Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment.
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.
Gommans
Willemijn M
WM
Department of Therapeutic Gene Modulation, University of Groningen, Groningen, The Netherlands.
McLaughlin
Pamela M J
PM
Lindhout
Beatrice I
BI
Segal
David J
DJ
Wiegman
D J
DJ
Haisma
Hidde J
HJ
van der Zaal
Bert J
BJ
Rots
Marianne G
MG
D000951:Antigens, Neoplasm / Q000235:genetics*; D001483:Base Sequence; D015815:Cell Adhesion Molecules / Q000235:genetics*; D017931:DNA Primers; D015536:Down-Regulation; D000071858:Epithelial Cell Adhesion Molecule; D015972:Gene Expression Regulation, Neoplastic; D006801:Humans; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D015202:Protein Engineering; D015183:Restriction Mapping; D014157:Transcription Factors / Q000235:genetics* / Q000627:therapeutic use*; D016335:Zinc Fingers / Q000235:genetics*
D000951:Antigens, Neoplasm; D015815:Cell Adhesion Molecules; D017931:DNA Primers; C499379:EPCAM protein, human; D000071858:Epithelial Cell Adhesion Molecule; D014157:Transcription Factors