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Engineering polydactyl zinc-finger
transcription factors
Roger R. Beerli1,2 and Carlos F. Barbas, III1*
The availability of rapid and robust methods for controlling gene function is of prime importance not only for
assigning functions to newly discovered genes, but also for therapeutic intervention. Traditionally, gene function has been probed by often-laborious methods that either increase the level of a gene product or decrease
it. Advances now make it possible to rapidly produce zinc-finger proteins capable of recognizing virtually any
18 bp stretch of DNA—a sequence long enough to specify a unique address in any genome. The attachment
of functional domains also allows the design of tailor-made transcription factors for specific genes. Recent
studies demonstrate that artificial transcription factors are capable of controlling the expression of endogenous genes in their native chromosomal context with a high degree of specificity in both animals and plants.
Dominant regulatory control of expression of any endogenous gene can be achieved rapidly and can be also
placed under chemical control. A wide range of potential applications is now within reach.
Phenotypic variation results not only from changes in genes themselves but also from changes in the coordination of gene expression and
in expression levels that together tremendously broaden the phenotypic spectrum available to an organism. Transcription is controlled by
transcription factors. Natural transcription factors usually have at least
two functional domains, a DNA-binding domain and an effector
domain. The DNA-binding domain acts as a targeting device to localize
the protein to a specific site on chromosomal DNA, whereas the effector domain functions to direct the localization of specific enzymes to
this site, ultimately enabling transcription of the gene to be up- or
downregulated1. Attempts at artificial control of gene expression are
often based on the application of this fundamental principle.
To this end, large numbers of effector domains have been described
that mediate either gene activation2 or repression3. These domains are
usually modular and retain their function when attached to a heterologous DNA-binding domain. However, for the construction of designer
transcription factors, DNA-binding domains with customized specificities have become available only recently. This review summarizes
recent progress in the production of zinc-finger DNA-binding
domains, describes the first successful applications of artificial transcription factors, and discusses the potential use of these proteins in
research and therapy.
Engineering zinc-finger DNA-binding specificity
The Cys2-His2 zinc-finger domain represents the most common DNAbinding motif in eukaryotes and the second most frequently encoded
protein domain in the human genome4–6. Some 700 or more of our
30,000–40,000 genes encode zinc-finger domains of this type, ∼2% of
our genes. Although these approximately 30 amino acid domains can
have functions beyond DNA binding, they typically function by binding 3 base pairs of DNA sequence. This structurally simple ββα domain
is stabilized by hydrophobic interactions and the coordination of a zinc
ion by the eponymous cysteine and histidine residues7. Cys2-His2 zincfinger domains are particularly well suited for the construction of syn-
1The
thetic transcription factors as they are commonly arranged as covalent
tandem repeats, allowing the recognition of extended asymmetrical
sequences. This modularity in both structure and function is the key
advantage of zinc-finger proteins (ZFPs) compared with other types of
DNA-binding domains that typically recognize DNA as dimers and use
nonmodular recognition domains8.
Sequence-specific DNA recognition in Cys2-His2 zinc-finger
domains is achieved by presentation of an α-helical reading head into
the major groove of the double helix. The first x-ray crystal structure of
a ZFP bound to DNA was that of the murine three-finger transcription
factor Zif268 and revealed that base-specific interactions are made by
amino acids protruding from the N terminus of the α-helix7,9. Contacts
are primarily made with one strand of the DNA double helix, with
positions –1, 3, and 6 of each finger (with respect to the start of the αhelix) contacting the 3′-, middle, and 5′-nucleotides of a 3 bp subsite,
respectively.
To further elucidate the principles of zinc-finger DNA recognition,
rational design10–12, the selection of large combinatorial libraries displayed on phage13–17, and a combination of these two approaches have
been applied18–20. Indeed, phage display of ZFPs was one of the first
applications of protein phage display proposed21. Selection protocols
often lead to impressive amino acid conservation for recognition of the
same nucleotide in different target sequences13–20. Most notably, an
arginine was typically found at positions –1 and 6 for recognition of a 3′
and 5′ guanine, respectively.
Other, albeit less impressive, amino acid conservations have also
been observed. However, although these studies laid the foundation for
future applications of designer ZFPs, early hopes that DNA recognition
could be explained by a simple recognition code of one amino acid to
one nucleotide have not materialized. DNA binding is more complex,
and the residues flanking the primary contact residues at positions –1,
3, and 6 play important roles in specificity, both allowing specific interactions to be made and excluding bases from the recognition site18–20.
