none of these variants are as active as their counterparts with mutations in the N-terminal TetR moiety. The F86Y mutation increased sc-rtTA activity ~3-fold, and the addition of the F67S, G138D, or V9I plus G138D mutations further increased activity ~2-fold. These results demonstrate that the activity of sc-rtTA can be improved by mutations in either TetR moiety, but mutations in the N-terminal TetR domain have a larger impact on activity than the same mutations in the C-terminal domain.
New sc-rtTA variants improve dox-control in combined activation/repression system
Since the background expression from tetO-controlled minimal promoters can be actively suppressed by tTS [8, 9], we cotransfected C33A cells with a tTS expression plasmid (tTSS ). The presence of tTS indeed reduced the background activity more than 20-fold (~0.3% residual activity in the absence of dox; Fig. 5). Administration of dox inactivates tTS and activates the (sc-)rtTAs. The dox-induced activity of rtTA and the wild-type sc-rtTA was however ~5-fold reduced by the presence of tTS. This may be due to heterodimerization between the tTS and rtTA or sc-rtTA molecules . The dox-induced activity of the new sc-rtTA variants was not affected by the presence of tTS. The low background and high dox-induced activity observed when combining the new sc-rtTA variants with tTS results in a high inducibility (809–1193 fold), whereas combining the wild-type sc-rtTA or regular rtTA with tTS yields a much lower inducibility (99 and 273 fold, respectively). The highest inducibilty is obtained with the F67S/F86Y-mutated sc-rtTA variant, which was also the most active and dox-sensitive variant when tested without tTS.
We recently identified amino acid substitutions in rtTA that greatly improve the transcriptional activity and dox-sensitivity of the transactivator . In this study, we tested whether these mutations similarly affect other TetR-based transactivators. The results demonstrate that whereas none of the mutations that we tested improved tTA activity, all mutations did enhance sc-rtTA activity significantly. The F67S/F86Y-mutated sc-rtTA proved to be the most active and dox-sensitive single-chain variant and was as active and dox-sensitive as the original dimeric rtTA.
Combining the rtTA and sc-rtTA variants with the repressor tTS reduced the background activity of the CMV-7tetO promoter. The dox-induced activity of the regular rtTA and the wild-type sc-rtTA was also reduced in the presence of tTS. This may be due to heterodimerization between the tTS and rtTA or sc-rtTA molecules, which are all based on the TetR class B from transposon Tn10. For sc-TetR and for sc-tTS, heterodimer formation is not observed , but this was not tested for sc-rtTA variants. The dox-induced activity of the new sc-rtTA variants was not affected by the presence of tTS. The introduced mutations may have modified the sc-rtTA dimerization surface indirectly, as none of the altered amino acids contribute directly to dimerization, or they may have improved intramolecular folding, thus preventing binding to tTS. This resistance to interference by other TetR-based transregulators makes the new sc-rtTA variants very useful in applications where multiple TetR-based regulatory systems are used simultaneously.
The fact that the mutations do not similarly affect tTA and rtTA is probably due to structural differences between the two proteins. In tTA, the natural TetR conformation and dox-response are preserved. In rtTA, the TetR part contains four amino acid substitutions that cause a reciprocal dox-response. These amino acid substitutions do not only confer the reverse phenotype, but also result in a 100-fold reduced dox-sensitivity . This suggests that the structure of the mutated TetR is not optimal for the function of rtTA. The mutations identified in the viral evolution experiments may compensate for such unwanted tTA to rtTA structural changes, and thus specifically enhance rtTA activity. While the V9I mutation in the DNA-binding domain is likely to increase the dox-induced binding affinity of rtTA for the tetO site, the F67S, F86Y, and G138D mutations positioned in the regulatory core domain may increase the binding affinity for dox or facilitate the structural transition to the DNA-binding form [24, 26]. In contrast, the structure of TetR in tTA is the product of natural evolution of bacterial Tc-resistance regulation. Apparently, introduction of the selected mutations into this already-optimized sequence has no (V9I and F67S) or a detrimental effect (F86Y and G138D). The reduced dox-responsiveness of the F86Y and G138D mutated tTAs is in agreement with previous TetR-mutation studies demonstrating that amino acid substitutions at positions 86 and 138 can cause an induction-deficient phenotype .
Both the transactivators rtTA and sc-rtTA are activated by dox. However, the induced activity of sc-rtTA is reduced when compared with rtTA . This is probably due to structural differences between these two proteins. First, sc-rtTA contains only one activation domain per active molecule, whereas the rtTA dimer contains two activation domains. Second, the two TetR parts of sc-rtTA are connected by a peptide linker. Although the linker was designed to be flexible, it may still interfere with the function of the TetR moieties. Our results demonstrate that the sc-rtTA activity can be significantly improved by introduction of mutations that enhanced rtTA activity, suggesting that sc-rtTA folds very similarly to rtTA.
The most active sc-rtTA variant in this study was obtained by introducing beneficial mutations in both TetR domains. The single-chain characteristic of sc-rtTA allows the modification of a single TetR part. We demonstrated that sc-rtTA can be improved by mutations in only one of the TetR moieties, and that the introduction of the mutations in the second TetR domain has an additive effect. This suggests that dox binding to one of the effector-binding pockets does not affect binding to the second one, which is in agreement with published data showing that the binding of two Tc molecules to a TetR dimer occurs without significant cooperativity . The sc-rtTA variants with beneficial mutations in the N-terminal TetR domain appeared to be more active than the variants with the same mutations in the C-terminal TetR part. This may indicate that the induction of the C-terminal TetR domain contributes less to the induced activity of sc-rtTA. Possibly, it binds less efficiently to the tetO site due to the close proximity of the DNA-binding domain to the linker peptide.
