An Arabidopsis AT-hook motif nuclear protein mediates somatic embryogenesis and coinciding genome duplication.

Plant somatic cells can be reprogrammed into totipotent embryonic cells that are able to form differentiated embryos in a process called somatic embryogenesis (SE), by hormone treatment or through overexpression of certain transcription factor genes, such as BABY BOOM (BBM). Here we show that overexpression of the AT-HOOK MOTIF CONTAINING NUCLEAR LOCALIZED 15 (AHL15) gene induces formation of somatic embryos on Arabidopsis thaliana seedlings in the absence of hormone treatment. During zygotic embryogenesis, AHL15 expression starts early in embryo development, and AH15 and other AHL genes are required for proper embryo patterning and development beyond the globular stage. Moreover, AHL15 and several of its homologs are upregulated and required for SE induction upon hormone treatment, and they are required for efficient BBM-induced SE as downstream targets of BBM. A significant number of plants derived from AHL15 overexpression-induced somatic embryos are polyploid. Polyploidisation occurs by endomitosis specifically during the initiation of SE, and is caused by strong heterochromatin decondensation induced by AHL15 overexpression.

Zinc Finger Artificial Transcription Factor-Mediated Chloroplast Genome Interrogation in Arabidopsis thaliana.

The large majority of core photosynthesis proteins in plants are encoded by nuclear genes, but a small portion have been retained in the plastid genome. These plastid-encoded chloroplast proteins fulfill essential roles in the process of photochemistry. Here, we report the use of nuclear-encoded, chloroplast-targeted zinc finger artificial transcription factors (ZF-ATFs) with effector domains of prokaryotic origin to modulate the expression of chloroplast genes, and to enhance the photochemical activity and growth characteristics of Arabidopsis thaliana plants. This technique was named chloroplast genome interrogation. Using this novel approach, we obtained evidence that ZF-ATFs can indeed be translocated to chloroplasts of Arabidopsis plants, can modulate their growth and operating light use efficiency of PSII, and finally can induce statistically significant changes in the expression levels of several chloroplast genes. Our data suggest that the distortion of chloroplast gene expression might be a feasible approach to manipulate the efficiency of photosynthesis in plants.

Agrobacterium-Mediated Transformation of Yeast and Fungi.

Two decades ago, it was discovered that the well-known plant vector Agrobacterium tumefaciens can also transform yeasts and fungi when these microorganisms are co-cultivated on a solid substrate in the presence of a phenolic inducer such as acetosyringone. It is important that the medium has a low pH (5-6) and that the temperature is kept at room temperature (20-25 °C) during co-cultivation. Nowadays, Agrobacterium-mediated transformation (AMT) is the method of choice for the transformation of many fungal species; as the method is simple, the transformation efficiencies are much higher than with other methods, and AMT leads to single-copy integration much more frequently than do other methods. Integration of T-DNA in fungi occurs by non-homologous end-joining (NHEJ), but also targeted integration of the T-DNA by homologous recombination (HR) is possible. In contrast to AMT of plants, which relies on the assistance of a number of translocated virulence (effector) proteins, none of these (VirE2, VirE3, VirD5, VirF) are necessary for AMT of yeast or fungi. This is in line with the idea that some of these proteins help to overcome plant defense. Importantly, it also showed that VirE2 is not necessary for the transport of the T-strand into the nucleus. The yeast Saccharomyces cerevisiae is a fast-growing organism with a relatively simple genome with reduced genetic redundancy. This yeast species has therefore been used to unravel basic molecular processes in eukaryotic cells as well as to elucidate the function of virulence factors of pathogenic microorganisms acting in plants or animals. Translocation of Agrobacterium virulence proteins into yeast was recently visualized in real time by confocal microscopy. In addition, the yeast 2-hybrid system, one of many tools that have been developed for use in this yeast, was used to identify plant and yeast proteins interacting with the translocated Agrobacterium virulence proteins. Dedicated mutant libraries, containing for each gene a mutant with a precise deletion, have been used to unravel the mode of action of some of the Agrobacterium virulence proteins. Yeast deletion mutant collections were also helpful in identifying host factors promoting or inhibiting AMT, including factors involved in T-DNA integration. Thus, the homologous recombination (HR) factor Rad52 was found to be essential for targeted integration of T-DNA by HR in yeast. Proteins mediating double-strand break (DSB) repair by end-joining (Ku70, Ku80, Lig4) turned out to be essential for non-homologous integration. Inactivation of any one of the genes encoding these end-joining factors in other yeasts and fungi was employed to reduce or totally eliminate non-homologous integration and promote efficient targeted integration at the homologous locus by HR. In plants, however, their inactivation did not prevent non-homologous integration, indicating that T-DNA is captured by different DNA repair pathways in plants and fungi.