Practically, this means that design alone is not sufficient and that selec-
Skaggs Institute for Chemical Biology and the Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037. 2Current address: Cytos
Biotechnology AG, Wagistrasse 21, 8952 Zurich-Schlieren, Switzerland. *Corresponding author (carlos@scripps.edu).
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3F
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ED
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Figure 1. Zinc-finger–based DNA recognition and modification
devices. (A) Transcription of transgenes and endogenous target genes
can be regulated by fusing designer ZFPs recognizing asymmetrical
DNA sequences of varying length to transcriptional effector domains.
Transcription factors containing three zinc-finger domains and
recognizing 9 base pairs (bp) of DNA sequence have been described
to mediate gene activation or repression22,35,51,52. (B) For genomic
specificity, transcription factors containing six zinc-finger domains that
recognize 18 bp of sequence have been reported20,22,26. (C) Genes can
be placed under the control of a chemical inducer by fusion of a ZFP
with the ligand-binding domain of a nuclear hormone receptor, a
process involving the formation of homodimers. The dimeric form
recognizes 18 bp of DNA through the interaction of paired three-finger
proteins23,57. (D) Monomeric gene switches that directly bind 18 bp of
DNA sequence are produced by fusion of six zinc-finger domains and
an effector domain to a designed single-chain nuclear hormone
receptor ligand-binding domain23. (E) Peptides can be selected from
libraries for the dimerization of proteins containing two zinc-finger
domains25. (F) Various types of other interaction domains, such as the
leucine zippers of Jun and Fos, have been used to direct the
heterodimerization of two zinc-finger proteins24. (G) Fusion with an
endonuclease domain from Fok1 mediates dimerization that restores
nuclease activity capable of directing the introduction of sequencespecific double-strand breaks, for example, to stimulate homologous
recombination60. 2F, two-finger protein; 3F, three-finger protein; 6F, sixfinger protein; ED, effector domain; LBD, nuclear hormone receptor
ligand-binding domain.
ED
3F
6F
E
F
2F
2F
Fos
Jun
2F
2F
G
Fok1
tion strategies must remain an integral part in the modification of zincfinger DNA-binding specificity. Domains obtained through selective
approaches are, however, not fail-safe with respect to specificity and
often benefit from refinement through a rational design cycle18–20.
domain within a zinc-finger protein can permit a rapid assessment
of any given domain’s specificity29,30. This approach is conceptionally similar to the ELISA-based assays that are typically employed in
these studies but allows a more rapid and quantitative assay of binding against a much larger array of oligonucleotides. For the assessment of the binding specificity of more than one binding domain,
selection of the preferred binding site from random sequence pools
remains the method of choice as many billions of sequences can be
probed this way31.
Polydactyl proteins
Parallel selection
Just how many fingers are required to specify a unique address in a
genome as complex as ours? Assuming random base distribution, any
given 16 bp sequence will only occur once every 4.3 billion nucleotides
and would be sufficiently long to specify a unique address in the 3.5 billion bp human genome. An 18 bp address would be specific within 68
billion bp—or 20 human genomes—and could be targeted by a polydactyl protein containing six zinc-finger domains22. One might also
argue that chromatin infrastructure serves to protect most of the
genome from DNA-binding proteins and the target sequence length
required for specific regulation in the chromosomal context is much
lower. In any case, the genomic specificity of any designed transcription
factor needs to be probed empirically.
Specificity can also be obtained by combinatorial interactions of
ZFPs with shorter recognition sequences on the target gene provided
that the individual constituents alone are inactive. Binding can either
be independent, if the two 9 bp sites are far apart, or mediated by specific dimerization domains, if the two sites are close23,24. Novel synthetic peptides suitable for the heterodimerization of ZFPs have also
recently been reported25. The use of a single six-finger protein, however,
has the important advantage that it requires only one gene to be delivered18,20,22,23,26–28.
A variety of zinc-finger-based recognition devices have now been
reported (see Fig. 1). Three phage display strategies for the construction
of such polydactyl proteins have been described and involve either parallel, sequential, or bipartite selection (Fig. 2). All methods have distinct
advantages and disadvantages when compared with one another, and
the protein products from any given methodology should be rigorously characterized8. The recent development of rapid DNA array assays as
applied to DNA–protein interactions should assist in this analysis.
