Molecular functions of the TLE tetramerization domain in Wnt target gene repression

Abstract

Wnt signaling activates target genes by promoting association of the co-activator β-catenin with TCF/LEF transcription factors. In the absence of β-catenin, target genes are silenced by TCF-mediated recruitment of TLE/Groucho proteins, but the molecular basis for TLE/TCF-dependent repression is unclear. We describe the unusual three-dimensional structure of the N-terminal Q domain of TLE1 that mediates tetramerization and binds to TCFs. We find that differences in repression potential of TCF/LEFs correlates with their affinities for TLE-Q, rather than direct competition between β-catenin and TLE for TCFs as part of an activation-repression switch. Structure-based mutation of the TLE tetramer interface shows that dimers cannot mediate repression, even though they bind to TCFs with the same affinity as tetramers. Furthermore, the TLE Q tetramer, not the dimer, binds to chromatin, specifically to K20 methylated histone H4 tails, suggesting that the TCF/TLE tetramer complex promotes structural transitions of chromatin to mediate repression.

Figure 1. Structure of the TLE1 Q domain

1. The primary structure of human TLE1 showing domain boundaries.

2. The structure of the TLE1 tetramerization domain comprising residues 23–136 in chains A (light blue) and B (red), and 23–133 in chains C (orange) and D (green) are shown; the remaining residues are disordered. Hydrophobic side chains are shown in stick representation, and the N- and C-termini marked.

3. Hydrophobic interactions between chain C (orange) and chain D (green) involving the α2 and α3 helices. The 120° orientation between α2 and α3 is fixed by a salt bridge between E118 of one chain and R84 of its partner.

4. Glutamine residues (shown in stick representation) are mostly clustered on helices α2 and α3.

5. Close-up of the TLE1-Q tetramer interface.

6. SAXS analysis of TLE1_20–156. The solid black line shows the experimentally determined scattering curve of TLE1_20–156 at 5 mg/ml. The R_g obtained from the Guinier plot = 70 Å, and D_max = 227 Å as calculated with Autognom (Semenyuk & Svergun, 1991). These values match closely the R_g = 69 Å and D_max = 217 Å calculated from the extended tetramer model (green line; χ² = 3.1) versus R_g = 35 Å and D_max = 162 Å for the compact tetramer (red line; χ² = 6.9; see Supplementary Fig S1B).

7. Gel filtration of the TLE1_20–156 tetramer and L26D, I29D dimer mutant. Purified proteins were run on a Superdex S200 gel filtration column. Due to their large hydrodynamic radius (axial ratio ˜10:1), the proteins run at apparent molecular weights of 173 kDa, and 53 kDa.

Figure 2. Reporter activity of TCF/LEF proteins correlates with affinity for TLE1

1. Primary structure of the TCF-LEF family of proteins, showing the N-terminal β-catenin binding domain, the context-dependent regulatory domain (CRD), and the HMG box that binds DNA. TCF3 and TCF4 also have extensions that arise from alternative splicing. Residue numbers for domain boundaries are shown for the XTCF3 protein used in this study. The equivalent sequence numbers of other family members can be found in Supplementary Fig S2.

2. Luciferase reporter assay in HEK293 cells (left) and Cos1 cells (right) shows differential abilities of transfected HA-tagged TCF/LEFs to activate the Wnt reporter SuperTOPFlash when co-transfected with β-catenin. The graphs for each cell line are representative of three replicates. The Western blots (anti-HA) shown in the insets reveal the relative expression levels of the transfected TCF/LEF constructs and show that the activity differences are not related to protein levels. The band marked * is a breakdown product of TCF3. Error bars represent the standard deviation of triplicate measurements.

3. Fluorescence anisotropy analysis of TLE1_20–156 binding to different TCF/LEFs. Increasing amounts of TCF/LEF ligands were titrated against labeled TLE1_20–156. The change in anisotropy was measured and plotted as a function of concentration. Error bars represent standard deviation of triplicate measurements.

Figure 3. TLE_120-156 tetramer and dimer can bind TCF3 and TCF4 in vitro, but only the tetramer represses transcription in vivo

1. Binding of TLE1_20–156 tetramer to the TCF3_(1–330): β-catenin complex or TCF3_1–330 measured by fluorescence anisotropy. Error bars represent standard deviation of n = 3.

2. Binding of TLE1_20–156 dimer mutant to and TCF3_1–330 and TCF4_1–330. Error bars represent standard deviation of n = 3.

3. Overexpression of TCF3 leads to significant repression of reporter activity in HEK293T cells. Cells were treated with 50 ng of purified Wnt3a 24 h after co-transfection with or without TCF3. Error bars represent standard deviation of n = 3.

4. TLE tetramer but not the dimer can repress reporter activity in HEK293T cells. Cells were treated with 100 ng of purified Wnt3a 24 h after co-transfection with β-catenin, TCF3 and indicated amounts of DNA constructs encoding either TLE1 dimer or TLE1 tetramer. Error bars represent standard deviation of n = 3. Inset Western blot shows levels of transfected TLE1 tetramer or dimer. Immunofluorescence demonstrating nuclear localization of transfectred TLE1 constructs is shown in Supplementary Fig S3.

Figure 4. TLE_120-156 tetramer but not the dimer binds to chromatin

A Binding between fluorescently labeled tri-nucleosomes and TLE1_20–156 tetramer (square), or TLE_20–156 dimer (open circles). The FRET signal is plotted as a function of Tri-nucleosome concentration. Error bars represent standard deviation of triplicate measurements. B Electrostatic surface representation of the TLE1_20–156 tetramerization domain, contoured between −15 k_BT and +15 k_BT. The regions shown in blue are positively charged and the regions in red are negatively charged. C, D TLE1_20–156 interaction with histone tails. TLE1_20–156 tetramer (C) or dimer (D) were incubated with the increasing concentration of biotinylated histone tails for 16 h at 4°C. The complexes were run on a native gel and probed with NeutrAvidin-800. The highly basic histone tails enter the gel only if bound to the TLE fragment. Gel bands were quantified using ImageJ and used to determine apparent K_D values. The H4-hyperacetylated tails are labeled as H4Ac and the H4K20-trimethylated tails are represented as H4K20Me3.

Figure 5. Model for TLE1 in Wnt signaling

See text for detailed description.