Polydactyl Zinc Finger Research Articles

Artificial Zinc-Finger Transcription Factor of A20 Suppresses Restenosis in Sprague Dawley Rats after Carotid Injury via the PPARα Pathway.

The inhibition of inflammation and vascular smooth muscle cell (VSMC) proliferation is an ideal strategy to suppress intimal hyperplasia after percutaneous transluminal angioplasty (PTA). Evidence has indicated that overexpression of A20 suppresses neointima formation, but its low transfection efficiency limits its application. Hence, we upregulated A20 expression via transfection of rAd.ATF (recombinant adenovirus vector of artificial transcription factor) and rAd.A20 in rat carotid arteries after balloon dilatation (in vivo) and isolated VSMCs (in vitro). In vivo, we found that after rAd.ATF and rAd.A20 transfection, A20 expression was markedly increased, whereas proliferating cell nuclear antigen (PCNA) and nuclear factor κB p65 (NF-κBp65) protein levels were significantly decreased, and intimal hyperplasia and secretion of proinflammatory factors were significantly reduced when compared with empty vector and saline control groups. Most importantly, the rAd.ATF-treated group showed more significant inhibition on intimal hyperplasia and expression of PCNA than the rAd.A20-treated group. In vitro, compared with the control group, transfection of rAd.ATF and rAd.A20 significantly increased A20 expression, which upregulated the proliferator-activated receptor (PPAR) level for both mRNA and protein, and reduced migration and proliferation of VSMCs and lipopolysaccharide (LPS)-induced inflammation. Furthermore, the PPARα agonist GW6471 could partially restore the effect of A20 on VSMCs. Our findings indicate that the ATF of A20 inhibits neointimal hyperplasia and, therefore, constitutes a novel potential alternative to prevent restenosis.

Deciphering the DNA code for the function of the Drosophila polydactyl zinc finger protein Suppressor of Hairy-wing.

Polydactyl zinc finger (ZF) proteins have prominent roles in gene regulation and often execute multiple regulatory functions. To understand how these proteins perform varied regulation, we studiedDrosophila Suppressor of Hairy-wing [Su(Hw)], an exemplar multifunctional polydactyl ZF protein. We identified separation-of-function (SOF) alleles that encode proteins disrupted in a single ZF that retain one of the Su(Hw) regulatory activities. Through extended in vitro analyses of the Su(Hw) ZF domain, we show that clusters of ZFs bind individual modules within a compound DNA consensus sequence. Through in vivo analysis of SOF mutants, we find that Su(Hw) genomic sites separate into sequence subclasses comprised of combinations of modules, with subclasses enriched for different chromatin features. These data suggest a Su(Hw) code, wherein DNA binding dictates its cofactor recruitment and regulatory output. We propose that similar DNA codes might be used to confer multiple regulatory functions of other polydactyl ZF proteins.

Sequence-Specific Biosensors Report Drug-Induced Changes in Epigenetic Silencing in Living Cells

Treatment with demethylating drugs can induce demethylation and reactivation of abnormally silenced tumor suppressor genes in cancer cells, but it can also induce potentially deleterious loss of methylation of repetitive elements. To enable the observation of unwanted drug effects related to loss of methylation of repetitive DNA, we have developed a novel biosensor capable of reporting changes in DNA accessibility via luminescence, in living cells. The biosensor design comprises two independent modules, each with a polydactyl zinc finger domain fused to a half intein and to a split-luciferase domain that can be joined by conditional protein splicing after binding to adjacent DNA targets. We show that an artificial zinc finger design specifically targeting DNA sequences near the promoter region of the L1PA2 subfamily of Line-1 retroelements is able to generate luminescent signals, reporting loss of epigenetic silencing and increased DNA accessibility of retroelements in human cells treated with the demethylating drugs decitabine or 5-azacytidine.

Polydactyl zinc finger proteins: software and hardware for genomes

Polydactyl zinc finger proteins: software and hardware for genomes

Design of a novel polydactyl zinc finger with binding specificity for the proximal promoter region of PLN in the proposed treatment of cardiomyopathy.

