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Originally published In Press as doi:10.1074/jbc.M602843200 on September 22, 2006

J. Biol. Chem., Vol. 281, Issue 47, 35742-35746, November 24, 2006
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KRAB Can Repress Lentivirus Proviral Transcription Independently of Integration Site*Formula

Yannick Bulliard, Maciej Wiznerowicz, Isabelle Barde, and Didier Trono1

From the 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

Received for publication, March 27, 2006 , and in revised form, September 13, 2006.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
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.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Epigenetic mechanisms are central to the control of genome expression. About 75 amino acids in length, KRAB is a transcriptional repression domain found in close to one-third of the several hundreds of zinc finger proteins encoded by the human genome. KRAB acts by triggering the formation of heterochromatin in the vicinity of its binding site and as such can repress either Pol I (polymerase I), Pol II, or Pol III promoters (1). Underlying this effect is the KRAB-mediated recruitment of a multimolecular complex comprising a variety of chromatinmodifying activities that can lead to histone deacetylation, histone methylation, and in some cases DNA methylation (2–4). However, whether these effects can be exerted anywhere in the genome or whether particular regions are "immune" to such repression is unknown.

The regulatory properties of KRAB can be exploited to engineer externally controllable gene expression systems. The tTR-KRAB protein, in which the tetracycline transrepressor (tTR)2 from Escherichia coli Tn10 is fused to the KRAB domain of human Kox1, can modulate transcription from an integrated promoter juxtaposed with tet operator (tetO) sequences (5). The tTR moiety of this protein confers drug controllability to the system, as its DNA binding can be regulated with doxycycline in either "Tet off" or "Tet on" configurations depending on the tTR variant used. Taking advantage of this approach, we recently generated KRAB-sensitive lentiviral vectors that can govern tightly controlled gene expression and knock down both in vitro and in vivo (6, 7). Lentiviruses have a net tendency to integrate genes that are transcribed (8). In such regions, chromatin is found in an open conformation and would be supposedly hostile to heterochromatin formation. In this study, we asked whether KRAB was efficient at inducing heterochromatization in the wide variety of lentiviral integration sites, even in the rather adverse context of active genes.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
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. Cell cultures, transfections, virus production, purification, and transductions were performed as previously described (7). After transduction, cultures were split into two, one being kept in Dulbecco's modified Eagle's medium, the other one in Dulbecco's modified Eagle's medium +1 µg/ml Dox for 72 h.

Sequencing of IRES-tTR-KRAB—PCR amplification was performed on purified genomic DNA using the primers 5'-CTGCTGCCCGACAACCAC-3' complementary to the 3'-end of GFP and 5'-CGCTATGTGAATACGCTGCT-3' complementary to the 5'-end of the woodchuck hepatitis post-transcriptional regulatory element using high-fidelity PfuTurbo polymerase (Stratagene). After amplification, 3'-deoxyadenosine was added by incubation with Taq polymerase (Qiagen) for 30 min at 72 °C. Amplification products were directly subcloned in TOPO TA vectors (Invitrogen) and sequenced using M13 forward and M13 reverse primers, as well as 5'-CATGCTTTACATGTGTTTAG-3' to cover the 1.9 kb of the insert.

Immunoblotting—Cells collected from confluent 10-cm dishes were used for protein extraction using standard procedures. 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). Tubulin-{alpha} was recognized by 1:1000 anti-tubulin-{alpha} mouse monoclonal antibody (Sigma) and by 1:5000 anti-mouse horseradish peroxidase-conjugated as secondary antibody (Amersham Biosciences). Detection of peroxidase activity was performed using Supersignal Pico (Pierce).


Figure 1
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  		FIGURE 1.
Schematic representation of the single vector construct. The vector was designed in a autoregulatory fashion such that in the absence of Dox, tTR-KRAB can bind to tetO and repress transcription of the bicistronic mRNA. SIN, self-inactivating long terminal repeat; cPPT, central polypurine tract; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; CAG, hybrid cytomegalovirus-actin-globin promoter.