MeioSeed: a CellProfiler-based program to count fluorescent seeds for crossover frequency analysis in Arabidopsis thaliana.

BACKGROUND: The formation of crossovers during meiosis is pivotal for the redistribution of traits among the progeny of sexually reproducing organisms. In plants the molecular mechanisms underlying the formation of crossovers have been well established, but relatively little is known about the factors that determine the exact location and the frequency of crossover events in the genome. In the model plant species Arabidopsis, research on these factors has been greatly facilitated by reporter lines containing linked fluorescence marker genes under control of promoters active in seeds or pollen, allowing for the visualization of crossover events by fluorescence microscopy. However, the usefulness of these reporter lines to screen for novel modulators of crossover frequency in a high throughput manner relies on the availability of programs that can accurately count fluorescent seeds. Such a program was previously not available in scientific literature. RESULTS: Here we present MeioSeed, a novel CellProfiler-based program that accurately counts GFP and RFP fluorescent Arabidopsis seeds with adjustable detection thresholds for fluorescence intensity, making use of a robust seed classifier which was trained by machine learning in Ilastik. Using the previously published reporter line Col3-4/20 as an example, we explain the use of MeioSeed and the steps taken to optimize the thresholding settings of the program to fit the published model for recombination frequency and transgene segregation. The use of MeioSeed is illustrated by investigating salt stress as a novel abiotic trigger for changes in crossover frequency in Col3-4/20 (♂) × Ler-0 (♀) F1 hybrids. Salt stress was found to trigger increases in crossover frequency between the marker genes of up to 70% compared to the control treatment without salt stress. Genotyping of control and salt treated populations revealed that the changes in crossover frequency were not limited to the region between the marker genes, but that fluctuations in crossover frequency are likely to occur genome-wide after treatment with high salt concentrations. CONCLUSIONS: MeioSeed allows for the high throughput recognition and counting of fluorescent Arabidopsis seeds and can facilitate the screening for novel abiotic and biotic modulators of crossover frequency using reporter lines in Arabidopsis.

High-resolution magic angle spinning NMR studies for metabolic characterization of Arabidopsis thaliana mutants with enhanced growth characteristics.

Developing smart crops which yield more biomass to meet the increasing demand for plant biomass has been an active area of research in last few decades. We investigated metabolic alterations in two Arabidopsis thaliana mutants with enhanced growth characteristics that were previously obtained from a collection of plant lines expressing artificial transcription factors. The metabolic profiles were obtained directly from intact Arabidopsis leaves using high-resolution magic angle spinning (HR-MAS) NMR. Multivariate analysis showed significant alteration of metabolite levels between the mutants and the wild-type Col-0. Interestingly, most of the metabolites that were reduced in the faster-growing mutants are generally involved in the defence against stress. These results suggest a growth-defence trade-off in the phenotypically engineered mutants. Our results further corroborate the idea that plant growth can be enhanced by suppressing defence pathways.

An Arabidopsis mutant with high operating efficiency of Photosystem II and low chlorophyll fluorescence.

