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

Abstract

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.

FIG. 1.

A 30-amino-acid motif in the TCF E-tail is highly conserved and related to a sequence in two heterologous transcription factors. (A) A general domain structure of LEF/TCFs includes the N-terminal β-catenin binding domain, the high-mobility group DNA binding domain (which also includes the nuclear localization signal [HMG box + NLS]), and the alternatively spliced E-tail. Partial alignment of homologous E-tail sequences in TCF orthologs and the unrelated Huntington's Disease Binding Proteins 1 and 2 shows a high level of sequence conservation of basic, aromatic, and cysteine residues. Regions of sequence conservation are shaded, and a consensus sequence is shown below the alignment, with the four conserved cysteines highlighted in red. hTCF-1E and hTCF-4E are human family members; dTCF, pop-1, hydra, and Ciona represent the single TCF orthologs in Drosophila, C. elegans, H. magnipapillata, and Ciona intestinalis, respectively. (B) Three luciferase reporter plasmids used in this study comprise a multimerized Wnt response element (core sequence shown in red) next to the minimal herpesvirus tk promoter.

FIG. 2.

The E-tail is necessary to activate transcription from weak Wnt response elements. Cos-1 cells were transiently transfected with TOP (A), LOP (B), or LOP/TOP (C) luciferase reporters with expression vectors for the indicated LEF/TCFs and β-catenin (cotransfected in all conditions). (D) TOP and LOP were cotransfected with increasing concentrations (200, 400, 800, and 1,000 ng) of LEF-VP16 expression vector. At the highest concentration of LEF-VP16, activation of LOP was increased twofold. Error bars represent standard deviations derived from the results of three or more experiments.

FIG. 3.

A cysteine motif in the E-tail cooperates with the HMG DNA binding domain for LEF1 regulation. (A) Activation of Wnt responsive reporter plasmids LEF1, TOP, LOP, and LOP/TOP requires the HMG DNA binding domain. A two-amino-acid substitution in the HMG DNA binding domain (KK changed to EE) prevents reporter gene activation even though the E-tail remains the wild type. The E-tail alone is not sufficient to recruit β-catenin to any of the reporter plasmids (β-cat/Gal4/E-tail denotes a fusion protein in which the Gal4 DNA binding domain is fused at the N terminus to the β-catenin binding domain and at the C terminus to the E-tail). (B) Transient transfection assays showed that coexpression of increasing amounts of a Gal4/E-tail fusion protein (Gal4 DNA binding domain fused to the 138-amino-acid E-tail of TCF-1E) does not inhibit the ability of TCF-1E and β-catenin to activate transcription from the Wnt response elements in the LEF1 promoter 1. (C) A novel cysteine motif is required for LEF1 regulation but not TOP reporter regulation. Cos cells were cotransfected with β-catenin and mutant TCF-1E expression vectors and the LEF1 reporter plasmid (left panel) or the TOPtk reporter plasmid (right panel). Mutations are single amino acid substitutions in the positions indicated in red type in an E-tail alignment (shown below). Error bars indicate standard deviations for the results of three experiments. Mock, mock treatment.

FIG. 4.

The E-tail is a new type of DNA binding domain. (A) Overlay sensorgrams for SPR analysis of TOP, LOP, or mutant LOP sequence (MOP) binding to immobilized GST E-tail. DNAs were injected at concentrations ranging from 10 nM to 100 nM over immobilized GST/E-tail at a flow rate of 40 μl/min at 25°C. Results are expressed in resonance units (RU) as a function of time in seconds. Increases in RU indicate binding of the double-stranded oligonucleotides to the GST/E-tail-modified surface. (B) Binding competition with a concentration of up to 1,000 nM of either single-stranded DNA or double-stranded DNA was performed with GST/E-tail bound to 100 RUs of immobilized DNA. (C) Equivalent concentrations of purified, recombinant GST/E-tail wild-type protein and GST/E-tail^mt mutant protein were compared with respect to their binding activities with 1,800 RUs of immobilized LOP DNA. The E-tail mutation carries the CRARF substitution shown in Fig. 3C. Sensorgrams are representative of specific interactions.

