Figure 5: Regulation of CDH5 by TFZFs in several human cancer cell lines.
Blue, cells infected with a pMX construct containing the DNA binding domain of VE-1 (A–E) or VE-8 (F) and the VP64 activator domain, stained with anti-CD144 and analyzed by FACS. Red, cells infected with the pMX vector containing the same DNA-binding domain but linked to the KRAB repression domain (SKD), and stained with anti-CD144 (anti-VE-cadherin). Green, level of VE-cadherin expressed on mock-infected cells; stippled lines, cells stained in the absence of primary antibody.
Among all the TFZFs tested, the promoter-binding TFZFs were able to regulate CDH5 in all cell lines tested, as expected for direct regulation of the promoter. Those TFZFs that did not transactivate the promoter in the reporter assay (such as 144-4, 144-5, and 144-13) showed different activation profiles that varied depending on the cell line examined. Some of these TFZFs could bind regulatory regions located in the large 5' introns of CDH5, or even regulatory regions of upstream genes, perhaps encoding tissue-specific factors involved in controlling CDH5 expression. Candidates for these indirect targets include some members of the ETS family, including ETS1, ERG, and FLI1 (refs. 23, 24). However, database searches showed that at most 14 of 18 bp within these regions had identity to predicted TFZF targets. A search for 6ZF-binding sites in the human genome identified target sites matching between 13 and 18 bp (see Supplementary Tables 1 and 2 online). Within the CDH5 locus, 13–14 bp matches were identified. Although further investigation is required to understand their in vivo significance, these results suggest that 6ZF proteins could use a subset of the 18 bp sites to interact with genomic sites.
In summary, we present a method to identify functional DNA-protein interactions involved in the activation of target genes in human cells by screening large combinatorial libraries of TFZFs. We characterized clones selected from 3ZF and 6ZF libraries that were able to induce an endothelial specific marker, VE-cadherin, in a non-endothelial cancer cell line A431. A population of selected TFZFs was able to directly transactivate the CDH5 promoter by binding both a proximal and a distal promoter region. In addition, we showed that these TFZFs could regulate their target gene in a variety of human cancer cell lines. The advantages of libraries of small TFZF, such as 3ZF libraries, include high representation of individual members and the possibility of binding multiple sites in one or more regulatory regions, a mode of regulation analogous to the action of natural transcription factors. Highly complex libraries of the 6ZF type have low representation of each individual TFZF clone but potentially higher specificity. These TFZFs could recognize low-frequency, potentially unique sites that are sufficient to activate or repress the target gene. Used in combination with current technologies such as DNA microarrays and chromatin immunoprecipitations, they could be useful for identifying genes and defining pathways. Recent studies in transgenic tobacco and Arabidopsis thaliana plants indicate that zinc-finger technology can be applied to whole organisms27, 28. Thus, this methodology represents a genetic tool for the selection or screening of gain-of-function and loss-of-function phenotypes at the level of the cell or organism based on direct gene regulation or on more complex changes in transcriptional programs.
Experimental protocol
Construction of TFZF libraries.
The 3ZF library was created by overlapping PCR using 23 different ZF1s, 21 ZF2s, and 19 ZF3s mixed into the PCR reaction (see Supplementary Experimental Protocol online). All DNAs used as templates for PCR were SP1 variants containing specific zinc-finger 
-helices selected and characterized in our laboratory8, 10, 12. These templates were cloned and sequenced in pMalc2 (New England Biolabs, Beverly, MA). The final (F1 + F2 + F3) PCR product was digested with SfII and cloned in the pComb3X vector29. The resulting pComb3X-3ZF library vector was used to construct the 6ZF library as follows. First, 10 
g of pComb3X-3ZF library vector was digested with AgeI and NheI and ligated with 3 g of XmaI- and NheI-digested inserts to generate the pComb3X-6ZF library vector. Both 3ZF and 6ZF library inserts were digested with SfII and subcloned into the retroviral vector pMX-IRES-GFP, containing the VP64 activation domain5. The final sizes of the 3ZF and 6ZF libraries in the retroviral vector were 3.52 
105 and 5.3 107, respectively.
Screening for functional TFZF activators in A431 cells and flow cytometry.
