Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor - PubMed (original) (raw)

Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor

T Nomura et al. Genes Dev. 1999.

Abstract

The N-CoR/SMRT complex containing mSin3 and histone deacetylase (HDAC) mediates transcriptional repression by nuclear hormone receptors and Mad. The proteins encoded by the ski proto-oncogene family directly bind to N-CoR/SMRT and mSin3A, and forms a complex with HDAC. c-Ski and its related gene product Sno are required for transcriptional repression by Mad and thyroid hormone receptor (TRbeta). The oncogenic form, v-Ski, which lacks the mSin3A-binding domain, acts in a dominant-negative fashion, and abrogates transcriptional repression by Mad and TRbeta. In ski-deficient mouse embryos, the ornithine decarboxylase gene, whose expression is normally repressed by Mad-Max, is expressed ectopically. These results show that Ski is a component of the HDAC complex and that Ski is required for the transcriptional repression mediated by this complex. The involvement of c-Ski in the HDAC complex indicates that the function of the HDAC complex is important for oncogenesis.

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Figures

Figure 1

Binding of c-Ski to N-CoR/SMRT. (A) Yeast two-hybrid assay. The three repressor domains (RI, RII, and RIII) and the mSin3-, Ski-, and nuclear hormone receptor (NHR)-binding domains in N-CoR molecule are indicated on the top. The regions encoded by the clones isolated in the yeast two-hybrid screening with c-Ski as a bait are referred to as Ski-binding domains (SBD). The plasmids used for the interaction assay are shown below. Three transformants harboring the plasmids indicated on the left were isolated, and their β-galactosidase activities were measured. The data represent an average of the results obtained using three transformants and are shown with standard deviations. (B) Coimmunoprercipitation. Whole-cell lysates were prepared from 293T cells transfected with a mixture of c-Ski (wild-type or ARPG mutant) and N-CoR–Flag (wild-type or mutant lacking SBD) expression plasmids, and a samples from the lysates were directly used for Western blotting with the anti-c-Ski antibodies. Whole-cell lysates were also immuno-precipitated by anti-Flag or anti-β-galactosidase antibody as a control, and the immuno-complex was analyzed by Western blotting using anti-c-Ski antibodies. (C) GST pull-down assay. In vitro-translated SMRT (left) or N-CoR (wild-type and SBD deletion mutant) (right) bound to GST–Ski were analyzed by SDS-PAGE followed by autoradiography. In the input lanes, the amount of protein was 10% of that used for the binding assay.

Figure 1

Figure 2

Complex formation between c-Ski, N-CoR, mSin3A, and HDAC. (A) Identification of N-CoR-binding domain in c-Ski. GST pull-down assay was performed using in vitro-translated c-Ski and GST–SBD. The various forms of c-Ski used are indicated. SnoN encoded by one of the sno mRNA species is shown below. The cysteine-rich region is indicated by a shaded box. The results of binding assays are summarized at right. (Bottom) In vitro-translated c-Ski used (input) and c-Ski bound to GST–N-CoR were analyzed by SDS-PAGE followed by autoradiography. In the input lanes, the amount of c-Ski was 10% of that used for the binding assay. (B) Binding of mSin3A to the carboxy-terminal region of c-Ski. GST pull-down assay was performed using in vitro-translated mSin3A and GST fusion proteins containing various portions of c-Ski. The various forms of c-Ski used are indicated. (Lower left) Various GST–Ski fusion proteins bound to glutathione beads were analyzed by SDS-PAGE followed by coomassie blue staining. (Lower right) In vitro-translated mSin3A used (input) and mSin3A bound to GST–Ski were analyzed by SDS-PAGE followed by autoradiography. (C) Coimmunoprecipitation of c-Ski with N-CoR, Sin3, and HDAC. (Left) Lysates from 293 cells were immunoprecipitated with anti-c-Ski antibodies or normal IgG. The immunocomplex was analyzed by Western blotting using anti-N-CoR, anti-Sin3A, and anti-HDAC1 antibodies. (Right) The immunocomplex was prepared using anti-c-Ski antibodies or IgG from 293 cells transfected with the c-Ski expression plasmid, and used for the HDAC assays. As a positive control, the immunocomplex prepared using the anti-HDAC1 antibody from 293 cells transfected with the HDAC1 expression plasmid was used.