The overall light energy to biomass conversion efficiency of plant photosynthesis is generally regarded as low. Forward genetic screens in Arabidopsis have yielded very few mutants with substantially enhanced photochemistry. Here, we report the isolation of a novel Arabidopsis mutant with a high operating efficiency of Photosystem II (φPSII) and low chlorophyll fluorescence from a library of lines harboring T-DNA constructs encoding artificial transcription factors. This mutant was named Low Chlorophyll Fluorescence 1 (LCF1). Only a single T-DNA insertion was detected in LCF1, which interrupted the expression of the full length mRNA of the gene At4g36280 (MORC2). We demonstrate that the high φPSII and low levels of chlorophyll fluorescence were due to a decrease in PSII:PSI ratio. Although LCF1 plants had decreased rosette surface area and biomass under normal growth conditions, they contained more starch per gram fresh weight. The growth defect of LCF1 was alleviated by low light and short day conditions, and growth could even be enhanced after a period of dark-induced senescence, showing that the plant can utilize its excess photosynthetic conversion capacity as a resource when needed.

Enhancement of Arabidopsis growth characteristics using genome interrogation with artificial transcription factors.

The rapidly growing world population has a greatly increasing demand for plant biomass, thus creating a great interest in the development of methods to enhance the growth and biomass accumulation of crop species. In this study, we used zinc finger artificial transcription factor (ZF-ATF)-mediated genome interrogation to manipulate the growth characteristics and biomass of Arabidopsis plants. We describe the construction of two collections of Arabidopsis lines expressing fusions of three zinc fingers (3F) to the transcriptional repressor motif EAR (3F-EAR) or the transcriptional activator VP16 (3F-VP16), and the characterization of their growth characteristics. In total, six different 3F-ATF lines with a consistent increase in rosette surface area (RSA) of up to 55% were isolated. For two lines we demonstrated that 3F-ATF constructs function as dominant in trans acting causative agents for an increase in RSA and biomass, and for five larger plant lines we have investigated 3F-ATF induced transcriptomic changes. Our results indicate that genome interrogation can be used as a powerful tool for the manipulation of plant growth and biomass and that it might supply novel cues for the discovery of genes and pathways involved in these properties.

Genome interrogation for novel salinity tolerant Arabidopsis mutants.

Soil salinity is becoming an increasingly large problem in agriculture. In this study, we have investigated whether a capacity to withstand salinity can be induced in the salinity sensitive plant species Arabidopsis thaliana, and whether it can be maintained in subsequent generations. To this end, we have used zinc finger artificial transcription factor (ZF-ATFs) mediated genome interrogation. Already within a relatively small collection Arabidopsis lines expressing ZF-ATFs, we found 41 lines that were tolerant to 100 mM NaCl. Furthermore, ZF-ATF encoding gene constructs rescued from the most strongly salinity tolerant lines were indeed found to act as dominant and heritable agents for salinity tolerance. Altogether, our data provide evidence that a silent capacity to withstand normally lethal levels of salinity exists in Arabidopsis and can be evoked relatively easily by in trans acting transcription factors like ZF-ATFs.

Enhanced targeted integration mediated by translocated I-SceI during the Agrobacterium mediated transformation of yeast.

Agrobacterium mediated transformation (AMT) has been embraced by biotechnologists as the technology of choice to introduce or alter genetic traits of plants. However, in plants it is virtually impossible to predetermine the integration site of the transferred T-strand unless one is able to generate a double stranded break (DSB) in the DNA at the site of interest. In this study, we used the model organism Saccharomyces cerevisiae to investigate whether the Agrobacterium mediated translocation of site-specific endonucleases via the type IV secretion system (T4SS), concomitantly with T-DNA transfer is possible and whether this can improve the gene targeting efficiency. In addition to that, the effect of different chromatin states on targeted integration, was investigated. It was found that Agrobacterium mediated translocation of the homing endonuclease I-SceI has a positive effect on the integration of T-DNA via the homologous repair (HR) pathway. Furthermore, we obtained evidence that nucleosome removal has a positive effect on I-SceI facilitated T-DNA integration by HR. Reversely; inducing nucleosome formation at the site of integration removes the positive effect of translocated I-SceI on T-DNA integration.