 
Immunostaining—Cells used for NGFR staining were harvested with phosphate-buffered saline, washed, and resuspended in phosphate-buffered saline supplemented with 2% fetal calf serum and 20 mM sodium azide. NGFR protein was detected with 20 µl 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.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
We transduced 105 293T human kidney epithelial cells with a lentiviral vector expressing GFP and the tTR-KRAB repressor from an internal ribosomal entry site (IRES) containing bicistronic mRNA and harboring tetO binding sites in its long terminal repeats, rendering it inactive (Fig. 1). We used a low multiplicity of infection to ensure that most cells contained only one provirus. We then examined the drug controllability of GFP expression in the resulting population. In the presence of doxycycline (Dox), which prevents binding of tTR-KRAB to the DNA and allows proviral expression, 6.6% of the cells expressed GFP. This means that we produced a heterogeneous population containing more than 6,000 putative different integration sites. Upon removal of the drug, 0.09% of the cells remained GFP positive (Fig. 2, left). This non-repressible population thus represented ~1.4% of the transduced cells. Through two rounds of sequential drug addition and removal coupled with stringent cell sorting, we isolated the repressible and the non-repressible cells to a degree of purity approximating 98% (Fig. 2, right), with a remaining 2% of repressible cells in the irrepressible population and vice versa. The phenotype of either subpopulation was stable over time without noticeable change after more than 10 passages.

The phenotype of the non-repressible population could result either from resistance of the corresponding lentiviral integrants to KRAB-mediated repression or from mutations in the provirus, for instance in the sequence governing expression of the repressor or in its TetO binding sites. To differentiate between these possibilities, we first compared tTR-KRAB expression in the repressible and non-repressible subpopulations by Western blotting (Fig. 3). In the drug-controllable cells, levels of repressor were high in the presence of Dox and very low in the absence of drug, as expected from the auto-regulatory configuration of the vector. In contrast, in the non-repressible population, levels of tTR-KRAB were unaffected by Dox and the tTR-specific antibody detected a series of smaller than full-length bands. We thus extracted DNA from both the repressible and the irrepressible subpopulations, amplified the IRES-tTR-KRAB cassette by PCR, and sequenced the resulting products. Of 20 DNA clones sequenced from the irrepressible population, 19 contained either point mutations or deletions within this region of the provirus that could be grouped in 9 distinct patterns (Table 1 and supplemental Fig. S1). In three of these, G-to-A point mutations were detected that led to missense or nonsense mutations in tTR-KRAB. In the other six cases, deletions ranging in size from a single nucleotide to the entire IRES-tTR-KRAB cassette were present. In one of them, a 14-bp sequence was inserted, a type of event previously reported in deleted proviruses (9). In the two cases of fully deleted IRES-tTR-KRAB cassette, the likely causal recombination could be traced to a 30-bp CTAG repeat flanking this sequence. This is consistent with the low processivity of reverse transcriptase, which promotes deletions by homologous recombination (10). Most of the mutations or deletions were recorded in more than one DNA clone, suggesting that we identified the majority of the genotypes responsible for the nonrepressible phenotype. Nevertheless, the range of IRES-tTR-KRAB sequence patterns thereby detected (Table 1) does not account for all the tTR-KRAB-related bands identified by Western blot (Fig. 3). Although some of them might be degradation products of large size proteins, it could also be that the PCR-based approach introduced a bias, for instance in favor of shorter products, as suggested by a trend toward the more frequent recovery of deleted versus mutated clones. Noteworthy, only 1 IRES-tTR-KRAB DNA clone of 20 amplified from the non-repressible subpopulation had a wild-type sequence. We cannot exclude that it originated from a cell that was truly nonrepressible owing to another mechanism, including mutations in tetO or immunity to chromatin repression. However, the low frequency of recovery of such a clone is within the 2% contamination rate of the non-repressible subpopulation by repressible cells. Finally, no mutation or deletion was recorded in 6 IRES-tTR-KRAB DNA clones amplified from the repressible population. Therefore, we conclude that, in the model under study the tTR-KRAB control system was functional over the diversity of lentivector integration sites.


