Tetracycline-regulated gene expression mediated by a novel chimeric repressor that recruits histone deacetylases in mammalian cells

Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be develo ped. Histone deacetylases have recently been shown to play an important rol e in regulating gene expression. Here we investigated whether a more tightl y controlled expression could be achieved by using a novel chimeric repress or that recruits histone deacetylases to a tetracycline-responsive promoter . This chimeric repressor was engineered by fusing the tetracycline repress or (TetR) with an mSin3-interacting domain of human Mad1 and was shown to b ind the tetO(2) element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly int eract with mSin3 of the historic deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chim eric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor sy stem exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HE K 293 cells, the chimeric repressor system was shown to exert a tight contr ol of green fluorescent protein expression in a doxycycline dose- and time- dependent fashion. Therefore, this novel chimeric repressor provides an eff ective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studi es.