Artificial transcription factor-mediated regulation of gene expression.

The transcriptional regulation of endogenous genes with artificial transcription factors (TFs) can offer new tools for plant biotechnology. Three systems are available for mediating site-specific DNA recognition of artificial TFs: those based on zinc fingers, TALEs, and on the CRISPR/Cas9 technology. Artificial TFs require an effector domain that controls the frequency of transcription initiation at endogenous target genes. These effector domains can be transcriptional activators or repressors, but can also have enzymatic activities involved in chromatin remodeling or epigenetic regulation. Artificial TFs are able to regulate gene expression in trans, thus allowing them to evoke dominant mutant phenotypes. Large scale changes in transcriptional activity are induced when the DNA binding domain is deliberately designed to have lower binding specificity. This technique, known as genome interrogation, is a powerful tool for generating novel mutant phenotypes. Genome interrogation has clear mechanistic and practical advantages over activation tagging, which is the technique most closely resembling it. Most notably, genome interrogation can lead to the discovery of mutant phenotypes that are unlikely to be found when using more conventional single gene-based approaches.

Involvement of Rad52 in T-DNA circle formation during Agrobacterium tumefaciens-mediated transformation of Saccharomyces cerevisiae.

Agrobacterium tumefaciens cells carrying a tumour inducing plasmid (Ti-plasmid) can transfer a defined region of transfer DNA (T-DNA) to plant cells as well as to yeast. This process of Agrobacterium-mediated transformation (AMT) eventually results in the incorporation of the T-DNA in the genomic DNA of the recipient cells. All available evidence indicates that T-strand transfer closely resembles conjugal DNA transfer as found between Gram-negative bacteria. However, where conjugal plasmid DNA transfer starts via relaxase-mediated processing of a single origin of transfer (oriT), the T-DNA is flanked by two imperfect direct border repeats which are both substrates for the Ti-plasmid encoded relaxase VirD2. Yeast was used as a model system to investigate the requirements of the recipient cell for the formation of T-DNA circles after AMT. It was found that, despite the absence of self-homology on the T-DNA, the homologous repair proteins Rad52 and Rad51 are involved in T-DNA circle formation. A model is presented involving the formation of T-DNA concatemers derived from T-strands by a process of strand-transfer catalysed by VirD2. These concatemers are then resolved into T-DNA circles by homologous recombination in the recipient cell.

Zinc finger artificial transcription factor-based nearest inactive analogue/nearest active analogue strategy used for the identification of plant genes controlling homologous recombination.

In previous work, we selected a particular transcription factor, designated VP16-HRU, from a pool of zinc finger artificial transcription factors (ZF-ATFs) used for genome interrogation. When expressed in Arabidopsis thaliana under control of the ribosomal protein S5A promoter, the RPS5A::VP16-HRU construct led to a 200- to 300-fold increase in the frequency of somatic intrachromosomal homologous recombination (iHR). Because the expression of each ZF-ATF leads to a large number of transcriptional changes, we designed a strategy employing a collection of structurally similar ZF-ATFs to filter out the transcriptional changes relevant to the phenotype by deep sequencing. In that manner, 30 transcripts were found to be consistently induced in plants with enhanced homologous recombination (HR). For 25 of the cognate genes, their effect on the HR process was assessed using cDNA/gDNA expression constructs. For three genes, ectopic expression indeed led to enhanced iHR frequencies, albeit much lower than the frequency observed when a HR-inducing ZF-ATF was present. Altogether, our data demonstrate that despite the large number of transcriptional changes brought about by individual ZF-ATFs, causal changes can be identified. In our case, the picture emerged that a natural regulatory switch for iHR does not exist but that ZF-ATFs-like VP16-HRU act as an ectopic master switch, orchestrating the timely expression of a set of plant genes that each by themselves only have modest effects, but when acting together support an extremely high iHR frequency.

