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• W2034932027 abstract "Article4 March 2014free access Molecular functions of the TLE tetramerization domain in Wnt target gene repression Jayanth V Chodaparambil Jayanth V Chodaparambil Departments of Structural Biology and Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Kira T Pate Kira T Pate Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA Search for more papers by this author Margretta R D Hepler Margretta R D Hepler Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Becky P Tsai Becky P Tsai Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA Search for more papers by this author Uma M Muthurajan Uma M Muthurajan Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Karolin Luger Karolin Luger Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Marian L Waterman Marian L Waterman Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA Search for more papers by this author William I Weis Corresponding Author William I Weis Departments of Structural Biology and Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Jayanth V Chodaparambil Jayanth V Chodaparambil Departments of Structural Biology and Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Kira T Pate Kira T Pate Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA Search for more papers by this author Margretta R D Hepler Margretta R D Hepler Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Becky P Tsai Becky P Tsai Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA Search for more papers by this author Uma M Muthurajan Uma M Muthurajan Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Karolin Luger Karolin Luger Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA Search for more papers by this author Marian L Waterman Marian L Waterman Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA Search for more papers by this author William I Weis Corresponding Author William I Weis Departments of Structural Biology and Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA Search for more papers by this author Author Information Jayanth V Chodaparambil1, Kira T Pate2, Margretta R D Hepler3, Becky P Tsai2, Uma M Muthurajan3, Karolin Luger3, Marian L Waterman2 and William I Weis 1 1Departments of Structural Biology and Molecular & Cellular Physiology, Stanford University School of Medicine, Stanford, CA, USA 2Department of Microbiology and Molecular Genetics, University of California Irvine, Irvine, CA, USA 3Department of Biochemistry and Molecular Biology, Colorado State University, Fort Collins, CO, USA *Corresponding author. Tel: +1 650 725 4623; Fax: +1 650 723 8464; E-mail: [email protected] The EMBO Journal (2014)33:719-731https://doi.org/10.1002/embj.201387188 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info 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. Synopsis Bill Weis and colleagues present an unusual tetramer fold for the N-terminal domain of the Groucho/TLE1 repressor. Their functional results propose chromatin binding, rather than competition for TCF as mechanism for Wnt-target gene repression. The N-terminal tetramerization domain of the transcriptional co-repressor TLE1 forms an extended, interdigitated dimer of dimers TLE1 binds the repressive TCF3 and TCF4 proteins more strongly than the activating TCF1 and LEF1 proteins There is no direct competition between TLE and ß-catenin for TCF/LEF binding, suggesting that other factors mediate the switch between repression and activation of Wnt target genes The TLE N-terminal domain can bind chromatin through its interaction with K20 methylated H4 tails. Introduction Wnt growth factors regulate cell fate determination, patterning during embryonic development and tissue renewal in adults (Logan & Nusse, 2004; Polakis, 2007). In the Wnt/β-catenin pathway, binding of a Wnt protein to cell surface receptors inhibits destruction of the transcriptional co-activator β-catenin. The stabilized β-catenin translocates to the nucleus where it interacts with sequence-specific DNA binding TCF/LEF (T-Cell Factor/Lymphoid Enhancer Factor) proteins at Wnt-responsive elements in the promoter region of target genes. β-Catenin acts as a scaffold for the