The discovery of zinc fingers and their development for practical applications in gene regulation

An account is given of the discovery in 1985 of the classical Cys(2)His(2) (C2H2) zinc finger, arising from the interpretation of biochemical studies on the interaction of the Xenopus protein transcription factor IIIA with 5S RNA, and of subsequent structural studies on its 3D structure and its interaction with DNA. Each finger constitutes a self-contained domain stabilized by a zinc ion ligated to a pair of cysteines and a pair of histidines, and by an inner structural hydrophobic core. This work showed not only a new protein fold but also a novel principle of DNA recognition. Whereas other DNA binding proteins generally make use of the two-fold symmetry of the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly in tandem to recognize nucleic acid sequences of different lengths. This modular design offers a large number of combinatorial possibilities for the specific recognition of DNA (or RNA). It is therefore not surprising that this zinc finger is found widespread in nature, in 3% of the genes of the human genome. It had long been the goal of molecular biologists to design DNA binding proteins for control of gene expression and we have adopted the zinc finger design and principle for this purpose. We demonstrated that the zinc finger design is ideally suited for such purposes, discriminating between closely related DNA sequences both in vitro and in vivo, and we have therefore adapted this natural design for engineering zinc finger proteins for targeting specific genes. The first example of the potential of the method was published in 1994 when a three-finger protein was constructed to block the expression of an oncogene transformed into a mouse cell line. In the same paper we also showed that we could activate a reporter gene by targeting a nine base pair promoter which we had inserted. Thus by fusing zinc finger peptides to repression or activation domains, genes can be selectively switched off or on. By combining the targeting zinc fingers with other effector or functional domains e.g. from nucleases or integrases, to form chimeric proteins, genomes can be manipulated or modified. Several applications of such engineered zinc finger proteins are described here, including some of potential therapeutic importance.