Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein‐2 promoter as a novel anti‐cancer treatment

Abstract

Zinc finger protein transcription factors (ZFP-TFs) are emerging as powerful novel tools for the treatment of many different diseases. ZFPs are DNA-binding motifs and consist of modular zinc finger domains. Each domain can be engineered to recognize a specific DNA triplet, and stitching six domains together results in the recognition of a gene-specific sequence. Inhibition of gene expression can be achieved by fusing a repressor domain to these DNA-binding motifs. In this study, we engineered ZFP-TFs to downregulate the activity of the epithelial glycoprotein-2 (EGP-2) promoter. The protein EGP-2 is overexpressed in a wide variety of cancer types and EGP-2 downregulation has been shown to result in a decreased oncogenic potential of tumor cells. Therefore, downregulation of EGP-2 expression by ZFP-TFs provides a novel anti-cancer therapeutic. Using a straightforward strategy, we engineered a 3-ZFP that could bind a 9 bp sequence within the EGP-2 promoter. After the addition of a repressor domain, this 3-ZFP-TF could efficiently downregulate EGP-2 promoter activity by 60%. To demonstrate the flexibility of this technology, we coupled an activation domain to the engineered ZFP, resulting in a nearly 200% increase in EGP-2 promoter activity. To inhibit the endogenous EGP-2 promoter, we engineered 6-ZFP-TFs. Although none of the constructed ZFP-TFs could convincingly modulate the endogenous promoter, efficient and specific inhibition of the exogenous promoter was observed. Overall, ZFP-TFs are versatile bi-directional modulators of gene expression and downregulation of EGP-2 promoter activity using ZFP-TFs can ultimately result in a novel anti-cancer treatment.

Persistent downregulation of the pancarcinoma‐associated epithelial cell adhesion molecule via active intranuclear methylation

“…Targeting of the EpCAM promoter by engineered zinc finger protein transcription factors (ZFPs) has already been demonstrated by us. 21 Methyltransferases fused to zinc finger proteins targeting predetermined sites in the DNA to repress gene expression have been reported. 12,22,23 Engineering ZFPs targeted to the EpCAM promoter 21 fused to M.SssI as an effector domain, provides a powerful tool to achieve targeted methylation.…”

“…21 Methyltransferases fused to zinc finger proteins targeting predetermined sites in the DNA to repress gene expression have been reported. 12,22,23 Engineering ZFPs targeted to the EpCAM promoter 21 fused to M.SssI as an effector domain, provides a powerful tool to achieve targeted methylation. The ZFP binds specific to the promoter where after the enzyme will methylate only the CpGs close to the ZFP target sequence.…”

Analysis of an artificial zinc finger epigenetic modulator: widespread binding but limited regulation

“…Unlike designer genomic nucleases or RNAi-based approaches, toggle switches can also upregulate silenced gene expression. For example, KRAB and VP64 effector domains were able to downregulate or upregulate the EGP-2 promoter, respectively (21). …”

Future of the Genetic Code

“…MicroRNAs (miRNAs), small interfering RNAs (siRNAs), and peptide motifs such as zinc fingers, play important roles in gene regulation and represent potential therapeutic agents [75,76,77,78]. Nuclease-resistant nucleobase-peptides and nucleobase-proteins comprising both regular peptide segments and polyamide nucleic acid (PNA) [79] segments can be synthesized through the encoded incorporation of NCAAs bearing U(T), C, A, and G nucleobase sidechains.…”