Figure 1.
Constitutive TCF/LEF activity in hematopoietic tumor cells in the absence of β-catenin stabilization.
(A) The indicated hematopoietic tumor cells were transduced with wild-type (Top) or mutant (Fop) TCF/LEF firefly luciferase reporter and a renilla luciferase virus, and the TCF/LEF transcriptional activity was calculated by dividing the TOP/renilla ratio by the FOP/renilla ratio. The mean values (± s.e.m., n>5) of at least two independent experiments are shown. (B) Lack of β-catenin stabilization in K562, Jurkat and Ramos cells, as measured by GST-E-cadherin pull-down. Ovarian PA1 cancer cells and breast immortalized AB5 cells were used as positive and negative control, respectively. (C) β-catenin or γ-catenin knockdown in Ramos cells did not affect TCF/LEF activity. Ramos cells containing Top or Fop luciferase reporter were infected with β-catenin or γ-catenin shRNA and the TCF/LEF reporter activity was measured (lower panel). β-catenin and γ-catenin down-regulation was assessed by immunoblot (upper panel). (D) Ramos and U937 cells containing the tet transactivator (tTA) with or without a lentiviral inducible vector containing DN-TCF4 fused to GFP were cultured for 24 hrs in the presence or the absence of doxycycline (dox), and the proportion of GFP positive cells was assessed by FACS analysis. (E) The cells in D were used in colony formation assay in the presence or the absence of dox. (F) Effects of DN-TCF4 induction in Ramos cells on the expression of LEF1 and Axin 2 by quantitative real-time PCR. (G) mRNA levels of Axin 2 and LEF1 in human primary hematopoietic tumors. For each sample, the qPCR values were normalized with those obtained for the negative control, U937 cells. The types of malignancy corresponding to each sample are listed in the table: MCL, mantle cell lymphoma; CLL, B-cell chronic lymphocytic leukemia; splMZL, splenic marginal zone lymphoma; FLN, follicular lymphoma; HLN, Hodgkin's lymphoma.
Figure 2.
β-catenin-independent transcriptional activity of TCF1/LEF1 factors.
(A) Empty pcDNA3HA vector or increasing amounts of HA-tagged TCF1, LEF1 or TCF4 constructs were co-transfected with SuperTop or SuperFop and renilla luciferase plasmids in 293T cells and the Top/Fop ratio was calculated. (B–C) Effects of a decoy construct containing TCF1 β-catenin binding domain (BCBD) on Wnt3a- and TCF1-induced TCF/LEF reporter activity. 293T cells were co-transfected with SuperTop and renilla reporter plasmids, together with the Wnt3a, TCF1 and BCBD vectors as indicated. (D–E) Mutant TCF1 (mtTCF1; D21A, E29K) is unable to bind to β-catenin. (D) 293T cells were transfected with the indicated HA-tagged TCF1 or TCF4 constructs and the myc-tagged β-catenin, followed by immunoprecipitation using anti-HA antibody and immunoblot with anti-myc or anti-HA antibodies. (E) 3T3 cells were co-transfected with β-catenin, the indicated amounts of TCF1, d36TCF1 or mtTCF1, together with SuperTop and renilla luciferase plasmids. (F) Effects of TCF1 and mtTCF1 expression on TCF/LEF reporter activity. 293T cells were co-transfected with SuperTop and renilla luciferase plasmids and different amounts of TCF1 or mtTCF1, followed by reporter assay.
Figure 3.
TCF1 activity in Xenopus laevis explants and embryos.
(A) Wnt3a, wild-type and β-catenin-independent mutants of TCF1, but not TCF4, induce expression of the canonical Wnt pathway-responsive gene Xnr3 in ectodermal (animal cap) explants. RT-PCR analysis of animal caps dissected at late blastula stages and cultured until stage 10.5. EF1-α is used as a loading control. The –RT lane contains all reagents except reverse transcriptase, and is used as a negative control. (B) Graph depicting embryonic perturbation by Wnt3a, TCF4, and wild-type and mutant TCF1. Embryos were injected in the ventral marginal zone at early cleavage stages and cultured until late tailbud stages. For (A) and (B), 1 ng (TCF4 high) or 500 pg (TCF1, del65TCF1, TCF1m, TCF4low, Wnt3a) of each RNA was injected, as listed. (C) Representative embryos recorded in the graph shown in (B). Dorsal views; anterior is to right. All embryos in (C) were injected with RNA encoding del65TCF1. Staining with the 12/101 antibody, which recognizes a somite specific epitope, was used to assess axis duplication.
Figure 4.
Mapping of the TCF1 domains involved in β-catenin-independent transcriptional activity.
(A) Diagram of the different constructs used for the mapping. (B–D) 293T cells were co-transfected with the SuperTop reporter, the renilla plasmid and the indicated constructs, followed by reporter assay two days after transfection. (B) TCF1 N-terminal domain is required, but not sufficient, for β-catenin-independent full transcriptional activity. (C) Deletion of either aa 101–211 or 212–299 inhibits the β-catenin-independent transcriptional activity of TCF1. (D) Transcriptional activity of two different TCF1 domains fused to the Gal4 DNA binding domain. 293T cells were co-transfected with a Gal4 responsive reporter and the indicated TCF1-Gal4 fusion constructs, followed by reporter assay. (E) Expression of TCF1 aa 37–122, but not aa 100–211, inhibits TCF1-induced reporter activity. 293T cells were co-transfected with the Supertop reporter and the indicated TCF1-Gal4 fusion constructs, in the presence or the absence of TCF1, followed by reporter assay two days after transfection.
Figure 5.
ATF2 transcription factors cooperate with TCF1/LEF1 to stimulate TCF/LEF activity.
(A–F) 293T cells were co-transfected with the Supertop reporter, the renilla plasmid and the indicated TCF/LEF or AP1 transcription factors, followed by TCF/LEF reporter assay two days after transfection. (A) c-Jun and JunB inhibition of TCF1-induced reporter activity. (B–C) ATF2 synergizes with TCF1 (B) and LEF1 (C) to increase TCF/LEF activity. (D) ATF7 enhances TCF1- and LEF1-induced reporter activity. (E–F) CREB5 cooperated with TCF1 (E) and LEF1 (F). (G) myc-tagged LEF1 co-immunoprecipitates with Flag-tagged CREB5 in 293T cells. (H) Endogenous ATF2 co-immunoprecipitates with Flag-tagged TCF1 in 293T cells. Cells transduced with lentiviral shATF2 were used as a negative control and immunoblot with anti-Axin antibody was used as loading control for the total lysate.
Figure 6.
ATF2 and ATF7 knockdown in Ramos cells decreases TCF/LEF activity, Axin 2 expression and cell growth.
(A–C) Ramos cells containing the TCF/LEF firefly and renilla luciferase lentiviral reporters were transduced with shATF2 and shATF7 or control lentiviral vectors. ATF2 and ATF7 down-regulation assessed by immunoblot analysis. (A) shATF2/7 inhibits constitutive TCF/LEF reporter activity in Ramos cells. (B) Knockdown of ATF2 and ATF7 reduces the expression of the Wnt target gene Axin 2 in Ramos cells as measured by quantitative real-time PCR. The results normalized to the control represent the mean values ± SEM of three independent experiments. (*) P<0.05; (**) P<0.001 compared with control (two-way Anova test) (C) shATF2/7 inhibits Ramos cell colony formation. (D) Model for β-catenin-dependent and β-catenin-independent canonical Wnt signaling. See the text for details.