TETRACYCLINE-REVERSIBLE SILENCING OF EUKARYOTIC PROMOTERS

A tetracycline-controlled transrepressor protein has been engineered t o silence transcriptional activities of eukaryotic promoters that are stably integrated into the chromatin of human cells. By fusing the KRA B domain of human Kox1 to the Tet repressor derived from Tn10 of Esche richia coli, a tetracycline-controlled hybrid protein (TetR-KRAB) was generated and constitutively expressed in HeLa cells. The TetR-KRAB pr otein binds to tet operator (tetO) sequences in the absence but not in the presence of tetracycline. When TetR-KRAB bound to te10 sequences upstream of the immediate-early promoter-enhancer of human cytomegalov irus (CMV), the expression of a CMV-driven luciferase reporter constru ct (ptet07-CMV-L) was repressed in transient transfection experiments, This silencing was found to operate on different promoters and from t etO sequences placed more than 3 kb from the transcriptional start sit e. We constructed a stable, doubly transfected cell line (TIS-10) carr ying a chromosomally integrated ptet07-CMV-L reporter construct acid e xpressing the TetR-KRAB protein. Upon addition of tetracycline, lucife rase expression was induced more than 50-fold above the baseline level , with half-maximal induction by 2 days. Furthermore, a protein of aro und 110 kDa was found to coimmunoprecipitate with the TetR-KRAB fusion protein. This protein might play a role as an adaptor protein mediati ng the silencing exerted by the TetR-KRAB protein. The TetR-KRAB silen cing system should be useful as a genetic switch far regulating the ex pression of chromosomally integrated heterologous and endogenous genes present in mammalian genomes.