Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily

Abstract

Some members of nuclear hormone receptors, such as the thyroid hormone receptor (TR), silence gene expression in the absence of the hormone. Corepressors, which bind to the receptor's silencing domain, are involved in this repression. Hormone binding leads to dissociation of corepressors and binding of coactivators, which in turn mediate gene activation. Here, we describe the characteristics of Alien, a novel corepressor. Alien interacts with TR only in the absence of hormone. Addition of thyroid hormone leads to dissociation of Alien from the receptor, as shown by the yeast two-hybrid system, glutathione S-transferase pull-down, and coimmunoprecipitation experiments. Reporter assays indicate that Alien increases receptor-mediated silencing and that it harbors an autonomous silencing function. Immune staining shows that Alien is localized in the cell nucleus. Alien is a highly conserved protein showing 90% identity between human and Drosophila. Drosophila Alien shows similar activities in that it interacts in a hormone-sensitive manner with TR and harbors an autonomous silencing function. Specific interaction of Alien is seen with Drosophila nuclear hormone receptors, such as the ecdysone receptor and Seven-up, the Drosophila homologue of COUP-TF1, but not with retinoic acid receptor, RXR/USP, DHR 3, DHR 38, DHR 78, or DHR 96. These properties, taken together, show that Alien has the characteristics of a corepressor. Thus, Alien represents a member of a novel class of corepressors specific for selected members of the nuclear hormone receptor superfamily.

FIG. 1

d-Alien interacts with TR in a hormone-sensitive manner. The C termini of TRα, RXRα, and RARα were used as bait (lex fusion) and d-Alien was used as the activator, with or without the cognate ligands, in yeast two-hybrid experiments as described by Gyuris et al. (32). Interaction leads to activation of the cotransfected reporter (lacZ gene). As controls, the bait alone (lex vector) or the empty activator vector (C) were used.

FIG. 2

Alien is highly conserved in evolution. h-Alien is aligned with the d-Alien homologue and translated open reading frames of C. elegans (C.e.) and Ricinus communis (R.c.). Identical amino acids to h-Alien are indicated by vertical lines, and related amino acids are shown in boldface type. Sequence comparison between h-Alien and d-Alien revealed 90% identity and about 95% similarity at the amino acid level. The peptide sequence for antibody generation is indicated by a box. Two incomplete open reading frames have been retrieved from C. elegans (Alien 1 and 2), and one partial open reading frame has been retrieved from R. communis. Missing amino acids are indicated by dots.

FIG. 3

(A) Alien interacts specifically with TR but not with RAR, RXR, or GR. h-Alien was tested for interaction with TRα f.l., the C termini of the TRα, TRβ, the oncogene v-erbA, RARα, RXRα, and GRα in the presence or absence of the cognate hormones. Yeast two-hybrid experiments were performed as described for Fig. 1. As controls, the parental expression vectors were used. The hormones used were TRIAC (10⁻⁶ M) for TR and v-erbA; retinoic acids (10⁻⁵ M) for RAR and RXR; and triamcinolon diacetate (10⁻⁶ M) for GR. (B) Alien interacts with the silencing domains of both RAR (RARΔC60) and EcR. Yeast two-hybrid experiments were performed as described in the legend to Fig. 1 in the absence of hormone, with or without h-Alien, with the C terminus of EcR, with hRARα, and with C-terminal deletions of hRARα: a 43-aa deletion (RARΔC43), lacking the RAR F-region, and a 60-aa deletion (RARΔC60), lacking both the receptor F-region and the AF2-AD/τ4/τc. (C) Interaction of Alien with TR is hormone sensitive in vitro. GST pull-down experiments were performed with bacterially expressed GST or GST-hTRβ fusion and in vitro-translated, ³⁵S-labeled h-Alien. The ligand TRIAC (5 × 10⁻⁸ M) was added to the reaction mixture in the indicated lanes. (D) Alien interacts with a subset of Drosophila NHRs. GST pull-down assays were performed with bacterially expressed GST, GST–d-Alien, or GST–h-Alien incubated with various in vitro-translated, ³⁵S-labeled NHRs from mammals or Drosophila. The input lane shows 10% of total input. Luciferase served as a negative control. GST–h-Alien was used for the human receptors, and GST–d-Alien was used for the Drosophila receptors. (E) Schematic presentation of the Alien-receptor interaction of the GST pull-down experiments. Results obtained in the experiment in Fig. 3D were plotted as the percentage of bound receptor compared to the input of each nuclear receptor. Interaction was observed with TR, EcR, and FTZ-F1, weak interaction was observed with COUP-TF1 and SVP, and no significant interaction was observed with RXR, USP, DHR 3, DHR 38, DHR 78, and DHR 96. (F) Alien is complexed with TR in the absence of hormone in vivo. Coimmunoprecipitations were performed with HA-tagged TRα c.t. (top) expressed in COS1 cells and endogenous Alien, using anti-HA-antibody for coimmunoprecipitation and anti-Alien antibody for Western analysis. Transfected COS1 cells were treated with or without thyroid hormone (10⁻⁶ M) for one day prior to harvest. Nonspecific bands with a lower migration rate appear in both lanes with similar intensity, while Alien (arrow) is complexed only in the absence of ligand with TR. TRα f.l. complexed with Alien was used in coimmunoprecipitation experiments (bottom). HA-tagged full-length TRα from transfected COS cells coprecipitates Alien only in the absence of ligand. Extracts from HA-tag-transfected cells, untransfected cells, and added hormone in extracts transfected with full-length TRα did not coprecipitate Alien. In vitro-translated [³⁵S]methionine-labeled h-Alien is shown as a migration control.

