All Tcf HMG box transcription factors interact with Groucho-related co-repressors

Abstract

Tcf/Lef family transcription factors are the downstream effectors of the Wingless/Wnt signal transduction pathway. Upon Wingless/Wnt signalling, beta-catenin translocates to the nucleus, interacts with Tcf (1-3) and thus activates transcription of target genes (4,5). Tcf factors also interact with members of the Groucho (Grg/TLE) family of transcriptional co-repressors (6). We have now tested all known mammalian Groucho family members for their ability to interact specifically with individual Tcf/Lef family members. Transcriptional activation by any Tcf could be repressed by Grg-1, Grg-2/TLE-2, Grg-3 and Grg-4 in a reporter assay. Specific interactions between Tcf and Grg proteins may be achieved in vivo by tissue- or cell type-limited expression. To address this, we determined the expression of all Tcf and Grg/TLE family members in a panel of cell lines. Within any cell line, several Tcfs and TLEs are co-expressed. Thus, redundancy in Tcf/Grg interactions appears to be the rule. The 'long' Groucho family members containing five domains are repressors of Tcf-mediated transactivation, whereas Grg-5, which only contains the first two domains, acts as a de-repressor. As previously shown for Drosophila Groucho, we show that long Grg proteins interact with histone deacetylase-1. Although Grg-5 contains the GP homology domain that mediates HDAC binding in long Grg proteins, Grg-5 fails to bind this co-repressor, explaining how it can de-repress transcription.

Figure 1

Domain structure of (A) Tcf and (B) Grg constructs. Tcf proteins contain a centrally located DNA-binding HMG box and the N-terminal β-catenin interaction domain. As previously reported (6), the Grg interaction domain is located between these two domains. The Grg proteins 1–4 contain five distinct domains: Q, GP, CCN, SP and WD40 regions (51), while the Grg-5 protein consists only of the Q and GP domains.

Figure 2

Mapping of the Grg–Tcf interaction domain and Grg multimerisation domain. The different bait Grg-5 constructs used in the yeast two-hybrid assay are shown on the left. The ability of these constructs to interact with prey molecules Tcf-1 (4-359) or Grg-5 (1-197) and allow growth of yeast on selective plates is indicated. The minimal domain of Grg-5 required for interaction with Tcf resides in the first 106 amino acids. Further truncation of this domain results in loss of interaction.

Figure 3

All ‘long’ Grg proteins can repress transactivation mediated by all Tcf family members. Mouse B cell line IIAI.6 was co-transfected with the Tcf reporter construct (containing either wild-type Tcf binding sites, TOP-TK, or mutated Tcf binding sites, FOP-TK, as a negative control) and a specific Tcf family member and β-catenin. This results in transactivation and increase of luciferase activity. Upon addition of increasing amounts of any of the Grg family members, the luciferase activity decreases proportionally. (A) Repression of Tcf-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (B) Repression of Lef-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (C) Repression of Tcf-3-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (D) Repression of Tcf-4-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. Transfections were performed in duplicate, and results from one representative independent experiment are depicted in each case. Luciferase values were corrected for the efficiency of transfection using the internal Renilla transfection control pRNL-TK by determining the luciferase/Renilla ratio. This ratio is given on the y-axis, and was arbitrarily set at 1 for the sample in which the TOP-TK reporter construct alone was transfected.

Figure 3

All ‘long’ Grg proteins can repress transactivation mediated by all Tcf family members. Mouse B cell line IIAI.6 was co-transfected with the Tcf reporter construct (containing either wild-type Tcf binding sites, TOP-TK, or mutated Tcf binding sites, FOP-TK, as a negative control) and a specific Tcf family member and β-catenin. This results in transactivation and increase of luciferase activity. Upon addition of increasing amounts of any of the Grg family members, the luciferase activity decreases proportionally. (A) Repression of Tcf-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (B) Repression of Lef-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (C) Repression of Tcf-3-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (D) Repression of Tcf-4-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. Transfections were performed in duplicate, and results from one representative independent experiment are depicted in each case. Luciferase values were corrected for the efficiency of transfection using the internal Renilla transfection control pRNL-TK by determining the luciferase/Renilla ratio. This ratio is given on the y-axis, and was arbitrarily set at 1 for the sample in which the TOP-TK reporter construct alone was transfected.

Figure 3

All ‘long’ Grg proteins can repress transactivation mediated by all Tcf family members. Mouse B cell line IIAI.6 was co-transfected with the Tcf reporter construct (containing either wild-type Tcf binding sites, TOP-TK, or mutated Tcf binding sites, FOP-TK, as a negative control) and a specific Tcf family member and β-catenin. This results in transactivation and increase of luciferase activity. Upon addition of increasing amounts of any of the Grg family members, the luciferase activity decreases proportionally. (A) Repression of Tcf-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (B) Repression of Lef-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (C) Repression of Tcf-3-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (D) Repression of Tcf-4-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. Transfections were performed in duplicate, and results from one representative independent experiment are depicted in each case. Luciferase values were corrected for the efficiency of transfection using the internal Renilla transfection control pRNL-TK by determining the luciferase/Renilla ratio. This ratio is given on the y-axis, and was arbitrarily set at 1 for the sample in which the TOP-TK reporter construct alone was transfected.