Heart disease is the leading cause of death worldwide. Cardiomyopathy, a form of heart disease, is one of the many areas of clinical interest and is characterized as a disease of the heart myocardium. Phospholamban (PLN) is one of the many proteins linked with this disease. Its primary function in the cell is the inhibition of the sarco(endo)plasmic reticulum Ca2+ ATPase (SERCA2) calcium transporter located in muscle tissue. There is a direct causal link between when PLN is over-expressed, super-inhibitory or non-existent and the development of various forms of cardiomyopathy. In an effort to counteract the development of cardiomyopathy, we have designed a novel zinc finger protein named Dandy, which specifically binds form the –77 to -95 region of the proximal promoter of the PLN gene. This protein is expected to transiently block the binding of the NFY promoter and thus, the initiation of transcription. Blocking initiation of transcription should result in an overall decrease in the concentration of PLN. Dandy is a zinc finger protein made up of two three-fingered peptides. It recognizes a 18 bp sequence with a 1 bp gap in the middle. Tertiary structure predictions of Dandy show that the protein has the intended structure of the design. More clinical research into the specific binding interactions will need to be completed before in vivo function and overall efficacy of Dandy is elucidated.

Live cell imaging of repetitive DNA sequences via GFP-tagged polydactyl zinc finger proteins

Several techniques are available to study chromosomes or chromosomal domains in nuclei of chemically fixed or living cells. Current methods to detect DNA sequences in vivo are limited to trans interactions between a DNA sequence and a transcription factor from natural systems. Here, we expand live cell imaging tools using a novel approach based on zinc finger-DNA recognition codes. We constructed several polydactyl zinc finger (PZF) DNA-binding domains aimed to recognize specific DNA sequences in Arabidopsis and mouse and fused these with GFP. Plants and mouse cells expressing PZF:GFP proteins were subsequently analyzed by confocal microscopy. For Arabidopsis, we designed a PZF:GFP protein aimed to specifically recognize a 9-bp sequence within centromeric 180-bp repeat and monitored centromeres in living roots. Similarly, in mouse cells a PZF:GFP protein was targeted to a 9-bp sequence in the major satellite repeat. Both PZF:GFP proteins localized in chromocenters which represent heterochromatin domains containing centromere and other tandem repeats. The number of PZF:GFP molecules per centromere in Arabidopsis, quantified with near single-molecule precision, approximated the number of expected binding sites. Our data demonstrate that live cell imaging of specific DNA sequences can be achieved with artificial zinc finger proteins in different organisms.

Human immunodeficiency virus type 1 incorporated with fusion proteins consisting of integrase and the designed polydactyl zinc finger protein E2C can bias integration of viral DNA into a predetermined chromosomal region in human cells.

In vitro studies using fusion proteins consisting of human immunodeficiency virus type 1 integrase (IN) and a synthetic polydactyl zinc finger protein E2C, a sequence-specific DNA-binding protein, showed that integration of retroviral DNA can be biased towards a contiguous 18-bp E2C-recognition site. To determine whether the fusion protein strategy can achieve site-specific integration in vivo, viruses were prepared by cotransfection and various IN-E2C fusion proteins were packaged in trans into virions. The resulting viruses incorporated with the IN-E2C fusion proteins were functional and capable of performing integration at a level ranging from 1 to 24% of that of viruses containing wild-type (WT) IN. Two of the more infectious viruses, which contained E2C fused to either the N (E2C/IN) or to the C (IN/E2C) terminus of IN, were tested for their ability to direct integration into a unique E2C-binding site present within the 5' untranslated region of erbB-2 gene on human chromosome 17. The copy number of proviral DNA was measured using a quantitative real-time nested-PCR assay, and the specificity of directed integration was determined by comparing the number of proviruses within the vicinity of the E2C-binding site to that in the whole genome. Viruses containing IN/E2C fusion proteins had sevenfold higher preference for integrating near the E2C-binding site than those viruses containing WT IN, whereas viruses containing E2C/IN had 10-fold higher preference. The results indicated that the IN-E2C fusion protein strategy is capable of directing integration of retroviral DNA into a predetermined chromosomal region in the human genome.

