Zinc finger proteins for the regulation of gene expression

Aaron Klug
MRC Laboratory of Molecular Biology
Hills Road
Cambridge  CB2 2QH UK

Zinc finger proteins of the classical Cys₂His₂ type are the most frequently used class of transcription factor and account for about 3% of genes in the human genome.  The zinc finger motif was discovered during biochemical studies on the transcription factor TFIIIA, which regulates the 5S ribosomal RNA genes of Xenopus laevis.

The C₂H₂ motif consists of a sequence of about 30 amino acids containing two histidines, two cysteines and three hydrophobic residues, which are all at conserved positions.  It forms a small, independently folded domain stabilized by Zn²⁺, which can be used repeatedly in a modular tandem fashion to achieve sequence-specific recognition of DNA.  The domains all have the same structural framework, but achieve chemical distinctiveness through variations in key residues.  The modular design thus offers a large number of combinatorial possibilities for DNA recognition, so it is no wonder that zinc-finger domains are so widely found in nature.

Structurally, the domain is composed of a b-hairpin and a a-helix pinned together by Zn²⁺.  In the canonical Zif268 docking arrangement, the primary contacts are made by the a-helix, which binds in the DNA major groove through primary hydrogen-bond interactions from helical positions – 1, 3 and 6 to three successive bases on one strand of the DNA, and also through a secondary interaction from helical position 2 to the other strand.

We have used the technique of phage display to create a large library (or repertoire) of different zinc fingers from which selections are made for binding to a given DNA sequence triplet.  From this database there have been elucidated elements of recognition rules that relate the amino acid sequence of a finger motif to its preferred DNA binding site.

Control of gene expression, using such specifically selected combinations of three zinc finger peptides, was originally demonstrated by the specific inhibition of an oncogene in a mouse cancer cell line and also, conversely, by activating particular genes in expression plasmids.  These experiments showed that zinc finger DNA binding domains can be engineered de novo to recognize given promoter DNA sequences, and regulate their activity.

Three zinc fingers recognize nine base pairs, a sequence which would occur randomly several times in a large genome.  However six fingers linked together would recognise a DNA sequence 18 basepairs in length, sufficiently long to constitute a rare address in the human genome.  We have learned how to engineer longer runs of zinc fingers which can target longer DNA sequences.  By adding functional groups to the engineered DNA binding domains, eg silencing or activation domains, novel transcription factors can be generated to up or downregulate expression of a target gene.  Some recent applications by ourselves and others will be described:

(i) the disruption of the infective cycle of infection by herpes simplex virus
(ii) inhibition of HIV-1 expression
(iii) activating the expression of erythropoietin (EPO) in a human kidney cell line
(iv) activating the expression of vascular endothelial growth factor (VEGF) in a human cell line, and in an animal model.

Postscript  While it has long been known that TFIIIA binds RNA as well as DNA, there is now increasing evidence that zinc fingers are more widely used to recognize RNA.  However the molecular basis of the recognition has remained elusive.  We have recently determined the X-ray structure of a zinc finger RNA complex which reveals two modes of zinc finger binding, both different from that for DNA.

References

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Papworth, M., Moore, M., Isalan, M. Minczuk, M., Choo, Y. & Klug, A. (2003).  Inhibition of herpes simplex virus 1 gene expression by designer zinc finger transcription factors.  PNAS, 100, 1621-1626.

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Lu, D., Searles, M.A. & Klug, A. (2003).  Crystal structure of a zinc finger RNA complex reveals two modes of molecular recognition.  Nature, 426, 96-100.