Plant Mol Biol (2010) 72:621–630
DOI 10.1007/s11103-010-9600-0
Negative regulation of the RTBV promoter by designed zinc finger
proteins
M. Isabel Ordiz • Laurent Magnenat •
Carlos F. Barbas III • Roger N. Beachy
Received: 2 September 2009 / Accepted: 8 January 2010 / Published online: 19 February 2010
Ó Springer Science+Business Media B.V. 2010
Abstract The symptoms of rice tungro disease are caused
by infection by a DNA-containing virus, rice tungro
bacilliform virus (RTBV). To reduce expression of the
RTBV promoter, and to ultimately reduce virus replication,
we tested three synthetic zinc finger protein transcription
factors (ZF-TFs), each comprised of six finger domains,
designed to bind to sequences between -58 and ?50 of the
promoter. Two of these ZF-TFs reduced expression from
the promoter in transient assays and in transgenic Arabidopsis thaliana plants. One of the ZF-TFs had significant
effects on plant regeneration, apparently as a consequence
of binding to multiple sites in the A. thaliana genome.
Expression from the RTBV promoter was reduced by
*45% in transient assays and was reduced by up to 80% in
transgenic plants. Co-expression of two different ZF-TFs
did not further reduce expression of the promoter. These
experiments suggest that ZF-TFs may be used to reduce
replication of RTBV and thereby offer a potential method
for control of an important crop disease.
Electronic supplementary material The online version of this
article (doi:10.1007/s11103-010-9600-0) contains supplementary
material, which is available to authorized users.
M. I. Ordiz R. N. Beachy (&)
Donald Danforth Plant Science Center, 975 N. Warson Road,
St. Louis, MO 63132, USA
e-mail: Rnbeachy@danforthcenter.org
L. Magnenat C. F. Barbas III
The Skaggs Institute for Chemical Biology and the Department
of Molecular Biology, The Scripps Research Institute, BCC-550,
North Torrey Pines Road, La Jolla, CA 92037, USA
Present Address:
L. Magnenat
Merck Serono S.A., 9 Chemin des Mines, 1202 Geneva,
Switzerland
Keywords Gene regulation Repression RTBV promoter Transcription factors Synthetic zinc fingers
Introduction
Rice tungro disease (RTD) is caused by co-infection by
rice tungro bacilliform virus (RTBV) and rice tungro
spherical virus (RTSV). RTBV is the primary causal agent
of disease symptoms while RTSV provides functions that
permit transmission of the disease agents by the green
leafhopper. RTBV is a pararetrovirus and its genome of
double stranded DNA is replicated primarily in phloem and
phloem associated tissues. The genome of RTBV has been
well characterized as has its transcriptional promoter (Yin
and Beachy 1995; Yin et al. 1997a) and the transcription
factors that bind to and activate its expression. The ‘E’
fragment of the promoter (nucleotides -164 to ?45) is
sufficient to confer high-level, tissue-specific gene
expression. Previous studies of the promoter characterized
the cis elements that confer phloem-specific expression and
several transcription factors that are important for expression. The cis elements include a GATA motif [nucleotides
(nt) -143 to -135], AS1-like (ASL) Box (nt -98 to -79),
Box II (nt -53 to -39), and Box I (nt -3 to ?8) (He et al.
2000, 2001, 2002; Yin and Beachy 1995; Yin et al. 1997a).
Box II plays an essential role in expression of genes driven
by the RTBV promoter and two host transcription factors
that bind to the Box II cis element have been identified and
partially characterized: RF2a (Dai et al. 2003; Yin et al.
1997b) and RF2b (Dai et al. 2004). RF2a and RF2b bind to
and activate transcription from the RTBV promoter (Dai
et al. 2003, 2004, 2006; Petruccelli et al. 2001; Yin et al.
1997b). A goal of our research is to reduce replication of
123
622
RTBV, and therefore mitigate disease symptoms, by
reducing RTBV gene expression.
The Cys2His2 class of zinc finger proteins (ZFPs) is the
most abundant family of transcription factors in Arabidopsis thaliana; of 176 known ZFPs, 33% are conserved
amongst other eukaryotes and 81% are plant specific
(Englbrecht et al. 2004). These proteins have a wide range
of functions and can bind to other proteins and to RNA and
DNA. Cys2His2 proteins have a bba fold that most typically recognizes three contiguous base pairs (bp) of DNA
sequence. This property has been used to design synthetic
zinc finger protein transcription factors (ZF-TFs) that can
regulate gene expression in a variety of different organisms
(reviewed in Blancafort et al. 2004).
Several recent reports have shown that synthetic ZF-TFs
can be used to regulate gene expression in plants. In earlier
studies we used a well-characterized six-finger ZF-TF, 2C7
(Liu et al. 1997), and its cognate DNA binding site in transient assays in plant protoplasts and transgenic plants (Ordiz
et al. 2002; Stege et al. 2002) to alter gene expression.
Expression of a reporter gene was enhanced by a 2C7 activator protein in BY-2 protoplasts; furthermore, the distance
between the TATA box and the 2C7 binding site affected the
efficiency of ZF-TF regulation of gene expression (Ordiz
et al. 2002; Stege et al. 2002). We also showed that a ZF-TF
activator could be applied to cause tissue specific expression
of a target gene in transgenic tobacco plants (Ordiz et al.
2002). Stege et al. (2002) explored the use of both activation
and repression domains with the 2C7 ZF-TF protein,
reporting that the SID domain of the human MAD protein
linked to a ZF-TF repressed gene expression by fivefold.
Guan et al. (2002) designed ZF-TFs to regulate the
promoter of the Apetala3 (Ap3) gene in A. thaliana and
used the AP1 promoter to drive expression of AP3-ZFP-TF
and demonstrated control of expression of the endogenous
gene. Other groups have used ZF-TFs to regulate plant
metabolism (Holmes-Davis et al. 2005; Sanchez et al.
2006; Van Eenennaam et al. 2004; Wu et al. 2004). Others
have used chemically inducible promoters to control
expression of ZF-TFs (Beerli et al. 2000a, b; Sanchez et al.
2002). New applications of sZFP nucleases to cleave,
repair, induce mutations, and effect homologous recombination in plants and other organisms, including Drosophila,
Caenorhabditis elegans, Xenopus, and mammalians cells,
have recently been described (Cai et al. 2009; Kandavelou
and Chandrasegaran 2009; Lloyd et al. 2005; Shukla et al.
