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.
Highlights
Epigenetic mechanisms are central to the control of genome expression
KRAB protein, in which the tetracycline transrepressor2 from Escherichia coli Tn10 is fused to the KRAB domain of human Kox1, can modulate transcription from an integrated promoter juxtaposed with tet operator sequences [5]
This study demonstrates that KRAB-mediated transcriptional repression is functional and robust over a wide spectrum of genomic loci, including over 6,000 integrants that are representative of the full range of sites targeted by lentiviral integration
Summary
Vector Construction and Production—pLVCT-tTRKRAB was described previously [7]. pLVC-tTRKRAB was obtained by cloning tTR-KRAB into pLVC [7]. pLVET-NGFR was derived from pLV-TH/siGFP [6] by removing H1-siGFP. Amplification products were directly subcloned in TOPO TA vectors (Invitrogen) and sequenced using M13 forward and M13 reverse primers, as well as 5Ј-CATGCTTTACATGTGTTTAG-3Ј to cover the 1.9 kb of the insert. TTR-KRAB protein was detected using anti-tetracycline repressor rabbit polyclonal antibody (Mobitec) with a 1:1000 dilution in a solution of 5% powdered milk and 0.2% Tween 20 in phosphate-buffered saline, followed by incubation with secondary anti-rabbit horseradish peroxidase-conjugated antibody diluted 1:5000 (Amersham Biosciences). NGFR protein was detected with 20 l of phycoerythrin-conjugated anti-NGFR monoclonal antibody (BD Biosciences) per 106 cells for 1 h at 4 °C in the dark and extensively washed before analysis by fluorescence-activated cell sorting
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