Modulation of transcriptional regulation by LEF-1 in response to Wnt-1 signaling and association with beta-catenin

Abstract

Wnt signaling is thought to be mediated via interactions between beta-catenin and members of the LEF-1/TCF family of transcription factors. Here we study the mechanism of transcriptional regulation by LEF-1 in response to a Wnt-1 signal under conditions of endogenous beta-catenin in NIH 3T3 cells, and we examine whether association with beta-catenin is obligatory for the function of LEF-1. We find that Wnt-1 signaling confers transcriptional activation potential upon LEF-1 by association with beta-catenin in the nucleus. By mutagenesis, we identified specific residues in LEF-1 important for interaction with beta-catenin, and we delineated two transcriptional activation domains in beta-catenin whose function is augmented in specific association with LEF-1. Finally, we show that a Wnt-1 signal and beta-catenin association are not required for the architectural function of LEF-1 in the regulation of the T-cell receptor alpha enhancer, which involves association of LEF-1 with a different cofactor, ALY. Thus, LEF-1 can assume diverse regulatory functions by association with different proteins.

FIG. 1

Transcriptional activation by LEF-1 in response to Wnt-1 signaling. (A) LEF-1 stimulates reporter gene activity in the presence of Wnt-1. NIH 3T3 cells were transiently transfected with 0.8 μg of a LEF-1–CAT reporter gene construct, together with expression plasmids encoding Wnt-1 (1.5 μg) or LEF-1 (amount as indicated). The LEF-1–CAT reporter gene contained either multimerized wt or mutant LEF-1 binding sites. For these and subsequent transfection assays, the levels of CAT activity were normalized for the expression of a cotransfected Rous sarcoma virus–β-galactosidase expression plasmid. Fold activation was quantitated relative to the level of CAT activity from cells transfected with the reporter gene alone. All the transfection experiments were performed at least twice, and the results of a representative experiment are shown here. Error bars represent standard errors of the mean. (B) β-Catenin activates transcription in collaboration with LEF-1. NIH 3T3 cells were transiently transfected with a LEF-CAT reporter gene, together with LEF-1 and β-catenin cDNA expression plasmids as indicated. mut, mutant.

FIG. 2

Delineation of the β-catenin interaction domain in LEF-1. (A) Sequence comparison of the amino termini of mouse LEF-1 (64), chicken TCF-1 (9), Xenopus TCF-3 (38), Drosophila pangolin/dTCF (7, 69), and C. elegans POP-1 (29). Identical residues are indicated by white letters, and conserved residues are shaded. (B) Alanine substitution mutagenesis of the β-catenin interaction domain in LEF-1. The mutations in the amino terminus of LEF-1 are indicated in the diagram, and their effects on the interaction with β-catenin in vitro are summarized. (C) Electrophoretic mobility shift assay of DNA binding by wt LEF-1 and various mutant forms of LEF-1, alone or in association with purified recombinant His₆–β-catenin. In vitro-translated LEF-1 polypeptides were incubated with a ³²P-labeled DNA probe containing a LEF-1 binding site in the absence or presence of β-catenin as indicated. The protein-DNA complexes were separated by electrophoresis through a native 6% polyacrylamide gel and visualized by autoradiography. Translation of the Δ56 mutant of LEF-1 also yielded a slightly smaller protein product, presumably by initiation at a downstream ATG. (D) Mutations in the β-catenin interaction domain of LEF-1 reduce the transcriptional activation by LEF-1 in association with β-catenin. Neuro2A cells were transiently cotransfected with a LEF-CAT reporter gene and wt or mutant LEF-1 expression plasmids, alone or together with a β-catenin expression plasmid.

FIG. 2

Delineation of the β-catenin interaction domain in LEF-1. (A) Sequence comparison of the amino termini of mouse LEF-1 (64), chicken TCF-1 (9), Xenopus TCF-3 (38), Drosophila pangolin/dTCF (7, 69), and C. elegans POP-1 (29). Identical residues are indicated by white letters, and conserved residues are shaded. (B) Alanine substitution mutagenesis of the β-catenin interaction domain in LEF-1. The mutations in the amino terminus of LEF-1 are indicated in the diagram, and their effects on the interaction with β-catenin in vitro are summarized. (C) Electrophoretic mobility shift assay of DNA binding by wt LEF-1 and various mutant forms of LEF-1, alone or in association with purified recombinant His₆–β-catenin. In vitro-translated LEF-1 polypeptides were incubated with a ³²P-labeled DNA probe containing a LEF-1 binding site in the absence or presence of β-catenin as indicated. The protein-DNA complexes were separated by electrophoresis through a native 6% polyacrylamide gel and visualized by autoradiography. Translation of the Δ56 mutant of LEF-1 also yielded a slightly smaller protein product, presumably by initiation at a downstream ATG. (D) Mutations in the β-catenin interaction domain of LEF-1 reduce the transcriptional activation by LEF-1 in association with β-catenin. Neuro2A cells were transiently cotransfected with a LEF-CAT reporter gene and wt or mutant LEF-1 expression plasmids, alone or together with a β-catenin expression plasmid.

FIG. 3

Nuclear localization of endogenous β-catenin in NIH 3T3 cells is dependent on expression of LEF-1 and on Wnt signaling. (A) NIH 3T3 cells were transiently transfected with an expression plasmid encoding wt or mutant (m1) LEF-1. The LEF-1 expression plasmids were transfected alone (−Wnt-1) or together with a Wnt-1 expression plasmid (+Wnt-1). LEF-1 and endogenous β-catenin were detected by indirect immunofluorescence. (B) Transient expression of LEF-1 and a CD8–β-catenin fusion protein in NIH 3T3 cells. The fusion protein was detected by indirect immunofluorescence with anti-CD8 antibody (top panel), anti-β-catenin antibody (middle panel), or anti-HA antibody (bottom panel). Endogenous β-catenin was also visualized with anti-β-catenin antibody (middle panel).