Microarrays containing all possible 3 bp binding sites for a given
Work in our laboratory has focused on the development and use of the
parallel selection strategy. The basic assumption of this approach is that
zinc-finger domains are functionally independent and can therefore be
recombined with one another in any desired sequence (Fig. 2A). Thus,
a complete zinc-finger domain complement of the genetic code would
consist of 64 domains specific for each DNA triplet. Stitching together
predefined zinc-finger domains in the appropriate sequence then
allows any DNA sequence to be targeted. Zinc fingers can either be
linked together using the canonical linker22, or using one or more noncanonical linkers27,28,32. The use of some noncanonical linker peptides
has been reported to lead to improvements in DNA affinity and specificity for certain proteins. While a consensus in the field has not yet
been formed as to the most optimal noncanonical linker and (perhaps
more importantly) the most general strategy for domain linking, a variety of approaches all seem to work sufficiently well. Ideally, no further
selection of the assembled protein is needed in this approach.
To date, we have described zinc-finger domains specific for half the
genetic code. These domains bind the 5′-GNN-3′ and 5′-ANN-3′
(where N is any one of the four nucleotides) families of DNA triplets
and were produced by phage display selection protocols followed by
optimization through systematic site-directed mutagenesis studies18–20.
Although these domains constitute half of the possible domains, it is
important to consider how many polydactyl proteins these domains
can access. The 5′-GNN-3′ set of domains alone is sufficient for the
construction of 1.7 × 107 new proteins binding the 5′-(GNN)6-3′ family of DNA sequences. Assuming random base distribution, 5′-(GNN)63′ sequences should occur approximately every 2,048 bp; however, as
eukaryotic promoter regions are relatively G+C rich, 5′-(GNN)6-3′
sequences are more abundant and multiple sites are found in most
human promoters. For instance, a 1,400 bp fragment of the human
3F
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C
erbB-2 promoter contains 11 A
sequences of 5′-(GNN)6-3′ (see
Fig. 3). Practically, this means
that every gene might be regulated by targeting 5′-(GNN)6-3′
sequences.
Although the number of suitable target sites in a given gene of
interest may be sufficient, not
every target may be available for
binding by an artificial transcription factor because of local chromatin structure, covalent modification, or occupation of the site Figure 2. Strategies for the production of ZFPs with desired DNA-binding specificity. (A) Parallel selection. (B)
by an endogenous DNA-binding Sequential selection. (C) Bipartite selection. Asterisks indicate preselected libraries. See text and references for
protein. If we now consider that details.
addition of the 5′-(ANN)-3′
Selection of this library ensures that the new finger two works well in
binding domains20 provide targeting of 5′-(RNN)6-3′ (where R = G or A) sequences, more than one
the context of the finger one selected in the previous round. In the final
billion transcription factors become accessible. Binding sites for these
step, the last remaining anchor finger is discarded and a randomized
proteins should occur every 32 bp, and an analysis of the 1,400 bp
finger three is attached to the C terminus, again followed by selection.
human erbB-2 promoter fragments reveals the presence of 77 such
In this manner, each finger of the new three-finger protein is selected in
sites. Thus, with just half the set, ∼27,000 times more transcription facthe context of its neighboring finger, preventing any potential problems
tors can be constructed than there are genes in the human genome. To
caused by target site overlap.
date, the parallel selection approach is the only one validated at the level
The target specificity of these three-finger proteins is similar to that
of endogenous gene regulation.
of stitched proteins8,35,37. Although sequential selection undoubtedly is
a useful method, it does require the generation and selection of six zincThe parallel selection approach for the production of new specificifinger libraries for each protein, making this approach inaccessible to
ties has the important advantage that it uses predefined domains and
most laboratories. Sequential selection may be essential for targeting
should not require additional design or selection, making it extremely
some sequences wherein no flexibility exists in target choice, for examrapid and accessible to any laboratory. However, it should be noted that
ple, in using the zinc finger to protect a specific site in the genome to
complete functional independence of zinc-finger domains oversimpliprobe the function of an endogenous factor binding at the same site.
