We describe methods for generating artificial transcription factors capable of up- or downregulating the expression of genes whose promoter regions contain the target DNA sequences. To accomplish this, we screened zinc fingers derived from sequences in the human genome and isolated 56 zinc fingers with diverse DNA-binding specificities. We used these zinc fingers as modular building blocks in the construction of novel, sequence-specific DNA-binding proteins. Fusion of these zinc-finger proteins with either a transcriptional activation or repression domain yielded potent transcriptional activators or repressors, respectively. These results show that the human genome encodes zinc fingers with diverse DNA-binding specificities and that these domains can be used to design sequence-specific DNA-binding proteins and artificial transcription factors.
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We thank Randall Reed for providing yeast strain yWAM2 and plasmids pRS315HIS and pPC86. We also thank S.I. Lee, S.I. Kim, J.W. Kim, and H.R. Lee for technical assistance and K. LaMarco for carefully reading our manuscript. This study was supported by a grant of the National Research Laboratory Program from the Ministry of Science & Technology, Republic of Korea (M1-0104-00-0048).
The research described in the paper was at least partially funded by ToolGen, Inc., a privately-held company, and most of the authors are employees of ToolGen, Inc. J.-S. K. is one of the co-founders of the company and is a major shareholder.
Zinc “fingerprints”. The DNA-binding specificities of selected zinc fingers fused to fingers 1 and 2 of Zif268 were determined in yeast cells using 64 LacZ reporter sets, each of which contained one of the 64 triplet subsites. “+” indicates a positive control that contains the third finger of Zif268 and a reporter plasmid containing its subsite sequence, GCG. “−” indicates a negative control that contains only the first and second fingers of Zif268 and a control reporter plasmid that lacks the Zif268 binding sequence. (PDF 20 kb)
Nucleotide sequence of the polylinker site of the P3 expression plasmid and the construction of plasmids encoding chimeric 3-finger proteins. (A) Nucleotide sequence of the polylinker site of the P3 expression plasmid. (B) The DNA encoding each zinc finger domain was cloned into the P3 vector to form “single finger” vectors. Equal amounts of each “single finger” vector were combined to form a pool. The pool was separately digested with two sets of enzymes: AgeI and XhoI, and XmaI and XhoI. After phosphatase treatment for 30 min, vectors from the AgeI and XhoI digested pool were ligated to the nucleic acid segments released from the vector by the XmaI and XhoI digestion. These segments each encode a single zinc finger domain. Ligation of the nucleic acid segments to the digested vector formed vectors that encode two zinc finger domains. After transformation into E. coli, the ligation products yielded ˜1.4 × 104 transformants, thereby forming a 2-finger library. Subsequently, the 2-finger library was digested with AgeI and XhoI. The digested vector, which retains the nucleic acid sequences encoding two zinc finger domains, was ligated to the pool of 1-finger fragments prepared as above by digestion with XmaI and XhoI. The products of this ligation were transformed into E. coli to yield about 2.4 × 105 independent transformants, and a library of plasmids encoding 3-finger proteins was isolated from the transformants. ZF1, one-finger protein; ZF2, two-finger protein; ZF3, three-finger protein. The arrows indicate the coding regions of ZF1-ZF3. (PDF 42 kb)
Promoters of reporter constructs used in transfection analyses. The DNA sequences of the reporter constructs are aligned to show the location of the zinc finger binding sites. The upper line (Control plasmid) shows the sequence of the synthetic promoter containing the initiator element, and bases are numbered with respect to the start of transcription. In the lower, reporter plasmid sequence, NNNNNNNNN indicates a cognate binding site for the specific ZFP. Identical nucleotides are shown as “−”, and a blank means that the corresponding nucleotide is deleted. An oligomer containing the specific 9-bp binding site for each ZFP replaced the 14-bp segment located 12-bp downstream from the transcription start site in the control plasmid. (PDF 21 kb)
Western blot analysis. Plasmids encoding representative ZFPs were transfected into HEK293 cells. After growing the cells in culture for 2 days, protein extracts were prepared and analyzed by Western blotting. These ZFPs and their fold repression values are described in Supplementary Table 2. The numbers of engineered zinc fingers and human zinc fingers that constitute these three-finger ZFPs are indicated. (PDF 960 kb)
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Bae, K., Do Kwon, Y., Shin, H. et al. Human zinc fingers as building blocks in the construction of artificial transcription factors. Nat Biotechnol 21, 275–280 (2003). https://doi.org/10.1038/nbt796
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