In previous studies we have developed Cys 2 -His 2 zinc finger domains that specifically recognized each of the 16 5-GNN-3 DNA target sequences and could be used to assemble six-finger proteins that bind 18-base pair DNA sequences (Beerli, R. R., Dreier, B., and Barbas, C. F., III (2000) Proc. Natl. Acad. Sci. U. S. A. 97, 1495-1500). Such proteins provide the basis for the construction of artificial transcription factors to study gene/function relationships in the post-genomic era. Central to the universal application of this approach is the development of zinc finger domains that specifically recognize each of the 64 possible DNA triplets. Here we describe the construction of a novel phage display library that enables the selection of zinc finger domains recognizing the 5-ANN-3 family of DNA sequences. Library selections provided domains that in most cases showed binding specificity for the 3-base pair target site that they were selected to bind. These zinc finger domains were used to construct 6-finger proteins that specifically bound their 18-base pair target site with affinities in the pM to low nM range. When fused to regulatory domains, these proteins containing various numbers of 5-ANN-3 domains were capable of specific transcriptional regulation of a reporter gene and the endogenous human ERBB-2 and ERBB-3 genes. These results suggest that modular DNA recognition by zinc finger domains is not limited to the 5-GNN-3 family of DNA sequences and can be extended to the 5-ANN-3 family. The domains characterized in this work provide for the rapid construction of artificial transcription factors, thereby greatly increasing the number of sequences and genes that can be targeted by DNA-binding proteins built from pre-defined zinc finger domains.The study of protein-DNA interactions is central to our understanding of the regulation of genes and the flow of genetic information characteristic of life. One practical application of the development of a protein-DNA recognition system is the construction of artificial transcription factors that might be used to purposefully regulate gene expression. We have demonstrated that gene expression can be specifically altered through the use of designed polydactyl zinc finger transcription factors that bind 18 base pairs (bp) 1 of DNA sequence. Because of their extended DNA recognition site, these proteins have the potential to be genome-specific transcriptional regulators (1, 2). Targeting of only 9 bp of sequence can also result in gene regulation wherein chromatin structure provides for an additional level of specificity (3, 4). Because a universal system for gene regulation would provide many new opportunities in basic and applied biology and medicine, the development of such a system is of considerable interest.Two key features have made Cys 2 -His 2 zinc finger domains the most promising DNA recognition motifs for the construction of artificial transcription factors, modular structure, and modular recognition. Each domain consists of ϳ30 amino acids and folds into a ␤␤␣ ...

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.The ability to rapidly prepare proteins with predefined specificities for DNA sequences could enable a wide range of technologies that might be used, for example, to direct the expression of genes or to physically modify genes and genomes. To develop a universal system for gene regulation, much effort has been applied to the development of artificial transcription factors based on polydactyl zinc finger proteins (1-3). Such a system might have considerable impact on biology and biotechnology and offer a new approach for treatment of diseases based on directed gene regulation. It has now been shown that gene expression can be specifically altered using artificial transcription factors based on polydactyl zinc finger proteins that bind to 18-bp target sites (1, 2). Targeting of sites as small as 9 bp can also provide some degree of regulatory specificity presumably through the aid of chromatin occlusion (4 -6). In addition to transcriptional regulation, novel zinc finger DNA binding specificities show tremendous promise in directing homologous recombination through their fusion with the Fok I nuclease domain (7,8).Zinc finger domains of the type Cys 2 -His 2 are a unique and promising class of proteins for the recognition of extended DNA sequences due to their modular nature. Each domain consists of ϳ30 amino acids folded into a ␤␤␣ structure stabilized by hydrophobic interactions and chelation of a zinc ion by the conserved Cys 2 -His 2 residues (9, 10). To date, the best-characterized protein of this family of zinc finger proteins is the mouse transcription factor Zif268. Each of the three zinc finger domains of Zif268 binds to a 3-bp subsite by insertion of the ␣-recognition helix into the major groove of the DNA double helix (11,12). To facilitate the rapid construction of DNA-bin...

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.

SummaryMalignant brain tumors, such as glioblastoma, are characterized by extensive angiogenesis and permeability of the blood-brain barrier (BBB). The infiltration of glioma cells away from the primary tumor mass is a pathological characteristic of glial tumors. The infiltrating tumor cells represent a significant factor in tumor recurrence following surgical debulking, radiation, and chemotherapy treatments. Vascular endothelial growth factor (VEGF)-mediated vascular permeability (VP) has been associated with the progression of glioma tumor growth and infiltration into surrounding normal brain parenchyma. While VEGF induces a robust VP response in control mice (src +/+ or src +/− ), the VP response is blocked in src −/− mice that demonstrate a 'leakage-resistant phenotype' in the brain. We used the Src-deficient mouse model to determine the role of Src in the maintenance of the BBB following orthotopic implantation and growth of glioma cells in the brain. Although solid tumor growth was the same in control and src −/− mice, the infiltrating component of glioma growth was reduced in src −/− mice. Characterization of the expression and localization of the extracellular matrix (ECM) protein fibrinogen was evaluated to determine the effect of a Src-mediated VP defect in the host compartment. These studies indicate that the reduced VP of host brain blood vessels of src −/− mice mediates a reduction in glioma cell invasion in a mouse brain tumor xenograft model.

