tetO Sequences Research Articles

Tetracycline-reversible silencing of eukaryotic promoters.

A tetracycline-controlled transrepressor protein has been engineered to silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRAB domain of human Kox1 to the Tet repressor derived from Tn10 of Escherichia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells. The TetR-KRAB protein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline. When TetR-KRAB bound to tetO sequences upstream of the immediate-early promoter-enhancer of human cytomegalovirus (CMV), the expression of a CMV-driven luciferase reporter construct (ptetO7-CMV-L) was repressed in transient transfection experiments. This silencing was found to operate on different promoters and from tetO sequences placed more than 3 kb from the transcriptional start site. We constructed a stable, doubly transfected cell line (TIS-10) carrying a chromosomally integrated ptetO7-CMV-L reporter construct and expressing the TetR-KRAB protein. Upon addition of tetracycline, luciferase expression was induced more than 50-fold above the baseline level, with half-maximal induction by 2 days. Furthermore, a protein of around 110 kDa was found to coimmunoprecipitate with the TetR-KRAB fusion protein. This protein might play a role as an adaptor protein mediating the silencing exerted by the TetR-KRAB protein. The TetR-KRAB silencing system should be useful as a genetic switch for regulating the expression of chromosomally integrated heterologous and endogenous genes present in mammalian genomes.

Tetracycline repressor-regulated gene repression in recombinant human cytomegalovirus

The tetracycline repressor (TetR)-regulated gene expression system from Escherichia coli was used to control gene expression in recombinant human cytomegalovirus (HCMV). To adapt the TetR system in HCMV, derivatives of the viral US11 (early) gene promoter, which controls the beta-glucuronidase reporter gene, were constructed by systematic insertion of the tetracycline operator (tetO) sequences. Gene expression from constructs containing two or three appropriately placed tetO sequences adjacent to the TATA box were efficiently repressed by a TetR-VP16 fusion protein (tTA) in a transient expression system. Efficient repression (50- to 120-fold) also occurred in tTA-expressing stably transfected human cells which were infected with recombinant HCMV containing a US11 promoter surrounded by three tetO sequences. The tTA-mediated gene repression was relieved in the presence of 1 microgram of tetracycline per ml. The results of this study are significant in three respects. (i) This is the first demonstration that a TetR-derived protein can be used to efficiently repress gene expression in a mammalian system. (ii) Efficient repression was dependent on the presence of the transcriptional activation domain from the herpes simplex virus type 1 VP16 protein. (iii) The ability to regulate gene expression in a controlled fashion in order to elucidate viral gene function is an important development in the HCMV field. The tTA-mediated gene repression system may be extremely useful for creating host-range mutants in essential genes in order to study their role in the HCMV replicative cycle, a system that is otherwise exceedingly difficult to genetically dissect.

Co-regulation of two gene activities by tetracycline via a bidirectional promoter.

Recently, we have described a regulatory system that allows the stringent control of individual gene activities in higher eukaryotic cell lines (1), in plants (2) and in animals (3,4). The essential components of this system are (i) an RNA polymerase II minimal promoter placed downstream of multiple operator sequences (tetO) of the Escherichia coli TnlO tetracycline resistance operon and (ii) a fusion between the Tet repressor (TetR) and the herpes simplex virus protein 16 (VP16), named tTA (1). In the absence of tetracycline (Tc), tTA binds to the tet operators to activate transcription from the minimal promoter, whereas in the presence of Tc its association and consequently its transcription activation is prevented. After the binding of tTA, minimal promoters derived from the cytomegalovirus IE promoter (PhCMv; 5) and fused to seven tetO sequences reach the remarkable strength of the parent promoter in HeLa cells when compared in transient expression assays (6). This high activation potential of tTA and the arrangement of its binding sites within PhCMV*-i [(!)'»see Fig. 1A] suggested the design of a bidirectional promoter which would allow the simultaneous regulation of two transcriptional units from centrally located multiple tetO sequences (Fig. 1A). Such a promoter should be useful for a number of experimental approaches. First, it may allow the co-regulation of the synthesis of two gene products in stoichiometric amounts, frequently a prerequisite for the production of heterodimeric (or hetero-oligomeric) proteins. Second, by fusing minimal promoters of differing efficiencies to the centrally located tetO sequences, two gene products may be co-regulated at different but defined levels. Third, by integrating an appropriate reporter gene at one side of the bidirectional promoter, the regulation of a not-readily-assayable gene of interest may be monitored via the reporter function. This latter possibility may also facilitate—at the cellular as well as at the organismal level—the screening for properly integrated expression units controlling the gene of interest.