632
Custom DNA-binding proteins come of age: polydactyl
zinc-finger proteins
David J Segal and Carlos F Barbas III*
Artificial transcription factors based on modified zinc-finger
DNA-binding domains have been shown to activate or repress
the transcription of endogenous genes in multiple organisms.
Advances in both the construction of novel zinc-finger proteins
and our understanding of the characteristics of a productive
regulatory site have fueled these achievements.
Figure 1
F1
Addresses
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
*e-mail: carlos@scripps.edu
F2
Current Opinion in Biotechnology 2001, 12:632–637
0958-1669/01/$ — see front matter
© 2001 Elsevier Science Ltd. All rights reserved.
Abbreviations
DBD
DNA-binding domain
ED
effector domain
F3
Introduction
The ability to wilfully manipulate gene expression holds
tremendous promise for both basic and applied research.
Primary methods for understanding gene function involve
enhancing or abolishing the expression of a gene, followed
by observation of phenotypic effects. The field of functional genomics, the effort to assign function to all of the
genes identified from the sequencing of the human and
other genomes, has become the driving force for the development of methodologies that are faster, simpler, and more
broadly applicable than classical techniques. Knowledge of
gene function coupled with improved methods to regulate
gene expression is also stimulating new approaches and
advances in the development of applications such as
animal models for human disease and novel therapeutics.
Creating a system that can both upregulate and downregulate gene expression requires control at the level of
transcription. Transcriptional regulation in all living cells is
mediated by protein transcription factors. These factors
are typically composed of at least two parts: a sequencespecific DNA-binding domain (DBD) and an effector
domain (ED) [1]. The DBD directs binding of the factor to
the promoter region of a particular gene or genes, whereas
the ED acts through protein–protein interactions to recruit
components of activation or repression complexes. Many
EDs have been shown to maintain their ability to activate
or repress transcription when attached to heterologous
DBDs [2,3]. Therefore, the task of creating an artificial
transcription factor can be reduced to attaching an ED to a
custom DNA-binding protein — one that can be targeted
to any desired gene. This review describes recent
Current Opinion in Biotechnology
The structure of a three-domain zinc-finger protein (based on [43]).
Fingers 1, 2 and 3 (labeled F1, F2 and F3, respectively) are shown as
ribbons. Zinc atoms are depicted as spheres. The protein wraps
through the major groove of the DNA (wire and shaded object).
advances in zinc-finger technology that now make custom
DNA-binding proteins readily available and details how
these proteins are being used to regulate the expression of
transgenes and endogenous genes.
Construction methodology
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 sequencespecific DNA-binding proteins [4]. Each 30 amino acid
residue domain or finger forms a stable ββα fold (Figure 1).
The N terminus of the α helix recognizes a small patch
of nucleotides in the major groove of the DNA, typically
three base pairs. Recognition of extended sequences is
achieved by linking the domains in tandem arrays. The
versatility of zinc-finger proteins in DNA recognition is
perhaps best reflected by its success in natural systems.
The Cys2-His2 zinc-finger domain is the most commonly
found DNA-binding motif in eukaryotes, and the second
most frequently encoded protein domain in the human
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
633
Figure 2
Representations of (a) parallel, (b) sequential
and (c) bipartite library construction methods.
Individual fingers and their approximate
recognition sites are color-matched. Anchor
fingers are in gray. Libraries of variant fingers
are circled. Fingers selected from the library
are then used in the next step of construction
(illustrated by arrows). Helices in (b) and (c)
have magenta and cyan shading to suggest
possible interactions between domains.
(a)
(b)
(c)
Current Opinion in Biotechnology
genome [5–7]. These features have attracted researchers
intent on creating novel, sequence-specific DNA-binding
proteins. The randomization and selection methodologies
that have allowed us to endow zinc fingers with new
binding specificities are also reminiscent of nature’s own
evolutionary forces, albeit a few million years faster.
Three successful selection methods have been described.
All have been based on the display of randomized zinc-finger proteins from the surface of filamentous bacteriophage,
although an alternative in vivo bacterial system has also
been described [8•]. The parallel and sequential selection
schemes have been compared recently [4,9]. In the parallel approach (Figure 2a), DNA-contacting residues in the
middle finger of a three-finger protein are randomized to
form a library of variants [10–13,14•,15••,16,17]. The two
flanking fingers are unmodified and serve to anchor and
appropriately orient the middle finger. Using phage display, variants are selected that recognize a new three-base
pair sequence in the middle of their binding site.
