title: Directing an artificial zinc finger protein to new targets by fusion to a non-DNA-binding domain.
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 bindingin vitro, it appears thatin vivoFDs 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 theVEGF-Apromoter 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.
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia.
authors.forename: Wooi F
authors.initials: WF
authors.lastname: Lim
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia.
authors.forename: Jon
authors.initials: J
authors.lastname: Burdach
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia.
authors.forename: Alister P W
authors.initials: AP
authors.lastname: Funnell
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia.
authors.forename: Richard C M
authors.initials: RC
authors.lastname: Pearson
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia.
authors.forename: Kate G R
authors.initials: KG
authors.lastname: Quinlan
authors.affiliation: School of Biotechnology and Biomolecular Sciences, University of New South Wales, NSW 2052, Australia m.crossley@unsw.edu.au.
authors.forename: Merlin
authors.initials: M
authors.lastname: Crossley
mesh_terms: D001665:Binding Sites; D002843:Chromatin / Q000378:metabolism; D004247:DNA / Q000737:chemistry / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D015894:Genome, Human; D057809:HEK293 Cells; D006801:Humans; D051741:Kruppel-Like Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D014157:Transcription Factors / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D042461:Vascular Endothelial Growth Factor A / Q000235:genetics; D016335:Zinc Fingers
chemical_list: D002843:Chromatin; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D042461:Vascular Endothelial Growth Factor A; D004247:DNA