2009; Wright et al. 2005; reviewed by Durai et al. 2005;
Porteus and Carroll 2005; Wu et al. 2007).
The goal of the current study was to identify ZF-TFs that
reduce expression of the RTBV promoter. We designed
three ZF-TFs to bind to sequences proximal to the TATA
box of the RTBV promoter. Data from transient assays in
tobacco BY-2 protoplasts and transgenic Arabidopsis
123
Plant Mol Biol (2010) 72:621–630
plants indicated that two ZF-TFs down-regulate the RTBV
promoter, causing a 40–80% decrease in the expression of
the genes driven by the promoter, while the third protein
had a negative effect on plant development.
Materials and methods
Sequences of RTBV promoters analyzed
EMBL accession numbers of the RTBV sequences compared in this study were AF113830 (G1), AF113831 (G2),
AF113832 (Ic), X57924 (Phi-1), M65026 (Phi-2), D10774
(Phi-3, Cabauatan et al. 1999), AF076470 (Serdang, Marmey et al. 1999), AF220561 (Chainat isolate, Nathwong
et al. 2000, unpublished), AJ314596 (West Bengal, Nath
et al. 2002), NC_001914 (Hay et al. 1991).
Assembly of zinc finger proteins and specificity
of protein binding
Open reading frames encoding ZF-TFs were generated by
PCR using domain sequences and methods described previously (Dreier et al. 2001, 2005; Segal et al. 1999).
ZF-TF2 was designed to bind nucleotides 50 AAGAG
AGCAAGAGAGGAG30 with the following zinc finger
DNA recognition helices (amino acids -1 to ?6 are shown
in brackets): F1-GAG (RSDNLVR), F2-GAG (RSDNLVR),
F3-AGA (QLAHLRA), F4-GCA (QSGDLRR), F5-AGA
(QLAHLRA), and F6-AAG (RKDNLKN). ZF-TF3 was
designed to bind 50 AGGGGCACACTGGTCATT30 with
recognition helices as follows: F1-ATT (HKNALQN),
F2-GTC (DPGALVR), F3-CTG (RNDALTE), F4-ACA
(SPADLTR), F5-GGC (DPGHLVR), and F6-AGG
(RSDHLAE). ZF-TF8 was designed to bind 50 GGAG
TCCAGGGGCACACT30 with recognition helices F1-ACT
(THIDLIR), F2-CAC (NLQHLGE), F3-GGG (RSDKLVR),
F4-CAG (RADNLTE), F5-GTC (DPGALVR), and F6-GGA
(QSSHLVR).
Assembled DNAs encoding three-finger proteins that
bind half-sites were subcloned by SfiI digestion and cloning
into a modified pMal-C2 bacterial expression vector (NEB,
MA) (Beerli et al. 1998), and joined as pairs using restriction
endonucleases for the construction of six-finger proteins.
Proteins were tested with multi-target specificity ELISA as
described (Magnenat et al. 2004; Segal et al. 1999). ZF-TF
proteins fused to maltose binding protein and hemaglutinin
tags were overexpressed in E. coli. Crude extracts of each
ZF-TF were obtained by freeze–thaw cycles. Twofold serial
dilutions of the extracts were tested individually for comparing their binding to different immobilized biotinylated
hairpin oligonucleotides (MWG-biotech) containing the
appropriate 18-bp target sequences. Bound protein was
Plant Mol Biol (2010) 72:621–630
detected with a mouse anti-maltose binding protein mAb
conjugated to alkaline phosphatase and substrate addition
(SIGMA, MO). Absorbance at 405 nm (OD405) was quantitated with SOFTmax 2.3.5 (Molecular Devices, CA),
background subtracted, normalized to a maximum binding
signal of 100% for each ZF-TF, and presented as the average
of duplicate experiments with standard deviations.
Proteins were tested in electrophoretic mobility shift
assays as described (Liu et al. 1997). The probes were
generated using double stranded oligonucleotides containing the recognition sites of the ZF-TF (described above)
labeled with c-[32P] ATP. The radioactive signal was taken
using a Typhoon PhosphoImager (Molecular Dynamics,
CA) and the data were analyzed using ImageQuant Software 5.2 (Molecular Dynamics, CA).
Plasmids for transfection assays
The plasmid RTBV-FL:GUS (Yin and Beachy 1995)
contains the full length RTBV promoter comprised of the
nucleotides (nt) -731 to ?45, RTBV-E:GUS (Yin and
Beachy 1995) contains RTBV promoter nt -164 to ?45
and RTBV-E*:GUS nt -164 to ?58; each was ligated with
the uidA open reading frame. The effector constructs were
expressed under the control of a constitutive cassava vein
mosaic virus (CsVMV) promoter (Verdaguer et al. 1998)
from plasmids designated ZF-TF2, ZF-TF3, ZF-TF8, ZFTF2S, ZF-TF3S, and ZF-TF8S; the latter three constructs
contain the SID transcription repression domain of the
human MAD protein fused to the N-terminus of the ZF-TF
protein. To develop the effector genes ZF-TF2S, ZF-TF3S,
and ZF-TF8S, the open reading frames were released from
vectors pcDNA-SIDZF-TF2, pcDNA-SIDZF-TF3, and
pcDNA-SIDZF-TF8 using the restriction enzymes NheI
and HindIII. ZF-TF2, ZF-TF3, and ZF-TF8 were obtained
from pcDNA-ZF-TF2VP64, pcDNA-ZF-TF3VP64, and
pcDNA-ZF-TF8VP64; the VP64 domain was removed
using AscI and PacI. The vector was reassembled using
HindIII and ApaI in order to clone the coding sequence into
a plasmid containing the CsVMV promoter. An SV40 large
T nuclear localization signal (NLS) was inserted in the
ZF-TFs between SID and the ZF-TF protein and in the
ZF-TFs at the C-terminal of the protein.
623
Gene constructs ZF-TF2-8 and ZF-TF2S-8S were created adding the ZF-TF8 and ZF-TF8S removed from their
respective plasmids with NotI and placed into the HindII
site in the polylinker of the binary vector already containing the ZF-TF2 and ZF-TF2S constructs.