Following selection and optimization of fingers that can
recognize each of the 64 possible three-base pair subsites,
the fingers can be assembled in any order necessary to
form new three- or six-finger proteins capable of binding
any desired sequence. With each domain specifying three
base pairs, a six-finger protein should have the capacity to
bind one of almost 70 billion unique 18 base-pair sites.
When presented with the 3.2 billion base pairs of DNA in
the human genome, a six-finger protein has the potential
to recognize a unique site.
As the selection process is not dependent on any particular
DNA target sequence, all 64 required domains could be
selected in parallel. An advantage of this method is that
once selection and optimization are complete, new
proteins can be constructed in a matter of hours using standard PCR methods. Consequently, this type of method has
been adopted by our laboratory [11,15••,18••] and for commercial purposes [19••,20••]. Potential limitations of this
approach lie in its underpinning assumptions: that each
domain recognizes only three base pairs and that the
domains can be assembled in any order. The major limitation of this strategy is seen in the phenomenon called
target-site overlap that occurs in Zif268. This occurs
because one of the anchor fingers of this protein actually
recognizes a four base-pair subsite, overlapping the subsite
of the middle finger and forcing specification of its
5′ nucleotide to be G or T [21]. Indeed, our early studies
bypassed these concerns by focusing on fingers capable of
recognizing members of the 5′-GNN-3′ set of DNA
sequences [12,14•]. The limitations imposed by target-site
overlap have, however, recently been overcome.
Sequential selection (Figure 2b) was developed to address
the concerns of target-site overlap [22••,23,24]. In this
method, a terminal finger of a three-finger protein is
randomized while the other two act as anchors. However,
following selection of a finger that recognizes a new DNA
subsite, one of the anchor fingers is removed and a new
library is appended to the previously selected finger. A
third cycle of removal, appendage addition, and selection
634
Pharmaceutical biotechnology
Figure 3
(a)
ED =
VP64
DBD =
Zinc finger
Activation
(b)
ED =
KRAB
DBD =
Zinc finger
Repression
Current Opinion in Biotechnology
Transcriptional activation or repression by a TFZF bound in the 5′ UTR of
its target gene. (a) A TFZF composed of a custom zinc-finger DBD and a
VP64 ED (a derivative of VP16 [11]) causes activation (indicated by a
bold arrow). (b) A TFZF composed of a custom zinc-finger DBD fused to
the KRAB ED leads to repression (indicated by a bold cross).
creates a sequence-specific three-finger protein. In this
way, the new finger is always selected in the ‘context’ of
the previous finger, with no assumptions about overlap or
modularity. A recent crystal structure of a sequentially
selected protein with its cognate DNA seems to validate
these concerns. Some fingers were found to recognize not
only four base pairs but five, and interdomain interactions
were observed [22••]. The primary disadvantage of this
system is that multiple rounds of library construction and
selection are required for each new DNA target. A six-finger protein would require six sequential construction and
selection steps, making the procedure extremely laborious
and beyond the technical reach of most laboratories.
Recently, a new method was reported that attempts to
embrace the virtues of the former two approaches while
overcoming their obstacles. The bipartite library approach
(Figure 2c) involves randomization of one-and-a-half fingers of a three-finger protein [25•,26]. This maximizes the
possibility for interdomain cooperativity. To circumvent
the technical limitations of cloning and displaying such a
large number of variants, randomization is limited to only a
subset of the 20 amino acids. Only two pre-constructed
libraries are required to create a new three-finger protein:
one randomized at the N terminus and one randomized at
the C terminus. After selection, the two libraries are
recombined and final sets of selections are performed to
obtain the optimal recombinant protein. Although assumptions are introduced by restricting the number, type, and
position of randomized amino acids, this method addresses
the concerns of target-site overlap while shortening
construction time, according to the authors, to 10–14 days.
All three methods produce proteins of comparable affinity
(when affinity data are normalized to that of the protein
Zif268) [4,25•]. Early pessimism that the parallel method
could produce only zinc fingers that recognize 5′-GNN-3′type sequences was recently dispelled with the report of
domains that bind 5′-ANN-3′ sequences [15••]. This was
accomplished by eliminating target-site overlap from the
offending anchor finger. Although fingers binding 5′-GNN-3′
or 5′-ANN-3′ subsites represent only half of the domains
required for comprehensive recognition, a six-finger site
recognizable by these domains should occur every 32 base
pairs. Using these 32 domains, over one billion different
six-finger proteins can now be constructed (i.e. 27 000 proteins for every gene in the human genome). Furthermore,
zinc-finger domains that bind the 5′-TNN-3′ and 5′-CNN-3′
subsites can also be prepared in this manner.
