Snail mediates E-cadherin repression by the recruitment of the Sin3A/histone deacetylase 1 (HDAC1)/HDAC2 complex

Abstract

The transcription factor Snail has been described as a direct repressor of E-cadherin expression during development and carcinogenesis; however, the specific mechanisms involved in this process remain largely unknown. Here we show that mammalian Snail requires histone deacetylase (HDAC) activity to repress E-cadherin promoter and that treatment with trichostatin A (TSA) is sufficient to block the repressor effect of Snail. Moreover, overexpression of Snail is correlated with deacetylation of histones H3 and H4 at the E-cadherin promoter, and TSA treatment in Snail-expressing cells reverses the acetylation status of histones. Additionally, we demonstrate that Snail interacts in vivo with the E-cadherin promoter and recruits HDAC activity. Most importantly, we demonstrate an interaction between Snail, histone deacetylase 1 (HDAC1) and HDAC2, and the corepressor mSin3A. This interaction is dependent on the SNAG domain of Snail, indicating that the Snail transcription factor mediates the repression by recruitment of chromatin-modifying activities, forming a multimolecular complex to repress E-cadherin expression. Our results establish a direct causal relationship between Snail-dependent repression of E-cadherin and the modification of chromatin at its promoter.

Figures

FIG. 1.

TSA inhibits the Snail-mediated repression of the E-cadherin promoter. (A and B) E-cadherin promoter activity was analyzed in epithelial MCDK (A) and epidermal keratinocyte MCA3D (B) cells in the presence of plasmids pcDNA3 control and pcDNA-Snail. Where indicated, cells were treated with TSA (300 nM; white bars) or with ethanol (black bars) for 24 h after transfection. (C and D) The activity of the E-cadherin promoter was analyzed in MDCK-Snail (C and D) and spindle carcinoma CarB (C) cells. Cells were treated with TSA (300 nM; white bars) or with ethanol (black bars) for 24 h (C) or at the indicated time points (D) after transfection. Luciferase and Renilla activities were determined 24 h after transfection; the promoter activity is represented as the relative luciferuse units (RLU) for control untreated cells. Results represent the averages ± standard deviations of at least two independent experiments performed in duplicate. − (D), cells treated with ethanol for 24 h after transfection. (E) RT-PCR analysis of E-cadherin levels. The indicated cell lines were treated for 24 h with TSA (300 nM) (lanes marked with plus sign) or with vehicle (lanes marked with a minus sign); total RNA was extracted and subjected to RT-PCR analysis with specific E-cadherin primers. The levels of GAPDH were analyzed as a control of the amount of cDNA.

FIG. 2.

Histone acetylation and methylation analysis at the endogenous E-cadherin promoter in Snail-expressing and Snail-deficient cells. (A and B) ChIP analysis of the modification status of histones H3 and H4 at the endogenous E-cadherin promoter in MDCK-CMV (MDCK) and MDCK-Snail cells, with anti-acetyl-histone H3 (α-Ac-H3) and anti-acetyl-histone H4 (α-Ac-H4) and anti-dimethyl-K4 histone H3 (α-Met-K4-H3) and anti-dimethyl-K9 histone H3 (α-Met-K9-H3) antibodies. Where indicated, cells were treated with TSA (300 nM) for 24 h before formaldehyde cross-linking. The amplified dog E-cadherin promoter sequences in the input and the immunoprecitated bound and unbound fractions are shown in the upper panels. (C) ChIP assays of the histone H3 and H4 acetylation status at the endogenous E-cadherin promoter in mouse keratinocyte MCA3D, Pam212, and spindle CarB cells, with anti-acetyl-histone H3 (α-Ac-H3) and anti-acetyl-histone H4 (α-Ac-H4) antibodies. Where indicated, cells were treated with TSA (300 nM) for 24 h before formaldehyde cross-linking. The amplified mouse E-cadherin promoter sequences in the input (upper panels) and bound and unbound (lower panels) fractions are shown. Results from controls with no antibody (NAB), in which no amplification occurs, are also included for each cell line. Quantification of the amplified sequences in the immunoprecipitated fractions (represented as the ratio of bound to unbound fractions) with each antibody and corresponding cells lines and treatments is shown in the lower (A and B) and right (C) panels. Results represent the averages ± standard deviations of at least two experiments. B, bound; UB, unbound.

FIG. 3.

Snail interacts with the endogenous E-cadherin promoter and recruits HDAC activity. (A) MDCK-GFP and MDCK-GFP-Snail cells were analyzed by ChIP assays with anti-GFP (α-GFP) antibodies. Amplification of the endogenous dog E-cadherin promoter in the input and immunoprecipitated fractions of a control MDCK-GFP clone and two independently isolated MDCK-GFP-Snail clones is shown. (B) HDAC activity was determined in the α-GFP immunoprecipitated fractions obtained from HEK 293T cells transiently transfected with GFP (left lanes) and GFP-Snail (right lanes) vectors and either untreated (black bars) or treated with TSA (300 nM) (white bars) for 24 h.

FIG. 4.

