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Molecular and Cellular Biology, December 2007, p. 8352-8363, Vol. 27, No. 23
0270-7306/07/$08.00+0     doi:10.1128/MCB.02132-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

A Unique DNA Binding Domain Converts T-Cell Factors into Strong Wnt Effectors{triangledown}

Fawzia A. Atcha,1 Adeela Syed,2 Beibei Wu,1 Nate P. Hoverter,1 Noriko N. Yokoyama,1 Ju-Hui T. Ting,1 Jesus E. Munguia,1 Harry J. Mangalam,3 J. Lawrence Marsh,2* and Marian L. Waterman1*

Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, California 92697,1 Developmental Biology Center and Department of Developmental and Cell Biology, University of California, Irvine, Irvine, California 92697,2 Network and Academic Computing, University of California, Irvine, Irvine, California 926973

Received 14 November 2006/ Returned for modification 16 January 2007/ Accepted 5 September 2007

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 ß-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 ß-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.

* Corresponding author. Mailing address: Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697. Phone: (949) 824-2885. Fax: (949) 824-8598. E-mail for Marian L. Waterman: mlwaterm{at}uci.edu. E-mail for J. Lawrence Marsh: jlmarsh{at}uci.edu

{triangledown} Published ahead of print on 24 September 2007.

Molecular and Cellular Biology, December 2007, p. 8352-8363, Vol. 27, No. 23
0270-7306/07/$08.00+0     doi:10.1128/MCB.02132-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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