Polydactyl Zinc Finger Proteins Research Articles

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.

Polydactyl zinc finger proteins: software and hardware for genomes

Polydactyl zinc finger proteins: software and hardware for genomes

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.

Site-directed transposon integration in human cells.

The Sleeping Beauty (SB) transposon is a promising gene transfer vector that integrates nonspecifically into host cell genomes. Herein, we attempt to direct transposon integration into predetermined DNA sites by coupling a site-specific DNA-binding domain (DBD) to the SB transposase. We engineered fusion proteins comprised of a hyperactive SB transposase (HSB5) joined via a variable-length linker to either end of the polydactyl zinc-finger protein E2C, which binds a unique sequence on human chromosome 17. Although DBD linkage to the C-terminus of SB abolished activity in a human cell transposition assay, the N-terminal addition of the E2C or Gal4 DBD did not. Molecular analyses indicated that these DBD-SB fusion proteins retained DNA-binding specificity for their respective substrate molecules and were capable of mediating bona fide transposition reactions. We also characterized transposon integrations in the presence of the E2C-SB fusion protein to determine its potential to target predefined DNA sites. Our results indicate that fusion protein-mediated tethering can effectively redirect transposon insertion site selection in human cells, but suggest that stable docking of integration complexes may also partially interfere with the cut-and-paste mechanism. These findings illustrate the feasibility of directed transposon integration and highlight potential means for future development.

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.

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.

Safe delivery of therapeutic genes into specific chromosomal sites using engineered retroviral integrase

Gene therapy approaches that involve the permanent insertion of therapeutic genes into host chromosomal DNA have many desirable features and show considerable promise for success in the clinic. One major drawback of these approaches is that any unintended insertion events from the therapy can potentially have detrimental effects in patients, as demonstrated by the development of malignancies in both animal and human studies. Therefore, directing the integration of foreign genes into safe sites within the genome is highly desirable for these approaches. In retroviral-based vector systems, the viral enzyme integrase (IN) catalyzes the insertion of a desired transgene nonspecifically into the host cell genome. Efforts to engineer IN to recognize specific target DNA sequences within the genome, and thereby improve the safety of future generations of retroviral-based vectors, are described. Recent results using fusion protein constructs of IN and E2C, a designed polydactyl zinc-finger protein that specifically recognizes an 18-base pair DNA sequence, are highlighted in this review. Encouraging results have been generated in vitro, and additional studies are ongoing in mammalian cell systems. The long-term goal of these efforts is the development of effective retroviral vectors that can safely deliver therapeutics in a gene therapy setting.

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.

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.

Design of polydactyl zinc-finger proteins for unique addressing within complex genomes.

Zinc-finger proteins of the Cys2-His2 type represent a class of malleable DNA-binding proteins that may be selected to bind diverse sequences. Typically, zinc-finger proteins containing three zinc-finger domains, like the murine transcription factor Zif268 and the human transcription factor Sp1, bind nine contiguous base pairs. To create a class of proteins that would be generally applicable to target unique sites within complex genomes, we have utilized structure-based modeling to design a polypeptide linker that fuses two three-finger proteins. Two six-fingered proteins were created and demonstrated to bind 18 contiguous bp of DNA in a sequence-specific fashion. Expression of these proteins as fusions to activation or repression domains allows transcription to be specifically up- or down-modulated within human cells. Polydactyl zinc-finger proteins should be broadly applicable as genome-specific transcriptional switches in gene therapy strategies and the development of novel transgenic plants and animals.