KRAB Can Repress Lentivirus Proviral Transcription Independently of Integration Site*
- School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland and “Frontiers in Genetics” National Center for Competence in Research, University of Geneva, CH-1211 Geneva, Switzerland
- 1 To whom correspondence should be addressed: Ecole Polytechnique Fédérale de Lausanne, Station 15, 1015 Lausanne, Switzerland. E-mail: didier.trono{at}epfl.ch.
Abstract
The KRAB transcriptional repressor domain, commonly found in zinc finger proteins, acts by inducing the formation of heterochromatin. We previously exploited this property to achieve drug-regulated transgenesis and knock down by combining doxycycline-controllable KRAB-containing fusion proteins and lentiviral vectors. Here, we asked whether KRAB-induced repression is widespread or limited to specific regions of the genome. For this, we transduced cells with a lentiviral vector expressing a target reporter and a KRAB-containing transcriptional repressor from a bicistronic mRNA. We found that ∼1.4% of the resulting proviruses escaped repression. However, this phenotype could be reverted by expressing the KRAB-containing protein in trans. Accordingly, the irrepressible proviruses all contained, in the DNA sequence encoding the KRAB-containing effector or its upstream internal ribosomal entry site, mutations or deletions likely resulting from errors or recombination during reverse transcription. These results indicate that KRAB-induced transcriptional repression is robust and active over a variety of genomic contexts that include at least the wide range of sites targeted by lentiviral integration.
Footnotes
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↵2 The abbreviations used are: tTR, tetracycline transrepressor; IRES, internal ribosome entry site; tetO, tetracycline operator; Dox, doxycycline; GFP, green fluorescent protein; NGFR, nerve growth factor receptor; HIV, human immunodeficiency virus; TSA, trichostatin A.
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↵* This work was supported by the Swiss National Science Foundation and the Institut Clayton de la Recherche Geneva. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1.
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- Received March 27, 2006.
- Revision received September 13, 2006.
- The American Society for Biochemistry and Molecular Biology, Inc.