fies their binding mechanism in some cases. The four 5′-(GNG)-3′
Recently the crystal structure of a sequentially selected protein in
zinc-finger domains appear to specify a 4 bp site rather than the typical
complex with its TATA box target sequence has been reported38. This
3 bp site. This was first noted on inspection of the Zif268 structure, in
structure demonstrates how interwoven the interactions of zinc fingers
which an aspartate in position 2 of finger 2 contacts the binding site of
can be when this type of sequential selection strategy is applied, implyfinger 1, forcing that site to be either GNN or TNN (refs 7,9,33,34).
ing that proteins developed using the three methods described here will
Fortunately, several strategies can be used to overcome or avoid
likely use different mechanisms for the molecular recognition of DNA.
problems imposed by target site overlap resulting from the occurrence
of an aspartate at position 2 of the 5′-GNG-3′ domains. First, polyBipartite selection
dactyl proteins can be produced within the structural constraints
This recently developed method aims at combining the distinct advanimposed by target site overlap. Specifically, the exclusive use of 5′tages of the two approaches described above39. It makes use of a pair of
GNN-3′ or 5′-TNN-3′ domains avoids this problem, as does the placeprefabricated zinc-finger libraries, in each of which one-and-a-half finment of a 5′-GNN-3′- or 5′-TNN-3′-type finger located N-terminal of
gers of the three-finger ZFP Zif268 are randomized (Fig. 2C). Selection
any 5′-GNG-3′ finger in otherwise mosaic proteins constructed with
of these two libraries is carried out in parallel against DNA sequences in
5′-ANN-3′ or 5′-CNN-3′ domains20,26,35. Alternatively, if the necessity
arises to combine two fingers in an otherwise incompatible manner,
which either the first or the last 5 bp of the 9 bp Zif268 target site are
residues at the interface of the two α-helices including position 2 can be
exchanged against a sequence of interest. After phage display selection,
randomized, allowing selection of optimal amino acid residues on a
the two libraries are combined and further rounds of selection are percase-by-case basis36. The practical implications of target site overlap to
formed on the 9 bp sequence of interest. The bipartite selection
the parallel approach exist primarily on paper. In reality, the large numapproach has yielded several three-finger proteins binding predefined
ber of predefined zinc-finger domains currently available makes this
sequences in various regions of the HIV-1 promoter, with Kd values in
the nanomolar range. Though not extensively analyzed, these proteins
problem a practical nullity for gene targeting.
appear to have specificities similar to those produced using the parallel
Sequential selection
and sequential selection strategies.
The sequential selection protocol pioneered by the Pabo laboratory
The bipartite selection strategy represents a potential advance
addresses the problems imposed by target site overlap and cooperativiin the field of zinc-finger engineering, as not only concerns of tarty in zinc-finger DNA recognition (Fig. 2B). As the name implies,
get site overlap and zinc-finger cooperation are addressed, but also
DNA-binding specificities of individual zinc-finger domains are altered
the time required to evolve new specificities is dramatically shortsequentially in the context of the other zinc fingers, rather than in parened compared with sequential selection. Although this approach
allel17. Thus, finger three of the three-finger protein Zif268 is replaced
may allow the targeting of any DNA sequence, it is important to
by a finger one in which the critical amino acid residues have been rankeep in mind that it does involve many rounds of phage display
domized. This library is then selected in the context of the two original
selection. For the regulation of a specific gene of interest, typically
fingers, which serve as “anchors”. After selection, the N-terminal anchor
there is a lot of flexibility in the choice of the precise target site
finger is removed and a finger two library is attached to the C terminus.
location. As a result, the use of predefined domains that do not
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300
400
500
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structure and promoter
proximity. The distinct
advantage of a transcription
factor–based approach is
that both gene repression
and activation can be
orchestrated in trans.
Gene repression
6xGNN
With respect to gene repression, it is possible to make a
functional knockout of a
transcription start
gene by simply adding a
transgene rather than resorttranslation start
ing to breeding homozygous
knockout organisms using
Figure 3. Structure of a 1,400 bp human erbB-2 promoter fragment. The major site of transcription initiation (light gray
more traditional approaches.
triangle) and the position of the ATG translation initiation codon (dark gray triangle) are indicated. The fragment was
Theoretically, gene repressearched for the presence of suitable 18 bp target sequences. White circles, 5´-(GNN)6-3´ sites; black circles, 5′-(RNN)6sion by designer DNA-bind3′ sites (R = guanine or adenine).