Transient expression assays
Protoplasts isolated from suspension cultures of tobacco
BY-2 cells (N. tabacum L., cv. Bright Yellow-2) were
transfected via electroporation as described by Watanabe
et al. (1987). The protoplasts were co-transfected with a
mixture of DNAs, including 10 lg of reporter gene construct, 20 lg of effector DNA, 5 lg of internal control
plasmid containing the chimeric gene p35S:GFP, and
10 lg of herring sperm DNA using a discharge of 125 lF
and 300 V through disposable 0.4 cm cuvettes. Each
transient assay was repeated three times per experiment
and each experiment was conducted three times.
Plant transformation
Plasmids containing the effector constructs were transferred
by electroporation into A. tumefaciens strain GV3101.
Agrobacterium isolates that contained the respective plasmids were used to transform a homozygous line of A. thaliana Col-0 that contained the reporter gene RTBV-E:GUS
by the standard floral dip method (Clough and Bent 1998).
T1 seeds were germinated in Murashige and Skoog medium
(Murashige and Skoog 1962) containing glufosinateammonium (10 mg/l) and carbenicillin (250 mg/l) and
seedlings that survived the selection were grown in soil in a
growth chamber, allowed to self-fertilize, and T2 seeds were
collected. T2 seeds were germinated in selective medium
and seedlings that survived selection and exhibited a segregation ratio of 3: 1 for glufosinate resistance were grown
in soil in the growth chamber (200 lmol m-2 s-1 12 h
light/12 h dark, 22°C, 50% HR), allowed to self-fertilize,
and T3 seeds were collected. T3 seeds were germinated in
Murashige and Skoog medium containing glufosinateammonium and homozygous seedlings were grown in soil in
the growth chamber (200 lmol m-2 s-1 12 h light/12 h
dark, 22°C, 50% HR), allowed to self-fertilize, and T4 seeds
were collected.
Gene constructs for plant transformation
Fluorescence GUS assays
Gene constructs ZF-TF2, ZF-TF8, ZF-TF2S, and ZF-TF8S
were removed from their respective plasmids with NotI and
placed into the BamHI site in the polylinker of the vector
pGJ366. The vector pGJ366 is a modification of the plasmid pPZP200 (Hajdukiewicz et al. 1994) and provides
resistance of transgenic plants to glufosinate-ammonium
(BASTA).
GUS extraction buffer was added to homogenized leaf
samples from the transgenic plants and the mixture was
centrifuged. The supernatants were used to determine
enzymatic activity by the method of Jefferson et al. (1987).
Enzyme activity was determined by quantifying fluorescence using a spectrofluorometer (Spectramax Gemini,
123
624
Molecular Devices, CA) with 365-nm excitation wavelength and 455-nm emission wavelength. Green fluorescence protein (GFP) activity was determined by
quantifying fluorescence using 490 nm excitation wavelength and 530 nm emission wavelength. The concentration of total protein in the extract was measured by the
dye-binding method of Bradford (1976).
Histological GUS assay
Samples of leaf tissue were collected and stained with filter-sterilized 5-bromo-4-chloro-3-indolyl b-D-glucuronide
(X-Gluc, Glycosynth, UK) staining solution (50 mM
phosphate buffer, pH 7.0, 0.5 mM potassium ferricyanide,
0.5 mM potassium ferrocyanide, 0.2% Triton X-100, 0.5%
DMSO, 20% methanol, 2 mM EDTA, 1 mM X-Gluc).
After vacuum filtration samples were incubated at different
periods of time at 37°C. At the appropriate time, the
X-Gluc solution was replaced and chlorophyll was
removed with 70% ethanol. Tissues were examined with an
Olympus SZX12 microscope with bright-field optics.
Northern blot assays
RNA was purified from leaf tissues using TRIZOL (Invitrogen, CA) following the manufacturer’s instructions.
Total RNA (50 lg) was separated on a denaturing 1.5%
agarose gel made with Northern Max 10x Denaturing Gel
Buffer (Ambion, TX). The RNA was then transferred to
and cross linked to Hybond N-positive membrane (Amersham, PA). After washing the membrane with prehyb/
hybridization buffer (Ambion, TX), the membrane was
incubated with a radioactive DNA probe containing the
gene sequence encoding the ZF-TF8 protein and the first
600 bases pairs of the uidA gene. The membrane was
hybridized overnight at 42°C and later washed to remove
nonspecifically bound radioactivity. Images were taken of
the membrane using a Typhoon PhosphoImager (Molecular
Dynamics, CA) and the data analyzed using ImageQuant
Software 5.2 (Molecular Dynamics, CA).
Plant Mol Biol (2010) 72:621–630
AF113832 (Ic), X57924 (Phi-1), M65026 (Phi-2), D10774
(Phi-3, Cabauatan et al. 1999), AF076470 (Serdang, Marmey et al. 1999), AF220561 (Chainat isolate, Nathwong
et al. 2000, unpublished), AJ314596 (West Bengal, Nath
et al. 2002), NC_001914 (Hay et al. 1991). Based on evaluation of the consensus sequence of the promoter region,
several 18 bp target sites were chosen and three-six finger
ZF-TF sequences, referred to as ZF-TF2, ZF-TF3, and ZFTF8, were designed (Fig. 1 and supplementary Fig. A1).
Site selection was based on the availability of modular zinc
finger domains previously selected by phage display to bind
triplets represented in GNN, ANN, and CNN with reference
to individual sequence specificity and binding characteristics (Dreier et al. 2001, 2005; Segal et al. 1999). When
known sequences of Oryza sativa (Yu, 2002) and A. thaliana were searched for potential binding sites for these ZFTFs using the Basic Local Alignment Search Tool (BLAST)
none was found.
