Abstract
Classical C2H2 zinc finger proteins are among the most abundant transcription factors found in eukaryotes, and the mechanisms through which they recognize their target genes have been extensively investigated. In general, a tandem array of three fingers separated by characteristic TGERP links is required for sequence-specific DNA recognition. Nevertheless, a significant number of zinc finger proteins do not contain a hallmark three-finger array of this type, raising the question of whether and how they contact DNA. We have examined the multi-finger protein ZNF217, which contains eight classical zinc fingers. ZNF217 is implicated as an oncogene and in repressing the E-cadherin gene. We show that two of its zinc fingers, 6 and 7, can mediate contacts with DNA. We examine its putative recognition site in the E-cadherin promoter and demonstrate that this is a suboptimal site. NMR analysis and mutagenesis is used to define the DNA binding surface of ZNF217, and we examine the specificity of the DNA binding activity using fluorescence anisotropy titrations. Finally, sequence analysis reveals that a variety of multi-finger proteins also contain two-finger units, and our data support the idea that these may constitute a distinct subclass of DNA recognition motif.
Highlights
Classical C2H2 zinc finger proteins generally bind DNA via a three-finger motif
Our results demonstrate that a single two-finger motif rather than the usual three or four finger units is sufficient to allow ZNF217 and potentially other multi-zinc finger proteins to contact DNA
Zinc Fingers 6 and 7 Are Capable of Binding DNA—ZNF217 contains eight classical C2H2 zinc fingers arranged in two major clusters (Fig. 1)
Summary
Classical C2H2 zinc finger proteins generally bind DNA via a three-finger motif. Results: We have identified the DNA site recognized by ZNF217 and defined its mechanism of binding. It is notable, that a large number of zinc finger proteins do not have three or four tandem-arranged zinc fingers separated by the canonical linker sequences, raising the question of how these proteins identify their target elements and whether or not they are DNA-binding proteins at all. The mechanisms through which it operates are complex (26 –28), but one proposal has been that ZNF217 directly binds to and represses the E-cadherin gene promoter via a two-zinc finger domain that recognizes the consensus sequence CAGAAY [29]. Our results demonstrate that a single two-finger motif rather than the usual three or four finger units is sufficient to allow ZNF217 and potentially other multi-zinc finger proteins to contact DNA
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