Identification and Characterization of a Consensus DNA Binding Element for the Zinc Finger Transcription Factor TIEG/EGR α

Abstract

TGFβ-Inducible Early Gene (TIEG) and the alternatively-transcribed Early Growth Response Gene α (EGRα) share a Cys₂His₂ three-zinc finger region with high homology to Sp1 within its zinc finger region. Three-zinc finger transcription factors bind to GC-rich sequences, with small variations in consensus sequence between subfamilies. In this work, a consensus sequence was identified for TIEG/EGRα by expressing and purifying the zinc finger region of the protein, and using this to select a binding site from a random oligonucleotide library by iterative cycles of nitrocellulose filter binding and PCR. A fusion of the TIEG/EGRα with the VP16 activation domain supported transcription from this site when cloned in front of a heterologous promoter. Mutational analysis of the binding site identified a GT-rich core (5′-GGTGTG-3′) that was necessary for binding, with mutations outside of this region causing only a small to moderate decrease in binding.