binding of chromatin remodeling complexes and general transcription activator proteins, including the histone acetyltransferase CBP/p300 (Hecht et al, 2000; Takemaru & Moon, 2000) and thereby activates target genes. In the absence of Wnt and nuclear β-catenin, TCF/LEF proteins interact with transcriptional repressors of the Groucho (Gro/Grg)/Transducin-like enhancer of split (TLE) family (Arce et al, 2009; Cadigan & Waterman, 2012). TLE proteins regulate transcription in several signaling pathways including Ras, Notch, Wingless and Decapentaplegic (Hasson & Paroush, 2007; Turki-Judeh & Courey, 2012). TLE proteins interact with histone deacetylases (HDACs) (Chen et al, 1999; Arce et al, 2009), whose activity is important in generating transcriptionally silent chromatin. TLE proteins also bind to deacetylated histone tails (Palaparti et al, 1997; Flores-Saaib & Courey, 2000). Grg3, a mouse homolog of TLE1 can bind, condense and oligomerize chromatin (Sekiya & Zaret, 2007). TLE proteins contain a conserved N-terminal glutamine-rich (Q) domain, followed by a variable central domain sub-divided into a glycine-proline rich (GP) region, the CcN and serine-proline rich (SP) regions, and a C-terminal WD40 domain that interacts with multiple transcription factors (Buscarlet & Stifani, 2007) (Fig 1A). The Q domain mediates tetramerization of TLEs (Chen et al, 1998) and binding to TCF/LEF proteins (Brantjes et al, 2001), and is essential for chromatin oligomerization (Sekiya & Zaret, 2007). In Groucho, the Q domain can also form higher oligomers, with the tetramer as the predominant species (Kuo et al, 2011). The GP region binds directly to HDAC1 (Chen et al, 1999; Billin et al, 2000; Brantjes et al, 2001). The CcN region has consensus CK2 and Cdc2 phosphorylation sites that are modified in a cell cycle-dependent manner (Nuthall et al, 2002). The SP region is a substrate for the Ser/Thr kinases HIPK2 and MAPK, which appear to negatively regulate transcription repression activity (Choi et al, 2005; Hasson et al, 2005), and may also interact with basic helix-loop-helix transcription factors (Fisher et al, 1996; Jimenez et al, 1997). The C-terminal WD40 domain is essential for chromatin condensation (Sekiya & Zaret, 2007) and together with SP interacts with bHLH proteins. Figure 1. Structure of the TLE1 Q domain The primary structure of human TLE1 showing domain boundaries. 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. 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. Glutamine residues (shown in stick representation) are mostly clustered on helices α2 and α3. Close-up of the TLE1-Q tetramer interface. SAXS analysis of TLE120–156. The solid black line shows the experimentally determined scattering curve of TLE120–156 at 5 mg/ml. The Rg obtained from the Guinier plot = 70 Å, and Dmax = 227 Å as calculated with Autognom (Semenyuk & Svergun, 1991). These values match closely the Rg = 69 Å and Dmax = 217 Å calculated from the extended tetramer model (green line; χ2 = 3.1) versus Rg = 35 Å and Dmax = 162 Å for the compact tetramer (red line; χ2 = 6.9; see Supplementary Fig S1B). Gel filtration of the TLE120–156 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. Download figure Download PowerPoint TCF/LEF proteins contain an N-terminal β-catenin binding sequence, followed by a Context-dependent Regulatory Domain (CRD) and a High Mobility Group (HMG) domain that binds DNA (Fig 2A). Although all TCF/LEFs interact with β-catenin and TLE co-repressors, gene knockout studies in vertebrate model systems have shown that TCF/LEF family members function differently from one another depending on the developmental context. Particular TCF/LEFs act predominantly as either repressors or activators in developing Xenopus embryos (Kim et al, 2000; Liu et al, 2005; Standley et al, 2006; Hikasa et al, 2010), mouse embryonic stem cells (Merrill et al, 2004; Tam et al, 2008; Hikasa et al, 2010; Yi et al, 2011), stem cells in embryonic skin (Nguyen et al, 2009), hair follicles and intestine (van de Wetering et al, 1997; Korinek et al, 1998; Roose et al, 1999), and in cancer cells (Tang et al, 2008). The most common trends are for TCF3 and TCF4 to serve as