Effects of different zinc finger transcription factors on genomic targets

We have developed a novel vector system for the efficient assembly of polydactyl zinc fingers. Next to proteins that possess short canonical TGEKP linkers between all constituting zinc fingers we constructed proteins with longer, 12 amino acid linkers between two three-finger (3F) units and between three two-finger (2F) units. Fusions of these zinc finger domains with the VP16 activation domain were tested for their ability to regulate a repressed genomic locus containing contiguous or noncontiguous zinc finger binding sites in yeast. In contrast to other studies, which were mostly confined to in vitro tests, we did not obtain evidence that superior artificial zinc finger transcription factors need to contain longer linkers between individual fingers. For the regulation of genomic loci, canonical linkers within a highly regular backbone in combination with a contiguous 18 base pair DNA target site were found to provide a sound base for polydactyl zinc finger design.

Development of Zinc Finger Domains for Recognition of the 5′-CNN-3′ Family DNA Sequences and Their Use in the Construction of Artificial Transcription Factors

Considerable progress has been made in recent years in the design of transcription factors for the directed regulation of endogenous genes. Although many strategies involve selection methods that must be applied for each new target sequence, we have developed an approach based on linkage of predefined zinc finger domains that each recognize a three-base pair DNA sequence to construct artificial transcription factors that bind to a desired sequence. These domains can be assembled to recognize unique 18-base pair DNA sequences with high specificity. Here we report the development and characterization of zinc finger domains that bind to 15 of the 16 5'-CNN-3' subsites. These domains were created through a combination of phage display selection, site-directed mutagenesis, and de novo design. Furthermore, these domains were used to generate a highly specific six-finger protein targeting the ERBB-2 promoter. When fused to regulatory domains, this protein was capable of up- and down-regulating the expression of the endogenous ERBB-2 gene. With the addition of this collection of predefined zinc finger domains, most 5'-CNN-3'-, 5'-GNN-3'-, and 5'-ANN-3'-containing sequences can now be rapidly targeted for directed gene regulation and nuclease cleavage.

1101. Fusion Proteins Consisting of the Sleeping Beauty Transposase and the Polydactyl Zinc Finger Protein E2C Direct Transposon Integration into a Unique Human Chromosomal Sequence

The DNA transposon Sleeping Beauty (SB) can integrate efficiently into host cell genomes. Although this process makes these elements attractive vehicles for therapeutic gene delivery, the nonspecific nature of integration presents inherent hazards. To alleviate these risks, we have engineered a new class of fusion proteins comprised of a hyperactive SB transposase mutant (HSB5, 1,000% of SB10 activity) fused to either the N-terminus (SB/E2C) or C-terminus of the polydactyl zinc finger protein E2C (E2C/SB) in an attempt to direct transposon integration into predetermined chromosomal sites. Eleven different chimeric transposases containing variable-length inter-domain linkers were expressed in plasmid-transfected mammalian cells and tested for their ability to stably integrate a co-transfected neomycin-marked transposon. Despite equivalent steady-state levels in transfected cells, only fusions proteins with the E2C/SB configuration showed any significant level of integration activity, with the best chimera retaining |[sim]|5% of HSB5 activity (52% of SB10 activity). Importantly, E2C/SB-mediated integration was abrogated by single amino acid substitutions that inactivate the SB enzyme, suggesting that integration by the fusion protein was indeed SB-mediated. Remarkably, co-expressing limiting amounts of native HSB5 protein together with the fusion protein caused a strong synergistic increase in integration frequencies relative to cells expressing either protein alone. This suggests that chimeric transposases can function either alone or as mixed multimers within the tight constraints of a transposon-transposase complex. We also explored the DNA-binding capabilities of this chimera using an electrophoretic mobility shift assay. Results showed that the E2C/SB protein could bind specifically to both the 18-bp E2C recognition sequence and a canonical SB binding site, but that its affinity for the latter was markedly reduced relative to HSB5. This provides one explanation for the reduced activity of the fusion protein and suggests that the formation of mixed multimers may enhance integration activity by stabilizing the DNA-binding capabilities of the chimera. Finally, we studied target site selection by the E2C/SB fusion protein using an inter-plasmid transposition assay. We transfected HeLa cells with ampr target plasmids containing a single E2C-binding site, or mutant E2C site as a control, together with plasmids encoding the E2C/SB fusion protein and a kanamycin-marked transposon. We then prepared DNA from these cells, identified integrations by screening for a kanr/ampr phenotype in E.coli, and then compared insertion site distributions between the two groups. Sequence analysis of |[sim]|70 insertions from each group suggests that: (i) the E2C/SB fusion protein can bias integration near the E2C binding site via sequence-specific DNA binding, and (ii) that E2C/SB-mediated integrations represent true transpositions, as evidenced by the presence of flanking target site duplications. These proof-of-principle results demonstrate that E2C/SB fusion proteins offer an efficient and versatile framework for directing transposon integration into predetermined DNA sites.