The ZF-TFs were designed to bind in regions conserved
among different virus isolates and without overlap with
binding sites of known plant transcription factors (e.g.,
GATA, ASL, and TATA box). The binding sites for
ZF-TF3 and ZF-TF8 overlap with each other on sequences
proximal to the Box II cis element. Previous work revealed
that transcription factors RF2a and RF2b bind to Box II
(Dai et al. 2004; Yin et al. 1997b) and it was proposed that
the virus requires RF2a and RF2b, and perhaps other
Results
Design of ZF-TFs to bind the RTBV promoter
The aim of this study was to reduce expression of genes
driven by the promoter of RTBV using artificial zinc finger
transcription factors (ZF-TFs). We first compared the
sequences of ten different isolates of RTBV from various
regions of South and Southeast Asia where the virus is
endemic. EMBL accession numbers of the virus sequences
compared were AF113830 (G1), AF113831 (G2),
123
Fig. 1 Diagram of the E fragment of the RTBV promoter with the
location of the different sequences targeted by the designed zinc
finger proteins (ZF-TFs)
Plant Mol Biol (2010) 72:621–630
Transient reporter gene assays in protoplasts
A transient assay in tobacco BY-2 protoplasts was used to
assess the ability of the ZF-TFs to regulate expression of
the genes driven by the RTBV promoter in plant cells. The
reporter construct contained the full length RTBV promoter (RTBV-FL, nt 2731 to ?45) ligated with the uidA
(GUS) coding sequence to create RTBV-FL:GUS (Yin and
Beachy 1995). The ZF-TFs with and without the SID (S)
repressor domain (Ayer et al. 1996) fused at the amino
terminus were evaluated. The SID domain is known to act
as repressor in human cells and in some plant cells (Ayer
et al. 1996; Beerli et al. 1998; Stege et al. 2002). The
effector genes were placed under the control of the cassava
vein mosaic virus (CsVMV) promoter (Verdaguer et al.
1998). These constructs were designated ZF-TF2, ZF-TF3,
ZF-TF8, ZF-TF2S, ZF-TF3S, and ZF-TF8S (Fig. 3a).
The reporter gene was introduced (by electroporation)
alone or co-introduced with the effector constructs into
BY-2 protoplasts. Three independent experiments were
conducted, each with three samples per experiment. GUS
activity was measured 24 h after electroporation using a
quantitative GUS assay (Jefferson et al. 1987). As shown in
(A) 100
ZF-TF
protein
-
-T
F3
ZF
-T
ZF
-T
ZF
(B)
Probe
F8
zf-2
zf-3
zf-8
90
80
70
60
50
40
30
20
10
0
F2
RELATIVE BINDING
(O.D. 405 nm)
factors, for replication (Dai et al. 2006). Previous data
demonstrated that targeting ZF-TFs bearing repression
domains to the 50 UTR of the human ErbB2 and ErbB3
genes efficiently reduced expression of these promoters
(Beerli et al. 1998, 2000b). To determine if this was successful in plant cells ZF-TF2 was designed to bind a highly
conserved region within the 50 UTR of the primary RTBV
transcript (Fig. 1).
The expression vectors for the ZF-TFs were built using
PCR-based gene assembly with oligonucleotides containing the desired sequences. Three-finger proteins targeting
9-bp half-sites were first constructed by PCR overlap
extension and then assembled as pairs to produce proteins
that contain 6-finger domains. These ORFS were then
cloned into the pMAL expression vector and the ZF-TFs
were expressed in E. coli. The fusion proteins were tested
using multi-target DNA binding ELISA as described (Segal
et al. 1999). The binding specificity of each of the pMALZF-TF2, pMAL-ZF-TF3, and pMAL-ZF-TF8 fused to the
maltose binding protein was measured individually for
biotinylated hairpin-loop DNA oligonucleotides containing
the three different target sequences (Fig. 2a). Also, electrophoretic mobility shift assays were carried out using the
fusion proteins containing the ZF-TFs against the different
binding recognition sequences used as probes (Fig. 2b).
Even though zf-3 and zf-8 target sequences share 11
nucleotides, there was no cross-reaction with the heterologous ZF-TFs in these assays. Each ZF-TF shows excellent specificity for its target sequence (Fig. 2).
625
zf-2
zf-3
zf-8
2 3 8 -
2 3 8 -
2 3 8
Fig. 2 Specific binding of the recognition sequences with the ZF-TF.
a Multi-target DNA specificity ELISA of the different ZF-TFs. The
six-zinc finger proteins ZF-TF2, ZF-TF3 and ZF-TF8 were tested
individually for binding to each other DNA target site zf-2 (black
bars), zf-3 (grey bars) and zf-8 (white bars) in duplicate experiments.
The maximum binding signal for each protein was normalized to
100%. b Electrophoretic mobility shift assay using different ZF-TFs.
The duplex DNA oligonucleotides containing the difference recognition sequence zf-2, zf-3 and zf-8 were labeled with c [32P]ATP and
used as a probe. 50 lg of different ZF-TFs were used into gel shift
reactions with the probes containing the three different target
sequences
Fig. 3b and considering RTBV-FL:GUS as 100% of GUS
activity: protoplasts that also contained ZF-TF2 exhibited
60% GUS activity; ZF-TF2S, 57%; ZF-TF3S, 70%; ZFTF8, 67% and ZF-TF8S, 49%. In contrast ZF-TF3 did not
reduce expression of the reporter gene. ZF-TF2 and ZFTF8 reduced GUS activity between 35 and 40%. Constructs that contained the SID domain did not reduce GUS
production to a greater extent than ZF-TF2, while in the
case of ZF-TF3 reduction was minor, and in ZF-TF8 was
about 15%.
We conducted similar experiments using the RTBV-E*
promoter (nt -164 to ?58 (data not shown). Compared
with the amount of enzyme activity produced by RTBVE*:GUS (set at 100%), ZF-TF2 permitted 42% of GUS
activity; ZF-TF2S permitted 35%; ZF-TF3 permitted 80%;
ZF-TF3S permitted 72%; ZF-TF8 permitted 76% and ZFTF8S permitted 48%. In the case of the RTBV-E promoter
(nt -164/?45) (data not shown), and considering activity
produced by the construct RTBV-E:GUS as 100%, ZF-TF2
produced 50% of GUS activity; ZF-TF2S permitted 39%;
ZF-TF8 permitted 63% and ZF-TF8S permitted 43%. The
degree of reduction of gene expression was similar in the
RTBV-FL and the RTBV-E* promoter.
123
626
Fig. 3 Analysis of the effect of ZF-TFs on expression of the RTBV
promoter in tobacco BY-2 protoplasts. a Diagram of the RTBV full
length promoter (comprising nt -731 to ?45), E (nt -164 to ?45)
and E* (nt -164 to ?58) fused to the uidA gene and the plasmids
containing the CsVMV promoter driving the genes encoding ZF-TF2,
ZF-TF3, ZF-TF8, or ZF-TFs fused to the SID repressor domain, i.e.,
ZF-TF2S, ZF-TF3S and ZF-TF8S. b Expression of GUS activity in
BY-2 protoplasts transfected with the RTBV full length promoter and
plasmids containing the CsVMV promoter driving the genes encoding
ZF-TF2, ZF-TF3, ZF-TF8, ZF-TF2S, ZF-TF3S and ZF-TF8S. Quantification of GUS activity from extracts 24 h after transfection.
