The Krüppel-associated Box Repressor Domain Can Induce Reversible Heterochromatization of a Mouse Locus in Vivo*

  1. Didier Trono‡§,4
  1. From the ‡School of Life Sciences, École Polytechnique Fédérale de Lausanne (EPFL), 1015 Lausanne, Switzerland,
  2. the §Frontiers-in-Genetics National Center of Competence in Research,
  3. the Department of Genetics and Evolution, University of Geneva, Sciences III, 1211 Geneva, Switzerland, and
  4. the ‖Mammalian Development Laboratory, Max-Planck-Institute for Molecular Biomedicine, 48149 Münster, Germany
  1. ↵4 To whom correspondence should be addressed: EPFL, SV-DO Station 19, CH-1015 Lausanne, Switzerland. Tel.: 41216931734; Fax: 41216931735; E-mail: Didier.trono{at}epfl.ch.

  • ↵1 Present address: Division of Molecular and Cellular Oncology, Dept. of Medical Oncology, Dana-Farber Cancer Institute and Dept. of Medicine, Brigham and Women's Hospital and Harvard Medical School, Boston, MA 02115.

  • ↵2 Present address: Dept. of Genetics, Harvard Medical School, 77 Ave. Louis Pasteur, Boston, MA 02115.

  • ↵3 Present address: Developmental Biology Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, 69117 Heidelberg, Germany.

Background: The KRAB module mediates ectopic and drug-controllable transcriptional repression.

Results: Targeting of KRAB to a mouse gene body results in reversible heterochromatization and gene silencing in adult and embryonic cells.

Conclusion: KRAB binding to gene bodies does not induce stable DNA promoter methylation as previously thought.

Significance: These proof-of-principle experiments provide the basis for the development of novel KRAB-based tools in vivo.

Abstract

The study of chromatin and its regulators is key to understanding and manipulating transcription. We previously exploited the Krüppel-associated box (KRAB) transcriptional repressor domain, present in hundreds of vertebrate-specific zinc finger proteins, to assess the effect of its binding to gene bodies. These experiments revealed that the ectopic and doxycycline (dox)-controlled tet repressor KRAB fusion protein (tTRKRAB) can induce reversible and long-range silencing of cellular promoters. Here, we extend this system to in vivo applications and use tTRKRAB to achieve externally controllable repression of an endogenous mouse locus. We employed lentiviral-mediated transgenesis with promoterless TetO-containing gene traps to engineer a mouse line where the endogenous kinesin family member 2A (Kif2A) promoter drives a YFP reporter gene. When these mice were crossed to animals expressing the TetO-binding tTRKRAB repressor, this regulator was recruited to the Kif2A locus, and YFP expression was reduced. This effect was reversed when dox was given to embryos or adult mice, demonstrating that the cellular Kif2A promoter was only silenced upon repressor binding. Molecular analyses confirmed that tTRKRAB induced transcriptional repression through the spread of H3K9me3-containing heterochromatin, without DNA methylation of the trapped Kif2A promoter. Therefore, we demonstrate that targeting of tTRKRAB to a gene body in vivo results in reversible transcriptional repression through the spreading of facultative heterochromatin. This finding not only sheds light on KRAB-mediated transcriptional processes, but also suggests approaches for the externally controllable and reversible modulation of chromatin and transcription in vivo.

Footnotes

  • Received February 7, 2012.
  • Revision received April 30, 2012.
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  1. First Published on May 17, 2012 doi: 10.1074/jbc.M112.350884 The Journal of Biological Chemistry 287, 25361-25369.
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