www.nature.com/naturebiotechnology • MARCH 2003 • VOLUME 21 • nature biotechnology Human zinc fingers as building blocks in the construction of artificial transcription factors Kwang-Hee Bae 1,† , Young Do Kwon 1,2,† , Hyun-Chul Shin 1 , Moon-Sun Hwang 1 , Eun-Hyun Ryu 1 , Kyung-Soon Park 1 , Hyo-Young Yang 1 , Dong-ki Lee 1 , Yangsoon Lee 1 , Jinwoo Park 1 , Heung Sun Kwon 1 , Hyun-Won Kim 3 , Byung-Il Yeh 3 , Hyean-Woo Lee 3 , Soon Hyung Sohn 3 , Joonho Yoon 3 , Wongi Seol 1 , and Jin-Soo Kim 1, * Published online 18 February 2003; doi:10.1038/nbt796 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 acti- vation 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. RESEARCH ARTICLE Genes are regulated at the transcriptional level by transcription factors that interact with specific DNA regulatory elements to acti- vate or inhibit transcription initiation. Many transcriptional fac- tors have modular structures that consist of a DNA-binding domain and an effector (activation or repression) domain. The effector domains of many transcription factors remain active when transferred to heterologous DNA-binding domains 1 . This suggests that it may be possible to construct novel transcription factors by preparing DNA-binding domains with the desired DNA-binding specificities and then linking them to the appropri- ate effector domains. A wide range of organisms has transcription factors containing zinc-finger motifs, which bind to DNA in a sequence-specific man- ner. There are several types of zinc fingers, but members of the Cys 2 -His 2 class are ideal for generating artificial transcription fac- tors owing to their diversity and modular structure. For example, novel DNA-binding proteins have been created by using phage dis- play to alter the DNA-binding specificity of a zinc-finger protein (ZFP) 2–7 . Although phage display is an effective method, it is labor- intensive and time-consuming. Moreover, because phage display uses in vitro selection, the selected ZFPs might not function effi- ciently in vivo. With phage display, ZFPs are selected by binding to ‘naked’ DNA rather than to chromatin, whereas in eukaryotic cells DNA is packaged into nucleosomes to form chromatin. To circumvent these problems, we developed an approach for constructing DNA-binding proteins that recognize the desired DNA sites in eukaryotic cells. We isolated zinc-finger domains with diverse DNA-binding specificities by screening, in yeast, plas- mid libraries that encode zinc fingers derived from the human genome. These zinc fingers were then used as modular building blocks to construct novel DNA-binding proteins and, subsequent- ly, designer transcription factors. Results Zinc fingers derived from the human genome. We modified the yeast one-hybrid system 8–10 to select zinc fingers that exhibit diverse DNA-binding specificities. This system includes two essential com- ponents: a reporter plasmid and a plasmid that encodes a hybrid transcription factor (Fig. 1A). The hybrid transcription factor con- sists of two domains: a DNA-binding domain and a transcriptional activation domain. We used the Gal4 transcriptional activation domain fused to the Zif268 protein as our hybrid transcription factor. Zif268 is a zinc-finger protein that contains three zinc- finger domains; together, the three-finger protein binds specifical- ly to a 9 base pair (bp) DNA sequence (5′-GCG TGG GCG-3′). Each zinc finger of Zif268 recognizes a 3 bp subsite within the 9 bp com- pound binding site. To isolate zinc fingers with diverse DNA-binding specificities, we cloned human DNA segments that encode zinc fingers. The DNA segments were amplified using PCR. The PCR products were then cloned into a yeast expression plasmid that contained the Gal4- Zif268 hybrid transcription factor. In this library, zinc finger 3 (the C-terminal finger) of Zif268 was replaced with zinc-finger sequences amplified from the human genome (Fig. 1A). We used both yeast HIS3 and Escherichia coli LacZ as reporter genes. The reporter plasmids were constructed by inserting three copies of the target DNA sequences adjacent to the coding regions of the reporter genes. The target sequences have the format 5′-XXX TGG GCG-3′, where XXX corresponds to a 3 bp target subsite and TGG GCG corresponds to the DNA sequences recognized by finger 1 (the N-terminal finger) and finger 2 (the middle finger) of Zif268 (Fig. 1A). In total, two sets of 64 different reporter plasmids were prepared (one set for each of the two reporter genes). We identified zinc fingers with diverse DNA-binding specificities by isolating yeast cells in which transcription of the reporter genes 1 ToolGen Inc., 461-6 Jeonmin-Dong, Yusung-Gu, Daejeon, 305-390, South Korea. 2 School of Biological Sciences, Seoul National University, Seoul, 151-742, South Korea. 3 Department of Biochemistry and Institute of Basic Medical Sciences and IFBB, Yonsei University, Wonju College of Medicine, Wonju, 220-701, South Korea. *Corresponding author (e-mail: jsk@toolgen.com). † These authors contributed equally to this work. 275 © 2003 Nature Publishing Group http://www.nature.com/naturebiotechnology