FIG. 3

(A) Alien interacts specifically with TR but not with RAR, RXR, or GR. h-Alien was tested for interaction with TRα f.l., the C termini of the TRα, TRβ, the oncogene v-erbA, RARα, RXRα, and GRα in the presence or absence of the cognate hormones. Yeast two-hybrid experiments were performed as described for Fig. 1. As controls, the parental expression vectors were used. The hormones used were TRIAC (10⁻⁶ M) for TR and v-erbA; retinoic acids (10⁻⁵ M) for RAR and RXR; and triamcinolon diacetate (10⁻⁶ M) for GR. (B) Alien interacts with the silencing domains of both RAR (RARΔC60) and EcR. Yeast two-hybrid experiments were performed as described in the legend to Fig. 1 in the absence of hormone, with or without h-Alien, with the C terminus of EcR, with hRARα, and with C-terminal deletions of hRARα: a 43-aa deletion (RARΔC43), lacking the RAR F-region, and a 60-aa deletion (RARΔC60), lacking both the receptor F-region and the AF2-AD/τ4/τc. (C) Interaction of Alien with TR is hormone sensitive in vitro. GST pull-down experiments were performed with bacterially expressed GST or GST-hTRβ fusion and in vitro-translated, ³⁵S-labeled h-Alien. The ligand TRIAC (5 × 10⁻⁸ M) was added to the reaction mixture in the indicated lanes. (D) Alien interacts with a subset of Drosophila NHRs. GST pull-down assays were performed with bacterially expressed GST, GST–d-Alien, or GST–h-Alien incubated with various in vitro-translated, ³⁵S-labeled NHRs from mammals or Drosophila. The input lane shows 10% of total input. Luciferase served as a negative control. GST–h-Alien was used for the human receptors, and GST–d-Alien was used for the Drosophila receptors. (E) Schematic presentation of the Alien-receptor interaction of the GST pull-down experiments. Results obtained in the experiment in Fig. 3D were plotted as the percentage of bound receptor compared to the input of each nuclear receptor. Interaction was observed with TR, EcR, and FTZ-F1, weak interaction was observed with COUP-TF1 and SVP, and no significant interaction was observed with RXR, USP, DHR 3, DHR 38, DHR 78, and DHR 96. (F) Alien is complexed with TR in the absence of hormone in vivo. Coimmunoprecipitations were performed with HA-tagged TRα c.t. (top) expressed in COS1 cells and endogenous Alien, using anti-HA-antibody for coimmunoprecipitation and anti-Alien antibody for Western analysis. Transfected COS1 cells were treated with or without thyroid hormone (10⁻⁶ M) for one day prior to harvest. Nonspecific bands with a lower migration rate appear in both lanes with similar intensity, while Alien (arrow) is complexed only in the absence of ligand with TR. TRα f.l. complexed with Alien was used in coimmunoprecipitation experiments (bottom). HA-tagged full-length TRα from transfected COS cells coprecipitates Alien only in the absence of ligand. Extracts from HA-tag-transfected cells, untransfected cells, and added hormone in extracts transfected with full-length TRα did not coprecipitate Alien. In vitro-translated [³⁵S]methionine-labeled h-Alien is shown as a migration control.