Protoplasts were co-transfected with a mixture of 10 lg of the
reporter gene, 20 lg of plasmids encoding ZF-TFs, 5 lg of the 35SGFP plasmid and 10 lg of herring sperm DNA. Results are expressed
as amount of GUS activity compared with GFP activity and expressed
as a percentage of activity produced by the RTBV-FL:GUS plasmid.
The results are the average of three independent transfection reactions
±SD
Activity of the ZF-TFs in transgenic Arabidopsis plants
To evaluate the activity of the ZF-TFs in Arabidopsis, ZFTFs or ZF-TFSs were introduced into the vector pGJ366
and used to develop transgenic plants using BASTA as the
selectable agent. Constructs containing individual effectors, i.e., ZF-TF2, ZF-TF8, ZF-TF2S and ZF-TF8S, a
combination of two effectors (ZF-TF2-8 plus ZF-TF2S-8S)
were introduced into a line that contained the reporter gene
RTBV-E:GUS. The RTBV-E fragment comprises nt -164
to ?45 (Fig. 4a). Plants resistant to BASTA were grown in
a growth chamber and analyzed via PCR for the presence
123
Plant Mol Biol (2010) 72:621–630
Fig. 4 Effect of ZF-TFs on expression of RTBV-E:GUS in T1
transgenic Arabidopsis plants. a Diagram of the plasmids used to
transform a transgenic plant line that contains the RTBV-E (nt -164
to ?45):GUS reporter construct. The effector constructs contain the
CsVMV promoter fused to genes encoding single ZF-TFs; i.e., ZFTF2, ZF-TF8, ZF-TF2S or ZF-TF8S or two ZF-TFs, i.e., ZF-TF2-8 or
ZF-TF2S-8S. b GUS activity in leaves of 15 T1 Arabidopsis plants
(6 weeks old). The activity of the transgenic plant line RTBV-E:GUS
was normalized to 100%
of the reporter and effector genes. At least 15 transgenic T1
plants containing one of each of the different ZF-TFs were
analyzed at 6 weeks after planting.
The GUS activity in extracts of leaves from the line
containing the reporter gene only was defined as 100%. T1
plant lines that also contained ZF-TF2 exhibited an average
decrease of 60% in GUS activity. Plant lines that contained
ZF-TF8 showed an average decrease in GUS activity of
80%, whereas plant lines with ZF-TF2 plus ZF-TF8 (ZFTF2-8) showed an average decrease of 60%. ZF-TF2S
caused a slight increase in repression when compared with
ZF-TF2, although the SID domain did not increase the
repression by ZF-TF8 (Fig. 4b). The results of these studies
demonstrate that a suitable ZF-TF can cause a significant
reduction in GUS activity and that the SID domain does not
enhance the effect significantly.
The T1 plants were allowed to self-fertilize and T2 seeds
of plants containing ZF-TF8 and ZF-TF8S were studied.
We did not recover fertile plants from lines that produced
ZF-TF2. Fifteen seedlings from six different lines with
segregation ratios of 3:1 were grown for 6 weeks and GUS
activity was determined in leaf extracts. The repression of
GUS activity in these lines ranged from 20 to 60% (data
not shown).
Plant Mol Biol (2010) 72:621–630
T2 plants were allowed to self-fertilize and T3 seeds
were collected; homozygous seeds that produced seedlings
under selective conditions were analyzed. Sixteen seedlings each from six different homozygous lines were grown
in soil for 6 weeks, leaf extracts were obtained, and GUS
activity was determined. The GUS activity in the homozygous lines containing ZF-TF8 was reduced 50–80%
compared with the activity in extracts of the parent plant
line that contained only the reporter gene. GUS activity in
the line containing ZF-TF8 plus the SID domain (ZFTF8S) was reduced 20–30% (Fig. 5a). These studies confirmed that ZF-TF8 was capable of significantly reducing
expression of genes driven by the RTBV promoter.
627
(Fig. 4a). In these analyses a decrease in GUS activity was
correlated with reduced levels of cognate mRNA. Samples
analyzed from tissue that contained ZF-TF8S had levels of
uidA mRNA similar to the controls. Similarly, the levels of
GUS activity were only slightly reduced in plants that
expressed ZF-TF8S relative to plant lines that expressed
only the reporter gene. Plant lines with high levels of ZFTF8 mRNA had low levels of uidA mRNA and GUS
activity. Furthermore, there was a strong positive correlation between GUS activity (Fig. 5a) and the levels of uidA
mRNA (Fig. 5b), and low levels of GUS activity were
correlated with high levels of ZF-TF8 mRNA (Fig. 5b).
Histochemical analysis of GUS activity
Correlation of GUS activity with mRNA levels
RNA was isolated from leaves from the parent line and
from several lines that contain RTBV-E:GUS and ZF-TF8
or ZF-TF8S. Non-transgenic Col-0 was used as a negative
control in these studies. Total RNA (50 lg) was separated
in a denaturing gel and transferred to a nylon membrane.
The levels of expression of the ZF-TFs were determined
using a probe containing sequences from uidA and ZF-TF8
Tissues from six plants from homozygous lines containing
the reporter gene alone or with ZF-TF8 were collected and
GUS activity was detected by staining with 5-bromo-4chloro-3-indolyl b-D-glucuronide (X-Gluc). All samples
were stained for identical periods of time to avoid saturation of the reaction. Non-transgenic Col-0 plants were used
as negative control. We did not observe staining in any of
the tissues of the non-transgenic plants. In plants containing the GUS reporter gene alone or with ZF-TF8, stain was
observed primarily in the vascular system as reported in
tobacco and rice (Petruccelli et al. 2001; Yin et al. 1997b).
However, the intensity of staining was greater in plants
containing only the reporter plasmid than in plants that
produced the ZF-TF (Fig. 6). From this data we concluded
that the presence of ZF-TF8 under the control of a constitutive promoter altered the amount of GUS in cells in
vascular tissues. These observations are in agreement with
the quantitative assays reported above.