ing proteins can be achieved
in a number of ways, includrequire additional design or selection likely remains the fastest
ing the following: (1) fusing a DNA-binding domain to a transcriptionroute to gene regulation.
al repression domain; (2) targeting a site in the transcribed region of a
gene and interfering with transcription elongation; (3) targeting a
Alternative selection strategies
sequence in the immediate vicinity of the transcription initiation site
Typically, phage
display
strategies
require
multiple
and interfering with the assembly of the transcription machinery; or
enrichment/amplification cycles and are rather time-consuming,
(4) targeting a site upstream of a gene of interest that is naturally bound
especially if DNA-binding domains with new specificities have to
by a coactivator, thus preventing coactivator binding. The first three of
be evolved de novo. As an alternative to phage display, several
these strategies have been investigated on transiently transfected
genetic screens have been described that are suitable for the selecreporter plasmids, on integrated reporter constructs, or on endogenous
tion of new DNA-binding specificities.
genes. These approaches should provide access to the spectrum of gene
A genetic screen in bacteria has been used to produce mutants
knockdown to knockout phenotypes.
of the basic region-leucine zipper protein C/EBP that recognized
Our laboratory has concentrated on the first strategy using zinc-finsequences differing in two of its five half-site nucleotides40. In
ger–effector domain fusion proteins. One important advantage of this
addition, zinc-finger domains with altered DNA-binding specificistrategy is that it provides great flexibility in the choice of the target
ty can be identified in a genetic screen, as has recently been shown
sequence, as most effector domains act in a distance- and orientationby the Pabo laboratory41. In this work, a bacterial variant of the
independent manner. Furthermore, the use of effector domains allows
yeast two-hybrid system has been developed and employed for the
not only the downregulation of gene activity, but also upregulation or
selection of zinc-finger domains with novel specificities. The
activation (see below).
newly selected zinc finger variants have affinities and specificities
Several modular transcriptional repressor domains have been
comparable to those obtained by phage display17. Finally, new
described and can be incorporated into designed transcription factors.
DNA-binding specificities could also be produced in eukaryotic
These include the Krüppel-associated box (KRAB), a domain comcells, for example, using a yeast one-hybrid screen, a method commonly found at the N terminus of naturally occurring zinc-finger promonly used for the identification of DNA-binding proteins42,43.
teins44, and the mSin3 interaction domain (SID), a short domain found
at the N terminus of Mad that mediates repression via recruitment of a
Trans dominant gene regulation
histone deacetylase45. When fused to the six-finger protein E2C that was
For the practical regulation of a given gene, one simply requires a
designed to bind an 18 bp sequence in the 5′-untranslated region
ZFP to regulate its target gene and not other genes with sequences
(UTR) of the human proto-oncogene erbB-2, both domains mediated
related to the target gene. As a multitude of protein engineering
effective repression of an erbB-2 promoter luciferase reporter construct
studies have shown in other systems, affinity, specificity, and
in transiently transfected HeLa cells35. Regulation of endogenous genes
was explored using the six-finger proteins E2C and E3 fused to a KRAB
stability of most proteins can be further improved by in vitro
domain26. E3 binds to an 18 bp sequence in the 5′-UTR of the human
evolution methods. The designer transcription factor need not
erbB-3 gene that is identical to the E2C sequence in 15 out of 18 bases.
bind DNA with the highest possible affinity or specificity as long
These zinc-finger proteins bind their respective DNA targets with ∼0.5
as it can act to specifically regulate the target gene. There does,
nM affinity. Homologous targeting sites were chosen in the two genes
however, appear to be a threshold time for DNA occupancy necesto ascertain whether specific gene regulation was possible using ZFPs
sary for transcriptional signaling, but exceeding this occupancy
(i.e., independent regulation through closely related binding sites).
time does not appear to provide additional gains when effector
Significantly, studies demonstrated that the transcription factors effidomains are used. This threshold time appears to be met with prociently repressed their respective target genes in a highly specific manteins with DNA-binding affinities of ∼10 nM or better, and to date,
ner26. The lack of cross-regulation in this system demonstrates the very
endogenous gene regulation has been accomplished only with
high degree of specificity attainable using polydactyl zinc-finger transuch proteins.
scription factors. Moreover, the erbB-2 transcription factor was shown
While a six-finger protein provides the obvious mathematical
to regulate its endogenous gene target in mouse, monkey, and human
advantage in terms of specificity, a three-finger protein may have sufcells that conserve its binding site. Additional regulators of the endogeficient specificity when accounting for such factors as chromatin
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nous erbB-2 and erbB-3 genes have also been reported20.