Direct proliferative effects of estrogen (E 2 ) on estrogen receptor-positive tumors are well documented; however, the potential for E 2 to mediate effects selective for the host (i.e., angiogenesis, vascular permeability, or stromal effects), which influence tumor growth and/or metastasis, has received less attention. In this study, we examine the capacity for E 2 to promote tumor growth and/or metastasis independent of direct effects on tumor cells. In these studies, we distinguish host versus tumor compartment components of E 2 action in tumor growth and metastasis by analysis of E 2 -nonresponsive tumor cells implanted in ovariectomized (OVX) mice that contain s.c. implants of placebo (OVX) or E 2 -containing slowrelease pellets (OVX + E 2 ). We show that the D121 lung carcinoma cell line is E 2 -nonresponsive, and following s.c. implantation in OVX versus OVX + E 2 mice, E 2 action on the host compartment leads to an increase in spontaneous metastasis but not primary tumor growth or neovascularization. Similarly, experimental lung metastasis of E 2 -nonresponsive 4T1 mammary carcinoma cells also leads to increased tumor burden in the lungs of OVX + E 2 mice. These results suggest that the E 2 status of the host compartment influences late steps in tumor cell metastasis that can provide important insights into the role of E 2 in the tumor versus host compartments. (Cancer Res 2006; 66(7): 3667-72)

Signaling through the ErbB family of tyrosine kinase receptors in normal and cancer-derived cell lines contributes to cell growth and differentiation. In this work, we altered the levels of ErbB2 and ErbB3 receptors, individually and in combination, by using 6-finger and 12-finger synthetic zinc finger protein artificial transcription factors (ATFs) in an epidermoid squamous cell carcinoma line, A431. We successfully designed 12-finger ATFs capable of coregulating ErbB3 and ICAM-1 or ErbB2 and ErbB3. With ATFs, the effects of changes in ErbB2 and ErbB3 receptor levels were evaluated by using cell proliferation, cell migration, and cell signaling assays. Cell proliferation was increased when ErbB2 and ErbB3 were both overexpressed. Cell migration on collagen was decreased when ErbB2 was down-regulated, yet migration on laminin was significantly increased with ErbB3 overexpression. ErbB2 and ErbB3 overexpression also stimulated the phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. Our ATF approach has elucidated differences in ErbB receptor-mediated proliferation, migration, and intracellular signaling that cannot be explained merely by the presence or absence of particular ErbB receptors and emphasizes the dynamic nature of the ErbB signaling system. The transcription factor approach developed here provides a gene-economical route to the regulation of multiple genes and may be important for complex gene therapies.Signaling from ErbB tyrosine kinase receptors influences diverse aspects of a cell's biology that include growth, differentiation, migration, and apoptosis (29, 84). ErbB1 (EGFR/ HER1) was the first member of the family identified. Based on homology to ErbB1, three additional family members, ErbB2 (HER2/p185), ErbB3 (HER3), and ErbB4 (HER4), were identified (41,56,60,77). In normal development, binding of a growth factor ligand induces dimerization of ErbB receptors. Subsequently, the cytoplasmic tails are transphosphorylated. Each ErbB receptor has a unique pattern of phosphorylation sites that recruit various secondary signaling proteins (23,51,52,55,71). Ongoing research shows that the identity of the ligand bound, the amount of ligand, and the identities of dimers formed determine the activation of a particular intracellular signaling pathway such as the mitogen-activated protein kinase (MAPK), the stress-activated protein kinase, the protein kinase C, or the Akt pathway (53,61,72). The combination of at least 10 different ligands and 10 possible receptor dimers of the ErbB system form a signaling network essential for development (15,34).Various cancers, including those of the breast, head and neck, kidney, prostate, colon, pancreas, bladder, lung, and ovaries, are associated with overexpression of ErbB receptors (11,59,84). Research using breast cancer models has identified a dominant role for ErbB2 in tumor cell proliferation and metastasis (35,64,73,74). ErbB2 is the preferred dimerization partner for all ErbB receptors, and dimers containing ErbB2 have higher ligand af...

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