Human zinc fingers as building blocks in the construction of artificial transcription factors

Kwang-Hee Bae^{1, 4}, Young Do Kwon^{1, 2, 4}, Hyun-Chul Shin¹, Moon-Sun Hwang¹, Eun-Hyun Ryu¹, Kyung-Soon Park¹, Hyo-Young Yang¹, Dong-ki Lee¹, Yangsoon Lee¹, Jinwoo Park¹, Heung Sun Kwon¹, Hyun-Won Kim³, Byung-Il Yeh³, Hyean-Woo Lee³, Soon Hyung Sohn³, Joonho Yoon³, Wongi Seol¹ & Jin-Soo Kim^{1, 1}
 

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.
4. These authors contributed equally to this work.
Correspondence should be addressed to J -S Kim. e-mail: jsk@toolgen.com

Genes are regulated at the transcriptional level by transcription factors that interact with specific DNA regulatory elements to activate or inhibit transcription initiation. Many transcriptional factors 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 domains¹. 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 appropriate effector domains.

A wide range of organisms has transcription factors containing zinc-finger motifs, which bind to DNA in a sequence-specific manner. There are several types of zinc fingers, but members of the Cys₂-His₂ class are ideal for generating artificial transcription factors owing to their diversity and modular structure. For example, novel DNA-binding proteins have been created by using phage display 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 efficiently 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, plasmid 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, subsequently, 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 components: a reporter plasmid and a plasmid that encodes a hybrid transcription factor (Fig. 1A). The hybrid transcription factor consists 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 specifically 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 compound 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 was activated. The amino acid sequence of each selected zinc finger was deduced from the DNA sequence, and each zinc finger was named using the single-abbreviation of the four amino acid residues at positions -1, 2, 3, and 6 in the -helix of the zinc finger. These amino acid residues are expected to make contact with bases in the target DNA subsite^11-15.

Figure 1: In vivo screening system and zinc 'fingerprints'.
(A) Zinc-finger screening system in yeast. Zinc-finger domain 'A' recognizes the 3-bp target DNA subsite (designated XXX) upstream of the reporter gene, and transcription of the HIS3 or LacZ reporter gene is activated. As a result, the yeast grows on a medium lacking histidine and forms a blue colony when grown on a medium containing X-gal (see Fig. 1B). In contrast, zinc-finger domain 'B' does not recognize the target subsite. In this case, the reporter gene is not activated; thus the yeast does not grow on a medium lacking histidine and forms a white colony on a medium containing X-gal (see Fig. 1B). AD, transcriptional activation domain; 1, zinc finger 1 of Zif268; 2, zinc finger 2 of Zif268. (B) Zinc fingerprints. The DNA-binding specificities of selected zinc fingers (QSSR1, VSNV, and RSHR) that had been 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, which contains the third finger of Zif268 and a reporter plasmid containing its subsite sequence, GCG. '-' indicates a negative control, which contains only the first and second fingers of Zif268 and a control reporter plasmid with no binding sequence. The sequences of the functional 3 bp subsites for each of the ZFPs are shown in parentheses after the protein name, but can also be deduced from the positions of the blue colonies using the key in the bottom right. 'Fingerprints' for other zinc fingers are shown in Supplementary Figure 4 online.

The DNA-binding specificities of the selected zinc fingers in yeast cells were determined using the 64 LacZ reporter plasmids, each of which contains one of the 64 distinct triplet subsites within the 9 bp target DNA sequences (Fig. 1B). Many of the selected zinc fingers showed a unique DNA-binding signature. For example, cells harboring the plasmid that encodes the QSSR1 zinc finger fused to fingers 1 and 2 of the Zif268 protein and the Gal4 activation domain formed blue colonies only when mated with cells that contained the GTA and GCA reporter plasmids. This means that, under the conditions of the assay, the QSSR1 finger recognizes the GTA and GCA triplets but not the other 62 triplet subsites. After screening 2,000 zinc fingers present in our library, we obtained a total of 56 zinc fingers that showed distinct DNA-binding specificities in our yeast system (Table 1). As a group, these zinc fingers recognize 25 subsites.

Table 1: Lists, target subsites, and K_d values of selected human zinc-finger domains^a

For selected fingers, we compared the amino acid residues at the critical base-contacting positions with those expected from the zinc finger