Yen Choo

A rapid, generally applicable method to engineer zinc fingers illustrated by targeting the HIV-1 promoter

DNA-binding domains with predetermined sequence specificity are engineered by selection of zinc finger modules using phage display, allowing the construction of customized transcription factors. Despite remarkable progress in this field, the available protein-engineering methods are deficient in many respects, thus hampering the applicability of the technique. Here we present a rapid and convenient method that can be used to design zinc finger proteins against a variety of DNA-binding sites. This is based on a pair of pre-made zinc finger phage-display libraries, which are used in parallel to select two DNA-binding domains each of which recognizes given 5 base pair sequences, and whose products are recombined to produce a single protein that recognizes a composite (9 base pair) site of predefined sequence. Engineering using this system can be completed in less than two weeks and yields proteins that bind sequence-specifically to DNA with Kd values in the nanomolar range. To illustrate the technique, we have selected seven different proteins to bind various regions of the human immunodeficiency virus 1 (HIV-1) promoter.

Exploring the DNA-binding specificities of zinc fingers with DNA microarrays

A key step in the regulation of networks that control gene expression is the sequence-specific binding of transcription factors to their DNA recognition sites. A more complete understanding of these DNA–protein interactions will permit a more comprehensive and quantitative mapping of the regulatory pathways within cells, as well as a deeper understanding of the potential functions of individual genes regulated by newly identified DNA-binding sites. Here we describe a DNA microarray-based method to characterize sequence-specific DNA recognition by zinc-finger proteins. A phage display library, prepared by randomizing critical amino acid residues in the second of three fingers of the mouse Zif268 domain, provided a rich source of zinc-finger proteins with variant DNA-binding specificities. Microarrays containing all possible 3-bp binding sites for the variable zinc fingers permitted the quantitation of the binding site preferences of the entire library, pools of zinc fingers corresponding to different rounds of selection from this library, as well as individual Zif268 variants that were isolated from the library by using specific DNA sequences. The results demonstrate the feasibility of using DNA microarrays for genome-wide identification of putative transcription factor-binding sites.

Physical basis of a protein-DNA recognition code

Can a stereochemical recognition code explain sequence-specific protein-nucleic acid interactions? Whereas a code that is generally applicable to DNA-binding proteins of all known structural families is unattainable, the indications are that a code can describe at least some of the interactions of classical zinc fingers with DNA. The crystal structures of related zinc finger-DNA complexes reveal a remarkable mode of interaction that sets the framework for this code, and recent biochemical studies have elucidated the intermolecular contacts (contingent on this framework) that result in specificity.

Advances in zinc finger engineering

Recently developments have been made in engineering sequence-specific zinc finger DNA-binding proteins. Advances in this area will soon make it routine to target proteins to specific DNA sequences associated with any given gene. The primary interest is in the regulation of gene expression using customised transcription factors. However, modular catalytic domains are also being developed in order to engineer chimaeric proteins with customised restriction enzyme, methylase and integrase activity.

Repression of the HIV-1 5′ LTR promoter and inhibition of HIV-1 replication by using engineered zinc-finger transcription factors

Zinc finger domains are small DNA-binding modules that can be engineered to bind desired target sequences. Functional transcription factors can be made from these DNA-binding modules, by fusion with an appropriate effector domain. In this study, eight three-zinc-finger proteins (ZFPs) that bound HIV-1 sequences in vitro were engineered into transcription repressors by linking them to the Krüppel-associated box (KRAB) repressor domain (KOX1). One protein, ZFP HIVB-KOX, which bound to a 9-bp region overlapping two Sp1 sites, was found to repress a Tat-activated 5′ LTR cellular HIV-reporter assay to almost basal levels. A related six-finger protein, HIVBA′-KOX, was made to target all three Sp1 sites in the 5′ LTR promoter and efficiently inhibited both basal and Tat-activated transcription in unstimulated and mitogen-stimulated T cells. In contrast, a combination of two unlinked three-finger ZFPs, HIVA′-KOX and HIVB-KOX, which bind over the same region of DNA, resulted in less effective repression. Finally, HIVBA′-KOX was tested for its capacity to block viral replication in a cellular infection assay using the HIV-1 HXB2 strain. This ZFP was found to inhibit HIV-1 replication by 75% compared with control constructs, thus demonstrating the potential of this approach for antiviral therapy.