repressors, whereas TCF1 and LEF1 perform as activators. Differences in activating and repressing roles amongst TCF/LEFs map to the CRD of each protein in both genetic (Liu et al, 2005) and cell culture systems (Cadigan & Waterman, 2012), and deletion of the CRD eliminates repressive functions and increases transcription of target genes (Pereira et al, 2006; Tam et al, 2008; Nguyen et al, 2009). A study of Wnt-mediated Xenopus axis specification found that the repressor TCF3 is replaced by TCF1/β-catenin (Hikasa et al, 2010), with the switch between TCF3 and TCF1 apparently mediated by β-catenin scaffolded HIPK2 phosphorylation of residues in the TCF3 CRD. Taken together, these studies define different roles for individual TCF/LEFs in gene regulation and indicate a key role for the CRD in defining those differences. Figure 2. Reporter activity of TCF/LEF proteins correlates with affinity for TLE1 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. 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. Fluorescence anisotropy analysis of TLE120–156 binding to different TCF/LEFs. Increasing amounts of TCF/LEF ligands were titrated against labeled TLE120–156. The change in anisotropy was measured and plotted as a function of concentration. Error bars represent standard deviation of triplicate measurements. Download figure Download PowerPoint In this work, we address the molecular basis for the different roles of TCF/LEFs in gene repression and activation in Wnt signaling. The TCF/LEF CRD is important for repression and is the most highly divergent region of TCF/LEFs. The TLE Q domain binds to the TCF/LEF CRD and is essential for transcriptional repression (Brantjes et al, 2001). However, it is unclear whether TLEs interact equivalently with the different TCF/LEFs, and it is not known how the Q domain can be involved simultaneously with tetramerization and TCF/LEF binding, and chromatin oligomerization. We report the three-dimensional structure of the TLE1 Q domain and an analysis of its binding interactions. We find that different affinities for the four TCF/LEF proteins correlate directly with their relative repression activities. We show that there is no competition between β-catenin and TLE1 for TCF3 binding, and we demonstrate that the Q domain tetramer binds directly to chromatin through hypoacetylated and H4K20 tri-methylated histone H4 tails. These findings lead to a new model of the switch between repression and activation of Wnt target genes. Results The TLE Q domain is an interdigitated dimer of dimers A construct spanning residues 20–156 of human TLE1 (Fig 1A), which includes the N-terminal Q domain and a portion of the neighboring GP region, was purified and crystallized, and the structure determined at 2.9 Å resolution (Supplementary Table S1, Supplementary Fig S1A). The asymmetric unit of the crystal contains four protomers comprising the Q domain tetramer (Supplementary Fig S1B). Each TLE20-156 protomer consists of a 70-amino-acid α helix (α1) followed by two short helices (α2 and α3) (Fig 1B). The long helices of two protomers associate to form an extensive parallel coiled-coil dimer, mediated by hydrophobic residues at canonical heptad a and d positions between residues 24–94 (Fig 1B and C). The dimer is further stabilized by interactions of α2 and α3 with the partner protomer (Fig 1C). Specifically, α2 (residues 102–111) packs against the C-terminal portion of the partner α1. A small non-helical stretch leads into α3 (115–134), which is bent approximately 120° relative to α2. This orientation is fixed by a salt bridge between E118 of α3 and R84 in the partner α1, as well as hydrophobic contacts between the N-terminal portion of α3 and α1 (Fig 1C). A total of 5,520 Å2 of surface area is buried in the dimer interface. The glutamine residues that give rise to the name of this domain cluster on the surfaces of α2 and α3 (Fig 1D). The Q domain tetramer forms by antiparallel association of the N-terminal portions of the dimer helices (residues 21–36) with the equivalent residues of another dimer, which produces a 212-Å elongated dimer of dimers (Fig 1B and E). The dimer-dimer interaction is mediated by hydrophobic side chains