A Finger on the Pulse of Transcriptional Control

"I lost concentration and began to think of our scholarly daughter working at Yale on a project called Zinc Fingers scanning a protein with pseudopods each with a trace of zinc that latch on to our DNA and help determine what we become." --From Zinc Fingers, Peter Meinke "GREEN" FINGERS: Zinc finger- based artificial transcription factors (background) have been applied in plants such as Arabidopsis thaliana (foreground). Reprinted with permission, Curr Opin Plant Biol, 6:163-8, April 2003. For some, the mention of zinc fingers fosters tasty images like ladyfinger- layered tiramisu or thin pieces of crispy fried chicken. For Meinke, zinc fingers are pure poetry. But only time and clinical tests will tell if zinc fingers, DNA-binding motifs with a zinc-ion core, can herald a new treatment for coronary and peripheral artery disease. A new drug, proposed by researchers at Sangamo Biosciences, Richmond, Calif., is just one of many potential applications of a new type of genetic engineering tool known as artificial zinc-finger protein transcription factors (TFsZF or ZFP TFs). These factors, with their unparalleled specificity, modu- larity, and more natural expression profiles, are revolutionizing the way scientists manipulate genes. At the heart of this technology are zinc fingers themselves. Protruding from transcription factors like pseudopods from a cell, they target and grasp specific sequences of DNA. The regulatory domains, to which they're fused, turn genes on or off. First discovered in frogs by Aaron Klug and coworkers in 1985,1 scientists can now engineer ZFP TFs to do what no other artificial transcription factors can do. By mixing and matching zinc fingers, biologists can construct ZFP TFs that bind virtually any gene in any cell. Yet, real and perceived difficulties in the application of ZFP TFs have restricted their use largely to the experts. Pioneered by Klug, Carlos Barbas of The Scripps Research Institute, La Jolla, Calif., and Carl Pabo of Sangamo, the field of zinc-finger engineering has progressed remarkably in the past half year. Two groups of scientists from the academic and private sectors demonstrated the feasibility of using engineered ZFP TFs to regulate gene expression in vivo. In October 2002, Xuen Guan, Justin Stege, Barbas, and collaborators from both Scripps and the Torrey Mesa Research Institute, San Diego, demonstrated engineered ZFP TF regulation of an endogenous gene in a plant, Arabidopsis thaliana.2 One month later, Edward Rebar, Yan Huang, their Sangamo colleagues, and others from Yale University published a paper that used the same technology in an animal model.3 They also demonstrated the potential utility of ZFP TFs as a therapeutic agent for humans, in this case to stimulate blood vessel growth. This provided crucial preclinical evidence for a novel type of heart disease drug for which Sangamo plans to file an investigational new-drug application in 2004, according to Edward Lanphier, the company's CEO. That zinc fingers have risen to such fame comes as no surprise to Klug, of the Medical Research Council, Cambridge, UK. "I thought it was such a beautiful system," says Klug. "I didn't think such a good system would be 'wasted' on something as lowly as a frog gene." Although some tout ZFP TFs as the next best way to develop novel therapeutic agents, scientists still face some major biological and regulatory hurdles. Genomicists probably have the most to gain from the new technology, as ZFP TFs provide a powerful new approach to gene discovery. "For simple research purposes, it is an excellent tool," says Klug. Others, including Roger Beachy of the Donald Danforth Plant Science Center, St. Louis, agree: "It is a very powerful tool for learning about gene function." The technology also may benefit GM-friendly farmers (those who grow genetically modified food) and their industrial partners. Sangamo, with a monopoly on ZFP TF intellectual property, is developing ways to use ZFP TFs to improve crop yield, cut costs, and generate products with desirable phenotypes. MOLECULAR LEGO Individual zinc fingers are short stretches of 30 amino acids folded into compact structures. Most natural ZFP TFs have three fingers, but some have as many as 37, arranged one after another, like fingers on a hand, positioned in such a way that they can readily touch multiple, adjacent spots along the DNA double helix. The key to zinc fingers' success is their sequence-specific, modular structure. Barbas likens them to "molecular Lego blocks." With each zinc finger targeting a specific 3-base pair DNA sequence, a six-fingered protein, for example, should have the capacity to bind just one of nearly 70 billion unique 18-bp sequences. That's more than good enough to target a single gene in the 3.2 billion-bp human genome. Other ways to engineer transcription factors exist, but none of them match the combinatorial power of ZFP TFs. Zinc fingers also are the most common DNA-binding motif, which has surprised Klug: "I didn't expect it to be 1% or 2% of the human genome." About 700 human genes encode zinc-finger proteins. Arabidopsis thaliana has about 85 zinc- finger genes. But, not all zinc-containing, DNA-binding proteins are true zinc fingers, Klug warns. Early