FIG. 5.

The E-tail is a sequence-specific DNA binding motif in the context of full-length TCF-1. (A) EMSA with extracts from COS-1 cells expressing full-length histidine-tagged TCF-1E TCF-1E^WT or TCF1E^mt (10 μg protein) showed that the CR1 mutation in the E-tail (see Fig. 3C for sequence of mutation) decreases TCF-1E binding to an extended probe that encodes the second Wnt response element (bold italics in sequences at bottom of figure) in the LEF1 promoter and a GC-rich motif (underlined) seven nucleotides downstream of the core (LOP2+GC). A Western blot probed with antibody for the histidine tag shows equal levels of wild-type and mutant proteins in the extracts. (B) Purified recombinant GST/E-tail (GST/E-tail^WT) can bind directly to the LOP2 + GC probe, but a mutant GST/E-tail with the CR1 mutation is inactive for DNA binding (GST/E-tail^mt). A Coomassie-stained gel shows amounts of purified recombinant proteins equal to those used in the EMSA. (C) In a competition assay for binding to LOP2+GC, the indicated molar excess of cold competitor oligonucleotides shows that the downstream GC-rich sequence is not specifically recognized by recombinant purified GST/E-tail (GC^SC refers to a “scrambled” mutation of this GC motif). These data are consistent with the SPR experiments (Fig. 4), which show the E-tail binds double-stranded DNA in a non-sequence-specific manner. (D) Extracts of Colo320 colon cancer cells (15 μg protein), which express high levels of TCF-1E and TCF-4E, were used in a competition EMSA as described for panel C. In this competition with endogenous full-length TCF-E isoforms, mutation of the GC element (as in LOP+GC^SC and GC^SC) reduces the ability of the oligonucleotide to compete for binding to the LOP2+GC probe. Both the WRE and the GC element can compete for some but not all of the binding activity.

FIG. 6.

CASTing analysis of full-length LEF-1 and TCF-1E. (A) Crude cell extracts containing overexpressed, epitope-tagged LEF-1 and TCF-1E were used in a CASTing analysis with a randomly synthesized library (see Materials and Methods). Two independent CASTing analyses were performed (left and right panels). The number of independent sequences used in the sequence alignment is shown for each replicate. Alignments are summarized as sequence logos in which the height of the nucleotide designation represents the frequency of occurrence of that nucleotide. A small GC element was enriched with wild-type TCF-1E for the second replicate CASTing (34 out of 66 independent sequences contained the RCCG motif downstream with variable spacing between 0 to 3 nucleotides). The consensus sequence from a previous systematic evolution of ligands by exponential enrichment experiment is shown (TCF-1 HMG) (39) as well as an affinity profile obtained with a TCF-4/Renilla luciferase fusion protein (TCF-4 profile) (18). W and S are International Union of Pure and Applied Chemistry nomenclature for A or T nucleotides (W) and C or G nucleotides (S). (B) Nucleotide alignment of experimentally validated Wnt response elements. Information about these response elements was derived from studies of LEF1 (3), CDX1 (20), CMYC (19), cyclin D1 (35), AXIN 2 (24), and MMP7 (17). Matches to the TOP sequences are shaded gray; mismatched nucleotides are shown in red. RCCG motifs are boxed. Asterisks denote two weak WRE motifs in the human MMP7 promoter that do not respond to LEF-1/β-catenin when multimerized in a reporter similar to TOPtk and LOPtk (17). ¶ refers to a WRE core sequence that has been experimentally validated in a recent genome-wide survey of sequences occupied by β-catenin (46).

FIG. 7.

The E-tail is involved in regulation of colon cancer cell growth. The results of doxycycline (DOX)-induced expression of dominant-negative TCF-1E^WT and TCF-1E^mt in DLD-1 colon cancer cells are shown. Quantitation of cell number with or without doxycycline was performed using a sulforhodamine B cell proliferation assay (see Materials and Methods). Error bars depict standard deviations of the results obtained with eight replicates. The inset shows the results of a Western blot analysis of the induced levels of expression of dnTCF-1E^WT and dnTCF-1E^mt proteins.