163
Zinc fingers and a green thumb: manipulating gene
expression in plants
David J Segaly, Justin T Stegez and Carlos F Barbas III§
Artificial transcription factors can be rapidly constructed
from predefined zinc-finger modules to regulate virtually any
gene. Stable, heritable up- and downregulation of
endogenous genes has been demonstrated in transgenic
plants. These advances promise new approaches for creating
functional knockouts and conditional overexpression, and
for other gene discovery and manipulation applications in
plants.
Addresses
The Skaggs Institute for Chemical Biology and the Department of
Molecular Biology, The Scripps Research Institute, La Jolla,
California 92037, USA
y
Department of Pharmacology and Toxicology, University of Arizona,
Tucson, Arizona 85721, USA
z
Diversa Corporation, San Diego, California 92121, USA
§
The Scripps Research Institute, BCC-550, North Torrey Pines Road,
La Jolla, California 92037, USA
e-mail: carlos@scripps.edu
Correspondence: Carlos F Barbas III
Current Opinion in Plant Biology 2003, 6:163–168
This review comes from a themed issue on
Plant biotechnology
Edited by Wolf B Frommer and Roger Beachy
1369-5266/03/$ – see front matter
ß 2003 Elsevier Science Ltd. All rights reserved.
DOI 10.1016/S1369-5266(03)00007-4
Abbreviations
Ap3
Apetella3
SID
Sin3A interaction domain
TFsZF zinc-finger-based artificial transcription factor
Introduction
The manipulation of plant traits in basic plant biology
research and agricultural biotechnology would be greatly
facilitated if endogenous genes-of-interest could be
turned on or off in a controlled and selective manner.
Altering the expression of specific target genes could give
rise to healthier, hardier or more nutritious crop plants by
increasing disease/stress resistance, altering metabolic
pathways or repressing the production of anti-nutritive
proteins. Specific genes could be activated or repressed to
elucidate the complex interactions involved in many
important processes. In this review, we describe several
approaches to the manipulation of gene regulation in
plants, focusing in detail on one promising new method
that involves zinc-finger-based artificial transcription factors (TFsZF; Figure 1).
www.current-opinion.com
A variety of techniques have been developed to manipulate gene expression in plants. Increased expression of
endogenous genes is most commonly achieved through
transgene overexpression [1]. The introduction of tissuespecific and inducible promoters has improved the utility
of this approach, and efficient and robust plant transformation techniques have made the construction of transgenes
a relatively routine task. However, variable expression and
co-suppression of transgenes often complicate this process.
Furthermore, transgenes cannot accommodate alternative
splicing, which may be important for the appropriate
function of some transgenes [2].
Reducing or eliminating the expression of a gene in plants
is not as simple as overexpressing a gene. Gene disruption
by homologous recombination, tDNA insertions and chemical mutagenesis has been used successfully, but these
approaches are inefficient and time-consuming technologies. Several promising approaches for the repression of
gene expression in animal systems have been described,
operating either at the transcriptional or posttranscriptional
level [3–6]. Many of these techniques have also been
applied in plants. For example, one of the first demonstrations that hammerhead ribozymes could function as active
endoribonucleases in vivo was performed in plant protoplasts [7]. Triplex-forming oligonucleotides, which bound
to homopurine/homopyrimidine tracts in a maize promoter, have been shown to represses gene expression in vivo
[8]. Chimeric RNA–DNA oligonucleotides have been
used to cause site-specific base changes in episomal and
chromosomal targets in plant cells [9]. The use of antisense
RNA or DNA to reduce mRNA levels was first explored in
tobacco more than a decade ago [10]; today this approach
remains one of the most commonly used methods to
regulate genes in plants. More recently, double-stranded
RNA interference (RNAi) has been employed to produce
specific and heritable genetic repression in Arabidopsis [11].
Back to nature: gene regulation with
transcription factors
In nature, the expression of eukaryotic nuclear genes is
tightly regulated at both the transcriptional and the
translational level. Much of this control is achieved
through DNA-binding transcription factors. Transcription factors are modular proteins that typically consist
of a DNA-binding domain that localizes the protein to a
specific DNA address and an effector domain that directs
the type of activity to take place at the site [12,13].
