Fig 2: The Pygo-ChiLS interaction is important for transcriptional Wnt responses in human HEK293T cells.a TOPflash assays in wt or LDB-/- or Pygo-/- HEK293T cells incubated with 20 mM NaCl or LiCl ±4 h prior to cell lysis; fold changes (Y axis) represent mean values from 3 biological repeats; error bars indicate standard deviations; Unpaired two-tailed t test were used for statistical analyses. b TOPflash assays in wt or Pygo-/- HEK293T cells transiently transfected with wt or mutant Pygo2 for 24 h and incubated with 20 mM NaCl or LiCl ±6 h prior to cell lysis; fold changes (Y axis) represent mean values from 3 biological repeats; error bars indicate standard deviations; Unpaired two-tailed t test were used for statistical analyses. ev, empty vector control; ns, not statistically significant. c TOPflash assays in wt or LDB-/- HEK293T cells, transfected and incubated with NaCl or LiCl as in b; fold changes (Y axis) represent mean values from 3 biological repeats; error bars indicate standard deviations; Unpaired two-tailed t test were used for statistical analyses. Source data are provided as a Source Data file.

Fig 3: BCL9, Pygo2, LDB1 and SSBP2 form a stable complex in vitro.a Domain structures of key components of the human Wnt enhanceosome; the double slash indicates shortening of the extensive C-terminus typical of BCL9-related proteins, which contains the binding site for Groucho/TLE and is required for Wnt responses in flies and human cells25,32; ARM-repeat (ß-catenin Armadillo repeat), BID (ß-catenin-binding domain), HMG (high mobility group), HD1/2/3 (homology domain 1/2/3), NHD (N-terminal homology domain of Pygo comprising an NLS, the PPP cluster and NPF motif), PHD (plant homology domain), DD (dimerization domain), LCCD (LDB/Chip conserved domain), LID (LIM-interaction domain), LUFS (LUG/LUH, Flo8 and SSBP conserved domain). b Co-migration of BCL9-Pygo2 and LDB1-SSBP2 complexes (both consisting of full-length human proteins) on size-exclusion chromatography (using a Superose 6 increase 10/300 GL column). Experiments were independently performed three times with similar results. c Diagram of human wt or truncated BCL9 tested for interaction with LDB1-SSBP2. d Binding-affinities of full-length or truncated forms of Avi-tagged BCL9-Pygo2 with full-length LDB1-SSBP2 complex, as measured by BLI assays. The KD was calculated based on steady-state analysis and was presented as mean values ± standard deviations (SD). n = 4 of analyzed concentrations. e A schematic model of the Wnt enhanceosome in its OFF or ON state (see also refs. 32,52); the red dashed circle marks the Pygo2-LDB1-SSBP2 ternary complex whose crystal structure is reported here. Note also that the HMG domain of TCF bends DNA53,54, which may facilitate assembly of transcriptional complex. Source data are provided as a Source Data file.

Fig 4: Mutagenesis analysis of the Pygo2-ChiLS interface.a MBP pull-down assays between MBP-tagged LDB1, SSBP2 and GST-tagged wt or mutant Pygo2 bearing alanine substitutions of individual interface residues; GST, negative control. Experiments were independently performed three times with similar results. Labeled are the molecular weights (MW) of the corresponding proteins. b MBP pull-down assays as in a, but with mutant MBP-tagged LDB1 or SSBP2 bearing alanine substitutions of interface residues. Experiments were independently performed three times with similar results. Labeled are the molecular weights (MW) of the corresponding proteins. c Binding affinities between mutant Pygo2 and wt ChiLS, and vice versa, as measured by BLI assays. ND: no detectable binding. The KD was calculated based on steady-state analysis and was presented as mean values ± standard deviations (SD). n = 5 of analyzed concentrations, except for Pygo2 P77A and SSBP2 F60A (n = 4 of analyzed concentrations). Source data are provided as a Source Data file.

Fig 5: Specific interactions between Pygo2 and ChiLS.Coloring as in Fig. 3, except for green, marking second SSBP2 molecule. a Hydrophobic interactions formed between Pygo2 NPF and its cognate surface of ChiLS; left, ribbon representation; right, electrostatic surface representation. b Interface residues mediating interactions between Pygo2 PPP and SSBP2. c Hydrogen bonds and salt bridges between the negatively charged side chains of conserved glutamic acid (E) or aspartic acid (D) residues 3’ adjacent to Pygo2 NPF (see also Fig. 2a) and the guanidium or carboxamide groups in the sidechains of conserved arginine (R) or asparagine (N) residues of LDB1.