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  		TABLE 1
List of distinct patterns of mutation in the irrepressible population

 


Figure 2
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  		FIGURE 2.
Fluorescence-activated cell sorting analysis of 293T cells transduced with LVCT-tTRKRAB in the presence or absence of Dox. Left, initially transduced population. Right, non-repressible and repressible subpopulations isolated after two rounds of cell sorting. The gates illustrated on the left side are representative of those used for cell sorting.

 


Figure 3
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  		FIGURE 3.
Western blot of total proteins from repressible and non-repressible subpopulations using a tTR-specific antibody. Non-transduced 293T cells served as negative control.

 
Consistent with this conclusion and confirming that the phenotype of the non-repressible population was due to mutations in tTR-KRAB and not to cis-mediated resistance to heterochromatin formation, transduction of these cells with a second vector, expressing tTR-KRAB from the cytomegalovirus-actinglobin promoter, restored the drug controllability of transgene expression in 98% of them (Fig. 4, top). Therefore, the overall percentage of KRAB-repressible GFP-positive cells was 99.96%, that is, the 98% originally observed plus 98% of the 1.4% of initially irrepressible cells subsequently rescued by addition of a functional KRAB fusion protein. In both repressible and nonrepressible dually transduced populations, a higher fraction of cells remained GFP negative in the absence of Dox than in the singly transduced cells. This likely reflects the fact that in the second vector tTR-KRAB is constitutively expressed rather than auto-regulated and present at a high copy number. We also transduced the repressible and irrepressible cellular subpopulations with a vector expressing a tTR-KRAB-controllable NGFR cell surface marker (Fig. 4, bottom). In the repressible cells, NGFR was fully Dox sensitive (data not shown). In contrast, in the irrepressible subpopulation, NGFR expression could not be controlled by doxycycline, confirming that the phenotype of these cells was due to a non-functional trans repressing system.


Figure 4
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  		FIGURE 4.
Fluorescence-activated cell sorting analysis of the non-repressible population after transduction with either LVC-tTRKRAB (top) or LVET-NGFR (bottom). Addition of tTR-KRAB restores the repressibility of GFP expression, while the NGFR is also non-controllable in the native nonrepressible cells. Numbers indicate the fraction of cells in each gate.

 
When T lymphocytes are exposed to HIV, a small fraction of the cells that become infected fail to produce virions, because they harbor latent proviruses. Recent studies have shown that these latent proviruses are more commonly integrated in gene deserts, centromeric heterochromatin, or very highly expressed cellular genes yet that they can be induced by treatment with cytokines such as tumor necrosis factor {alpha} (11). Here, the GFP reporter was expressed not from the HIV long terminal repeat but from an internal cytomegalovirus-actin-globin promoter, and the targets cells were not T lymphocytes but kidney epithelial cells. Still, we wished to test whether our GFP-negative population contained latent proviruses and, if this were the case, whether these would be controlled by KRAB upon reactivation. For this, we selected GFP-negative cells in the presence of Dox to a degree of purity of 99.2% and subjected the resulting population to various drug treatments. We first observed that tumor necrosis factor {alpha} did not increase the percentage of GFP-positive cells (not illustrated), in striking contrast with what was previously observed in T lymphoid Jurkat cells transduced with a vector expressing GFP from the HIV long terminal repeat (11). We then treated the cells with TSA and 5-aza-dC, alone or in combination, to relieve possible silencing by histone deacetylation and/or DNA methylation (Fig. 5). These drugs had no significant effect in the presence of Dox, that is, when KRAB was sequestered away from the proviruses. The same result was obtained with 5 mM sodium butyrate, another known alleviator of epigenetic silencing (data not shown). We thus conclude that, in our system, the impact of latent proviruses was negligible.


Figure 5
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  		FIGURE 5.
Effects of 5-aza-dC and TSA on indicated populations transduced with LVCT-tTRKRAB in the absence or presence of Dox. GFP–indicates the subpopulation of cells obtained after eliminating GFP+ cells by two rounds of sorting. It is still contaminated by ~1% of GFP+ cells, which are fully revealed by Dox treatment. Treatments were for 48 h with 5 µM 5-aza-dC, 300 nM TSA, or 5 µM 5-aza-dC in combination with 200 nM TSA. For each population, ~6,000 cells were analyzed by fluorescence-activated cell sorting.

