Characterization of the E-box binding affinity to snag-zinc finger proteins.

Members of the Snail/Gfi-1 domain family of zinc finger proteins are known to recognize the E-box sequence CANNTG, such as that found in the promoter of E-cadherin, however, no studies have shown that the internal "NN" dinucleotides can play a role in different binding affinities. We show via gel shift assays that only the sequences CACCTG and CAGGTG can be recognized more strongly by the SNAG-ZFP members such as Slug, Smuc, Snail, and Scratch while the other combinations of the internal nucleotides were bound weakly. All 16 possible dinucleotide combinations were tested by competition EMSAs to determine their relative binding affinities. The Kd value for the best-binding sequences was approximately 1.25 x 10(-6) M, while the other interactions were less effective. Our study has shown for the first time how different internal dinucleotide combinations of the E-box can be recognized differently by different transcription factors and also sheds light into how this transcription factor binding site may participate in DNA-protein interactions.