title: KRAB can repress lentivirus proviral transcription independently of integration site.
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 approximately 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.
authors.affiliation: School of Life Sciences, Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland.
authors.forename: Yannick
authors.initials: Y
authors.lastname: Bulliard
authors.forename: Maciej
authors.initials: M
authors.lastname: Wiznerowicz
authors.forename: Isabelle
authors.initials: I
authors.lastname: Barde
authors.forename: Didier
authors.initials: D
authors.lastname: Trono
mesh_terms: D001665:Binding Sites; D002460:Cell Line; D002469:Cell Separation; D004268:DNA-Binding Proteins / Q000378:metabolism / Q000502:physiology*; D004318:Doxycycline / Q000494:pharmacology; D020868:Gene Silencing; D005822:Genetic Vectors; D016679:Genome, Viral; D006801:Humans; D016086:Lentivirus / Q000235:genetics* / Q000378:metabolism; D008957:Models, Genetic; D012333:RNA, Messenger / Q000378:metabolism; D012097:Repressor Proteins / Q000378:metabolism / Q000502:physiology*; D014158:Transcription, Genetic; D019076:Transgenes
chemical_list: D004268:DNA-Binding Proteins; D012333:RNA, Messenger; D012097:Repressor Proteins; C422407:ZNF350 protein, human; D004318:Doxycycline