393. Site-Directed Integration in Human Genome by Lentiviral Vectors Containing HIV-1 Integrase/Polydactyl Zinc Finger Fusion Proteins

Studies on site directed integration using fusion proteins may lead to a new approach for inserting exogenous genes at specific chromosomal sites and improve the therapeutic application of current retroviral and lentiviral vectors. Using an in trans method (Holmes-Son, et al, J Virol., 2000;74:11548), we incorporated the synthetic polydactyl zinc finger E2C fused to the N- (E2C/IN) or C- (IN/E2C) terminus of IN into HIV-1 virions. E2C contains six zinc finger domains and recognizes a unique contiguous 18-bp sequence (e2c) located within the 5'untranslated region of erbB2 gene on human chromosome 17. These viruses were infectious and capable of performing integration at a level ranging from 6 to 12% of viruses containing wild-type (WT) IN. Human Hela cells, an ovary epithelial cell line, were infected by viruses containing WT-IN or fusion proteins. Chromosomal DNA from infected cells was isolated three days post-infection and the number of proviruses was determined using a quantitative real-time PCR assay. The specificity of directed integration was quantified by comparing the number of proviruses near the E2C recognition site (between 100 bp upstream and downstream of e2c) to that of the whole genome. The number of proviruses around the e2c site was six to eleven times higher with viruses containing the E2C fusion proteins (IN/E2C or E2C/IN) than those containing the WT-IN. The results indicated that the IN-E2C fusion protein strategy is capable of directing the integration of lentiviral DNA into a predetermined site in human genome.

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

Zinc finger DNA-binding domains can be combined to create new proteins of desired DNA-binding specificity. By shuffling our repertoire of modified zinc finger domains to create randomly generated polydactyl zinc finger proteins with transcriptional regulatory domains, we developed large combinatorial libraries of zinc finger transcription factors (TF ZFs). Millions of TF ZFs can then be simultaneously screened in mammalian cells. Here, we successfully isolated specific TF ZFs that significantly positively and negatively modulate the transcription of the ICAM-1 gene in primary and cancer cells, which are relevant to ICAM-1 biology and tumor development. We show that TF ZFs can work in a general and in a cell-type specific manner depending on the regulatory domain and the zinc finger protein. We show that a TF ZF that interacts directly with the ICAM-1 promoter at an overlapping NF-κB binding enhancer can overcome or synergistically cooperate with NF-κB induction of ICAM-1. For this TF ZF, rational design was used to optimize the binding of the zinc finger protein to its DNA element and the resulting TF ZF demonstrated a direct correlation between increased affinity and efficiency of target gene regulation. Thus, combining library and affinity maturation approaches generated superior TF ZFs that may find further applications in therapeutic research and in ICAM-1 biology, and also provided novel mechanistic insights into the biology of transcription factors. Transcription factor libraries provide genome-wide approaches that can be applied towards the development of TF ZFs specific for virtually any gene or desired phenotype and may lead to the discovery of new genetic functions and pathways.

Fusion proteins consisting of human immunodeficiency virus type 1 integrase and the designed polydactyl zinc finger protein E2C direct integration of viral DNA into specific sites.

