Zinc-finger protein-targeted gene regulation: Genomewide single-gene specificity

Abstract

Zinc-finger protein transcription factors (ZFP TFs)D efects in transcriptional regulation underlie numerous disease states, most notably cancer (1). A major goal of current strategies for correcting such defects is to achieve sufficient specificity of action (2). Designed zinc-finger protein transcription factors (ZFP TFs) emulate natural transcriptional control mechanisms and therefore provide an attractive tool for precisely regulating gene expression. Accurate control of gene expression is important for understanding gene function (target validation) and for developing therapeutics to treat disease (3). We and others have used engineered ZFP TFs to either activate or repress a variety of endogenous gene targets (4-11). For these proteins, or any other gene-regulation technology, to succeed as tools in drug discovery or direct agents in the clinic, their specificity of action within the genome must be precise, a challenging criterion to meet given the size and complexity of the human genome. Recent studies with small interfering RNA (12, 13) and antisense DNA͞RNA (14) have illuminated the magnitude of the task of achieving single-gene specificity in regulating the human genome.We focus here on the use of ZFP TFs in the area of oncology and specifically on the emerging role of checkpoint kinase 2 (CHK2). CHK2 acts as a key integrator of DNA-damage signals regulating cell-cycle progression, DNA repair, and cell death by phosphorylating a variety of substrates, including the p53 tumor suppressor protein (15, 16). Here we show that a designed ZFP TF targeted to a unique 18-bp recognition sequence in the promoter of the CHK2 gene binds the intended site within chromatin and represses CHK2 transcription in vivo. Moreover, repression of CHK2 by this engineered ZFP TF occurs with remarkable specificity, while simultaneously reducing CHK2 protein to levels that functionally ablate the action of this kinase. Finally, we show that constitutive expression of the ZFP TF in telomerase-immortalized, untransformed human fibroblasts provides stable repression of the CHK2 gene and results in loss of DNA-damage-induced CHK2-dependent phosphorylation of p53 on Ser-20. These data demonstrate that ZFP TFs can be exquisitely specific, yet potent repressors of gene expression and, therefore, are potentially powerful reagents for target validation and therapeutic interventions in vivo.

Re-activation of a dormant tumor suppressor gene maspin by designed transcription factors

“…Thus, a designed 6ZF ATF will recognize 18-bp, providing high degree of specificity (Blancafort et al, 2004;Beltran et al, 2006). Indeed, previous reports have described that highly specific 6ZF ATFs are able to target unique sites in the human genome (Tan et al, 2003). We chose three 18-bp sites in the maspin proximal promoter as targets for our ATFs (Figure 1a).…”

Sequence-Enabled Reassembly of β-Lactamase (SEER-LAC): A Sensitive Method for the Detection of Double-Stranded DNA

“…These results identify the quality of the ZF-DNA interaction as an important determinant of SEER function. Generally, the specificity of designed zinc finger proteins has been shown to be excellent (9,11,25).…”

“…These results identify the quality of the ZF-DNA interaction as an important determinant of SEER function. Generally, the specificity of designed zinc finger proteins has been shown to be excellent (9,11,25).Substitution of a different custom zinc finger DNA-binding domain generated a new SEER that produced a signal only on the new cognate target. These results clearly demonstrate that the observed effects were not due to special properties of ZFs Zif268 and PBSII, but can be achieved using other custom ZFs, such as PE1A.…”

Gene-based therapies for dominantly inherited retinopathies

“…Such suppression agents can not only be targeted to RNA transcripts as is the case with antisense, ribozymes and RNAi, but can include targeting DNA and proteins using zinc-finger proteins (ZFPs), oligonucleotides to induce DNA correction, aptamers and antibodies among other methodologies. [23][24][25][26] For example, DNA-binding ZFPs can be used (alone or in conjunction with endonucleases for DNA cleavage) to bind specific DNA targets. In principle, binding of ZFPs to 5¢ promoter sequences can thus be utilised to suppress expression of a target gene.…”