Figure 3

All ‘long’ Grg proteins can repress transactivation mediated by all Tcf family members. Mouse B cell line IIAI.6 was co-transfected with the Tcf reporter construct (containing either wild-type Tcf binding sites, TOP-TK, or mutated Tcf binding sites, FOP-TK, as a negative control) and a specific Tcf family member and β-catenin. This results in transactivation and increase of luciferase activity. Upon addition of increasing amounts of any of the Grg family members, the luciferase activity decreases proportionally. (A) Repression of Tcf-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (B) Repression of Lef-1-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (C) Repression of Tcf-3-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. (D) Repression of Tcf-4-mediated transcriptional activation by Grg-1, TLE-2, Grg-3 and Grg-4. Transfections were performed in duplicate, and results from one representative independent experiment are depicted in each case. Luciferase values were corrected for the efficiency of transfection using the internal Renilla transfection control pRNL-TK by determining the luciferase/Renilla ratio. This ratio is given on the y-axis, and was arbitrarily set at 1 for the sample in which the TOP-TK reporter construct alone was transfected.

Figure 4

Expression of (A) hTCF-1, hLEF-1, hTCF-3, hTCF-4; and (B) TLE-1, TLE-2, TLE-3, TLE-4 and hAES was determined by RT–PCR using cDNA generated from a panel of cell lines. Reh (lane 1), Raji (lane 2), CEM (lane 3), Jurkat (lane 4), 293T (lane 5), HCT116 (lane 6), LS174T (lane 7), HT29 (lane 8), DLD-1 (lane 9), SW480 (lane 10), SW620 (lane 11), RKO (lane 12), T47D (lane 13), SKBR3 (lane 14) and H₂O control for the PCR (lane 15). The lane marked λ depicts the DNA marker (bacteriophage λ DNA digested with EcoRI and HindIII). Expression is tabulated for Tcfs and TLEs for the given cell lines. +, visible RT–PCR product; –, no visible RT–PCR product.

Figure 5

(A) Structure of the truncated XGrg-4-QGP molecule compared to that of Grg-5. (B) Alignment of the GP domains of long Grg homologues with mGrg-5. The amino acid residues that are unique for mGrg-5 when compared to Gmgrg-1, Mgrg-3 and XGrg-4 are depicted in bold. (C) XGrg-4-QGP functions as a repressor, but mGrg-5 functions as a de-repressor for Tcf-mediated transcriptional activation. Mouse B cell line IIAI.6 was co-transfected with the Tcf reporter construct (TOP-TK) and XTcf-3 and Armadillo (the Drosophila β-catenin homologue). This results in transactivation and increase of luciferase activity. This transactivation is de-repressed when Grg-5 is co-transfected, whereas the transactivation is repressed upon co-transfection with a truncation of XGrg-4 that only contains the Q and GP domains. Transfections were performed in duplicate, and luciferase values were corrected for the efficiency of transfection using the internal Renilla transfection control pRNL-TK by determining the luciferase/Renilla ratio. This ratio is given on the y-axis.

Figure 6

HDAC-1 associates with XGrg-4-QGP, but not with mGrg-5. 293T cells were transfected with either Myc-tagged XGrg-4 QGP or Myc-tagged Grg-5 alone (lanes 1 and 2), or with a Flag-tagged HDAC-1 (lanes 3 and 4). Cell lysates were prepared 24 h after transfection. (A) Extracts from transfected cells were immunoprecipitated (IP) with an anti-Myc antibody against Myc-XGrg-4-QGP and Myc-Grg-5, and the presence of Flag-HDAC-1 in the immunoprecipitate was assayed on immunoblot (IB) with anti-Flag antibody. In lanes 1 and 2, only Myc-tagged XGrg-4-QGP or Myc-Grg-5 were transfected. In lanes 3 and 4, Myc-tagged XGrg-4-QGP or Myc-Grg-5 were co-transfected with Flag-tagged HDAC-1. Lane 5 shows the presence of HDAC-1 in the co-transfection of Myc-Grg-5 and Flag-HDAC-1. Lanes 6 and 7 show an immunoblot with anti-Myc antibody, indicating the expression of the Myc-Grg-4-QGP and Myc-Grg-5 when co-transfected with Flag-HDAC-1. (B) Extracts from transfected cells were immunoprecipitated (IP) with an anti-Flag antibody against Flag-HDAC-1. The presence of Myc-XGrg-4-QGP and Myc-Grg-5 was assayed on immunoblot (IB) with anti-Myc antibody. In lanes 1 and 2 only Myc-tagged XGrg-4-QGP or Myc-Grg-5 were transfected. In lanes 3 and 4, Myc-tagged XGrg-4-QGP or Myc-Grg-5 were co-transfected with Flag-tagged HDAC-1. Lanes 5 and 6 show an immunoblot with anti-Myc antibody, indicating the expression of Myc-Grg-4-QGP and Myc-Grg-5 when co-transfected with Flag-HDAC-1. Bands corresponding to HDAC, Grg-5 and XGrg-4 QGP are indicated with arrows. Bands labelled ‘Ig’ indicate the heavy and light chains of the antibodies used for the immune precipitation.

Figure 7

Model for Tcf, long Grg and Grg-5 functions. Expression of Tcf target genes is activated when Tcf is associated with β-catenin and repressed when Tcf binds Groucho. Here we show that the function of Grg-5 provides another level of regulation. When in a complex, both the long repressor Grg and Grg-5 are bound to Tcf, a less competent repressor is formed, since less HDAC activity is tethered to the promotor. When Grg-5 replaces the longer repressor entirely from the complex with Tcf, repression is absent because HDAC activity is lacking.