on, researchers were calling any and every zinc- containing, DNA-binding domain a zinc finger. But only C2H2 (cysteine2-histidine2) zinc proteins are "true" zinc fingers. Even the zinc fingers in Meinke's poem weren't the real thing. Gretchen Meinke, the "scholarly daughter" who is now a protein crystallographer at Boston Biomedical Research Institute, explains that she was working on a project on nuclear hormone receptors, not zinc fingers. In addition to their combinatorial power, by mixing and matching regulatory domains with zinc fingers, scientists can engineer ZFP TFs that either activate or inhibit gene expression. The techniques allow only one or the other: transgenes to overexpress, or RNA interference to turn genes off. ZFP TFs can do both. Moreover, ZFP TFs act on endogenous genes. As Rebar, Huang, and their colleagues demonstrated, using nature's own creation has phenotypic advantages. Natural transcription of a single gene often results in a set of slightly different protein sequences, or splice variants. When used to overexpress endogenous genes, ZFP TFs produce all the splice variants in their proper ratios. The resulting phenotype is much more natural than that attained by transgenic expression, which produces only a single splice variant. PHOTOSYSTEMS REVEALED: DNA gets fingered: … Click for larger version (59K) ZINC GOES FOR THE GOLD Many researchers have been discouraged by reports that ZFP TF technology is difficult to use. Not so, says Barbas, who is generally credited with making the technology more accessible to the general research community. Most recently, he and collaborators devised a genome-wide, ZFP TF library approach for identifying zinc fingers that confer certain phenotypes.4 The approach's primary advantage over the "rational," targeted approach to ZFP TF design is this: ZFP TFs engineered in vitro often don't work in vivo because of unforeseen complications, such as chromatin structure. Screening a library in vivo, however, allows the researcher to home in immediately on the ZFP TF that works. Aseem Ansari, a biochemist at the University of Wisconsin who prefers the targeted approach, says the library method is easier, "and, in the end, may be the most productive for several applications." Barbas says the next biggest challenge is to spread the word about "how easy it is to construct these proteins." Klug agrees that it's an "easy" technology, although he cautions, "We do actually worry about design and selection." DNA structure is not absolutely fixed, and even a 1-angstrom discrepancy in structure can be vital when trying to make a 3-dimensional DNA-protein match. Also, what's easy for one lab may not be easy for another. Although Barbas has made the technology more user-friendly, Danford Center's Beachy, who has collaborated with Barbas on a few zinc-finger projects, says accessibility may still be a problem: "It would be difficult for the average biochemistry lab to [construct ZFP TFs] without tremendous investment. Not many labs have the technical expertise." Meanwhile, Klug says, more researchers worldwide are demonstrating the use of ZFP TFs. Although Sangamo's current focus is drug design and human thera- peutics, the company foresees agricultural applications, according to Peter Bluford, vice president of corporate development. For example, the implicated role of a vitamin E-rich diet in disease prevention has generated interest in creating commercial crops that produce seed oil with enriched vitamin E. ZFP TFs could be used to controlthe expression of the enzymes involved with vitamin E synthesis. Bluford, however, hesitates to predict what the first agricultural application of the new technology will be. Such golden opportunities for zinc fingers are several years out of reach. Leslie Pray (lpray@nasw.org) is a freelance writer in Northampton, Mass. References 1. D. Rhodes, A. Klug, "Zinc fingers," Scientific American, 268:56- 65, 1993. 2. X. Guen et al., "Heritable endogenous gene regulation in plants with designed polydactyl zinc finger transcription factors," Proc Natl Acad Sci, 99:13296- 301, 2002. 3. E.J. Rebar et al., "Induction of angiogenesis in a mouse model using engineered transcription factors," Nat Med, 8:1427-32, 2002. 4. P. Blancafort et al., "Scanning the human genome with combinatorial transcription factor libraries," Nat Biotech, 21:269-74, March 2003. function sendData() { document.frm.pathName.value = location.pathname; result = false if (document.frm.score[0].checked) result = true; if (document.frm.score[1].checked) result = true; if (document.frm.score[2].checked) result = true; if (document.frm.score[3].checked) result = true; if (document.frm.score[4].checked) result = true; if (!result) alert("Please select") return result; } .myradio{background-color : #94bee5} Please indicate on a 1 - 5 scale how strongly you would recommend this article to your colleagues? Not recommended 1 2 3 4 5 Highly recommended Please register your vote

A Finger on the Pulse of Transcriptional Control

Interested in reading more?

Become a Member of

"GREEN" FINGERS: Zinc finger- based artificial transcription factors (background) have been applied in plants such as Arabidopsis thaliana (foreground). Reprinted with permission, Curr Opin Plant Biol, 6:163-8, April 2003.