ZFN-mediated gene targeting of the Arabidopsis protoporphyrinogen oxidase gene through Agrobacterium-mediated floral dip transformation.

Previously, we showed that ZFN-mediated induction of double-strand breaks (DSBs) at the intended recombination site enhanced the frequency of gene targeting (GT) at an artificial target locus using Agrobacterium-mediated floral dip transformation. Here, we designed zinc finger nucleases (ZFNs) for induction of DSBs in the natural protoporphyrinogen oxidase (PPO) gene, which can be conveniently utilized for GT experiments. Wild-type Arabidopsis plants and plants expressing the ZFNs were transformed via floral dip transformation with a repair T-DNA with an incomplete PPO gene, missing the 5' coding region but containing two mutations rendering the enzyme insensitive to the herbicide butafenacil as well as an extra KpnI site for molecular analysis of GT events. Selection on butafenacil yielded 2 GT events for the wild type with a frequency of 0.8 × 10⁻³ per transformation event and 8 GT events for the ZFNs expressing plant line with a frequency of 3.1 × 10⁻³ per transformation event. Molecular analysis using PCR and Southern blot analysis showed that 9 of the GT events were so-called true GT events, repaired via homologous recombination (HR) at the 5' and the 3' end of the gene. One plant line contained a PPO gene repaired only at the 5' end via HR. Most plant lines contained extra randomly integrated T-DNA copies. Two plant lines did not contain extra T-DNAs, and the repaired PPO genes in these lines were transmitted to the next generation in a Mendelian fashion.

Zinc finger-mediated live cell imaging in Arabidopsis roots.

Following the elucidation of recognition codes, artificial zinc finger (ZF) domains can now be assembled to create custom-made DNA-binding proteins in which the alpha helix of each zinc finger mediates an interaction with 3 or 4 bp of DNA. A module of consecutive zinc finger domains, designated a polydactyl zinc finger (PZF) domain, is thus capable of binding an extended number of base pairs of DNA. Besides the multitude of utilities of PZF domains addressed in other chapters, we have shown that they can also be used for live cell imaging of repetitive DNA sequences in Arabidopsis, as well as in mouse cells by generating and expressing PZF:GFP fusion proteins (1). Here we provide a detailed protocol for the construction of such PZF:GFP reporter proteins using our established cloning vehicles, together with a protocol for their expression in plants in order to achieve in vivo labelling of repetitive DNA. Furthermore we provide an accurate quantification method for GFP signals using fluorescent beads (FluoSpheres). Single-molecule precision can be obtained using any confocal setup once the fluorescent beads have been calibrated against purified GFP. The methods can easily be adapted to meet the demands for other situations or for other experimental systems.

Agrobacterium-mediated T-DNA transfer and integration by minimal VirD2 consisting of the relaxase domain and a type IV secretion system translocation signal.

The VirD2 protein of Agrobacterium tumefaciens is essential for processing and transport of the T-DNA. It has at least three functional domains: a relaxase domain at the N terminus, a bipartite nuclear localization signal (NLS), and a sequence called omega at the C terminus. We confirm here that deletions of the C-terminal part of VirD2 led to lack of transfer of T-DNA but, for the first time, we report that virulence is restored when these truncations are supplemented at the C terminus by a short translocation signal from the VirF protein. The lack of virulence of C-terminal deletions suggests that the C-terminal part contains all or part of the translocation signal of VirD2. Using a novel series of mutant VirD2 proteins, the C-terminal half of VirD2 was further investigated. We demonstrate that the C-terminal 40 amino acids of VirD2, which include the NLS and omega, contain all or part of the translocation domain necessary for transport of VirD2 into plant cells, while another element is present in the middle of the protein. The finding that a type IV secretion system transport signal at the C terminus of VirD2 is necessary for virulence provides evidence for the role of VirD2 as a pilot protein driving translocation of the T-strand.