Snail associates with HDAC1/2 and the mSin3A corepressor through the SNAG domain. (A to D) HEK 293T cells were transiently transfected with Snail-HA and HDAC1-Flag constructs. Cell extracts were immunoprecipitated with anti-HA antibodies (α-HA) and control IgG, as indicated, and analyzed by Western blotting with anti-HDAC2 (α-HDAC-2), anti-HDAC3 (α-HDCA-3), anti-Flag (α-Flag), and anti-mSin3A (α-mSin3A) antibodies. (E) HEK 293T cells transiently transfected as above were immunoprecipitated with anti-mSin3A antibodies and analyzed by Western blotting with anti-HA. Cell extracts (HEK) were analyzed in parallel in all panels. (F) HEK 293T cells transiently transfected withSnail-HA, HDAC1-Flag, and mSin3A-myc constructs were immunoprecipitated with the indicated antibodies and analyzed by Western blotting with anti-HA antibodies (upper panel). Detection of IgG heavy chain is shown in the lower panel as an internal control. Input (10%) of whole cell extract was also analyzed in parallel. (G) Cell extracts obtained from HEK 293T cells transiently transfected as above were incubated with the indicated GST fusion proteins. The bound fractions from the glutathione-Sepharose beads and the input cell extract were analyzed by Western blotting with the indicated antibodies (upper panel). Analysis of the different recombinant GST fusion proteins used is shown in the lower panel. WB, Western blotting; IP, immunoprecipitation; Ab, antibody.

FIG. 5.

Snail colocalizes with HDAC1/2 and mSin3A in the nucleus. (A) MDCK (a to f), CarB (g to l), and HEK 293T (m to r) cells were transiently transfected with Snail-HA and HDAC1-Flag (a to c, g to i, and m to o) or with Snail-HA and HDAC2-Flag (d to f, j to l, and p to r) constructs and costained with anti-HA (red), anti-mSin3A (green), and anti-Flag (cyan) antibodies. Note the nuclear colocalization of the proteins (white to pink) in the merged images of the corresponding three channels presented in the lower panels. (B) CarB cells were transiently transfected with the Snail-HA construct and costained with anti-HA (a to c) and anti-HDAC1 (d), anti-HDAC2 (e), or anti-mSin3A (f). Merged images of the corresponding two channels show the nuclear colocalization of Snail-HA with endogenous HDAC1 (ad), endogenous HDAC2 (be), and endogenous mSin3A (ef). Bar, 5 μm.

FIG. 5.

Snail colocalizes with HDAC1/2 and mSin3A in the nucleus. (A) MDCK (a to f), CarB (g to l), and HEK 293T (m to r) cells were transiently transfected with Snail-HA and HDAC1-Flag (a to c, g to i, and m to o) or with Snail-HA and HDAC2-Flag (d to f, j to l, and p to r) constructs and costained with anti-HA (red), anti-mSin3A (green), and anti-Flag (cyan) antibodies. Note the nuclear colocalization of the proteins (white to pink) in the merged images of the corresponding three channels presented in the lower panels. (B) CarB cells were transiently transfected with the Snail-HA construct and costained with anti-HA (a to c) and anti-HDAC1 (d), anti-HDAC2 (e), or anti-mSin3A (f). Merged images of the corresponding two channels show the nuclear colocalization of Snail-HA with endogenous HDAC1 (ad), endogenous HDAC2 (be), and endogenous mSin3A (ef). Bar, 5 μm.

FIG. 6.

HDAC1/2 proteins are recruited at the endogenous E-cadherin promoter and cooperate with mSin3A in Snail-mediated repression of the promoter activity. (A and B) ChIP assays of HDAC1/2/3 at the endogenous E-cadherin promoter in MDCK-Snail (A, upper panel) and MDCK-CMV (A, lower panel) cells, and in spindle CarB (B, upper panel) and epidermal keratinocyte MCA3D (B, lower panel) cells. ChIP assays were performed as indicated in Materials and Methods with antibodies specific for HDAC1 (HD1), HDAC2 (HD2), and HDAC3 (HD3). The amplified sequences of the dog or mouse E-cadherin promoter detected in the input and in the immunoprecipitated bound and unbound fractions are shown. Results for controls with no antibody (NoAb), in which no amplification occurs, are also included for each cell line. (C) The activity of the E-cadherin promoter was analyzed in MDCK cells in the presence of the indicated Snail expression vectors (50 ng) and in the absence or presence of cotransfection with HDAC1 and/or mSin3A expression vectors (100 ng). Promoter activity was determined 24 h after transfection, as indicated in the legend of Fig. 1.

FIG. 7.

The ΔSNAG construct acts as a dominant negative of Snail in E-cadherin promoter repression. (A) The activity of the E-cadherin promoter in Snail-expressing CarB cells was more efficiently derepressed by the ΔSNAG than by the ΔNt Snail mutant. (B) E-cadherin promoter activity in Snail-deficient PDV cells. The repression of the E-cadherin promoter induced by cotransfection of Snail was fully relieved by the ΔSNAG mutant but not affected by the ΔNt mutant. Promoter activity was determined 24 h after transfection, as indicated in the legend of Fig. 1.