New mode of DNA binding of multi-zinc finger transcription factors: delta EF1 family members bind with two hands to two target sites

SIP1, a Smad-interacting protein, and delta EF1, a transcriptional represso r involved in skeletal and T-cell development, belong to the same family of DNA binding proteins. SIP1 and delta EF1 contain two separated clusters of zinc fingers, one N-terminal and one C-terminal, These clusters show high sequence homology and are highly conserved between SIP1 and delta EF1, Each zinc finger cluster binds independently to a 5'-CACCT sequence. However, h igh-affinity binding sites for full-length SIP1 and delta EF1 in the promot er regions of candidate target genes like Xenopus Xbra2, and human alpha 4- integrin and E-cadherin, are bipartite elements composed of one CACCT and o ne CACCTG sequence, the orientation and spacing of which can vary, Using tr ansgenic Xenopus embryos, we demonstrate that the integrity of these two se quences is necessary for correct spatial expression of a Xbra2 promoter-dri ven reporter gene. Both zinc finger clusters must be intact for the high-af finity binding of SIP1 to DNA and for its optimal repressor activity. Our r esults show that SIP1 binds as monomer and contacts one target sequence wit h the first zinc finger cluster, and the other with the second cluster. Our work redefines the optimal binding site and, consequently, candidate targe t genes for vertebrate members of the delta EF1 family.