In order to establish a productive infection, a retrovirus must integrate the cDNA of its RNA genome into the host cell chromosome. While this critical process makes retroviruses an attractive vector for gene delivery, the nonspecific nature of integration presents inherent hazards and variations in gene expression. One approach to alleviating the problem involves fusing retroviral integrase to a sequence-specific DNA-binding protein that targets a defined chromosomal site. We prepared proteins consisting of wild-type or truncated human immunodeficiency virus type 1 (HIV-1) integrase fused to the synthetic polydactyl zinc finger protein E2C. The purified fusion proteins bound specifically to the 18-bp E2C recognition sequence as analyzed by DNase I footprinting. The fusion proteins were catalytically active and biased integration of retroviral DNA near the E2C-binding site in vitro. The distribution was asymmetric, and the major integration hot spots were localized within a 20-bp region upstream of the C-rich strand of the E2C recognition sequence. Integration bias was not observed with target plasmids bearing a mutated E2C-binding site or when HIV-1 integrase and E2C were added to the reaction as separate proteins. The results demonstrate that the integrase-E2C fusion proteins offer an efficient approach and a versatile framework for directing the integration of retroviral DNA into a predetermined DNA site.

Evaluation of a modular strategy for the construction of novel polydactyl zinc finger DNA-binding proteins.

In previous studies, we have developed a technology for the rapid construction of novel DNA-binding proteins with the potential to recognize any unique site in a given genome. This technology relies on the modular assembly of modified zinc finger DNA-binding domains, each of which recognizes a three bp subsite of DNA. A complete set of 64 domains would provide comprehensive recognition of any desired DNA sequence, and new proteins could be assembled by any laboratory in a matter of hours. However, a critical parameter for this approach is the extent to which each domain functions as an independent, modular unit, without influence or dependence on its neighboring domains. We therefore examined the detailed binding behavior of several modularly assembled polydactyl zinc finger proteins. We first demonstrated that 80 modularly assembled 3-finger proteins can recognize their DNA target with very high specificity using a multitarget ELISA-based specificity assay. A more detailed analysis of DNA binding specificity for eight 3-finger proteins and two 6-finger proteins was performed using a target site selection assay. Results showed that the specificity of these proteins was as good or better than that of zinc finger proteins constructed using methods that allow for interdependency. In some cases, near perfect specificity was achieved. Complications due to target site overlap were found to be restricted to only one particular amino acid interaction (involving an aspartate in position 2 of the alpha-helix) that occurs in a minority of cases. As this is the first report of target site selection for designed, well characterized 6-finger proteins, unique insights are discussed concerning the relationship of protein length and specificity. These results have important implications for the design of proteins that can recognize extended DNA sequences, as well as provide insights into the general rules of recognition for naturally occurring zinc finger proteins.

Regulation of transgene expression in plants with polydactyl zinc finger transcription factors.

Designer zinc finger transcription factors (TFs(ZF)) have been developed to control the expression of transgenes and endogenous genes in mammalian cells. Application of TFs(ZF) technology in plants would enable a wide range of both basic and applied studies. In this paper, we report the use of TFs(ZF) to target a defined 18-bp DNA sequence to control gene expression in plant cells and in transgenic plants. A beta-glucuronidase reporter gene was activated by using the designed six-zinc finger protein 2C7 expressed as a fusion with the herpes simplex virus VP16 transcription factor activation domain. Reporter gene expression was activated 5- to 30-fold by using TFs(ZF) in BY-2 protoplasts, whereas expression was increased as much as 450 times in transgenic tobacco plants. Use of a phloem-specific promoter to drive expression of the TFs(ZF) resulted in activation of the reporter gene in vascular tissues. Transgenic tobacco plants that produce 2C7 transcription factors were phenotypically normal through two generations, suggesting that the factors exerted no adverse effects. This study demonstrates the utility of zinc finger technology in plants, setting the stage for its application in basic and applied agricultural biotechnology.

Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors.

