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

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.