and buries 3,320 Å2 of surface area. We confirmed that this unusual N-terminally interdigitated dimer of dimers is the solution structure by small-angle X-ray scattering (Fig 1F). This architecture is also consistent with previous biochemical characterization that revealed a frictional coefficient ratio f/f0 > 2 for the tetramer (Kuo et al, 2011). The residues that mediate the hydrophobic interactions in the tetramer interface are highly conserved amongst the TLE/Gro family (Supplementary Fig S1C), suggesting that this mode of tetramerization is an intrinsic and ancient feature. To eliminate the possibility the observed mode of association arises from removal of the first 19 TLE1 residues, we refined the structure against a 4-Å data set measured from virtually isomorphous crystals of TLE1–156. This structure showed that the first 20 residues of the Q domain are unstructured and not involved in tetramer formation (Supplementary Fig S1A). The elongated tetramer model was tested further by selectively destabilizing the tetramer interface. Leu26 and Ile29, which interact in the interface, were both changed to aspartate. The L26D/I29D mutant eluted from a size-exclusion chromatography column at a reduced apparent molecular weight corresponding to a prolate dimer relative to the wild-type tetramer (Fig 1G), and we therefore desginate this mutant the TLE120–156 dimer. The strong conservation of the Q domain tetramerization interface (Supplementary Fig S1C) suggests that heterotetramers of different family members might form to produce graded levels of repression. Heterotetramer formation could underlie the ability of AES/Grg5, a C-terminally truncated family member, to antagonize TLE repressive activity (Beagle & Johnson, 2010). TLE120-156 binds to TCF3 and TCF4 but not to LEF1 and TCF1 Invertebrates possess a single TCF (e.g., Drosophila Pangolin) that recruits Groucho for repressor activities, or Armadillo (β-catenin) for target gene activation (van de Wetering et al, 1997). In contrast, vertebrates have four family members, and as described above, TCF3 and TCF4 are often associated with repressive functions, whereas TCF1 and LEF1 are more associated with target gene activation (Kim et al, 2000; Merrill et al, 2004; Liu et al, 2005; Tang et al, 2008; Nguyen et al, 2009). This segregation of duties is not absolute, as all family members can bind to β-catenin for activation of transcription. To compare directly their potential for transcription activation, we assessed the relative activities of TCF1, LEF1, TCF3 or TCF4 with co-expressed β-catenin in a standard TOPflash luciferase reporter assay in two different cell lines. We observed that TCF1 and LEF1 directed the highest reporter activity, much more than TCF3 and TCF4, even though they are equivalently expressed (Fig 2B). These results extend similar findings in HEK293 cells (Brantjes et al, 2001), and indicate that there are inherent differences in the ability of TCF/LEFs to mediate transcription activation. β-catenin binds with similar high affinities (~20 nM KD) to the β-catenin binding domain of each TCF/LEF (Knapp et al, 2001; Sun & Weis, 2011), so we hypothesized that the differences in TOPflash activation reflect differences in their binding affinity for endogenous TLE repressor proteins. We used fluorescence anisotropy of labeled TLE120–156 to measure its affinity for purified mLEF1, hTCF1, xTCF3 and hTCF4 constructs lacking their HMG domains, a separate region that does not contribute to TLE interactions (Arce et al, 2009). TLE120-156 binds to TCF3 and TCF4 with KDs of 16 μm and 34 μM respectively, whereas it binds LEF1 and TCF1 with KDs of 195 μM and 1 mM (Fig 2C, Table 1). Although interactions of other regions in TCFs undoubtedly have a role in determining activating versus repressive activity, these results demonstrate that TCF3 and TCF4 can form repressive complexes with TLE1 more readily than can LEF1 and TCF1. Table 1. Binding affinities of TCF/LEFs to the TLE120–156 tetramer and L26D/I29D dimer. Tetramer Dimer Complex KD ± s.d. (μM) KD ± s.d. (μM) Binding to different TCF/LEF family members TLE1(20–156) : LEF1(1–296) 195 ± 7 N.D. TLE1(20–156) : TCF1(1–264) 1023 ± 465 