One conceptual approach to gene manipulation is the engineered expression of specific endogenous transcription
Current Opinion in Plant Biology 2003, 6:163–168
164 Plant biotechnology
Figure 1
proteins in DNA recognition is perhaps best reflected by
their success in natural systems. The Cys2-His2 zincfinger domain is the most common DNA-binding motif
in nature [23]. With 4500 examples identified, it is by far
the most frequently encoded protein domain in the
human genome [24], and examples have also been found
in various plants [24,25].
The rise and fall of universal recognition
codes
In canonical-type zinc fingers, the amino acids in positions 1, 2, 3, and 6 of the a-helix make base-specific
contacts [26]. Early observations of zinc-finger proteins
gave rise to speculation that a particular amino acid in
each position could recognize a particular base. The hope
was that a simple universal one-amino-acid to one-basepair (1aa:1bp) recognition code could be divined that
would allow the construction of new DNA-binding proteins. Research carried out over the past several years has
found, however, that simple recognition codes are insufficient to predict true binding specificity [26,27,28–
31,32]. The primary weakness of simple recognition
codes is that they fail to consider the influence of other
amino acids within the helix and between helices. As a
further complication, a given sequence can often be
recognized by helices containing different amino acids.
Zinc-finger-based artificial transcription factors (background) have been
applied in plants such as Arabidopsis (foreground).
factors that have evolved to control particular genes. The
whole-genome sequencing of Arabidopsis [14] and more
recently rice [15], combined with informatics-based analysis, has identified numerous putative plant transcription
factors [15,16]. However, the identification and characterization of the molecular targets of these transcription
factors is still at a very early stage. Consequently, it is not
yet possible to use them broadly as gene-specific tools to
regulate endogenous gene expression.
Recently, several studies have targeted endogenous
genes in cultured mammalian cells using synthetic TFsZF
[2,17–20,21]. Among the many naturally occurring
DNA-binding proteins, the Cys2-His2 zinc-finger domain
has emerged as the scaffold of choice for the design of
novel sequence-specific DNA-binding proteins [22].
Within these proteins, each 30-amino-acid domain, or
finger, forms a stable bba fold. The amino-terminus of
the a-helix typically recognizes a 3-bp subsite in the DNA
(Figure 2). The recognition of extended DNA sequences
is achieved by linking the domains into tandem arrays.
These target-recognizing domains can be found in arrays
of up to 37 repeats [23], facilitating the recognition of
extended DNA sequences. The versatility of zinc-finger
Current Opinion in Plant Biology 2003, 6:163–168
More recent efforts have focused on the randomization of
the entire recognition helix and the selection of proteins
with new binding specificities. Several successful construction methods have been described [32,33,34] and
compared in detail elsewhere [22,35,36]. The most
commonly used approach is based on the assembly of
pre-defined zinc-finger modules that have been previously selected and optimized [17,30,33]. Each module
originated as the middle finger in a three-finger protein,
which was subsequently selected to recognize a new 3-bp
binding site using phage-display technology. The
selected modules were further refined by site-directed
mutagenesis to provide optimal binding specificity. The
modules can be assembled in any order necessary to form
new three- or six-finger proteins. With each domain
specifying 3-bp, a six-finger protein should have the
capacity to bind one of almost 70 billion unique 18-base
pair sites. The human genome contains around 3.2 billion
base pairs of DNA, and so a six-finger protein has the
potential to recognize a unique site in the human genome. Only 64 modules are required to recognize all
possible 3-bp sites. To date, modules have been reported
that can bind a majority of these sites, enabling the
construction of more than one billion different six-finger
proteins, approximately 27 000 proteins for every gene
in the human genome. New proteins can now be
constructed by any molecular biology laboratory using
this modular assembly method, relying solely on the
PCR-based assembly of published zinc-finger modules
(Figure 2; [37]).