Discussion
Fig. 5 Effect of ZF-TF8 alone or with the SID repressor domain on
GUS expression, activity and mRNA levels, in homozygous Arabidopsis transgenic lines. a GUS activity in leaves of T3 homozygous
Arabidopsis plants (6 weeks old) containing the RTBV-E:GUS
plasmid and the CsVMV promoter fused to genes encoding the ZFTF8 alone or fused with the SID repressor, ZF-TF8S. b Northern blot
to determine the presence of uidA and ZF-TF8 mRNA. Leaf extracts
from the same plants were used to obtain total RNA. 50 lg of total
RNA were loaded in each lane from young leaves taken from multiple
plants from each line. The probe used was made from the first 600 bp
from the amino terminal end of the uidA gene and the full gene of ZFTF8. The GUS activity of the transgenic plant line RTBV-E:GUS was
normalized to 100%
The goal of this study was to determine the effectiveness of
artificial zinc finger transcription factors (ZF-TFs) to
reduce expression of genes driven by the RTBV promoter
in transgenic plants and plant cells. ZF-TFs have been
previously used in strategies to control replication and/or
infection by viruses, including the DNA virus herpes
simplex virus and the retrovirus HIV (Eberhardy et al.
2006; Kim et al. 2005; Papworth et al. 2003; Reynolds
et al. 2003; Segal et al. 2004), in mammalian cells. Sera
(2005) described a ZF-TF that restricts the replication of
the single-stranded-DNA-containing Geminivirus beet
curly top virus by binding to the origin of replication.
Three target regions in the RTBV promoter were
selected based on the consensus sequences of 10 isolates of
RTBV, and three-six-finger transcription factors, ZF-TF2,
ZF-TF3, and ZF-TF8, were designed to bind to these
123
628
Plant Mol Biol (2010) 72:621–630
Fig. 6 Histochemical
localization of GUS in wild type
Col-0 and transgenic
Arabidopsis plants containing
RTBV-E:GUS alone or plus the
ZF-TF8 driven by the
constitutive CsVMV promoter.
In leaf tissue, cross section of
stem tissue, whole roots, flowers
and siliques. GUS activity was
localized following staining of
tissue slices with X-Gluc and
visualizing the indigo dye
precipitates formed via light
microscopy
sequences. ZF-TFs offer high specificity in sequence
recognition and are anticipated to regulate expression of
target genes with minimal side effects. A search of genomic sequences of A. thaliana and O. sativa did not reveal
identical matches with the predicted binding sites of the
designed ZF-TFs. However, there were two potential
binding sites in the genome A. thaliana for ZF-TF2 that
each contained a single mismatch. Interestingly, we did not
recover viable T2 seeds from transgenic plants that produced ZF-TF2, a result that suggests that this protein, but
not ZF-TF3 or ZF-TF8, interfered with fertility, perhaps by
binding to DNA sequences that are important in these
processes. These results indicate the importance of careful
design and testing of ZF-TFs.
123
Binding sites for each of the ZF-TFs tested in this study
are proximal to the TATA Box of the promoter (Fig. 1).
The proximity of the target sequence to the TATA box was
critical to the efficacy of ZF-TFs in regulation of gene
expression in plants in other studies (Guan et al. 2002;
Ordiz et al. 2002; Stege et al. 2002). ZF-TF3 and ZF-TF8
binding sites are proximal to the Box II cis element of the
RTBV promoter, a region known to bind to transcription
factors RF2a and RF2b; these factors activate expression of
the promoter (Dai et al. 2004; Yin et al. 1997b). ZF-TF2
binds in the 50 UTR region of the genome-length transcript.
The efficacy of the ZF-TFs was first tested in BY-2
protoplasts in an attempt to predict their activity in whole
plants. ZF-TF2 reduced GUS activity by 40% compared to
Plant Mol Biol (2010) 72:621–630
controls, and addition of the SID domain (ZF-TF2S) did
not increase repression. ZF-TF3 did not repress GUS
activity except in constructs that contained the SID domain
(ZF-TF3S). ZF-TF8 repressed GUS activity by 35% and
addition of the SID domain (ZF-TF8S) increased repression to 50%. These data agree with previous studies that
indicated that the SID domain provides a modest
enhancement of repression of gene expression by ZF-TFs
in plant cells (Stege et al. 2002).
In T1 Arabidopsis plants that expressed ZF-TF2 and ZFTF8, GUS gene expression was reduced between 60 and
80%, i.e., greater than the effects observed in transient
assays. These results may indicate that repression is more
complete when the target gene is integrated into the host
genome vs as free DNA. In homozygous populations,
repression of the reporter gene ranged from 20 to 30%
among different transgenic lines that expressed ZF-TF8S
and between 50 and 80% among lines that contained ZFTF8. This data indicates a strong negative regulatory effect
of expression of uidA gene driven by the RTBV promoter
by the ZF-TF8 protein in the absence of the SID domain.
The data presented here support previous results that
location of the binding site of the ZF-TFs within the Box II
element provides efficient repression (Dai et al. 2006). The
fact that ZF-TF2 binds downstream of this element and was
effective may reflect some regulatory activity within this
area (He et al. 2000, 2002) or may imply that the area is
easily accessible to binding by the protein. In support of the
hypothesis that other regulatory proteins may bind in this
area and may compete with binding of the relevant ZF-TF,
we note that the repression of activity of ZF-TFs used in
this study was not significantly increased through fusion
with the SID repression domain. This result is in contrast to
the results reported by Beerli et al. (1998) and Guan et al.
(2002), where addition of the SID domain to ZF-TFs
substantially improved repression. Since the SID domain is
not an exceptionally potent repressor in plants, the activity
of ZF-TFs might be further improved by addition of a
different repression domain.
Based on these studies, we conclude that the RTBV
promoter can be negatively regulated using six-finger
ZF-TFs. The possibility that these or other ZF-TFs can
reduce replication of RTBV in transgenic rice plants
remains to be demonstrated, and presents a potential
application of ZF-TFs to control of an important plant
disease.
Acknowledgments We thank Dr. Jitender Yadav for his assistance
with the RNA work and Dr. Shunhong Dai for providing some
materials and for helpful discussions during the early stages of this
work. This study was supported by Department of Energy grant DOEFG02-99ER20355, and NASA grant NNJ04HG98G to RNB and by a
grant from The Skaggs Institute for Chemical Biology to CFB.
