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

Abstract

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.

The Protein-Binding Potential of C2H2 Zinc Finger Domains

“…Typically, synthetic proteins are generated by mixing and matching naturally occurring C2H2 domains to create a protein with novel DNA binding properties (Bae et al, 2003), or by altering specific residues within the framework of a standard C2H2 …”

Genome-Editing Technologies: Principles and Applications

“…The residues that facilitate DNA recognition are located within the a-helical domain and typically interact with three bps of DNA, with occasional overlap from an adjacent domain (Wolfe et al 2000). Using methods such as phage display (Choo and Klug 1994;Jamieson et al 1994;Wu et al 1995), a large number of zinc-finger domains recognizing distinct DNA triplets have been identified (Segal et al 1999;Dreier et al 2001Dreier et al , 2005Bae et al 2003). These domains can be fused together in tandem using a canonical linker peptide (Liu et al 1997) to generate polydactyl zinc-finger proteins that can target a wide range of possible DNA sequences (Beerli et al 1998(Beerli et al , 2000aKim et al 2009).…”

Toward a Functional Annotation of the Human Genome Using Artificial Transcription Factors

“…Human embryonic kidney (HEK) cell lines stably expressing ZFP-TFs were generated as follows: Plasmids encoding ZFP-TFs were stably introduced into FlpTRex-293 cell lines (Invitrogen) essentially as described in the manufacturer's protocol. Briefly, the HindIII-XhoI fragment from the pLFD-p65, pLFD-VP16, or pLFD-Kid vectors (Bae et al 2003), which contain DNA segments that encode ZFP-TFs, were subcloned individually into pcDNA5/FRT/TO (Invitrogen). The resulting plasmids were cotransfected along with pOG44 (Invitrogen) into FlpTRex-293 cells to induce a site-specific integration event.…”

“…Recent advances in the field of artificial transcription-factor technology allow one to build tens of thousands of active transcription factors with ease (Segal and Barbas III 2001;Bae et al 2003;Lee et al 2003). Zinc fingers are small DNA recognition motifs composed of ∼30 amino acid residues and a zinc ion.…”

“…Thousands of highly active ZFPs can be constructed simultaneously. For example, shuffling 20 domains to make three-finger proteins would yield 8000 (= 20 ‫ן‬ 20 ‫ן‬ 20) ZFPs in a single step (Bae et al 2003). ZFPs are then fused to either transcriptional activation domains, such as VP16 or p65 or repression domains such as KRAB, to build artificial transcription factors.…”