We generated a novel sc-rtTA variant that is more active and more dox-sensitive than the original sc-rtTA, and does not show any background activity in the absence of dox. This novel sc-rtTA is almost as active and dox-sensitive as the regular dimeric rtTA but is less sensitive to interference by other TetR-based transregulators, and may therefore be more suitable for applications where multiple TetR-based regulatory systems are used.
Construction of tTA variants
The plasmid pCMV-tTA contains the tTA2S gene cloned in the expression vector pUHD141-1/X . Mutations were introduced in pCMV-tTA by mutagenesis PCR  with the mutagenic primers (primer M) tTA-V9I (5'-GGACAAGAGCAAAATCATAAACTCTGCTCTGGA-3', mismatching nucleotide underlined), tTA-F67S (5'-CATACCCACTCCTGCCCCCTGGAAGGCGA-3'), tTA-F86Y (5'-GCGGAACAACGCCAAGTCATACCGCTGTGCT-3'), or tTA-G138D (5'-GTCCGCCGTGGACCACTTTACACTGGGCT-3') and the primers 5'-TGGAGACGCCATCCACGCT-3' (primer 1), 5'-TGAAATCGAGTTTCTCCAGGCCACATATGA-3' (primer 2), and 5'-TCACTGCATTCTAGTTGTGGT-3' (primer 3). Briefly, PCR reactions were performed with primer M plus primer 3, and with primer 1 plus primer 2. The PCR products were purified, mixed, and PCR amplified with primers 1 and 3 (see ref.  for details). The resulting mutated tTA genes were cloned as EcoRI-BamHI fragments into pCMV-tTA. All constructs were verified by sequence analysis.
Construction of sc-rtTA variants
The plasmids pCMV-rtTA and pCMV-sc-rtTA contain the rtTA2S-S2 and sc-rtTA2-S2 genes, respectively, cloned in the expression vector pUHD141-1/X [6, 18]. The sc-rtTA gene contains two TetR moieties and a single activation domain. To introduce mutations into the N-terminal tetR gene (with synthetic humanized codon usage), the EcoRI-BfuAI fragment of pCMV-sc-rtTA was replaced with the corresponding fragment of the appropriate pCMV-rtTA plasmid . Mutations were introduced into the C-terminal tetR gene (with original bacterial codon usage) by mutagenesis PCR  on pCMV-sc-rtTA with the mutagenic primers (primer M) sc-rtTA-V9I (5'-GGCTCTAGATCTCGTTTAGATAAAAGTAAAATCATTAACAGCGCA-3'), sc-rtTA-F67S (5'-AGGCACCATACTCACTCTTGCCCTTTA-3'), sc-rtTA-F86Y (5'-AACGCTAAAAGTTATAGATGTGCT-3'), or sc-rtTA-G138D (5'-CAGCGCTGTGGACCACTTTACTTTA-3') and the primers 5'-TAATCATATGTGGCCTGGAGAA-3' (primer 1), 5'-AGGCGTATTGATCAATTCAAGGCCGAATAAG-3' (primer 2), and 5'-TCACTGCATTCTAGTTGTGGT-3' (primer 3). The final PCR products were digested with BglII and SmaI and used to replace the corresponding fragment of pCMV-sc-rtTA. All constructs were verified by sequence analysis.
Cell culture and (r)tTA activity assay
The activity of tTA, tTS, rtTA and sc-rtTA was assayed in C33A cells (ATCC HTB31)  and HeLa X1/6 cells , which are HeLa-derived cells containing chromosomally integrated copies of the CMV-7tetO luciferase reporter construct pUHC13-3 . Cells were cultured at 37°C with 5% CO2 in Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum, minimal essential medium nonessential amino acids, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells were grown in 2-cm2 wells to 60% confluency and transfected with the pCMV-tTA, pCMV-tTS (pWHE122sB, ), pCMV-rtTA or pCMV-sc-rtTA expression plasmids and the plasmid pRL-CMV (Promega) by the calcium phosphate precipitation method. pRL-CMV expresses Renilla luciferase from the CMV promoter and was used as an internal control to allow correction for differences in transfection efficiency. 1 μg of the DNA mixture in 15 μl water was mixed with 25 μl of 50 mM HEPES (pH 7.1)-250 mM NaCl-1.5 mM Na2HPO4 and 10 μl of 0.6 M CaCl2, incubated at room temperature for 20 min, and added to the culture medium. The DNA mixture contained 8 ng pCMV-tTA and 2.5 ng pRL-CMV for the tTA activity assay, 20 ng pCMV-sc-rtTA or pCMV-rtTA and 2 ng pRL-CMV for the sc-rtTA or rtTA activity assay in HeLa X1/6 cells, and 5 ng pCMV-sc-rtTA or pCMV-rtTA, 20 ng pCMV-7tetO-luciferase (pUHC13-3) , 0.5 ng pRL-CMV and 200 ng pCMV-tTS (when indicated) for the sc-rtTA or rtTA activity assay in C33A cells. The DNA mixture was completed to 1 μg with pBluescript as carrier DNA. Cells were cultured after transfection for 48 hours at different dox (D-9891, Sigma) concentrations and then lysed in Passive Lysis Buffer (Promega). Firefly and Renilla luciferase activities were determined with the Dual-Luciferase Reporter Assay (Promega). The expression of firefly and Renilla luciferase was within the linear range and no squelching effects were observed. The activity of the transactivators was calculated as the ratio of the firefly and Renilla luciferase activities, and corrected for between-session variation as described .
This research was sponsored by the Technology Foundation STW (applied science division of NWO and the technology program of the Ministry of Economic Affairs, Utrecht, the Netherlands), and the initial development of sc-rtTA was sponsored by the Deutsche Forschungsgemeinschaft through SFB473.
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