Journal
MOLECULAR AND CELLULAR BIOLOGY
Volume 27, Issue 23, Pages 8352-8363Publisher
AMER SOC MICROBIOLOGY
DOI: 10.1128/MCB.02132-06
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Funding
- NCI NIH HHS [CA096878, CA-62203, CA108697, R01 CA096878, P30 CA062203, R01 CA108697] Funding Source: Medline
- NICHD NIH HHS [R01 HD036081, HD36081] Funding Source: Medline
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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.
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