FIG. 3

(A) Alien interacts specifically with TR but not with RAR, RXR, or GR. h-Alien was tested for interaction with TRα f.l., the C termini of the TRα, TRβ, the oncogene v-erbA, RARα, RXRα, and GRα in the presence or absence of the cognate hormones. Yeast two-hybrid experiments were performed as described for Fig. 1. As controls, the parental expression vectors were used. The hormones used were TRIAC (10⁻⁶ M) for TR and v-erbA; retinoic acids (10⁻⁵ M) for RAR and RXR; and triamcinolon diacetate (10⁻⁶ M) for GR. (B) Alien interacts with the silencing domains of both RAR (RARΔC60) and EcR. Yeast two-hybrid experiments were performed as described in the legend to Fig. 1 in the absence of hormone, with or without h-Alien, with the C terminus of EcR, with hRARα, and with C-terminal deletions of hRARα: a 43-aa deletion (RARΔC43), lacking the RAR F-region, and a 60-aa deletion (RARΔC60), lacking both the receptor F-region and the AF2-AD/τ4/τc. (C) Interaction of Alien with TR is hormone sensitive in vitro. GST pull-down experiments were performed with bacterially expressed GST or GST-hTRβ fusion and in vitro-translated, ³⁵S-labeled h-Alien. The ligand TRIAC (5 × 10⁻⁸ M) was added to the reaction mixture in the indicated lanes. (D) Alien interacts with a subset of Drosophila NHRs. GST pull-down assays were performed with bacterially expressed GST, GST–d-Alien, or GST–h-Alien incubated with various in vitro-translated, ³⁵S-labeled NHRs from mammals or Drosophila. The input lane shows 10% of total input. Luciferase served as a negative control. GST–h-Alien was used for the human receptors, and GST–d-Alien was used for the Drosophila receptors. (E) Schematic presentation of the Alien-receptor interaction of the GST pull-down experiments. Results obtained in the experiment in Fig. 3D were plotted as the percentage of bound receptor compared to the input of each nuclear receptor. Interaction was observed with TR, EcR, and FTZ-F1, weak interaction was observed with COUP-TF1 and SVP, and no significant interaction was observed with RXR, USP, DHR 3, DHR 38, DHR 78, and DHR 96. (F) Alien is complexed with TR in the absence of hormone in vivo. Coimmunoprecipitations were performed with HA-tagged TRα c.t. (top) expressed in COS1 cells and endogenous Alien, using anti-HA-antibody for coimmunoprecipitation and anti-Alien antibody for Western analysis. Transfected COS1 cells were treated with or without thyroid hormone (10⁻⁶ M) for one day prior to harvest. Nonspecific bands with a lower migration rate appear in both lanes with similar intensity, while Alien (arrow) is complexed only in the absence of ligand with TR. TRα f.l. complexed with Alien was used in coimmunoprecipitation experiments (bottom). HA-tagged full-length TRα from transfected COS cells coprecipitates Alien only in the absence of ligand. Extracts from HA-tag-transfected cells, untransfected cells, and added hormone in extracts transfected with full-length TRα did not coprecipitate Alien. In vitro-translated [³⁵S]methionine-labeled h-Alien is shown as a migration control.