Zinc finger transcription factors (TFs(ZF)) were designed and applied to transgene and endogenous gene regulation in stably transformed plants. The target of the TFs(ZF) is the Arabidopsis gene APETALA3 (AP3), which encodes a transcription factor that determines floral organ identity. A zinc finger protein (ZFP) was designed to specifically bind to a region upstream of AP3. AP3 transcription was induced by transformation of leaf protoplasts with a transformation vector that expressed a TF(ZF) consisting of the ZFP fused to the tetrameric repeat of herpes simplex VP16's minimal activation domain. Histochemical staining of beta-glucuronidase (GUS) activity in transgenic AP3GUS reporter plants expressing GUS under control of the AP3 promoter was increased dramatically in petals when the AP3-specific TF(ZF) activator was cointroduced. TF(ZF)-amplified GUS expression signals were also evident in sepal tissues of these double-transgenic plants. Floral phenotype changes indicative of endogenous AP3 factor coactivation were also observed. The same AP3-specific ZFP(AP3) was also fused to a human transcriptional repression domain, the mSIN3 interaction domain, and introduced into either AP3GUS-expressing plants or wild-type Arabidopsis plants. Dramatic repression of endogenous AP3 expression in floral tissue resulted when a constitutive promoter was used to drive the expression of this TF(ZF). These plants were also sterile. When a floral tissue-specific promoter from APETALA1 (AP1) gene was used, floral phenotype changes were also observed, but in contrast the plants were fertile. Our results demonstrate that artificial transcriptional factors based on synthetic zinc finger proteins are capable of stable and specific regulation of endogenous genes through multiple generations in multicellular organisms.

Condensation by DNA looping facilitates transfer of large DNA molecules into mammalian cells.

Experimental studies of complete mammalian genes and other genetic domains are impeded by the difficulty of introducing large DNA molecules into cells in culture. Previously we have shown that GST-Z2, a protein that contains three zinc fingers and a proline-rich multimerization domain from the polydactyl zinc finger protein RIP60 fused to glutathione S-transferase (GST), mediates DNA binding and looping in vitro. Atomic force microscopy showed that GST-Z2 is able to condense 130-150 kb bacterial artificial chromosomes (BACs) into protein-DNA complexes containing multiple DNA loops. Condensation of the DNA loops onto the Z2 protein-BAC DNA core complexes with cationic lipid resulted in particles that were readily transferred into multiple cell types in culture. Transfer of total genomic linear DNA containing amplified DHFR genes into DHFR(-) cells by GST-Z2 resulted in a 10-fold higher transformation rate than calcium phosphate co-precipitation. Chinese hamster ovarian cells transfected with a BAC containing the human TP53 gene locus expressed p53, showing native promoter elements are active after GST-Z2-mediated gene transfer. Because DNA condensation by GST-Z2 does not require the introduction of specific recognition sequences into the DNA substrate, condensation by the Z2 domain of RIP60 may be used in conjunction with a variety of other agents to provide a flexible and efficient non-viral platform for the delivery of large genes into mammalian cells.

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.

To create a universal system for the control of gene expression, we have studied methods for the construction of novel polydactyl zinc finger proteins that recognize extended DNA sequences. Elsewhere we have described the generation of zinc finger domains recognizing sequences of the 5'-GNN-3' subset of a 64-member zinc finger alphabet. Here we report on the use of these domains as modular building blocks for the construction of polydactyl proteins specifically recognizing 9- or 18-bp sequences. A rapid PCR assembly method was developed that, together with this predefined set of zinc finger domains, provides ready access to 17 million novel proteins that bind the 5'-(GNN)6-3' family of 18-bp DNA sites. To examine the efficacy of this strategy in gene control, the human erbB-2 gene was chosen as a model. A polydactyl protein specifically recognizing an 18-bp sequence in the 5'-untranslated region of this gene was converted into a transcriptional repressor by fusion with Kr uppel-associated box (KRAB), ERD, or SID repressor domains. Transcriptional activators were generated by fusion with the herpes simplex VP16 activation domain or with a tetrameric repeat of VP16's minimal activation domain, termed VP64. We demonstrate that both gene repression and activation can be achieved by targeting designed proteins to a single site within the transcribed region of a gene. We anticipate that gene-specific transcriptional regulators of the type described here will find diverse applications in gene therapy, functional genomics, and the generation of transgenic organisms.