N.D. TLE1(20–156) : TCF3(1–330) 16 ± 2 25 ± 5 TLE1(20–156) : TCF4(1–300) 34 ± 7 40 ± 8 Mapping the TCF3 binding site TLE1(20–156) : TCF3(1–65) N.D. TLE1(20–156) : TCF3(1–131) 33 ± 7 TLE1(20–156) : TCF3(131–330) 9 ± 1 TLE1(20–156) : TCF3(200–330) 126 ± 35 TLE1(20–156) : TCF3(∆131–162) 42 ± 6 Control binding to BSA TLE1(20-156) : Bovine Serum Albumin N.D. N.D. Standard deviation corresponds to n = 3, N.D.: not detected. β-catenin and TLE120–156 bind to distinct regions of TCF3 and do not compete for TCF3 We used TCF3 deletion constructs to define the region that binds to the TLE120–156 tetramer (Table 1). Our data indicate that the principal interaction region of TCF3 spans residues 65–200, with weaker contributions from residues between 200 and 330. This is consistent with earlier deletion studies (Daniels & Weis, 2005; Arce et al, 2009) as well as studies in Xenopus that highlighted this region in TCF/LEFs as the key difference for repression and activation roles (Liu et al, 2005; Nguyen et al, 2009). Shorter constructs designed to define the binding site more precisely proved too unstable to purify. Nonetheless, the data show the TLE120–156 interaction involves more than the region equivalent to residues 216–256 of LEF1 described previously (Daniels & Weis, 2005; Arce et al, 2009). Earlier studies indicated that β-catenin could compete directly with TLE1 Q domain for binding to LEF1, suggesting that the switch from repression to activation upon Wnt signaling is due to direct competition for TCF/LEF as the levels of nuclear β-catenin rise (Daniels & Weis, 2005). Our data confirm that the β-catenin binding domain of TCF3 (residues 1–65) does not contribute to TLE1 binding (Table 1). We also find that LEF1 binds very weakly to TLE1. As the previous study used shorter TLE protein constructs that were poorly soluble and sensitive to pH (Daniels & Weis, 2005), we retested the competition model using our well-behaved TLE120–156 fragment with repressive TCF3. Since the binding affinity is only 16 μM, we purified the β-catenin-TCF31–330 complex (KDs for β-catenin binding to the highly homologous LEF1 1–131 and TCF4 1–53 are 10 and 20 nM, respectively (Daniels & Weis, 2005; Fasolini et al, 2003; Sun & Weis, 2011)), and titrated increasing concentrations of this complex into labeled TLE120–156. The affinity of TLE120–156 for the β-catenin–TCF3 complex was 14 ± 1 μM, essentially the same as the TLE120–156-TCF3 interaction (Fig 3A). These data demonstrate that there is no competition between β-catenin and TLE120–156 and, consistent with the domain mapping data (Table 1), that the binding regions for β-catenin and TLE120–156 on TCF3 are independent of one another. Figure 3. TLE120–156 tetramer and dimer can bind TCF3 and TCF4 in vitro, but only the tetramer represses transcription in vivo Binding of TLE120–156 tetramer to the TCF3(1–330): β-catenin complex or TCF31–330 measured by fluorescence anisotropy. Error bars represent standard deviation of n = 3. Binding of TLE120–156 dimer mutant to and TCF31–330 and TCF41–330. Error bars represent standard deviation of n = 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. 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. Download figure Download PowerPoint The finding that TCF3–TLE1 complexes are exchanged for TCF1–β-catenin complexes at a Wnt target gene promoter during Xenopus development, and that HIPK2-mediated phosphorylation of sites within amino acids 131–160 of TCF3 facilitates this switch, suggested that TLE binding might require residues in this region (Hikasa et al, 2010). The sequence equivalent to TCF3 131–160 is similar in TCF4, but differs significantly from the equivalent region in TCF1 and LEF1 (Supplementary Fig S2). To test the role of this region in TLE1 binding, we made a TCF3 construct lacking residues 130–162. TLE120–156 bound to this deletion mutant with a KD of 42 μM, only slightly weaker than constructs containing this region. Thus, the non-phosphorylated 131–160 sequence contributes only modestly to TLE binding, and suggests that phosphorylation might non-specifically prevent binding by electrostatic repulsion, and/or promote binding of another factor that removes TLE from