ENGINEERING OF ZINC FINGER PROTEINS TO BIND DOUBLE-STRANDED DNA

Within the last 20 years, the understanding of the biology of the ‘classical’ or Cys2His2 zinc finger domain has progressed rapidly from the initial identification of the zinc finger as a repetitive zinc-binding motif in transcription factors to its use in biotechnology. The domain is the most abundant DNA-binding motif in the human genome and is a component of many key eukaryotic transcription factors involved in growth and development. Numerous structures now exist for this domain and its mode of action is known in a variety of zinc finger–DNA complexes. Application of this knowledge has led to the development of ‘designer’ transcription factors where zinc fingers have been engineered to bind desired DNA sequences. Recently, advances have been made in this field that potentially allow the targeting of any DNA site. Consideration of chromatin structure and the use of effector domains in these ‘designer’ transcription factors have made possible the regulation of a number of endogenous genes. These advances in the customised regulation of genes will be discussed in detail, as well as the potential to use these proteins in functional genomics and gene therapy applications. INTRODUCTION The expression of genes in all organisms is primarily controlled by the interactions of protein complexes with DNA. Transcription factors are important mediators of these interactions and generally contain one or more DNAbinding motifs. The structures of wellcharacterised DNA-binding domains such as the leucine zipper, helix-turn-helix, homeodomain and zinc finger show that these domains have similar modes of specific base recognition via amino acid side-chains protruding from an Æ-helix, known as a ‘recognition helix’, into the major groove of DNA. It was anticipated that these domains could be engineered into ‘designer’ transcription factors to target any DNA sequence for custom gene regulation. One domain, the zinc finger, emerged as the obvious choice to engineer because of its small size, simplicity of structure and its relatively simple interactions with DNA. Now, as biology enters the post-genomic era, with the sequences of many eukaryotic genomes unveiled, the technology to regulate genes using these ‘designer’ zinc finger transcription factors exists. This paper will first provide an overview of zinc finger structure and will then focus on the current status of efforts to regulate gene expression at will. Progress made towards using ‘designer’ transcription factors in the treatment of human disease will also be considered. STRUCTURE OF CYS2HIS2 ZINC FINGERS The zinc finger motif, originally recognised by Klug and co-workers, contains approximately 30 amino acids, which fold into a compact Æ domain stabilised by a zinc ion and a cluster of hydrophobic residues. The zinc ion is chelated between two cysteine residues within the -strands and two histidine residues in the Æ-helix. Hence, zinc fingers of this type are known as Cys2His2 zinc fingers. This domain is found in over 700 human proteins, making it the second most abundant motif found in the human 3 4 2 & HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N O M I C S A N D P R O T E O M I C S . VOL 1. NO 4. 342–355. JANUARY 2003 Downloaded from https://academic.oup. com/bfg/article-abstract/1/4/342/259612 by guest on 09 April 2018 genome and the most common domain used for DNA recognition; however, since the Cys2His2 zinc finger was first identified as a DNA-binding motif, numerous variations on this classical zinc finger have been characterised and implicated in roles as diverse as RNA binding, protein binding and even as zinc-sensing factors. These other functions of zinc fingers are also receiving much attention at present and are reviewed by Laity et al. Although high resolution structures had been determined for zinc finger domains, it was only following the elucidation of the crystal structure of the three zinc fingers of Zif268 bound to its DNA target site that the molecular interactions between protein and DNA could be understood in detail. In this structure, the recognition helix of each finger sits in the major groove of DNA such that residues at –1, 3 and 6 with respect to the start of the helix make specific base contacts to the 39, middle and 59 bases of the triplet (Figure 1). Later, it was noticed that the residue at position 2 (aspartate in all three fingers of Zif268) makes a ‘cross-strand’ interaction to a C (or A) which is the base opposite that contacted by the 6 position of the preceding zinc finger. Thus, each zinc finger binds an overlapping 4 base pair (bp) site (Figure 1), and the three-finger Zif268 peptide binds 9–10 bp of DNA. These cross-strand interactions can create synergy between neighbouring fingers for the recognition of the 59 base of each triplet-binding site, and have been explored in detail. In general, adjacent zinc fingers within a transcription factor are connected by the consensus linker peptide –TGEKP– . Such a high degree of conservation implies a significant role for this linker sequence in zinc finger function. Indeed, mutagenesis studies on the first three fingers of transcription factor IIIA (TFIIIA) demonstrated that mutations in this linker could reduce DNA-binding affinity up to 20-fold. Also, it has been shown that the disordered TGEKP-linker becomes structured on binding to DNA and forms a C-terminal helix-capping motif which helps stabilise the zinc finger–DNA complex. The importance of the linker sequence is further demonstrated by the natural Wilms’ tumour suppressor protein, WT1. This protein has two isoforms, characterised by the presence (+WTS) or absence (–WTS) of a three-amino acid insertion in the TGEKP-linker between the third and fourth fingers. The WT1– WTS isoform binds DNA through four fingers and is associated mainly with active transcription, whereas the WT1+WTS isoform binds DNA far more weakly, and is associated instead with regions of RNA processing. Cys2His2 zinc fingers have a simple Æ structure Zinc finger interactions with DNA are well understood for model proteins 3 zinc fingers bind 9– 10 bp of DNA Figure 1: A schematic representation of the interaction between a single zinc finger and its double-stranded DNA target site, as inferred from X-ray crystal structures 20,21 and biochemical studies. 22,23 The simple Æ structure of the zinc finger can clearly be seen. Straight arrows indicate contacts from zinc finger amino acid side-chains at helical positions –1, 3 and 6 to consecutive nucleotides on the top DNA strand. The curved arrow demonstrates a ‘cross-strand’ interaction from the amino acid side-chain at position 2 to the complementary DNA strand. Note that a single zinc finger interacts with double-stranded DNA over a 4 bp span & HENRY STEWART PUBLICATIONS 1473-9550. B R I E F I N G S I N F U N C T I O N A L G E N OM I C S A N D P R O T E O M I C S . VOL 1. NO 4. 342–355. JANUARY 2003 3 4 3 Recent developments in the engineering of zinc finger proteins Downloaded from https://academic.oup. com/bfg/article-abstract/1/4/342/259612 by guest on 09 April 2018 ENGINEERING OF ZINC FINGER PROTEINS TO BIND DOUBLE-STRANDED DNA Background Two approaches were originally used for zinc finger protein (ZFP) engineering in order to expand the DNA recognition code and to create zinc fingers that bind desired base triplets — a combinatorial approach using libraries of zinc fingers displayed on the surface of filamentous phage that were selected against target DNA sequences and a rational design approach that used databases to predict rules for amino acid–base interactions. Methods to design and select zinc finger units and assemble them to create three-finger DNA-binding domains have been reviewed thoroughly by the authors’ group and others and only recent advances will be discussed in detail here.