FIG. 4

Wnt and CD8–β-catenin increase the pool of cytosolic β-catenin but do not affect the total amount of β-catenin. (A) NIH 3T3 cells were transfected with an expression plasmid encoding Wnt-1 or CD8–β-catenin fusion protein. Cytosolic cell extract was prepared, and β-catenin was immunoprecipitated with a monoclonal anti-β-catenin antibody and visualized by immunoblot analysis. (B) Whole-cell lysates from transfected NIH 3T3 cells were prepared, and β-catenin expression was analyzed as described above. Numbers to the left of each panel show molecular mass in kilodaltons.

FIG. 5

Analysis of transcriptional activation domains in β-catenin. (A) Schematic diagram of the structure of wt and mutant forms of β-catenin with individual domains highlighted. The numbers indicate the amino acid positions in β-catenin. The proteins contain one or two epitope tags at their amino termini. The DP mutant form of β-catenin contains substitutions of Ser/Thr residues in the amino terminus that are phosphorylated and regulate protein stability (74). The amino acid changes, indicated by asterisks, are S33A, S37A, T40A, S45A, and S47A. The mutant nuclear localization signal (NLS) β-catenin protein contains a nuclear localization sequence at the amino terminus. (B) Steady-state protein expression of wt and mutant β-catenins in transfected Cos7 cells. β-Catenin expression plasmids were transiently transfected into Cos7 cells, and cytotosolic (lanes 1 to 5) or whole-cell (lanes 6 and 7) extracts were prepared and analyzed by SDS–7.5% PAGE. A monoclonal antibody against β-catenin was used to detect the level of protein expression. Closed circles indicate the positions of the exogenous β-catenins. The altered electrophoretic mobility of the exogenous β-catenins is likely due to the amino-terminal epitope tags. The molecular size markers are shown in kilodaltons. (C) Transcriptional properties of various forms of β-catenin. Neuro2A cells were transiently cotransfected with a LEF-CAT reporter gene together with expression plasmids encoding LEF-1 and one of the various forms of β-catenin as indicated.

FIG. 6

Both amino- and carboxyl-terminal regions of β-catenin contain transcriptional activation domains. (A) Amino- and carboxyl-terminal regions of β-catenin were fused in frame to the DNA-binding domain of the yeast GAL4 activator. Neuro2A cells were transiently transfected with a GAL-CAT reporter gene construct together with a plasmid expressing the GAL4 fusion protein as indicated. (B) Analysis of DNA binding by GAL4–β-catenin fusion proteins. Shown are the results of an electrophoretic mobility shift assay of 4 μg of nuclear extracts from Cos7 cells, transiently expressing GAL4–β-catenin fusion proteins, and a probe containing a GAL4 DNA binding site in the presence of 500 ng of salmon sperm DNA and 1 μg of poly(dI-dC).

FIG. 7

Transcriptional activation by β-catenin–LEF-1 fusion proteins. (A) Schematic diagrams of β-catenin–LEF-1 fusion proteins in which amino- or carboxyl-terminal domains of β-catenin were linked to either full-length LEF-1 (catN-LEF and catC-LEF) or the HMG domain of LEF-1 (catN-HMG and catC-HMG). The β-catenin-binding domain (βBD), context-dependent activation domain (CAD), and high-mobility-group domain (HMG) are indicated. The carboxyl-terminal domain of β-catenin was also linked to LEF-1 lacking the amino-terminal 56 residues (catC-LEFΔ56). (B) DNA binding by LEF-1 and β-catenin–LEF-1 fusion proteins. Shown are results of an electrophoretic mobility shift assay with 4 μg of nuclear extracts of Cos7 cells transfected with control vector (lane 2) or expression plasmids encoding LEF-1 (lane 3) or β-catenin–LEF-1 fusion proteins (lanes 4 to 7), catN-LEF (lane 5), catC-HMG (lane 6), and catN-HMG (lane 7). (C) Transcriptional activation by β-catenin–LEF-1 fusion proteins. Neuro2A cells were transiently transfected with a LEF-CAT reporter gene together with expression plasmids encoding the LEF-1 fusion proteins as indicated.

FIG. 8

LEF-1-interacting proteins confer distinct regulatory properties upon LEF-1. (A) Requirement of the β-catenin interaction domain in LEF-1 for transcriptional activation in response to Wnt signaling. NIH 3T3 cells were transiently cotransfected with a LEF-CAT reporter gene (0.8 μg) and a wt or mutant (m1) LEF-1 expression plasmid (450 ng), alone or together with a cytomegalovirus–Wnt-1 expression plasmid (1.5 μg). (B) LEF-1 in collaboration with ALY fails to activate the LEF-CAT reporter gene. Neuro2A cells were transiently transfected with the LEF-CAT reporter gene and 50 ng of LEF-1 expression plasmid, alone or together with ALY or β-catenin expression vectors as indicated. (C) The β-catenin interaction domain of LEF-1 is dispensible for the activation of the TCRα enhancer. HeLa cells were transiently transfected with a TCRα-CAT reporter gene (250 ng), expression plasmids for AML-1 and Ets-1 (200 ng each), and a wt or mutant (m1) LEF-1 expression plasmid (100 ng). (D) β-Catenin has no effect on TCRα enhancer function. A TCRα-CAT reporter gene was cotransfected with expression plasmids for AML-1, Ets-1, and LEF-1, as described for panel C, in the absence or presence of expression plasmids encoding ALY or β-catenin.