The use of DNA-binding domains lacking effector domains for gene
regulation has also been studied extensively. This strategy attempts to
place a steric blockade in the path of RNA polymerase. Klug and colleagues46 have transiently expressed a three-finger ZFP in BaF3 cells
previously made interleukin 3 (IL-3)-independent by integration of a
plasmid encoding a p190BCR-ABL transgene. The ZFP-transfected cells
reverted to IL-3 dependence through repression of p190BCR-ABL expression. These results are remarkable as the ZFP employed was designed
to bind deep within the transcribed region of the gene and had a rather
low affinity (Kd = 0.6 µM). Quantitative studies from the Pabo laboratory using ZFPs that bind 1,000 times more strongly have revealed at
most twofold repression of basal transcription and no repression of
activated transcription by binding within a transcribed gene47. Kang
and Kim48 have also reported no repression upon targeting a site >50
bp downstream of the transcriptional start site of a reporter gene, again
with a protein with far greater affinity for DNA. Consistent with these
reports, our own studies with the six-finger protein E2C that binds
within the transcribed region of the erbB-2 gene with subnanomolar
affinity, yielded a modest 30% repression when this ZFP was expressed
without a fused repression domain35. In contrast, fusion of E2C to a
KRAB domain could effect complete repression of the endogenous
gene. Taken together, these studies suggest that a polymerase blockade
mechanism is typically not very effective.
Pabo and coworkers47–49 have also evaluated the potential of repressing transcription by interfering with the assembly of the transcription
machinery (i.e., mechanism 3 described above). In initial transient
transfection studies, Zif268 binding sites were placed in an artificial
promoter, at various distances from the TATA box or the transcription
initiation site47. Substantial repression of both basal and activated transcription was found when Zif268 bound either close to the TATA box
or to the initiator element. Efficient repression by blocking access to the
TATA box of a synthetic promoter was also shown with previously
unknown fusion proteins consisting of Zif268 and the TATA-binding
protein connected by a flexible peptide linker49. Finally, inhibition of
transcription by targeting sites close to the initiator element of synthetic promoter constructs has been recently shown using six-finger proteins, either upon transient transfection27 or after integration of the
reporter construct into chromosomal DNA48.
Although these data convincingly demonstrate the feasibility of targeting the transcription initiation complex, downregulation of an
endogenous gene based on this mechanism has yet to be shown.
Importantly, the targeting of endogenous genes by this strategy has the
complications that many promoters lack a TATA box and that many
genes use multiple transcription initiation sites.
Gene activation
The zinc-finger strategy is not limited to gene repression, but is readily
adapted to gene activation. Upregulation of fetal hemoglobin, for
example, could impact sickle-cell anemia, while upregulation of
growth hormone could impact dwarfism and controlled regulation of
erythropoietin and vascular endothelial growth factor (VEGF) could
be important in cancer therapy as well as diabetes.
Two major mechanisms can be employed to upregulate expression
of a specific gene: first, a DNA-binding domain can be fused to a transcriptional activation domain, upregulating transcription by actively
recruiting the transcriptional machinery50; second, a DNA-binding
protein can be designed to compete with a transcriptional repressor
bound upstream of a gene of interest, leading to gene activation without the need for an effector domain.
The first strategy has the significant advantage that it allows flexibility in choosing the precise location of the target sequence. Using zincfinger-activation domain fusion proteins, our group has recently
shown efficient upregulation of endogenous genes26. The six-finger
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proteins E2C and E3 fused to the synthetic transcriptional activation
domain VP64, a derivative of the herpes simplex virus protein VP16,
efficiently upregulated the endogenous human erbB-2 and erbB-3
genes, respectively. Flexibility in target site location was demonstrated
by the finding that both target sites are located within the 5′-UTR of the
respective gene. Thus, efficient activation can be achieved by targeting
not only promoter sequences but also sites within the transcribed
region of a gene. Several additional six-finger activators of the endogenous human erbB-2 and erbB-3 genes have also been reported20.
Other studies have further demonstrated the potential of zinc-finger-based transcriptional activators. Panels of three-finger proteins
have been designed and constructed to bind in various locations in the
promoter regions of the human erythropoietin and VEGF-A genes.