Cellular gene regulation
The attachment of appropriate EDs to zinc-finger DBDs
creates potent transcriptional activators and repressors.
Activation domains such as VP16 [3] and p65 [27] and
repression domains such as KRAB [2] and SID [28] are
components of naturally occurring transcription factors. All
have been shown to regulate a variety of promoters in a
distance- and orientation-independent manner when
fused to heterologous DBDs. Fusion of these domains
with custom zinc-finger DBDs results in artificial
transcription factors (TFZF) that can upregulate and
downregulate genes (Figure 3) [11]. This is significant as,
for the first time, regulation is possible using unmodified,
genomic DNA sequences (i.e. without the pre-insertion of
artificial binding sites).
The ability to study and manipulate genes in their native
chromatin environment, a previously unapproachable task,
represents a major advance. Within the past year, TFZFs
have been shown to regulate the endogenous chromosomal
genes for ErbB-2 and ErbB-3 [15••,18••], Epo [20••],
VEGF-A [19••], and AP3 [29••,30••] (Table 1). The ErbB2-specific TFZF targeted a highly conserved sequence in
the gene, and was shown to function in human, mouse and
monkey cells [18••]. Furthermore, by using tetracycline
regulation, chemical control of an endogenous gene was
imposed. The AP3-specific TFZF functions in plant cell
culture and whole plants. The ErbB-2-specific TFZF was
shown to upregulate (with a VP16 derivative ED) or downregulate (with a KRAB ED) erbB-2, but not erbB-3. The
reverse was also true for the ErbB-3-specific TFZF. The
Custom DNA-binding proteins come of age: polydactyl zinc-finger proteins Segal and Barbas
635
Table 1
Endogenous genes regulated by zinc finger–effector domain fusions.
Gene product
Function
Target position
Number of fingers
Species
Activation
Repression
References
ErbB-2
Oncogene
5′-UTR
6
Human, monkey, mouse
✓
✓
[15••,18••]
ErbB-3
Oncogene
5′-UTR
6
Human
✓
✓
[15••,18••]
Epo
Erythropoiesis induction
Upstream
3
Human
✓
[20••]
VEGF-A
Angiogenesis induction
5′-UTR
3
Human
✓
[19••]
Flower development
5′-UTR
6
Arabidopsis
✓
Ap3
binding sites of these two factors differed by only three
out of 18 base pairs, demonstrating that regulation
was extremely specific. Endogenous gene expression was
reduced to background levels, suggesting that TFZF can
be used to make rapid, functional gene knockouts.
An issue of current importance in the field is to understand
the characteristics of a productive regulatory site. Zinc
fingers, even without EDs, have been shown to inhibit
transcriptional initiation if targeted very close to the initiation site [31,32]. Some of the most activating TFZFs were
targeted downstream of the initiation site, however,
demonstrating that they do not inhibit transcriptional elongation [18••,19••]. Affinity did not seem to correlate with
activity, as long as the affinity was less than 10 nM
[15••,18••–20••]. Local DNA topology and cellular binding
proteins, such as transcription factors and histones, present
challenges to in vivo target-site selection. Using DNase I
hypersensitivity as an indicator of chromatin accessibility,
TFZF targeted to DNase I-protected sites failed to
regulate the VEGF-A gene, whereas others targeted to
hypersensitive sites were activating [19••]. However, even
within accessible regions some TFZFs were more activating than others, suggesting chromatin accessibility is
necessary, but not sufficient, for productive regulation.
More studies will be needed to understand what works
best and why.
Conclusions
Our understanding of zinc-finger–DNA interactions continues to advance rapidly, augmented recently by reports
that have examined the effects of non-DNA-contacting
residues on affinity and specificity [33–38]. Studies such as
these, in conjunction with computer modeling [14•,15••]
and structural studies [22••,39•], are producing an increasingly clear picture of a recognition domain that can interact
with DNA in both simple and complex ways. The frontier
of zinc-finger research is now shifting beyond DNA-recognition to novel applications. Aside from gene regulation,
zinc-finger-based endonucleases were recently shown to
stimulate homologous recombination in eukaryotic cells
[40••]. Strategies for creating ligand-dependent TFZFs
have also been reported [41•,42•] (and reviewed in [9]),
while improved delivery systems are being investigated.
✓
[29••,30••]
With the barriers of sequence recognition and small-molecule regulation breached, zinc-finger transcription factors
are serious candidates as gene therapeutics. The robust
features of this technology that provide for the rapid
assembly of transcription factors from pre-defined zincfinger domains should also ensure their application in
functional genomics.
Acknowledgements
We thank Laurent Magnenat for his comments on this manuscript.
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637
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