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.
Atcha
Fawzia A
FA
Department of Microbiology and Molecular Genetics, University of California, Irvine, Irvine, CA 92697, USA.
Syed
Adeela
A
Wu
Beibei
B
Hoverter
Nate P
NP
Yokoyama
Noriko N
NN
Ting
Ju-Hui T
JH
Munguia
Jesus E
JE
Mangalam
Harry J
HJ
Marsh
J Lawrence
JL
Waterman
Marian L
ML
D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019556:COS Cells; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002522:Chlorocebus aethiops; D003110:Colonic Neoplasms / Q000473:pathology; D017124:Conserved Sequence; D003545:Cysteine / Q000378:metabolism; D004247:DNA / Q000378:metabolism*; D006801:Humans; D051821:Lymphoid Enhancer-Binding Factor 1 / Q000378:metabolism; D008969:Molecular Sequence Data; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D020218:Response Elements / Q000235:genetics; D013329:Structure-Activity Relationship; D051820:TCF Transcription Factors / Q000737:chemistry* / Q000378:metabolism*; D015533:Transcriptional Activation / Q000235:genetics; D051153:Wnt Proteins / Q000378:metabolism*
D051821:Lymphoid Enhancer-Binding Factor 1; D051820:TCF Transcription Factors; D051153:Wnt Proteins; D004247:DNA; D003545:Cysteine