Identification and characterization of a consensus DNA binding element for the zinc finger transcription factor TIEG/EGRalpha.
TGFbeta-Inducible Early Gene (TIEG) and the alternatively-transcribed Early Growth Response Gene alpha (EGRalpha) share a Cys(2)His(2) 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/EGRalpha 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/EGRalpha 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.
Chrisman
Holly R
HR
Department of Urology Research, Mayo Clinic/Foundation, Rochester, Minnesota 55905, USA.
Tindall
Donald J
DJ
D001483:Base Sequence; D001665:Binding Sites; D017124:Conserved Sequence; D004247:DNA / Q000378:metabolism*; D004252:DNA Mutational Analysis; D004268:DNA-Binding Proteins / Q000235:genetics* / Q000378:metabolism*; D051765:Early Growth Response Transcription Factors; D015723:Gene Library; D017405:Herpes Simplex Virus Protein Vmw65 / Q000235:genetics / Q000378:metabolism; D051741:Kruppel-Like Transcription Factors; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D011897:Random Allocation; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics* / Q000378:metabolism*; D016335:Zinc Fingers
D004268:DNA-Binding Proteins; D051765:Early Growth Response Transcription Factors; D017405:Herpes Simplex Virus Protein Vmw65; C098111:KLF10 protein, human; D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D004247:DNA