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

摘要

A long-standing goal of molecular biologists has been to construct DNA-bindingproteins for the control of gene expression. The classical Cys_2Нis_2(С_2Н_2) zinc finger design isideally suited for such purposes. Discriminating between closely related DNA sequences bothin vitro and in vivo, this naturally occurring design was adopted for engineering zinc finger proteins (ZFPs) to target genes specifically. Zinc fingers were discovered in 1985, arising from the interpretation of our biochemicalstudies on the interaction of the Xenopus protein transcription factor IIlA (TFIIIA) with 5SRNA. Subsequent structural studies revealed its three-dimensional structure and its interactionwith DNA. Each finger constitutes a self-contained domain stabilized by a zinc (Zn) ion ligatedto a pair of cysteines and a pair of histidines and also by an inner structural hydrophobic core.This discovery showed not only a new protein fold but also a novel principle of DNArecognition. Whereas other DNA-binding proteins generally make use of the 2-fold symmetryof the double helix, functioning as homo- or heterodimers, zinc fingers can be linked linearly intandem to recognize nucleic acid sequences of varying lengths. This modular design offers alarge number of combinatorial possibilities for the specific recognition of DNA (or RNA). It istherefore not surprising that the zinc finger is found widespread in nature, including 3% of thegenes of the human genome. The zinc finger design can be used to construct DNA-binding proteins for specificintervention in gene expression. By fusing selected zinc finger peptides to repression oractivation domains, genes can be selectively switched off or on by targeting the peptide to thedesired gene target. It was also suggested that by combining an appropriate zinc finger peptidewith other effector or functional domains, e.g. from nucleases or integrases to form chimaericproteins, genomes could be modified Qr manipulated. The first example of the power of the method was published in 1994 when a three-fingerprotein was constructed to block the expression of a human oncogene transformed into amouse cell line. The same paper also described how a reporter gene was activated bytargeting an inserted 9-base pair (bp) sequence, which acts as the promoter. Thus, by fusingzinc finger peptides to repression or activation domains, genes can be selectively switched offor on. It was also suggested that, by combining zinc fingers with other effector or functionaldomains, e.g. from nucleases or integrases, to form chimaeric proteins, genomes could bemanipulated or modified. Several applications of such engineered ZFPs are described here, including some oftherapeutic importance, and also their adaptation for breeding improved crop plants.