629
References
Ayer DE, Laherty CD, Lawrence QA, Armstrong AP, Eisenman RN
(1996) Mad proteins contain a dominant transcription repression
domain. Mol Cell Biol 16:5772–5781
Beerli RR, Segal DJ, Dreier B, Barbas CF III (1998) Toward
controlling gene expression at will: specific regulation of the
erbB-2/HER-2 promoter by using polydactyl zinc finger proteins
constructed from modular building blocks. Proc Natl Acad Sci
USA 95:14628–14633
Beerli RR, Schopfer U, Dreier B, Barbas CF 3rd (2000a) Chemically
regulated zinc finger transcription factors. J Biol Chem
275:32617–32627
Beerli RR, Dreier B, Barbas CF 3rd (2000b) Positive and negative
regulation of endogenous genes by designed transcription
factors. Proc Natl Acad Sci USA 97:1495–1500
Blancafort P, Segal DJ, Barbas CF 3rd (2004) Designing transcription
factor architectures for drug discovery. Mol Pharmacol 66:1361–
1371
Bradford MM (1976) A rapid and sensitive method for the
quantitation of microgram quantities of protein utilizing the
principle of protein-dye binding. Anal Biochem 72:248–254
Cai CQ, Doyon Y, Ainley WM, Miller JC, Dekelver RC, Moehle EA,
Rock JM, Lee YL, Garrison R, Schulenberg L, Blue R, Worden
A, Baker L, Faraji F, Zhang L, Holmes MC, Rebar EJ,
Collingwood TN, Rubin-Wilson B, Gregory PD, Urnov FD,
Petolino JF (2009) Targeted transgene integration in plant cells
using designed zinc finger nucleases. Plant Mol Biol 69:699–709
Clough SJ, Bent AF (1998) Floral dip: a simplified method for
Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743
Dai S, Petruccelli S, Ordiz MI, Zhang Z, Chen S, Beachy RN (2003)
Functional analysis of RF2a, a rice transcription factor. J Biol
Chem 38:36396–36402
Dai S, Zhang Z, Chen S, Beachy RN (2004) RF2b, a rice bZIP
transcription activator, interacts with RF2a and is involved in
symptom development of rice tungro disease. Proc Natl Acad Sci
USA 101:687–692
Dai S, Zhang Z, Bick J, Beachy RN (2006) Essential role of the Box
II cis element and cognate host factors in regulating the promoter
of Rice tungro bacilliform virus. J Gen Virol 87:715–722
Dreier B, Beerli RR, Segal DJ, Flippin JD, Barbas CF 3rd (2001)
Development of zinc finger domains for recognition of the
50 -ANN-30 family of DNA sequences and their use in the
construction of artificial transcription factors. J Biol Chem
276:29466–29478
Dreier B, Fuller RP, Segal DJ, Lund CV, Blancafort P, Huber A,
Koksch B, Barbas CF 3rd (2005) Development of zinc finger
domains for recognition of the 50 -CNN-30 family DNA
sequences and their use in the construction of artificial
transcription factors. J Biol Chem 280:35588–35597
Durai S, Mani M, Kandavelou K, Wu J, Porteus MH, Chandrasegaran
S (2005) Zinc finger nucleases: custom-designed molecular
scissors for genome engineering of plant and mammalian cells.
Nucleic Acids Res 33:5978–5990
Eberhardy SR, Goncalves J, Coelho S, Segal DJ, Berkhout B, Barbas
CF 3rd (2006) Inhibition of human immunodeficiency virus type
1 replication with artificial transcription factors targeting the
highly conserved primer-binding site. J Virol 80:2873–2883
Englbrecht CC, Schoof H, Bohm S (2004) Conservation, diversification and expansion of C2H2 zinc finger proteins in the
Arabidopsis thaliana genome. BMC Genomics 5:39–56
Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, Barbas CF
3rd, Briggs SP (2002) Heritable endogenous gene regulation in
123
630
plants with designed polydactyl zinc finger transcription factors.
Proc Natl Acad Sci USA 99:13296–13301
Hajdukiewicz P, Svab Z, Maliga P (1994) The small, versatile pPZP
family of Agrobacterium binary vectors for plant transformation.
Plant Mol Biol 25:989–994
Hay JM, Jones MC, Blakebrough ML, Dasgupta I, Davies JW, Hull R
(1991) An analysis of the sequence of an infectious clone of rice
tungro bacilliform virus, a plant pararetrovirus. Nucleic Acids
Res 19:2615–2621
He X, Hohn T, Futterer J (2000) Transcriptional activation of the rice
tungro bacilliform virus gene is critically dependent on an
activator element located immediately upstream of the TATA
box. J Biol Chem 275:11799–11808
He X, Futterer J, Hohn T (2001) Sequence-specific and methylationdependent and -independent binding of rice nuclear proteins to a
rice tungro bacilliform virus vascular bundle expression element.
