HIC1 attenuates Wnt signaling by recruitment of TCF-4 and beta-catenin to the nuclear bodies

Abstract

The hypermethylated in cancer 1 (HIC1) gene is epigenetically inactivated in cancer, and in addition, the haploinsufficiency of HIC1 is linked to the development of human Miller-Dieker syndrome. HIC1 encodes a zinc-finger transcription factor that acts as a transcriptional repressor. Additionally, the HIC1 protein oligomerizes via the N-terminal BTB/POZ domain and forms discrete nuclear structures known as HIC1 bodies. Here, we provide evidence that HIC1 antagonizes the TCF/beta-catenin-mediated transcription in Wnt-stimulated cells. This appears to be due to the ability of HIC1 to associate with TCF-4 and to recruit TCF-4 and beta-catenin to the HIC1 bodies. As a result of the recruitment, both proteins are prevented from association with the TCF-binding elements of the Wnt-responsive genes. These data indicate that the intracellular amounts of HIC1 protein can modulate the level of the transcriptional stimulation of the genes regulated by canonical Wnt/beta-catenin signaling.

Figure 1

HIC1 targets CtBP into the nuclear bodies. (A) A schematic representation of the human HIC1 constructs used in this study. tag, Flag or EGFP tag; BTB/POZ, the BTB/POZ domain; CtBP, CtBP-binding site; ZF, five C₂H₂ Krüppel-like zinc fingers. Right, Western blots of total cell extracts after transfection with the Flag-HIC1 constructs, probed with anti-Flag or with anti-α-tubulin as internal control. (B) Confocal microscopy images of CtBP(−/−) cells trasfected with constructs indicated on the left and subsequently stained with mouse anti-Flag and rabbit anti-CtBP antibody. The DRAQ5 nuclear stain was gained in the blue channel. Mutant HIC1-ΔCtBP polypeptide lacking the CtBP-interacting motif displays the punctuated expression of the wild-type HIC1 protein (compare (e) and (i)) but does not influence the distribution of CtBP (compare (b) and (j)). As seen in (j, k), a fraction of nuclear CtBP is still localized in the CtBP bodies (arrows in insets (c, k)), which evidently differ from the HIC1 bodies. Bar, 10 μm.

Figure 2

TCF-4 and HIC1 form nuclear protein complexes in mammalian cells. (A) TCF-4 expression constructs. The TCF-4mutCtBP polypeptide has a triple amino-acid substitution of each CtBP-binding motif as indicated. tag, myc or EGFP tag; β-cat, β-catenin interaction domain; TLE/Groucho, TLE/Groucho binding domain; CtBP, CtBP binding sites; HMG, DNA-binding domain. Right, Western blots of total cell extracts after transfection with the indicated TCF-4 constructs, probed with anti-TCF-4 or with anti-α-tubulin. (B) CtBP, HIC1 and TCF-4 colocalize in COS-7 cells. (C) Simultaneous interaction between CtBP, TCF-4 and HIC1 is essential for the efficient nuclear sequestration of TCF-4 into the HIC1 bodies. Confocal microscopy images of CtBP(−/−) and CtBP1(+) cells transfected with the indicated constructs (left) and stained with anti-Flag and anti-TCF-4 antibody. The right panel shows the overlap of fluorescence intensity peaks along profiles as indicated in the merged micrographs. The nuclear sequestration of TCF-4 by HIC1 is less efficient in CtBP(−/−) than in CtBP(+) cells (compare (e, f, g, h) to (i, j, k, l)). The formation of the TCF-4/HIC1 bodies in CtBP(+) cells strictly depends on the presence of the intact CtBP-binding sites in TCF-4 (m, n, o, p). Notice only a partial colocalization of TCF-4 and HIC1-ΔCtBP (q, r, s, t). Bar, 10 μm.

Figure 3

Association between TCF-4 and HIC1 in vivo. (A) Left, coimmunoprecipitations between endogenous TCF-4 and various exogenous HIC1 proteins in human 293 cells. Right, coimmunoprecipitations of endogenous HIC1 and TCF-4 in cells derived from mouse embryos on day 12.5 p.c. IP, immunoprecipitation; IB, immunoblotting; in lanes denoted ‘lysate' five percent of the total sample were loaded. (B) TCF-4 associates with HIC1 in CtBP(−/−) cells. Coimmunoprecipitation of endogenous TCF-4 with EGFP-HIC1 is specific for HIC1 as evidenced by no detectable interaction between TCF-4 and EGFP-nls in a control experiment (right).

Figure 4

In vitro interaction of TCF-4 and HIC1. (A) Structures of HIC1 and TCF-4 proteins used in the in vitro pull-down assays (see also the diagrams of the additional HIC1 and TCF-4 constructs depicted in Figures 1A and 2A). (B) Pull-down assays between bacterially expressed GST-fusion and in vitro translated proteins, as indicated. Ten percent of the total reactions were loaded in lanes denoted ‘input'. (C) Right, TCF-4/HIC1 interaction is resistant to DNase I treatment; left, the intact DNA-binding domain of HIC1 is not essential for the interaction with TCF-4.

Figure 5

HIC1 represses the Wnt-stimulated transcription. Reporter gene assay with the Wnt-responsive promoters. 293 cells were cotransfected with the reporters and the HIC1 constructs as indicated and stimulated for 24 h with Wnt3a-conditioned or control medium. Luciferase (firefly) activities were corrected for the efficiency of transfection using the internal control Renilla luciferase expression plasmid. The reporter activity in unstimulated mock-transfected cells was arbitrarily set to 1. The histograms represent mean values of triplicate experiments and SDs (standard deviations) are shown by error bars.