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Zinc fingers and a green thumb: manipulating gene expression in plants Segal, Stege and Barbas 165
Figure 2
(d)
(a)
(
(b)
F1
AP3 gene
)
(e)
5′-TAC TTC TTC AAC TCC ATC-3′
3′-ATG AAG AAG TTG AGG TAG-5′
F1
F2
F3
F4
F5
F2
F1
F3
F2
F4
F3
F4
F5
F5
F6
F6
SID-
F6
(c)
GAA
GAC
GAG
GAT
QSSNL VR
DPGNL VR
RSDNL VR
TSGNL VR
GCA
GCC
GCG
GCT
QSGDL RR
DCRDL AR
RSDDL VR
TSGEL VR
GGA
GGC
GGG
GGT
QRAHL ER
DPGHL VR
RSDKL VR
TSGHL VR
GTA
GTC
GTG
GTT
QSSSL VR
DPGAL VR
RSDEL VR
TSGSL VR
(f)
AP3 gene
SID-
(
)
Current Opinion in Plant Biology
Modular construction of artificial transcription factors. (a) The DNA sequence of the gene to be regulated (in this case AP3) is searched for (b) an
18-bp binding site that can be recognized using a combination of existing zinc-finger domains. Sequence-specific recognition domains (F1–F6) are
selected for each 3-bp subsite in the target sequence using (c) a table of optimized zinc-finger modules. (Note: 3-bp subsites in (c) are written 50 to 30 ,
those in (b) appear in the opposite orientation.) A more complete table is provided in [37]. (d) The coding region for the new DNA-binding protein is
assembled from overlapping oligonucleotide primers using PCR. (e) Appending an activation or repression domain (a SID repression domain is shown)
creates an artificial transcription factor that is capable of (f) endogenous gene regulation.
Into the plant
The attachment of an appropriate effector domain to a
zinc-finger protein creates potent transcriptional activators and repressors. Activation domains such as VP16 [38]
and p65 [39] and repression domains such as KRAB
(Krüpple-associated box) [40] and SID (Sin3A interaction
domain) [41] are components of naturally occurring transcription factors. All of these domains are able to regulate
a variety of mammalian promoters in a distance- and
orientation-independent manner when fused to zinc-finger proteins [2,17–20,21]. These domains exert their
effect by recruiting global activation and repression complexes to the promoter site. Because this recruitment
often involves specific protein–protein interactions, effector domains that are found in one cell type may not
function in a different cell type or species. For example,
the KRAB domain is a potent transcriptional repression
domain in mammalian systems. This domain is not found
in Drosophila melanogaster, Caenorhabditis elegans, Saccharomyces cerevisiae, or Arabidopsis thaliana [24], however, and
would therefore be a poor candidate for gene regulation in
plants. By contrast, the SID domain interacts directly
with the highly conserved Sin3A protein and its homologues [41], and is therefore a more appropriate candidate
to regulate endogenous plant genes.
Plant-specific TFsZF have a diverse range of potential
applications for agriculture. Until recently, however, two
crucial issues remained to be addressed before the
application of artificial plant-specific TFsZF could
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become a reality: the function and stability of TFsZF
in transgenic multicellular organs and the ability of these
genes to be stably inherited by subsequent generations.
Several studies on the regulation of plant genes by
TFsZF have been recently published. An in vitro assay
has been used to evaluate the activity of several different
TFsZF constructs on a variety of target reporter configurations in plant cells. The effective repression and
activation of reporter genes was dependent on the promoter strength and the location of the site that binds the
zinc-finger protein [42]. A b-glucuronidase (GUS)
reporter gene stably integrated in tobacco plants was
activated to high levels in transgenic plants using an
artificial TFsZF activator [43]. This activation was
stable over multiple generations, indicating that TFsZF
functions are stably inherited and non-toxic in plants.
These results were supported by evidence from a study
by Guan et al. [44] in which TFsZF were designed to
target the Apetella3 (Ap3) promoter in Arabidopsis for
activation or repression. When expressed in a tissuespecific manner, these reporters were able to activate or
repress an Ap3::GUS reporter in a stable, inheritable
manner. Furthermore, homeotic transformations of the
floral organs were evident in these transgenic plants,
consistent with the well-established model for Arabidopsis floral-organ identity [45]. These results indicate that
the Ap3-specific TFsZF is able to manipulate the expression of the endogenous AP3 gene (Figure 3). Again, the
TFsZF used in these studies were constructed from
predefined and engineered domains.
