Promoter-targeted phage display selections with preassembled synthetic zinc finger libraries for endogenous gene...

Regulation of endogenous gene expression has been achieved using synthetic zinc finger proteins fused to activation or repression domains, zinc finger transcription factors (TFZFs). Two key aspects of selective gene regulation using TFZFs are the accessibility of a zinc finger protein to its target DNA sequence and the interaction of the fused activation or repression domain with endogenous proteins. Previous work has shown that predicting a biologically active binding site at which a TF(ZF) can control gene expression is not always straightforward. Here, we used a library of preassembled three-finger zinc finger proteins (ZFPs) displayed on filamentous phage, and selected for ZFPs that bound along a 1.4 kb promoter fragment of the human ErbB-2 gene. Following affinity selection by phage display, 13 ZFPs were isolated and sequenced. Transcription factors were prepared by fusion of the zinc finger proteins with a VP64 activation domain or a KRAB repression domain and the transcriptional control imposed by these TFZFs was evaluated using luciferase reporter assays. Endogenous gene regulation activity was studied following retroviral delivery into A431 cells. Additional ZFP characterization included DNaseI footprinting to evaluate the integrity of each predicted protein:DNA interaction. The most promising TFZFs able to both up-regulate and down-regulate ErbB-2 expression were extended to six-finger proteins. The increased affinity and refined specificity demonstrated by the six-finger proteins provided reliable transcriptional control. As a result of studies with the six-finger proteins, the specific region of the promoter most accessible to transcriptional control by VP64-ZFP and KRAB-ZFP fusion proteins was elucidated and confirmed by DNaseI footprinting, flow cytometric analysis and immunofluorescence. The ZFP phage display library strategy disclosed here, coupled with the growing availability of genome sequencing information, provides a route to identifying gene-regulating TFZFs without the prerequisite of well-defined promoter elements.