A unique DNA binding domain converts T-cell factors into strong Wnt effectors.

Wnt regulation of gene expression requires binding of LEF/T-cell factor (LEF/TCF) transcription factors to Wnt response elements (WREs) and recruitment of the activator beta-catenin. There are significant differences in the abilities of LEF/TCF family members to regulate Wnt target genes. For example, alternatively spliced isoforms of TCF-1 and TCF-4 with a C-terminal "E" tail are uniquely potent in their activation of LEF1 and CDX1. Here we report that the mechanism responsible for this unique activity is an auxiliary 30-amino-acid DNA interaction motif referred to here as the "cysteine clamp" (or C-clamp). The C-clamp contains invariant cysteine, aromatic, and basic residues, and surface plasmon resonance (SPR) studies with recombinant C-clamp protein showed that it binds double-stranded DNA but not single-stranded DNA or RNA (equilibrium dissociation constant = 16 nM). CASTing (Cyclic Amplification and Selection of Targets) experiments were used to test whether this motif influences WRE recognition. Full-length LEF-1, TCF-1E, and TCF-1E with a mutated C-clamp all bind nearly identical WREs (TYYCTTTGATSTT), showing that the C-clamp does not alter WRE specificity. However, a GC element downstream of the WRE (RCCG) is enriched in wild-type TCF-1E binding sites but not in mutant TCF-1E binding sites. We conclude that the C-clamp is a sequence-specific DNA binding motif. C-clamp mutations destroy the ability of beta-catenin to regulate the LEF1 promoter, and they severely impair the ability of TCF-1 to regulate growth in colon cancer cells. Thus, E-tail isoforms of TCFs utilize two DNA binding activities to access a subset of Wnt targets important for cell growth.

A new beta-catenin-dependent activation domain in T cell factor.

Transcription of the lymphoid enhancer factor-1 (LEF1) gene is aberrantly activated in sporadic colon cancer, whereas this gene is not expressed in the normal adult colon. We have shown previously that promoter 1 of the LEF1 gene is activated by T cell factor (TCF)-beta-catenin complexes in transient transfection assays, suggesting that LEF1 is a target of the Wnt pathway in colon cancer. To further explore the link between LEF1 expression and the Wnt pathway, we studied two response elements in the promoter. Surprisingly we found that the LEF1 promoter is selectively activated by specific isoforms of the LEF/TCF transcription factor family that contain an alternative C-terminal "E" tail. These isoforms, TCF-1E and TCF-4E, activate the promoter in a beta-catenin-dependent manner. We show that a complete E-tail domain is necessary for full activity and delimits residues within two highly conserved peptide motifs within the tail that are required (KKCRARFG; WCXXCRRKKKC). These peptide motifs are not only conserved among the TCF family members but are also found in two newly identified DNA-binding proteins named papillomavirus binding factor and GLUT4 enhancer factor. This study thus identifies a new and unique set of motifs used by the Wnt pathway for target gene regulation.