Figure 6

HIC1 represses TCF/β-catenin signaling in DLD-1 adenocarcinoma cells. (A) Right, transgenic DLD/HIC1 cells growing at higher concentrations of a synthetic compound AP21967 (dimerizer) contain increasing amounts of the HIC1 protein as evidenced by Western blots of total cell extracts probed with anti-HIC1 antibody. Middle, HIC1 expression does not influence the protein levels of TCF-4 and β-catenin. Left, the constitutive activity of the TCF-dependent reporter pTOPFLASH is suppressed by increasing amounts of the HIC1 protein. Average luciferase light units per second (RLU/s) corrected for the efficiency of transfection determined as the luciferase/Renilla ratio from five experiments are given (right, pFOPFLASH values). (B) Colocalization of HIC1 with endogenous β-catenin. Confocal micrographs of DLD-1 cells transfected with the full-length Flag-HIC1 construct stained with anti-Flag and anti-β-catenin antibody. Bar, 10 μm. (C) HIC1 expression does not disrupt the binding between TCF-4 and β-catenin. Coimmunoprecipitation of endogenous TCF-4 with ectopically expressed β-catenin is not affected by co-expression of HIC1 (compare left and middle panel). The coimmunoprecipitation is specific for β-catenin as indicated by a control experiment using the EGFP-nls instead of β-catenin-EGFP fusion protein (right).

Figure 7

The nuclear HIC1 bodies in DAOY cells. (A) Increased expression of HIC1 mRNA and protein in the cell line DAOY after treatment with 1 μM 5-aza-2′-deoxycytidine for 6 days. The expression was analyzed by qRT–PCR (left) or by Western blotting using anti-HIC1 antibody. In lane 3, a lysate from 293 cells transfected with 0.5 μg of the Flag-HIC1 construct was loaded. (B) Confocal micrographs of DAOY cells treated with 5-aza-2′-deoxycytidine (c, d) or with a vehicle (a, b) stained with the affinity purified anti-HIC1 antibody. Bar, 10 μm.

Figure 8

HIC1 knockdown increases the TCF-mediated transcription. (A) Human primary cells WI38 were transfected with HIC1 siRNAs or control siRNAs and the changes in the levels of HIC1 mRNA or protein were tested 24 h post-transfection. Left, results of the qRT–PCR analysis. The relative abundance of the given mRNA in HIC1 siRNA versus control siRNA-transfected cells was derived from the average CT values of four independent experiments after normalizing to the levels of β-actin cDNA. Right, Western blots of nuclear extracts prepared from the indicated cells. Bottom, confocal micrographs of WI38 cells transfected with the indicated siRNAs and stained with the affinity purified anti-HIC1 antibody. Bar, 10 μm. (B) The activity of the Wnt-dependent promoter of the Axin2 gene is increased by HIC1 knockdown. Results of qRT–PCR analysis performed with cDNA generated from WI38 cells transfected with the indicated siRNAs upon 24-h stimulation with Wnt3a. Six PCR reactions were done for each primer set. The relative abundance of the indicated mRNA in Wnt3a-stimulated versus control cells was derived from the average CT values after normalizing to the levels of β-actin cDNA.

Figure 9

HIC1 sequesters TCF and β-catenin from the TCF-dependent promoters. (A) HIC1 blocks transcriptional activation of the Wnt signaling target genes. Results of qRT–PCR analyses performed with cDNA generated from 293 cells transfected with the indicated constructs upon 12- or 24-h stimulation with Wnt3a. Four to six PCR reactions were performed for each primer set. The relative abundance of the indicated mRNA in Wnt3a-stimulated versus control cells was derived from the average CT values after normalizing to the levels of β-actin cDNA. (B) HIC1 is not associated with the Sp5 promoter but sequesters TCF-4 and β-catenin from the TCF-specific DNA element of this promoter. ChIP analysis of chromatin isolated from 293 cells transfected with the indicated constructs. The diagram at the left represents real-time PCR values obtained with primers spanning the respective DNA element, normalized to the inputs. The image on the right depicts relevant PCR products after 29 cycles of amplification. (C) HIC1-EGFP blocks transcription of the Tenascin C promoter in DLD-1 cells. Left, results of qRT–PCR analyses performed with cDNA prepared from DLD/HIC1 cells growing in the presence of the dimerizer (25 nM; HIC1 induction) or without induction. Right, Western blot analysis of nuclear extracts isolated from DLD/HIC1 and WI38 cells. (D) HIC1 sequesters TCF-4 and β-catenin from the Tenascin C promoter. Left, ChIP analysis of chromatin isolated from DLD/HIC1 cells prior to and upon HIC1-EGFP induction. The diagram at the left represents real-time PCR values obtained with primers spanning the proximal TCF-binding element in the Tenascin C promoter, normalized to the inputs. The image on the right depicts relevant PCR products after 29 cycles of amplification. Right bottom, although HIC1-EGFP is not associated with the Tenascin C promoter, it binds its recognition element in the SIRT1 promoter.

Figure 10

A model for HIC1 suppression of the transcriptional response induced by Wnts. The regulation of a complex promoter integrating inputs from the Wnt and other signaling pathways is depicted. At low levels of HIC1 the activity of the promoter depends mainly on the Wnt signaling components. High levels of HIC1 uncouple the promoter from Wnt signaling. X depicts a hypothetical factor mediating besides TCF-4 the interaction between β-catenin and HIC1.