Journal of Molecular Biology
Regular articlePromoter-specific activation of gene expression directed by bacteriophage-selected zinc fingers1
Introduction
The differentiation and development of an organism is effected by a programme of differential gene expression which is ordered in space and time; this is controlled mainly at the level of transcription, by protein transcription factors which bind to regulatory promoter DNA sequences found adjacent to specific genes Mitchell and Tjian 1989, Nikolov and Burley 1997, Ptashne and Gann 1990, Roeder 1991. Artificially manipulating this programme would allow us to dissect developmental pathways, and to interfere with the aetiology of disease. Unfortunately, naturally occurring transcription factors are not normally suitable for applications where specific control over one gene is required, because the DNA sequences they bind are present in the promoters of many different genes (Kel et al., 1995). In addition, gene regulation by natural transcription factors often requires the concerted action of combinations of factors, which by themselves have little or no activity Herschlag and Johnson 1993, Milos and Zaret 1992, Reith et al 1994, Rivera et al 1993.
One approach to the artificial control of gene expression is to use transcription factors containing potent activation or repression domains, and engineered with new DNA-binding specificities for rare binding sites associated only with the target gene, but probably distinct from the natural promoter. This strategy is feasible because transcription factors are structurally modular, with different domains responsible for DNA binding and transcriptional regulation Mitchell and Tjian 1989, Pabo and Sauer 1992. These domains are often interchangeable such that chimaeric transcription factors with swapped characteristics can be easily engineered (Brent & Ptashne, 1985).
The manipulation of the DNA-binding specificity of transcription factors is less straightforward. Efforts in this field have focused on the zinc finger of the TFIIIA type: a small DNA-binding module found in eukaryotic transcription factors Klug and Rhodes 1987, Miller et al 1985. Zinc finger domains which bind to given DNA sequences in vitro can be selected by bacteriophage display (Choo and Klug 1994a, Jamieson et al 1994, Rebar and Pabo 1994, Greisman and Pabo 1997; for a review see Choo & Klug, 1995) and their specificity later assessed by screening for binding to 12 self-encoding DNA oligonucleotide libraries (Choo & Klug, 1994b). The DNA-binding characteristics of some phage-selected zinc fingers have been investigated in vitro, confirming that they function as well as their naturally occurring counterparts Choo and Klug 1994b, Jamieson et al 1994, Rebar and Pabo 1994. While the affinity of such phage-selected DNA-binding domains is quite high, usually falling in the nanomolar range for a three-finger peptide, the observed discrimination against closely related DNA sequences, by a two to tenfold increase in the apparent Kd per point mutation, is rather modest.
Hence, here we have questioned whether DNA binding by a phage-selected zinc finger domain is sufficiently discriminating to allow operations on specific DNA sequences in vivo. We used a CAT transactivation assay to investigate the ability of a model phage-selected DNA-binding domain to discriminate its target from a set of closely related promoter sequences. We found that this was possible with a high degree of specificity, but only when the intracellular concentration of zinc finger transcription factor was optimised. These results warrant the use of phage-selected DNA-binding domains to direct in vivo DNA manipulation, in particular the control of gene expression, but invoke the need for controlled delivery of the DNA-binding domain in order to achieve specific effects.
Section snippets
In vitro DNA specificity of phage-selected zinc fingers
A phage display library of the three-finger DNA-binding domain of Zif268 has been described, in which fingers 1 and 3 have the wild-type sequence but finger 2 is randomised in eight positions which could function in DNA binding (Choo & Klug, 1994a). Affinity selections from this library were previously performed using the nine base-pair long Zif268 binding site, , in which the underlined three-base subsite contacted by finger 2 was substituted by a different triplet. Many selections
Discussion
Promoter-specific activation of gene expression has been described in cell culture, using a representative phage-selected DNA-binding domain which was converted to a transcription factor by fusion with the activation domain from VP16. Significant activation of gene expression was recorded by the CAT assay when one binding site for the artificial transcription factor was present upstream of the reporter gene, and under these circumstances no non-specific activation of gene expression was
In vitro DNA-binding assays
Zinc finger phage selections and binding site signatures were performed as described Choo and Klug 1994a, Choo and Klug 1994b. The method for the calculation of apparent Kd for ZN1 was exactly as described by Choo & Klug (1994b).
Eukaryotic expression constructs
Genes to be cloned into expression plasmids were generated by the polymerase chain reaction (PCR) using phage DNA as the template. PCR primers included appropriate initiation and termination codons and restriction sites for cloning. Expression constructs were generated
Acknowledgements
We thank D. Martı́n-Zanca for advice, L. Fairall for suggesting improvements to the manuscript, J. Girdlestone for the pMCAT3 vector, I. Sadowski for plasmids pG5EC and pM1VP16, and V.N. Rao for plasmid pESP-SVTEXP. Research in I. S. G.’ s laboratory is supported by Fundación Internacional José Carreras (FIJC-94/INT), European Commission (BMH4-CT96-0375) and Fundación Cientı́fica of the AECC.
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Edited by T. Richmond