Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression - PubMed (original) (raw)

Design of TATA box-binding protein/zinc finger fusions for targeted regulation of gene expression

J S Kim et al. Proc Natl Acad Sci U S A. 1997.

Abstract

Fusing the TATA box-binding protein (TBP) to other DNA-binding domains may provide a powerful way of targeting TBP to particular promoters. To explore this possibility, a structure-based design strategy was used to construct a fusion protein, TBP/ZF, in which the three zinc fingers of Zif268 were linked to the COOH terminus of yeast TBP. Gel shift experiments revealed that this fusion protein formed an extraordinarily stable complex when bound to the appropriate composite DNA site (half-life up to 630 h). In vitro transcription experiments and transient cotransfection assays revealed that TBP/ZF could act as a site-specific repressor. Because the DNA-binding specificities of zinc finger domains can be systematically altered by phage display, it may be possible to target such TBP/zinc finger fusions to desired promoters and thus specifically regulate expression of endogenous genes.

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Figures

Figure 1

Figure 1

Structure-based design of TBP/ZF. The cocrystal structures of the Zif268:DNA (14) and the TBP:TATA box complexes (9) were aligned by superimposing phosphates in several different registers. In the model shown above, the NH2-terminal end of Zif268 was 23 Å away from the COOH-terminal end of TBP. We created the TBP/ZF fusion protein by adding a NH2-(Gly-Gly-Gly-Ser)2Gly-COOH polypeptide linker to join the two molecules. The alignment of binding sites used in this modeling study suggested that TBP/ZF would bind tightly to the sequence 5′-GCGTGGGCGNNNNTATATAAA-3′.

Figure 2

Figure 2

Determination of dissociation rate constants. (A) Probe DNA sequences used in gel shift assays. The TATA boxes and the Zif268-binding site are underlined. The sequence of only one strand is shown. (B) Example of gel shift assay used to determine dissociation rate constants. yTBP (5 μg/ml) or TBP/ZF (6 μg/ml) was incubated with labeled probe DNAs (0.1 nM) for 1 h at room temperature. To begin measurement of dissociation rates, a large excess of unlabeled probe DNA (final concentration, 1 μM) was added to each incubation mixture at time t = 0. Aliquots were removed at indicated times and analyzed by gel electrophoresis. Samples were loaded on the gel at different times, and thus the bands appear staggered. (C) The fraction of labeled probe DNA bound by protein was quantified by PhosphorImager (Molecular Dynamics) analysis, and normalized to the fraction bound at time t = 0. The natural log of the normalized fraction bound was plotted against time, and the dissociation rate was determined from the slope. (D) Models indicating how the orientationof the TBP moiety of TBP/ZF on the TATA box may be controlled by flanking Zif268-binding sites. The direction of transcription relative to the TATA box is shown with an arrow. (The “x” over the lower arrow indicates that this TBP orientation cannot support transcription.)

Figure 3

Figure 3

In vitro transcription analysis. Biotinylated DNA fragments containing the promoters (TATA, TATA/CGC, and GCG/TATA) upstream of a G-free cassette were immobilized on streptavidin-coupled paramagnetic beads and used as transcription templates. yTBP at 6 μg/ml (lanes 1–3 and 7–9) or TBP/ZF at 8 μg/ml (lanes 4–6 and 10–12) was preincubated with each template (0.1 nM) for 1 h at room temperature. Then, supernatants were removed, and excess amounts (1 μM each) of competitor DNA oligonucleotides (GCG and TATA from Fig. 2_A_) were added to the preincubation mixture. After incubating 24 h at 4°C, the beads were washed to remove proteins that dissociated from the templates, and human transcription factors (TFIIB, -IIE, -IIF, and -IIH), RNA polymerase II, and substrate nucleotides were added to initiate transcription. yTBP was also added to a final concentration of 0.2 μg/ml in lanes 7–12. The transcripts were analyzed by urea gel electrophoresis.

Figure 4

Figure 4

Transient cotransfection assay. Human 293 cells were cotransfected, using the calcium phosphate precipitation method with (i) 1 μg of expression plasmid encoding yTBP or TBP/ZF, (ii) 5 μg of activator plasmid, GAL4-VP16, (iii) 0.5 μg of β-galactosidase expression plasmid (pCMVβ) as an internal control, (iv) 1 μg of a reporter plasmid (derived from pGL3-Basic) encoding the firefly luciferase gene, and (v) variable amount of the carrier plasmid (pUC19) to keep the total amount of transfected DNA at 20 μg. Each reporter construct had five GAL4-binding sites upstream of one of the promoter sequences (TATA, TATA/CGC, or GCG/TATA) used in the in vitro transcription assay (Fig. 3). In a parallel assay of basal transcription, GAL4-VP16 was omitted. Luciferase activity was measured 2 days after transfection and was normalized (i) with respect to β-galactosidase activity (to correct for transfection efficiency), and (ii) to the corresponding value from the cells transfected with blank expression vector, pcDNA3 (which was set to an arbitrary value of 104). The absence or presence of GAL4-VP16 is indicated. The data represent an average of three independent experiments, and the standard error of the mean is shown.

Figure 5

Figure 5

Transcriptional repression by TBP/ZF and ΔTBP/ZF in vivo. Transient cotransfection assays were used to determine whether TBP/ZF and ΔTBP/ZF could affect VP16-activated transcription from the TATA/GCG promoter. The results were compared with those from the TATA/CGC promoter (Fig. 4). The data represent an average of three independent experiments.

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