Upon fusion with VP16 or p65 activation domains, the zinc-finger
transcription factors were capable of upregulating expression from the
endogenous chromosomal loci51,52. These reports demonstrate the efficacy of three-finger proteins in activation and the manner in which
chromatin structure can effectively enhance their specificity. The activation of endogenous VEGF-A illustrates an important advantage of
this approach over simple cDNA delivery to enhance gene expression.
The VEGF-A transcript is naturally processed into three major splice
variants, and proper functional responses require the appropriate relative levels of these variants to be expressed. To date, this has only been
achieved with zinc-finger activation of the endogenous gene.
Regulating the regulators
Designer transcription factors with tailored DNA-binding specificity
facilitate useful strategies for manipulating gene expression, both in
cultured cells and in whole organisms. However, constitutive regulation
of a given target gene may not always be desirable and reversible regulation of target gene expression, preferentially by a small chemical inducer, would broaden application. Inducible gene regulation is particularly
desirable in a gene therapy setting, as it could limit side effects and
enhance safety by rendering the modulation of gene activity reversible.
One way of regulating an endogenous gene in an inducible manner
is to place the expression of the designed transcription factor under the
control of an inducible promoter. Several inducible expression systems
have been described23,53–55; one of the most prominent involves the use
of an expression cassette regulated by a tetracycline-controlled transactivator53. We recently have demonstrated that even endogenous genes
can be placed under chemically regulated control. The tetracycline-regulated expression system was adapted for the control of the endogenous erbB-2 gene in human cervical carcinoma cells26. Stably integrated
genes encoding the repressor E2C–KRAB or the activator E2C–VP64
were induced by removal of the tetracycline analog doxycycline, leading
to repression or activation of the endogenous erbB-2 gene. Level of the
chemical inducer correlated well with the level of gene product.
One complication of inducible promoter systems is that they require
the delivery of two genes: one encoding the ZFP under the control of an
inducible promoter, the other encoding the regulatory protein.
Delivery of multiple vectors is a laborious process and a hurdle in a
gene therapy setting. For the inducible control of target gene expression, it would be much more simple and efficient to regulate the activity of the transcription factor itself, rather than its expression.
Members of the nuclear hormone receptor protein superfamily are
prototypical ligand-regulated transcription factors and offer features
that can be exploited in protein engineering56. Thus, to create chemically regulated zinc-finger transcription factors, our group23 has prepared
fusion proteins containing ligand-binding domains (LBDs) derived
from progesterone, estrogen, and ecdysone receptors. These types of
zinc-finger fusion proteins could prove useful not only for targeting
endogenous genes, but also for the inducible regulation of transgenes.
To achieve inducible regulation of gene expression, we have fused
designed ZFPs with novel DNA binding specificities to a transcription•
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al activation domain, as well as to the LBDs derived from either the
estrogen or progesterone receptor (Fig. 1C). Together with optimized
minimal promoters, the progesterone- and estrogen-based transcription factors provide 4-hydroxytamoxifen- or RU486-inducible expression systems with induction ratios of up to three orders of magnitude23.
As distinct DNA-binding specificities were used, these inducible systems are functionally independent and can be selectively switched on
within the same cell, allowing the concomitant regulation of multiple
transgenes.
The therapeutic potential of an estrogen-based zinc-finger system
has now been demonstrated in vivo with the regulation of an adenovirus-delivered endostatin transgene in a mouse model57. In this system, endostatin expression could be controlled by the administration
of low levels of 4-hydroxytamoxifen to the mice. As the LBDs of nuclear
hormone receptors mediate dimerization of their fused three-finger
proteins, these transcription factors act on symmetrical 18 bp sites, sites
long enough also for the specific targeting of endogenous genes.
To further enhance the potential of ligand-dependent zinc-finger
transcription factors for the regulation of endogenous genes, our group
has used two LBDs connected by a flexible peptide linker. In this system, the response to the chemical inducer results in an intramolecular
rearrangement, rather than dimerization, leading to transcriptionally
active proteins (Fig. 1D). The significant advantage of this type of
fusion protein is that it allows targeting of extended asymmetrical
sequences, which enables ligand-dependent regulation of any promoter. By obviating the need to deliver multiple genes, these monomeric
single-chain regulators hold great promise for the inducible control of
gene expression.
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nous as well as foreign genes. The production of viral gene products
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