Binding of ZEB to CtBP mediates transcriptional repression. The region of ZEB corresponding to amino acids 1–1120, 302–903, and 700–776 were fused to the DNA binding domain of the yeast Gal4 protein and tested for its ability to repress transcription of the SV40 promoter/enhancer. Two micrograms of the Gal4 PM1 empty vector, 2 μg of wild-type G-ZEB-700–776 (amino acids 700–776), 2 μg of G-ZEB-700–776-mut (amino acids 700–776 with mutation of the CtBP site at 734), 2 μg of G-ZEB3 mut (amino acids 700–776 with mutation of CtBP sites at 705, 734, and 767), 3 μg of G-ZEB-302–903 (amino acids 302–903), 4 μg of G-ZEB-1–1120 (full-length ZEB), 2 μg of wild-type G-zfh-1–765-821 (amino acids 765–821) and 2 μg of G-zfh-1–765-821-mut (amino acids 765–821 with a mutation of the CtBP site at 792) were cotransfected with 0.8 μg of a reporter containing the SV40 enhancer/promoter (18). Transfection and assessment of CAT activity was performed as described in Materials and Methods.

CtBP and ZEB must interact at the promoter to repress transcription. Soluble ZEB (amino acids 700–776, without the Gal4 DNA binding domain) is not able to repress transcription of the reporter containing Gal4 binding sites upstream of the SV40 promoter/enhancer. Two micrograms of either G-ZEB (amino acids 700–776 of ZEB fused to Gal4 binding domain), G-ZEB-3 mut (amino acids 700–776 of ZEB fused to Gal4 binding domain but with all three CtBP sites at positions 705, 734, and 767 mutated) or soluble ZEB were cotransfected with 0.8 μg of the SV40 promoter/enhancer reporter. However, overexpression of soluble ZEB and soluble snail inhibit repression by G-ZEB (amino acids 700–776) bound to the promoter. Increasing amounts of the soluble ZEB expression vector (2.3 and 4.7 μg) or soluble snail expression vector (6.2 μg) were cotransfected with 0.8 μg of a SV40 enhancer/promoter reporter and 2 μg of the G-ZEB (amino acids 700–776) expression vector.

ZEB and zfh-1 bind to CtBP-1 and CtBP-2. (A) ZEB and zfh-1 contain CtBP binding sites. Drosophila zfh-1 and ZEB from various vertebrate species (h: human; ck: chicken; hms: hamster; ms: mouse and rat) contain a PLDLS CtBP binding sequence in their repressor domain. In addition, vertebrates contain two additional PLDLS-like sequences, which vary slightly from species to species. (B) Flag-tagged constructs for snail, the repressor domains of ZEB and zfh-1 (the region between both zinc finger domains) as well as the DNA binding domains (C-terminal zinc fingers) of ZEB (DB-ZEB) and zfh-1 (DB-zfh-1) were cotransfected in C33a cells with myc-tagged CtBP-1. After 48 hr, cells were lysed and after immunoprecipitation with Flag antibody, binding to CtBP-1 was detected by Western blot using 9E10 anti-myc mAb as described in Materials and Methods. Ten percent of the lysate was run without immunoprecipitation as input control. Blots then were stripped and incubated with anti-Flag antibody to detect levels of snail, ZEB and zfh-1. (C) As in B but using myc-tagged CtBP-2.

(A) Mutation of all three CtBP binding sites in ZEB abolishes binding to CtBP. Flag-tagged expression vectors for snail, the DNA binding domain of ZEB, and the region of ZEB between amino acids 700 and 776 (either wild type or mutated in all three CtBP binding sites, as described in Fig. 2) were cotransfected along with myc-tagged CtBP-1. Cells were collected, and binding to CtBP-1 was detected by Western blot using 9E10 anti-myc mAb as described in Materials and Methods. The blot then was stripped and reprobed with anti-Flag antibody to check expression levels of the Flag proteins. (B) Consensus binding sequence for CtBP. Amino acids at positions −2 and +1 are constant in all proteins and species (outlined). The residue at position 0 is also highly conserved with Asp and Asn in most cases. The residues at positions −1 and +2 show great variability. The size of the residue indicates the frequency for the presence of that residue. The consensus sequence was established by accounting all proteins so far known to interact with CtBP proteins: E1A regions of adenovirus types 2, 5, and 12, CtIP, ZEB in all species where it has been cloned (see Fig. 1A), BKLF, zfh-1, hairy, snail, and knirps.