Figure 2

Figure 3

Multiple repressor domains in c-Ski. (A) Transcriptional repression by Gal4–Ski fusion proteins. (Top) The structure of Ski is schematically shown. The Gal4–Ski fusion proteins consisting of the Gal4 DNA-binding domain and the various regions of c-Ski are indicated. The transcriptional repression by various Gal4–Ski fusions was measured by cotransfection assays using the luciferase reporter containing the Gal4-binding sites, and the results are indicated below by bar graphs. The average degree of repression compared with that of the Gal4 DNA-binding domain alone is represented along with the standard deviation. The shaded bars indicate significant repressor activity. The results of the cotransfection assays are summarized at right. (++, +, −) Strong, weak, and no repressor activity, respectively. (B) Enhancement of repressor activity of Ski by N-CoR. Cotransfection experiments were done as described above using a small amount of Gal-Ski expression plasmid. The effect of wild-type or mutant N-CoR lacking the amino-proximal repressor domain (N-CoRΔR) on Gal4–Ski-induced transcriptional repression was examined by cotransfection of the N-CoR expression plasmid. (C) Inhibition of the Gal4–Ski-induced repression by SBD. The effect of SBD on Gal4–Ski-induced transcriptional repression was examined by cotransfection.

Figure 3

Figure 4

Disruption of nuclear dot-like structure of N-CoR by Ski mutants. (A) The plasmid to express wild-type N-CoR–Flag or either of three kinds of c-Ski (wild type and two mutants) was transfected into 293T cells, cells were immunostained with anti-Flag or anti-c-Ski antibodies. The stainings were visualized by FITC-conjugated secondary antibodies. (B_–_D) A mixture of the c-Ski [wild type (B) or a mutant lacking the amino-proximal N-CoR-binding domain (C) or a mutant lacking the carboxy-proximal coiled–coil region (D)] and the N-CoR–Flag expression plasmid was transfected into 293T cells, and cells were immunostained with anti-Flag (left) and anti-c-Ski antibodies (middle). The N-CoR- and c-Ski-stainings were visualized by FITC- and rhodamine-conjugated secondary antibodies, respectively. (Right panels) The signals for N-CoR and c-Ski are superimposed.

Figure 5

Abrogation of Mad- or TRβ-induced transcriptional repression by anti-Ski/Sno antibodies. The Gal4 site-containing TK–lacZ reporter was injected with the expression plasmid for Gal4 DNA-binding domain alone or with Gal4–Mad, Gal4–TRβ, or Gal4–δEF1 expression plasmid into the nuclei of Rat-1 cells. The GFP vector was co-injected for the identification of the injected cells. The affinity-purified anti-Ski and anti-Sno antibodies were co-injected with the Gal4–Mad, Gal4–TRβ, or Gal4–δEF1 expression plasmid. The expression of lacZ was monitored by X-gal staining. Typical photomicrographs of green fluorescence of GFP and X-gal staining for the indicated constructs and antibodies are shown (A). Experiments were repeated three (Gal4–Mad) or two times (Gal4–TRβ and Gal4–δEF1), and the number of injected cells in each experiment was 70–240. The lacZ+ cells were quantified based on the percentage of injected cells that stained blue, and the results are presented as bar graphs with the standard deviation (B). The shaded bar shows the significant abrogation of the Mad- or TRβ-induced transcriptional repression by coinjection of both anti-Ski and anti-Sno antibodies.

Figure 6

Inhibition of the Mad- or TRβ-induced transcriptional repression by v-Ski. (a) Summary of the cotransfection assays. The structures of the various forms of Ski, whose capacity to abrogate the transcriptional repression by Gal–Mad, Gal–TRβ, or Gal4–δEF1 was examined, are shown. The results of the cotransfection assays shown in B and C are summarized as almost complete abrogation of transcriptional repression (++) and partial abrogation of transcriptional repression (+). (B_–_D) Cotransfection assays. A mixture of Gal4 site-containing luciferase reporter, the Gal4–Mad (B), Gal4–TRβ (C), Gal4–δEF1 (D) or Gal4 expression plasmid, and the effector plasmid encoding various forms of Ski was transfected into CV-1 cells, and the luciferase activity was measured. The amounts of effector plasmid were 4 (+) or 8 μg (++). The data shown are an average of two experiments with standard deviations. The shaded bar indicates the relative luciferase activity >0.7.

Figure 7

Ectopic expression of ODC in _ski_-deficient embryos. Frontal sections were prepared from c-ski heterozygous and homozygous mutant embryos at 9.5 dpc, and analyzed with the TUNEL assay (a,b). Apoptotic cells in the neuroepithelium that had incorporated labeled dUTP are indicated by arrows. The signals of in situ hybridization (c,d) or immunostaining for ODC (e,f) are shown. Cells that overexpressed ODC are indicated by arrows.

Figure 8

Schematic model of the HDAC complex. N-CoR/SMRT directly binds to mSin3, which interacts with HDAC. The amino-terminal region of c-Ski binds to N-CoR through the carboxy-terminal region of N-CoR. Skip also binds to the amino-terminal region of c-Ski. The carboxy-terminal region of c-Ski binds to mSin3A. The nuclear hormone receptor is linked to the HDAC complex by direct binding to N-CoR, whereas a transcriptional repressor Mad is linked to the HDAC complex via mSin3.

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Ski is a component of the histone deacetylase complex required for transcriptional repression by Mad and thyroid hormone receptor - PubMed (original) (raw)