J Biol Chem 276:2644–2651
He X, Futterer J, Hohn T (2002) Contribution of downstream
promoter elements to transcriptional regulation of the rice tungro
bacilliform virus promoter. Nucleic Acids Res 30:497–506
Holmes-Davis R, Li G, Jamieson AC, Rebar EJ, Liu Q, Kong Y, Case
CC, Gregory PD (2005) Gene regulation in planta by plantderived engineered zinc finger protein transcription factors. Plant
Mol Biol 57:411–423
Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions bglucuronidase as a sensitive and versatile gene fusion marker in
higher plants. EMBO J 6:3901–3907
Kandavelou K, Chandrasegaran S (2009) Custom-designed molecular
scissors for site-specific manipulation of the plant and Mammalian genomes. Methods Mol Biol 544:617–636
Kim YS, Kim JM, Jung DL, Kang JE, Lee S, Kim JS, Seol W,
Shin HC, Kwon HS, Van Lint C, Hernandez N, Hur MW
(2005) Artificial zinc finger fusions targeting Sp1-binding sites
and the trans-activator-responsive element potently repress
transcription and replication of HIV-1. J Biol Chem 280:
21545–21552
Liu Q, Segal DJ, Ghiara JB, Barbas CF 3rd (1997) Design of
polydactyl zinc-finger proteins for unique addressing within
complex genomes. Proc Natl Acad Sci USA 94:5525–5530
Lloyd A, Plaisier CL, Carroll D, Drews GN (2005) Targeted
mutagenesis using zinc-finger nucleases in Arabidopsis. Proc
Natl Acad Sci USA 102:2232–2237
Magnenat L, Blancafort P, Barbas CF 3rd (2004) In vivo selection of
combinatorial libraries and designed affinity maturation of
polydactyl zinc finger transcription factors for ICAM-1 provides
new insights into gene regulation. J Mol Biol 341:635–649
Marmey P, Bothner B, Jacquot E, de Kochko A, Ong CA, Yot P,
Siuzdak G, Beachy RN, Fauquet CM (1999) Rice tungro
bacilliform virus open reading frame 3 encodes a single 37kDa coat protein. Virology 253:319–326
Murashige T, Skoog F (1962) A revised medium for rapid growth and
bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Nath N, Mathur S, Dasgupta I (2002) Molecular analysis of two
complete rice tungro bacilliform virus genomic sequences from
India. Arch Virol 147:1173–1187
Ordiz MI, Barbas CF 3rd, Beachy RN (2002) Regulation of transgene
expression in plants with polydactyl zinc finger transcription
factors. Proc Natl Acad Sci USA 99:13290–13295
Papworth M, Moore M, Isalan M, Minczuk M, Choo Y, Klug A
(2003) Inhibition of herpes simplex virus 1 gene expression by
designer zinc-finger transcription factors. Proc Natl Acad Sci
USA 100:1621–1626
Petruccelli S, Dai S, Carcamo R, Yin Y, Chen S, Beachy RN (2001)
Transcription factor RF2a alters expression of the rice tungro
bacilliform virus promoter in transgenic tobacco plants. Proc
Natl Acad Sci USA 98:7635–7640
123
Plant Mol Biol (2010) 72:621–630
Porteus MH, Carroll D (2005) Gene targeting using zinc finger
nucleases. Nat Biotechnol 23:967–973
Reynolds L, Ullman C, Moore M, Isalan M, West MJ, Clapham P, Klug
A, Choo Y (2003) Repression of the HIV-1 50 LTR promoter and
inhibition of HIV-1 replication by using engineered zinc-finger
transcription factors. Proc Natl Acad Sci USA 100:1615–1620
Sanchez JP, Ullman C, Moore M, Choo Y, Chua NH (2002)
Regulation of gene expression in Arabidopsis thaliana by
artificial zinc finger chimeras. Plant Cell Physiol 43:1465–1472
Sanchez JP, Ullman C, Moore M, Choo Y, Chua NH (2006)
Regulation of Arabidopsis thaliana 4-coumarate:coenzyme-A
ligase-1 expression by artificial zinc finger chimeras. Plant
Biotech J 4:103–114
Segal DJ, Dreier B, Beerli RR, Barbas CF III (1999) Toward
controlling gene expression at will: selection and design of zinc
finger domains recognizing each of the 50 -GNN-30 DNA target
sequences. Proc Natl Acad Sci USA 96:2758–2763
Segal DJ, Goncalves J, Eberhardy S, Swan CH, Torbett BE, Li X,
Barbas CF 3rd (2004) Attenuation of HIV-1 replication in
primary human cells with a designed zinc finger transcription
factor. J Biol Chem 279:14509–14519
Sera T (2005) Inhibition of virus DNA replication by artificial zinc
finger proteins. J Virol 79:2614–2619
Shukla VK, Doyon Y, Miller JC, DeKelver RC, Moehle EA, Worden
SE, Mitchell JC, Arnold NL, Gopalan S, Meng X, Choi VM,
Rock JM, Wu YY, Katibah GE, Zhifang G, McCaskill D,
Simpson MA, Blakeslee B, Greenwalt SA, Butler HJ, Hinkley
SJ, Zhang L, Rebar EJ, Gregory PD, Urnov FD (2009) Precise
genome modification in the crop species Zea mays using zincfinger nucleases. Nature 459:437–441
Stege JT, Guan X, Ho T, Beachy RN, Barbas CF 3rd (2002)
Controlling gene expression in plants using synthetic zinc finger
transcription factors. Plant J 32:1077–1086
Van Eenennaam AL, Li G, Venkatramesh M, Levering C, Gong X,
Jamieson AC, Rebar EJ, Shewmaker CK, Case CC (2004)
Elevation of seed alpha-tocopherol levels using plant-based
transcription factors targeted to an endogenous locus. Metab Eng
6:101–108
Verdaguer B, de Kochko A, Fux CI, Beachy RN, Fauquet C (1998)
Functional organization of the cassava vein mosaic virus
(CsVMV) promoter. Plant Mol Biol 37:1055–1067
Watanabe Y, Mushi T, Okada Y (1987) Infection of tobacco protoplasts
with in vitro transcribed tobacco mosaic virus RNA using an
improved electroporation method. FEBS Lett 219:65–69
Wright DA, Townsend JA, Winfrey RJ Jr, Irwin PA, Rajagopal J,
Lonosky PM, Hall BD, Jondle MD, Voytas DF (2005) Highfrequency homologous recombination in plants mediated by
zinc-finger nucleases. Plant J 44:693–705
Wu YM, Mao X, Wang SJ, Li RZ (2004) Improving the nutritional
value of plant foods through transgenic approaches. Sheng Wu
Gong Cheng Xue Bao 20:471–476
Wu J, Kandavelou K, Chandrasegaran S (2007) Custom-designed
zinc finger nucleases: what is next? Cell Mol Life Sci 64:2933–
2944
Yin Y, Beachy RN (1995) The regulatory regions of the rice tungro
bacilliform virus promoter and interacting nuclear factors in rice
(Oryza sativa L.). Plant J 7:969–980
Yin Y, Chen L, Beachy R (1997a) Promoter elements required for
phloem-specific gene expression from the RTBV promoter in
rice. Plant J 12:1179–1188
Yin Y, Zhu Q, Dai S, Lamb C, Beachy RN (1997b) RF2a, a bZIP
transcriptional activator of the phloem-specific rice tungro
bacilliform virus promoter, functions in vascular development.
EMBO J 16:5247–5259
Yu JI (2002) A draft sequence of the rice genome (Oryza sativa L.
ssp. Indica). Science 296:79–92
Supplementary figure A1. DNA sequence alignment and consensus within the promoter
region of 10 different virus isolates of Rice Tungro Bacilliform Virus (RTBV). Identical
regions are in red, conserved blocks are in orange and regions of low homology are in
green. Positions of known plant transcription factors (ASL and TATA boxes, virus ciselements Boxes I and II) (thin arrows) and 18 bp target sites of designed six-zinc finger
transcription factors ZF-TF2, -3 and -8 (thick arrows) are represented.
1