ZFN-induced mutagenesis and gene-targeting in Arabidopsis through Agrobacterium-mediated floral dip transformation.

Zinc-finger nucleases (ZFNs) are artificial restriction enzymes, custom designed for induction of double-strand breaks (DSBs) at a specific locus. These DSBs may result in site-specific mutagenesis or homologous recombination at the repair site, depending on the DNA repair pathway that is used. These promising techniques for genome engineering were evaluated in Arabidopsis plants using Agrobacterium-mediated floral dip transformation. A T-DNA containing the target site for a ZFN pair, that was shown to be active in yeast, was integrated in the Arabidopsis genome. Subsequently, the corresponding pair of ZFN genes was stably integrated in the Arabidopsis genome and ZFN activity was determined by PCR and sequence analysis of the target site. Footprints were obtained in up to 2% of the PCR products, consisting of deletions ranging between 1 and 200 bp and insertions ranging between 1 and 14 bp. We did not observe any toxicity from expression of the ZFNs. In order to obtain ZFN-induced gene-targeting (GT), Arabidopsis plants containing the target site and expressing the ZFN pair were transformed with a T-DNA GT construct. Three GT plants were obtained from approximately 3000 transformants. Two of these represent heritable true GT events, as determined by PCR, Southern blot analysis and sequencing of the resulting recombined locus. The third plant showed an ectopic GT event. No GT plants were obtained in a comparable number of transformants that did not contain the ZFNs. Our results demonstrate that ZFNs enhance site-specific mutagenesis and gene-targeting of Agrobacterium T-DNA constructs delivered through floral dip transformation.

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.

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment.

Zinc finger protein transcription factors (ZFP-TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA-binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene-specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA-binding motifs. In this study, we engineered ZFP-TFs to downregulate the activity of the epithelial glycoprotein-2 (EGP-2) promoter. The protein EGP-2 is overexpressed in a wide variety of cancer types and EGP-2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP-2 expression by ZFP-TFs provides a novel anti-cancer therapeutic. Using a straightforward strategy, we engineered a 3-ZFP that could bind a 9 bp sequence within the EGP-2 promoter. After the addition of a repressor domain, this 3-ZFP-TF could efficiently downregulate EGP-2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP-2 promoter activity. To inhibit the endogenous EGP-2 promoter, we engineered 6-ZFP-TFs. Although none of the constructed ZFP-TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP-TFs are versatile bi-directional modulators of gene expression and downregulation of EGP-2 promoter activity using ZFP-TFs can ultimately result in a novel anti-cancer treatment.

Employing libraries of zinc finger artificial transcription factors to screen for homologous recombination mutants in Arabidopsis.

A library of genes for zinc finger artificial transcription factors (ZF-ATF) was generated by fusion of DNA sequences encoding three-finger Cys(2)His(2) ZF domains to the VP16 activation domain under the control of the promoter of the ribosomal protein gene RPS5A from Arabidopsis thaliana. After introduction of this library into an Arabidopsis homologous recombination (HR) indicator line, we selected primary transformants exhibiting multiple somatic recombination events. After PCR-mediated rescue of ZF sequences, reconstituted ZF-ATFs were re-introduced in the target line. In this manner, a ZF-ATF was identified that led to a 200-1000-fold increase in somatic HR (replicated in an independent second target line). A mutant plant line expressing the HR-inducing ZF-ATF exhibited increased resistance to the DNA-damaging agent bleomycin and was more sensitive to methyl methanesulfonate (MMS), a combination of traits not described previously. Our results demonstrate that the use of ZF-ATF pools is highly rewarding when screening for novel dominant phenotypes in Arabidopsis.

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.