Nanog interferes with the binding of β-catenin to Tcf7 in vitro and in vivo.
(A) Nanog interacts with Tcf7 through its N terminal. Different Myc-tagged Nanog were constructed and co-transfection with HA-Tcf7 in HEK293T cells. Among all the mutated types of Nanog, only the N-terminal truncated Nanog (Nanog-ΔN) could not coprecipitate with Tcf7, indicating that Nanog physically interacts with Tcf7 through its N terminal. (B) Tcf7 interacts with Nanog through its GroBD. Different Myc-tagged Tcf7 were constructed and co-transfection with HA-Nanog in HEK293T cells. Among all the mutated types of Tcf7, only the GroBD deleted Tcf7 (Tcf7_ΔGroBD) could not coprecipitate with Nanog, indicating that Tcf7 binds with Nanog through GroBD. (C) Nanog and β-catenin competitively binds with Tcf7. Co-transfection of increasing amount of Nanog decreases the interaction between β-catenin and Tcf7 in a dose-dependent manner. When increased amount of Nanog was transfected into Tcf7 and β-catenin co-transfected cells, decreased amount of β-catenin could be coprecipitated. The molecular weight of HA-Nanog is around 55 Kda, and HA-β-catenin is around 100 KDa, so we could distinguish the 2 anti-HA bands by different protein sizes. (D) Co-transfection of Ctnnbip1 increased the binding affinity between Nanog and Tcf7. Two different amounts of HA-ctnnbip1 were co-transfected with Tcf7, Nanog and β-catenin in HEK293T cells; because Ctnnbip1 was overexpressed, an increased amount of Nanog was coprecipitated by myc-Tcf7. The molecular weight of HA-ctnnbip1 is around 10 KDa. Note that HA-β-catenin level was reduced when Ctnnbip1 was overexpressed. (E) Co-immunoprecipitation assay showed that increased amount of endogenous β-catenin could interact with Tcf7 in MZnanog mutants compared with WT embryos. Embryos were collected at 4 hpf. (F) The intensity ratio of β-catenin to Tcf7 in panel E. Error bars, mean ± SD, *P < 0.05. The P values in this figure were calculated by Student t test. The underlying data in this figure can be found in S1 Data. GroBD, Groucho binding domain; HEK293T cells, human embryonic kidney 293T cells; MZnanog, maternal -zygotic mutant of nanog; hpf, hours post fertilization; WT, wild type.