Designing DNA-binding proteins on the surface of filamentous phage

The strategy of molecular evolution by phage display recently has been applied to the study of interactions between protein and DNA. This technology will imminently enable DNA-binding proteins to be made to measure. In the first instance, this will greatly advance our understanding of protein-DNA interactions, but in the long term, it is expected to yield powerful tools for use in medicine and research.

Inhibition of herpes simplex virus 1 gene expression by designer zinc-finger transcription factors

The herpes simplex virus 1 (HSV-1) replicative cycle begins by binding of the viral activator, VP16, to a set of sequences in the immediate-early (IE) gene promoters. With the aim of inhibiting this cycle, we have constructed a number of synthetic zinc-finger DNA-binding peptides by using recently reported methods. Peptides containing either three or six fingers, targeted to a viral promoter, were engineered as fusions with a KOX-1 transcription repression domain. These proteins bound to the HSV-1 IE175k (ICP4) promoter, in vitro, with nanomolar or subnanomolar binding affinity. However, in a chloramphenicol acetyltransferase reporter system, only the six-finger protein was found to repress VP16-activated transcription significantly. Thus the longer array of zinc fingers is required to compete successfully against VP16, one of the most powerful natural activators known. We found that the HSV-1 replication cycle can be partially repressed by the six-finger peptide with the viral titer reduced by 90%.

Rapid, high-throughput engineering of sequence-specific zinc finger DNA-binding proteins.

Publisher Summary Zinc fingers engineered to recognize predetermined DNA sequences have been used to control gene expression and also to target catalytic domains, such as restriction enzymes, methylases, and integrases, to specific regions of DNA. The use of these proteins as transcription factors alone has potential applications in functional genomics, gene target validation, engineering of animals or plants with improved phenotypes, and human therapy. This chapter illustrates the use of a new, rapid, and widely applicable zinc finger-engineering strategy that produces three-finger domains that bind a wide variety of DNA sequences. This engineering strategy is described, placing particular emphasis on methodological aspects, in order to illustrate the increase in the speed and throughput of zinc finger engineering. Although the interactions of zinc fingers with DNA have been studied in order to classify the protein–DNA contacts into a “recognition code” that could be used for zinc finger design, this code does not allow the engineering of zinc fingers that bind to any given DNA sequence.

Tailor-made zinc-finger transcription factors activate FLO11 gene expression with phenotypic consequences in the yeast Saccharomyces cerevisiae.

Cys2His2 zinc fingers are eukaryotic DNA-binding motifs, capable of distinguishing different DNA sequences, and are suitable for engineering artificial transcription factors. In this work, we used the budding yeast Saccharomyces cerevisiae to study the ability of tailor-made zinc finger proteins to activate the expression of the FLO11 gene, with phenotypic consequences. Two three-finger peptides were identified, recognizing sites from the 5′ UTR of the FLO11 gene with nanomolar DNA-binding affinity. The three-finger domains and their combined six-finger motif, recognizing an 18-bp site, were fused to the activation domain of VP16 or VP64. These transcription factor constructs retained their DNA-binding ability, with the six-finger ones being the highest in affinity. However, when expressed in haploid yeast cells, only one three-finger recombinant transcription factor was able to activate the expression of FLO11 efficiently. Unlike in the wild-type, cells with such transcriptional activation displayed invasive growth and biofilm formation, without any requirement for glucose depletion. The VP16 and VP64 domains appeared to act equally well in the activation of FLO11 expression, with comparable effects in phenotypic alteration. We conclude that the functional activity of tailor-made transcription factors in cells is not easily predicted by the in vitro DNA-binding activity.

Directed Differentiation of Embryonic Stem Cells Using a Bead-Based Combinatorial Screening Method

We have developed a rapid, bead-based combinatorial screening method to determine optimal combinations of variables that direct stem cell differentiation to produce known or novel cell types having pre-determined characteristics. Here we describe three experiments comprising stepwise exposure of mouse or human embryonic cells to 10,000 combinations of serum-free differentiation media, through which we discovered multiple novel, efficient and robust protocols to generate a number of specific hematopoietic and neural lineages. We further demonstrate that the technology can be used to optimize existing protocols in order to substitute costly growth factors with bioactive small molecules and/or increase cell yield, and to identify in vitro conditions for the production of rare developmental intermediates such as an embryonic lymphoid progenitor cell that has not previously been reported.