Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression

Abstract

Background: Drosophila Groucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLE's modify Wnt signaling is by direct competition with beta-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.

Methods: We used in vitro protein interaction assays with the LEF/TCF family member LEF-1, and in vivo assays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.

Results: Mapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay. Drosophila Groucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1*beta-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated beta-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.

Conclusion: Our data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with beta-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.

Figure 1

Schematic representation of the LEF-1 and LEF-1 deletion constructs, Groucho and APC. A) A LEF-1 protein schematic highlights four features: the β-catenin binding domain (βBD; aa1-67), the context dependent regulatory domain (CRD; aa67-296), an alternative exon in the CRD (black bar; aa214-241) and the high mobility group DNA binding domain (HMG; aa296-384). LEF-1 deletions tested in a GST pulldown assay are also depicted. B) General domain structure of Groucho/TLE proteins and truncated AES isoforms. The Q domain (aa1-132) is required for tetramerization and the GP domain interacts with HDACs. Three additional domains of protein-protein interaction are shown: the CcN, SP and WD-repeat domains. C) Schematic of Adenomatous polyposis coli (APC) and APC mutations found in Familial Adenomatous Polyposis (FAP) cancer cell of type I (DLD-1, SW480, and Colo 320) and type II (HT-29) and Hereditary Non-Polyposis Colon Cancer (HNPCC) colon cancer cell lines (LS174T and HCT116) [29,34]. Asterisks indicate nuclear export signals and diamonds indicate nuclear localization signals. The MCR (mutation cluster region) refers to the most common site of truncating mutations found in colon cancer. This region overlaps a region comprised of 15 and 20 amino acid repeats that bind beta-catenin. [28,48].

Figure 2

Groucho binds to LEF-1 via a motif in the CRD and the HMG DNA binding domain. A) Glutathione beads bound with either GST-Groucho (middle panel) or GST (bottom panel) were tested for interaction with in vitro translated ³⁵S LEF-1 deletion constructs (translations shown in the top panel refer to deletions depicted in Fig. 1). B) Additional LEF-1 N-terminal deletions 1–234 and 1–191 (lanes 2 and 3) were not able to interact with GST-Groucho. Again, LEF-1 1–256 (lane 1) strongly interacts with Groucho but a deletion mutant lacking the entire CRD but retaining the HMG (Δ110–295, lane 4) weakly interacts. A GST negative control pull-down shows that LEF-1 does not interact with GST or the beads. Coomassie stained SDS-PAGE gel of purified GST-Groucho (lane 1) or GST alone (lane 2). C) Amino acid alignment of the putative Groucho binding sequence (GBS) and flanking sequences in LEF-1 and other human LEF/TCF family members as well as D. melanogaster and C. elegans worm orthologs, pangolin and POP-1 respectively. The LEF/TCF Groucho binding sequence described in this study, FS/T xxx I/L/V, is similar to the eh1 motif FxIxxIL [20]

Figure 3

Groucho and AES repress LEF-1/β-catenin mediated activation of transcription. Luciferase activity of a transiently transfected TOPFlash reporter assay using COS-1 cells. The TOPFlash (100 ng) reporter with three multimerized LEF/TCF DNA consensus sequences in front of the c-fos minimal promoter was activated 5 and 3-fold by LEF-1•β-catenin complexes (200 and 400 ng of expression plasmid respectively). This level of activation was set equal to one for comparison to the amount of TOPflash activity measured in the presence of Groucho or AES expression vector (250 and 500 ng as shown). Increasing amounts of Groucho or AES expression vector cause increasing repression of reporter activity. All data was normalized with a co-transfected CMV β-galactosidase reporter. Error bars represent the standard deviation among three experiments. Student t test was performed and p values are represented by asterisks. One asterisk represents a value < 0.05 and two asterisks represent a value of < 0.01. Western analysis of Groucho, AES and TLE-2 expression shows a dose-dependent increase in protein expression (insets).

Figure 4

The Groucho Binding Motif in LEF-1 is necessary for repression. Luciferase activity in transiently transfected COS-1 cells. The TOPFlash or Super TOPFlash reporters were co-transfected with the indicated LEF-1 deletion constructs (200 ng) and β-catenin (400 ng) with increasing amounts of Groucho expression vector (indicated). Repression is maintained when activated by LEF-1 lacking the alternative exon (Δ214–241; left panel). Repression is also maintained when residues next to the GBM are changed (250/255). Repression is attenuated when all eight residues of the GBM are altered (right panel, center). Error bars represent the spread between duplicates and is representative of more than four replicate experiments.

Figure 5

Groucho and AES recruit HDAC for transcription repression. LEF-1•β-catenin activated TOPFlash was repressed by Groucho or AES (1000 ng) via transient transfection in COS-1 cells. Treatment with the HDAC inhibitor trichostatin A (50 ng/ml) relieved repression. Data is reported as described in Figure 3. Three independent experiments are shown. The error bars represent the spread of the duplicates for each experiment.

Figure 6

Groucho action in colon carcinoma derived cells. A) Western analysis of endogenous TLE expression in colon cancer cell lines. Whole cell extracts from the indicated colon cancer cell lines were analyzed by western blotting. Blots were probed with rat monoclonal antisera with pan specificity for the TLE family of proteins. Longer exposure of the blot reveals low levels of TLE protein in HCT-116 cell line (data not shown). B) Groucho activity was assessed in colon cancer cell lines. SW480, Colo320 and DLD-1 cells are type I APC mutant cells (left panels). LS174T, HCT116 and HT-29 cells are wild type APC or type II APC mutant cells (right panels). Refer to Figure 1 for the corresponding APC mutations in these cells. TOPFlash reporter plasmid was co-transfected with either a dominant negative TCF-1 expression vector (400 ng) or increasing amounts of Groucho expression vector. Luciferase activity was assayed 24 hours post transfection and activities normalized to TOPFlash reporter alone. The error bars show the standard deviation derived from three independent experiments.