O-GlcNAc transferase is critical for transducin-like enhancer of split (TLE)-mediated repression of canonical Wnt signaling

Abstract

The Drosophila Groucho protein and its mammalian orthologues the transducin-like enhancers of split (TLEs) are critical transcriptional corepressors that repress Wnt and other signaling pathways. Although it is known that Groucho/TLEs are recruited to target genes by pathway-specific transcription factors, molecular events after the corepressor recruitment are largely unclear. We report that association of TLEs with O-GlcNAc transferase, an enzyme that catalyzes posttranslational modification of proteins by O-linked N-acetylglucosamine, is essential for TLE-mediated transcriptional repression. Removal of O-GlcNAc from Wnt-responsive gene promoters is critical for gene activation from Wnt-responsive promoters. Thus, these studies identify a molecular mechanism by which Groucho/TLEs repress gene transcription and provide a model whereby O-GlcNAc may control distinct intracellular signaling pathways.

Keywords: Gene Silencing; Groucho/TLE; O-GlcNAc; OGT; Signal Transduction; Transcription Repressor; Wnt Pathway.

FIGURE 1.

Physical interaction between OGT and TLEs. A, coimmunoprecipitation analysis. COS-7 cells were transfected with HA-tagged OGT or His-tagged TLE1 or -2 expression vectors and immunoprecipitated with α-HA antibody, followed by immunoblotting with α-HA and α-His antibodies. B–E, mapping interaction domains in OGT and TLEs. TLE proteins and their deletion mutants were synthesized in reticulocyte lysate and were incubated with bacterially produced full-length OGT and deletion mutants. F, schematic representation of primary structures of TLE and OGT and the identified interactions. CcN, a region containing phosphorylation sites for Cdc2 and casein kinase 2; SP, serine/proline-rich domain; WD, WD40-repeat domains in tandem.

FIGURE 2.

OGT mediates transcriptional repression by TLEs. A, TLE can function independent of HDACs. HepG2 cells were transfected with a luciferase reporter with Gal4 DNA-binding sites and the indicated plasmids and were treated with 500 nm TSA. B, OGT acts via the Q or WD domains of TLE to repress transcription. HepG2 cells were transfected with a luciferase reporter with Gal4 DNA-binding sites and the indicated plasmids in the presence of 500 nm TSA. C, OGT overexpression potentiates TLE repression. Cells were transfected with the luciferase reporter and increasing amounts of the Gal4-TLE1/2 plasmids in the absence or presence of the OGT plasmid. D, TLE repression requires OGT catalytic activity. A catalytically dead OGT mutant (OGTcd) was compared with wild-type OGT (OGTwt) in a luciferase assay. Error bars, S.E.

FIGURE 3.

In vivo association of OGT with TLE at the TCF/LEF locus in the absence of Wnt activation. A and B, OGT associates with TLE1/2 in the absence of Wnt in 293 cells. C and D, OGT associates with LEF and TLEs in the absence of Wnt in 293 cells. In the presence of Wnt, activated β-catenin (β-catenin*) associates with TCF/LEF. E, ChIP demonstrates the association of OGT, TLE1/2, and LEF at TCF binding sites on Wnt-responsive promoters (cyclin D1 and c-MYC) in the absence of Wnt1 conditioned media in 293 cells. In the presence of Wnt conditioned media, activated β-catenin (B-cat*) and LEF are associated with the promoters. As a control, OGT, TLE1, and TLE2 were constitutively bound to the COl2A1 gene. IP, immunoprecipitation. Error bars, S.E.

FIGURE 4.

Gene repression in the Wnt pathway requires O-GlcNAc. A, Western blot analysis of knockdown of OGT and OGA in HEK293 cells using pSUPER expressing short hairpin RNA sequences listed in Table 2 and corresponding changes in global O-GlcNAc levels. B, Western blot analysis of Wnt-responsive genes in the absence and presence of Wnt1 conditioned media. C, knocking down OGT expression by shRNA significantly increases Wnt-responsive promoter activity. The pSUPER vector expressing shRNA against OGT was transfected into HEK293 cells, together with a Wnt-responsive luciferase reporter (TOPFLASH) or a non-responsive promoter (FOPFLASH). For a control, pSUPER encoding a shRNA against EGFP sequence was used. The β-galactosidase assay was used as a control for transfection efficiency. D, OGT shRNA elevates expression of endogenous Wnt target genes in the presence of Wnt. HEK293 cells were transfected with OGT shRNA or control shRNA. E, shRNA against OGA prevents activation by Wnt using the TOP/FOPFLASH reporter system. F, shRNA knockdown of OGT enhances and shRNA knockdown of OGA prevents expression of Wnt target genes analyzed by Western blot. G–L, shRNA knockdown of OGT enhances and shRNA knockdown of OGA prevents expression of Wnt target genes analyzed by quantitative real-time RT-PCR. Gene expression was normalized to GAPDH. Error bars, S.E.

FIGURE 5.

AES relieves TLE repression by inhibiting TLE-OGT interactions. A, AES competes with OGT for TLE1 binding. TLE1 and increasing amounts of AES were co-incubated with GST-OGT fusion protein. B and C, GST pull-down analyses revealed the interaction of AES with TLE1 but not OGT. D, AES specifically relieves Q domain-mediated repression. Cells were transfected with the plasmids for Gal4-TLE1 or its deletion mutants, together with OGT and AES either individually or in combination. Error bars, S.E.

FIGURE 6.

Proposed model representing the role of OGT at the TCF/LEF locus. OGT contributes to transcriptional repression through interaction with Groucho/TLEs. Human AES may displace OGT to evoke derepression. An activating signal is necessary for binding of β-catenin to LEF, and the removal of O-GlcNAc residues from transcriptional coactivators and other proteins involved in activation is essential for activation of the canonical Wnt locus.