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J. Biol. Chem., Vol. 276, Issue 48, 45168-45174, November 30, 2001

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Tetracycline-regulated Gene Expression Mediated by a Novel Chimeric Repressor That Recruits Histone Deacetylases in Mammalian Cells^*

Wei Jiang, Lan Zhou^§, Benjamin Breyer, Tao Feng^§, Hongwei Cheng, Rex Haydon, Akira Ishikawa^¶, and Tong-Chuan He

From the Molecular Oncology Laboratory, Department of Surgery, The University of Chicago Medical Center, Chicago, Illinois 60637, the ^§ Department of Biochemistry and Molecular Biology, Chongqing University of Medical Sciences, Chongqing 400046, China, and the ^¶ Department of Orthopaedic Surgery, Yamagata University School of Medicine, 2-2-2, Iida-Nishi, Yamagata 990-9585, Japan

Regulated gene expression will provide important platforms from which gene functions can be investigated and safer means of gene therapy may be developed. Histone deacetylases have recently been shown to play an important role in regulating gene expression. Here we investigated whether a more tightly controlled expression could be achieved by using a novel chimeric repressor that recruits histone deacetylases to a tetracycline-responsive promoter. This chimeric repressor was engineered by fusing the tetracycline repressor (TetR) with an mSin3-interacting domain of human Mad1 and was shown to bind the tetO₂ element with high affinity, and its binding was efficiently abrogated by doxycycline. The chimeric repressor was shown to directly interact with mSin3 of the histone deacetylase complex. This inducible system was further simplified by using a single vector that contained both a chimeric repressor expression cassette and a tetracycline-responsive promoter. When transiently introduced into mammalian cells, the chimeric repressor system exhibited a significantly lower basal level of luciferase activity (up to 25-fold) than that of the TetR control. When stably transfected into HEK 293 cells, the chimeric repressor system was shown to exert a tight control of green fluorescent protein expression in a doxycycline dose- and time-dependent fashion. Therefore, this novel chimeric repressor provides an effective means for more tightly regulated gene expression, and the simplified inducible system may be used for a broad range of basic and clinical studies.

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