 
Interestingly, in the absence of Dox, that is, when KRAB was bound to the DNA, TSA and to a lesser extent 5-aza-dC induced the appearance of some GFP-positive cells in the KRAB-repressible population (Fig. 5). This is consistent with the previous demonstration that these two drugs can reactivate the expression of genes silenced by the KRAB repressor complex (2, 4, 12).


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
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. A series of studies have revealed that lentiviral integration favors active transcription units as well as Alu elements and regions that correlate with high gene density and have a high GC content (8, 13, 14). Lentiviral integration sites are apparently also often flanked by matrix attachment regions, which are cis-regulatory elements involved in stabilizing gene expression (15). In contrast, heterochromatic centromeres and telomeres are disfavored for HIV integration (14). Our results thus indicate that KRAB-mediated repression is fully effective in the context of highly active genes, which could have been postulated to be a rather adverse environment where chromatin is actively maintained in an opened conformation.

The KRAB-mediated induction of heterochromatin formation stems from the recruitment of a multimolecular chromatin-remodeling complex. Interestingly, one of the components of this complex, SETDB1, is an H3K9 methyltransferase that, contrary to other members of its family, is not blocked by H3K4 methylation, a mark of open chromatin (3). This could explain the independence of KRAB activity toward integration site chromatin context. Locus control regions are cis-acting elements that ensure active transcription by protecting against chromatin-mediated repression (16). Given the strong potency of KRAB, and to understand better chromatin regulation at the boundary between active/open and inactive/closed regions, it would be interesting to examine the ability of KRAB-containing zinc finger proteins to overcome the influence of locus control regions. Relatedly, it remains to be seen whether KRAB-mediated silencing brought about by lentiviral transduction extends to genes that host the integrants. It has been demonstrated that KRAB-induced repression can extend up to 3.6 kb from its DNA binding site (4, 5). Repression or not of the targeted gene might thus depend on where integration occurs within its sequence. In that respect, KRAB binding sites introduced through murine leukemia virus (MLV)-based vectors might be revealing, because MLV tends to integrate in and around promoters whereas HIV avoids these regions, targeting instead the transcribed part of genes (13).

Affinity and off-rate are also important factors for repression potency. This is exemplified by the greater potency of KRAB fusion proteins containing six rather than three zinc fingers to inhibit herpes simplex virus 1 gene expression in a VP16 competition assay (17). Fusion of KRAB with the DNA-binding domain of a mammalian transcription factor, PAX3, also exhibited good repression capacities (4, 18). This indicates that the robustness of KRAB in our system does not solely depend on the binding capacity of the tTR moiety.

Our finding that the vast majority, if not all, of the few cells transduced with LVCT-tTRKRAB that did not respond to doxycycline control owed their phenotype to mutations or deletions in IRES-tTR-KRAB has practical implications. We could indeed reduce the leakiness of the system to negligible levels by transducing the non-repressible population with a second vector expressing tTR-KRAB. This is of particular interest if the hereby-described approach is used for the in vivo regulation of potentially toxic genes. With at least two copies of a KRAB-expressing vector in each cell, leakage should be virtually eliminated, because it is very unlikely that both copies will contain a non-functional trans repressor sequence. Furthermore, vectors could be optimized by eliminating direct repeats to decrease the risk of deletion by recombination during reverse transcription. Our results thus support the further exploration of tTR-KRAB-controlled gene transfer and knock down for clinical applications and for obtaining the tight control of pathogenic proteins in animal models of human diseases.


   FOOTNOTES
 
* 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. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Fig. S1. Back

1 To whom correspondence should be addressed: Ecole Polytechnique Fédérale de Lausanne, Station 15, 1015 Lausanne, Switzerland. E-mail: didier.trono{at}epfl.ch.

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. Back


   ACKNOWLEDGMENTS
 
We thank Stephanie Jost, Alexander Dorr, and Johan Jackobsson for their help, Fritz Mueller from the University of Fribourg for critical evaluation of the manuscript, and the entire laboratory for motivation.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

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