The pMX-IRES-GFP-3ZF library and pMX-IRES-GFP-6ZF library DNAs were transfected into 293 packaging cells5 using Lipofectamine Plus (Invitrogen, Carlsbad, CA) according to the manufacturer's directions. The product retroviral particles were used to infect 5 105(3ZF library) or 108 (6ZF library) A431 cells. At 48 h after infection, these cells were stained with ten different primary antibodies (5 g/ml) specific for different cell surface markers: anti-CD15 (clone 2F3; BD, PharMingen, San Diego, CA), anti-ERBB-2 (clone SP77; ref. 5), anti-ERBB-3 (clone SPG1, NeoMarkers, Fremont, CA), anti-CD104 (clone 450–9D), anti-CD144 (clone 55–7H1, PharMingen), anti-CD54 (clone HA58, PharMingen), anti-CD58 (clone 1C3, PharMingen), anti-CD95 (Clone DX2, PharMingen), anti-EGF (Santa Cruz Biotechnology, Santa Cruz, CA), anti-CD49f (clone GoH3, PharMingen) and secondary antibodies conjugated to phycoerythrin (PE, 1:100 dilution, Jackson ImmunoResearch, West Grove, PA). Next, 5 105 to 106 GFP+PE+ infected cells (3ZF library) or 107 GFP+PE+ infected cells (6ZF library) were sorted using a FACSVantage (BD, PharMingen), and the DNA encoding the pool of TFZFs was recovered by PCR using the primers pMXf2 (forward) 5'-TCAAAGTAGACGGCATCG-3' and VP64AscB (backward) 5'-TCGTCCAGCGCGCGTCGGCGCG-3', and cloned again into the pMX vector. PCR was typically carried out using 50 ng–1 g of genomic DNA and a program of 1 cycle of 5 min at 94 °C; 35 cycles of 30 s at 94 °C, 2 min at 52 °C and 2 min (3ZF library) or 3 min (6ZF library) at 72 °C cycles; and a final cycle of 10 min at 72 °C. Independent selections were done for each cell-surface marker. The selections were repeated for three (3ZF library) and four rounds (6ZF library). DNA from individual clones was prepared and used to prepare virus to infect A431 cells. These cells were analyzed by flow cytometry using ten different antibodies as described above. For downregulation analysis, zinc fingers were subcloned into pMX-IRES-GFP-SKD vector (containing the KRAB repression domain, SKD; ref. 5) and infections were carried out as described above. The cell lines A431, HeLa, and SKBR-3 were cultured as described5, cell line MDA-MB-435s was obtained from the American Type Culture Collection (Manassas, VA), and cell lines C8161 and HT29 were a generous gift from R.A. Reisfeld of the Scripps Research Institute.
RNA extraction and RT-PCR. RNA from A431-infected cells and HUVEC cells (Clonetics, San Diego, CA) were extracted with the Tri reagent method (MRC, Cincinnati, OH). cDNA was made using a RT-PCR kit (Invitrogen, Carlsbad, CA). PCR was made using CDH5-specific primers25: VE-CAD-f (forward) 5'-CCGGCGCCAAAAGAGAGA-3' and VE-CAD-b (backward) 5'-CTCCTTTTCCTTCAGCTGAAGTGGT-3'. Expression of GAPDH (encoding glyceraldehyde-3-phosphate dehydrogenase) was measured as a loading control using the primers GAPDH-f (forward) 5'-CCATGTTCGTCATGGGTGTGA-3' and GAPDH-b (backward) 5'-CATGGACTGTGGTCATGAGT-3'. CDH5 mRNA levels were normalized relative to TFZFs using primers NLSseq-F (forward) 5'-CCGAAAAAGAAACGCAAAGTTGGG-3' and pMXB (backward) 5'-CAGAATTTCGACCACTGTGC-3', which amplify VP64. PCR conditions were 1 cycle of 3 min at 94 °C; 20–30 cycles of 1 min at 94 °C, 2.5 min at 52 °C, and 2 min at 72 °C; and 1 cycle of 5 min at 72 °C. PCR products were visualized in a 1% (CDH5) or 1.5% agarose gel (GAPDH) and quantified using ImageQuant 1.2. The 1-kbp CDH5-specific PCR product was sequenced and shown to correspond to the expected CDH5 sequence.
Luciferase assays. The human CDH5 promoter fragment (-2486 to +24) was amplified from A431 cells by PCR using the primers cdh5pro-f3 (forward) 5'-GAGGAGGAGGAGGAGGGTACCGGGGCCCAAGAAAT
CTGCATATTC-3' and cdh5pro-b2 (backward)
5'-GAGGAGGAGGAGGAGAGATCTTGTTTCTGTTCC
GTTGGACTGC-3'). The products were sequenced and cloned into pGL3basic (Promega, Madison, WI). Next, 100 ng of reporter construct, 75 ng of TFZF cloned in pcDNA3 (Invitrogen), and 100 ng of CMV-LacZ reporter were transiently cotransfected in A431 cells. Luciferase activities were measured using a luciferase reporter assay system (Promega). Transfection efficiencies were normalized with the 
-galactosidase reporter system (Galacto-Light Plus kit; Tropix, Bedford, MA). Data represent the average of 6–12 experiments. Point mutations in the promoter were introduced by PCR using high-fidelity enzyme (Roche, Indianapolis, IN) and verified by DNA sequencing.
In vitro analysis of TFZF binding, mobility-shift experiments, and CAST.
These assays were done as described previously11, 30 (see Supplementary Experimental Protocol online).
Note: Supplementary information is available on the Nature Biotechnology website.
Received 16 September 2002; Accepted 3 January 2003; Published online 18 February 2003.
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