Current Opinion in Plant Biology 2003, 6:163–168
166 Plant biotechnology
Figure 3
(b)
AP1::VP64::AP3
Ap3 activator
p
VP64-
(a)
f
Activation of Ap3 in sepals causes
partial sepal → petal transformation
se
pe
ca
st
Wildtype flower
(c)
SID-
AP1::SID::AP3
Ap3 repressor
Repression of Ap3 in petals causes
partial petal → sepal transformation
Current Opinion in Plant Biology
(a) In wildtype flowers, AP1 is expressed in the sepals (se) and petals (pe) whereas AP3 is expressed in the petals and stamen (st). When AP1 alone is
expressed in a floral organ, a sepal is formed. When AP1 and AP3 are expressed together, a petal is formed. (b) Expression of an Ap3-specific
activator (VP64::AP3) with the Ap1 promoter (in sepals and petals) causes a partial homeotic transformation of the sepals into petals. (c) Expression of
an Ap3-specific repressor (SID::AP3) with the Ap1 promoter (again in sepals and petals) causes a partial homeotic transformation of the petals into
sepals. Photographs reproduced from [44]. ca, carpel.
Conclusions
Recent reports demonstrate the potential of artificial
transcription factor technology to target specific plant
genes for up- or downregulation. Further research will
focus on the identification of potent, robust, plant-specific
activation and repression domains, and on the optimization of TFsZF design to provide effective, specific regulation of target gene(s). Once this approach has been
demonstrated to be a robust technology that can be used
to manipulate the expression of any given plant gene, it
will have many applications in agricultural biotechnology.
Repressing the expression of anti-nutritive or allergenic
proteins would increase the value and quality of many
important crop plants. The expression of virus-specific
TFsZF could provide effective resistance to plant viruses
that replicate through double-stranded DNA intermediates. TFsZF could also be used to activate the expression of
key genes that are involved in disease and stress resistance
to make more hardy, productive crop plants. As the genes
involved in metabolite biosynthesis are characterized,
TFsZF could be used to make tissue-specific changes to
improve the flavor or nutritional value of many plants.
TFsZF could be designed to alter the expression of key
regulatory gene(s), and used alongside information from
Current Opinion in Plant Biology 2003, 6:163–168
genome sequencing to dissect the complex genetic interactions involved in many important processes such as
disease/stress resistance, morphogenesis and metabolite
biosynthesis. Taken a step further, complex physiological
changes could be achieved through the tissue-specific or
inducible expression of a TFsZF that is designed to control
a pathway or family of genes. Biosynthetic pathways
might be engineered at the level of transcription, with
competing pathways silenced in an orchestrated fashion.
Future studies will also take advantage of libraries of
combinatorial transcription factors with predefined specificities. In this approach, libraries of TFsZF are introduced into cells potentially to turn every gene in the
genome either off or on/up within a population of cells in
which each cell is modulated by a unique transcription
factor. This forward-genetic approach then allows cells
that display a desired phenotype to be selected. The
TFsZF responsible for the desired phenotypic modification can then be recovered. This method has further
potential as a powerful tool for gene discovery. As the
18-bp binding site for six-finger TFsZF can be deduced
from their primary sequence, their unique genomic binding sites and associated gene of action can be identified.
The use of libraries of three-finger TFsZF that recognize
9-bp binding sites would make the elucidation of the
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Zinc fingers and a green thumb: manipulating gene expression in plants Segal, Stege and Barbas 167
target gene more challenging. However, the reduced
specificity of three-finger TFsZF in comparison with
six-finger TFsZF potentially allows several genes or gene
families to be regulated potentially by a single TFZF.
The use of three-finger TFsZF may then induce more
complex phenotypes. The power of this combinatorial
approach is that potent transcriptional regulators can be
obtained even when the targeted gene(s) is unknown.
The potential of this approach has been demonstrated in
cell lines in which endogenous regulators for a variety of
human genes have been selected. In plants, the combinatorial approach may be applied at the organismal level
[46]. Libraries of TFsZF could readily be delivered using
Agrobacterium transformation to create large libraries of
whole plants that could be screened or selected for novel
plant phenotypes.
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44. Guan X, Stege J, Kim M, Dahmani Z, Fan N, Heifetz P, Barbas CF III,
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Nat Biotechnol 2003, in press.
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gene regulation that uses libraries of TFsZF in human cells. This approach
allows libraries of TFsZF to be applied for forward genetics in cells and
whole organisms.
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