TCF. Tetramerization is essential for repression in vivo The TLE120–156 L26D/I29D mutant selectively disrupts the tetramer without destroying the dimeric building block (Fig 1G), so we tested whether the dimer can still interact with TCF/LEF proteins. TLE120–156 mutant dimer binds to TCF3 and TCF4 with the same affinity as the wild-type tetramer (Fig 3B). This demonstrates that the dimer confers full binding to TCF3 and TCF4, and it suggests that the stoichiometry of TLE-TCF interaction is unlikely to be 4:1 TLE:TCF as reported (Daniels & Weis, 2005). We next tested whether the TLE1 dimer can function as a co-repressor for transcription using a standard TOPflash assay. Co-expression of TCF3 and β-catenin caused modest repression of TOPflash even in the absence of Wnt activation (Fig 3C), presumably because TCF3 was preferentially working through endogenous TLE proteins. When wild-type TLE1 was co-expressed, strong repression was observed (Fig 3C and D), whereas co-expression of the TLE1 dimer mutant showed little repression (Fig 3D). These data indicate that tetramerization of the TLE1 Q domain has additional critical functions in repression beyond binding to TCF proteins. TLE120–156 binds directly to chromatin Grg3, a mouse homolog of TLE1, binds to, condenses and oligomerizes chromatin into a higher-order structure. The compaction/condensation activity (short-range or cis interactions between nucleosomes) depends on the C-terminal WD domain, whereas the Q domain is needed for oligomerization (long-range or trans interactions) that create higher-order, transcriptionally silent chromatin structure. However, the molecular basis of Q domain involvement in this process is not clear (Sekiya & Zaret, 2007). As Q domain tetramerization is important for transcription repression in vivo (Fig 3D) but not for TCF/LEF binding, we tested binding of wild-type TLE120–156 tetramer or the TLE120-156 dimer to tri-nucleosomes using a FRET-based assay. Trinucleosome arrays provide a minimal system to study nucleosome interactions in a chromatin context (Winkler et al, 2011). The tetramer bound to tri-nucleosomes with a KD of 423 nM, whereas the dimer did not approach saturation, preventing accurate affinity determination and indicating a weak interaction with KD > 2 μM (Fig 4A). Figure 4. TLE120–156 tetramer but not the dimer binds to chromatin A. Binding between fluorescently labeled tri-nucleosomes and TLE120–156 tetramer (square), or TLE20–156 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 TLE120–156 tetramerization domain, contoured between −15 kBT and +15 kBT. The regions shown in blue are positively charged and the regions in red are negatively charged. C, D. TLE120–156 interaction with histone tails. TLE120–156 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 KD values. The H4-hyperacetylated tails are labeled as H4Ac and the H4K20-trimethylated tails are represented as H4K20Me3. Download figure Download PowerPoint The surface electrostatic potential of the TLE1 tetramer features a positively charged region at the N-terminal tetramerization region separating strongly negatively charged surfaces that run along the parallel dimer (Fig 4B). We hypothesized that the acidic surface of the Q domain binds to highly basic histone tails. The binding of synthetic biotinylated histone tails to the TLE120–156 tetramer and dimer mutant was analyzed using native gel shifts to obtain relative binding affinities. The TLE120–156 tetramer bound to unmodified H4 and H4 trimethylated at lysine 20 (H4K20Me3; associated with heterochromatin) tails with apparent KDs of 42 and 13 μM, respectively (Fig 4C). The dimer also bound to these tails (Fig 4D). Binding to H2A, H2B and H3 was significantly weaker for both the tetramer and dimer (Fig 4C and D). The increase in affinity of H4K20Me3 versus unmodif" @default.
• W2034932027 created "2016-06-24" @default.
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• W2034932027 date "2014-03-03" @default.
• W2034932027 modified "2023-10-18" @default.
• W2034932027 title "Molecular functions of the TLE tetramerization domain in Wnt target gene repression" @default.
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