1. Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain
- Author
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Merlin Crossley, Wooi F. Lim, Richard C. M. Pearson, Alister P. W. Funnell, Jon Burdach, and Kate G. R. Quinlan
- Subjects
Vascular Endothelial Growth Factor A ,0301 basic medicine ,Recombinant Fusion Proteins ,Kruppel-Like Transcription Factors ,Biology ,DNA-binding protein ,03 medical and health sciences ,Protein structure ,Recognition sequence ,Genetics ,Humans ,Promoter Regions, Genetic ,Transcription factor ,Zinc finger ,Binding Sites ,Genome, Human ,Gene regulation, Chromatin and Epigenetics ,Zinc Fingers ,Promoter ,DNA ,DNA-binding domain ,Fusion protein ,Chromatin ,Protein Structure, Tertiary ,Cell biology ,DNA-Binding Proteins ,HEK293 Cells ,030104 developmental biology ,Protein Binding ,Transcription Factors - Abstract
Transcription factors are often regarded as having two separable components: a DNA-binding domain (DBD) and a functional domain (FD), with the DBD thought to determine target gene recognition. While this holds true for DNA binding in vitro, it appears that in vivo FDs can also influence genomic targeting. We fused the FD from the well-characterized transcription factor Krüppel-like Factor 3 (KLF3) to an artificial zinc finger (AZF) protein originally designed to target the Vascular Endothelial Growth Factor-A (VEGF-A) gene promoter. We compared genome-wide occupancy of the KLF3FD-AZF fusion to that observed with AZF. AZF bound to the VEGF-A promoter as predicted, but was also found to occupy approximately 25 000 other sites, a large number of which contained the expected AZF recognition sequence, GCTGGGGGC. Interestingly, addition of the KLF3 FD re-distributes the fusion protein to new sites, with total DNA occupancy detected at around 50 000 sites. A portion of these sites correspond to known KLF3-bound regions, while others contained sequences similar but not identical to the expected AZF recognition sequence. These results show that FDs can influence and may be useful in directing AZF DNA-binding proteins to specific targets and provide insights into how natural transcription factors operate.
- Published
- 2015