FIG. 3

(A) Alien interacts specifically with TR but not with RAR, RXR, or GR. h-Alien was tested for interaction with TRα f.l., the C termini of the TRα, TRβ, the oncogene v-erbA, RARα, RXRα, and GRα in the presence or absence of the cognate hormones. Yeast two-hybrid experiments were performed as described for Fig. 1. As controls, the parental expression vectors were used. The hormones used were TRIAC (10⁻⁶ M) for TR and v-erbA; retinoic acids (10⁻⁵ M) for RAR and RXR; and triamcinolon diacetate (10⁻⁶ M) for GR. (B) Alien interacts with the silencing domains of both RAR (RARΔC60) and EcR. Yeast two-hybrid experiments were performed as described in the legend to Fig. 1 in the absence of hormone, with or without h-Alien, with the C terminus of EcR, with hRARα, and with C-terminal deletions of hRARα: a 43-aa deletion (RARΔC43), lacking the RAR F-region, and a 60-aa deletion (RARΔC60), lacking both the receptor F-region and the AF2-AD/τ4/τc. (C) Interaction of Alien with TR is hormone sensitive in vitro. GST pull-down experiments were performed with bacterially expressed GST or GST-hTRβ fusion and in vitro-translated, ³⁵S-labeled h-Alien. The ligand TRIAC (5 × 10⁻⁸ M) was added to the reaction mixture in the indicated lanes. (D) Alien interacts with a subset of Drosophila NHRs. GST pull-down assays were performed with bacterially expressed GST, GST–d-Alien, or GST–h-Alien incubated with various in vitro-translated, ³⁵S-labeled NHRs from mammals or Drosophila. The input lane shows 10% of total input. Luciferase served as a negative control. GST–h-Alien was used for the human receptors, and GST–d-Alien was used for the Drosophila receptors. (E) Schematic presentation of the Alien-receptor interaction of the GST pull-down experiments. Results obtained in the experiment in Fig. 3D were plotted as the percentage of bound receptor compared to the input of each nuclear receptor. Interaction was observed with TR, EcR, and FTZ-F1, weak interaction was observed with COUP-TF1 and SVP, and no significant interaction was observed with RXR, USP, DHR 3, DHR 38, DHR 78, and DHR 96. (F) Alien is complexed with TR in the absence of hormone in vivo. Coimmunoprecipitations were performed with HA-tagged TRα c.t. (top) expressed in COS1 cells and endogenous Alien, using anti-HA-antibody for coimmunoprecipitation and anti-Alien antibody for Western analysis. Transfected COS1 cells were treated with or without thyroid hormone (10⁻⁶ M) for one day prior to harvest. Nonspecific bands with a lower migration rate appear in both lanes with similar intensity, while Alien (arrow) is complexed only in the absence of ligand with TR. TRα f.l. complexed with Alien was used in coimmunoprecipitation experiments (bottom). HA-tagged full-length TRα from transfected COS cells coprecipitates Alien only in the absence of ligand. Extracts from HA-tag-transfected cells, untransfected cells, and added hormone in extracts transfected with full-length TRα did not coprecipitate Alien. In vitro-translated [³⁵S]methionine-labeled h-Alien is shown as a migration control.

FIG. 3

(A) Alien interacts specifically with TR but not with RAR, RXR, or GR. h-Alien was tested for interaction with TRα f.l., the C termini of the TRα, TRβ, the oncogene v-erbA, RARα, RXRα, and GRα in the presence or absence of the cognate hormones. Yeast two-hybrid experiments were performed as described for Fig. 1. As controls, the parental expression vectors were used. The hormones used were TRIAC (10⁻⁶ M) for TR and v-erbA; retinoic acids (10⁻⁵ M) for RAR and RXR; and triamcinolon diacetate (10⁻⁶ M) for GR. (B) Alien interacts with the silencing domains of both RAR (RARΔC60) and EcR. Yeast two-hybrid experiments were performed as described in the legend to Fig. 1 in the absence of hormone, with or without h-Alien, with the C terminus of EcR, with hRARα, and with C-terminal deletions of hRARα: a 43-aa deletion (RARΔC43), lacking the RAR F-region, and a 60-aa deletion (RARΔC60), lacking both the receptor F-region and the AF2-AD/τ4/τc. (C) Interaction of Alien with TR is hormone sensitive in vitro. GST pull-down experiments were performed with bacterially expressed GST or GST-hTRβ fusion and in vitro-translated, ³⁵S-labeled h-Alien. The ligand TRIAC (5 × 10⁻⁸ M) was added to the reaction mixture in the indicated lanes. (D) Alien interacts with a subset of Drosophila NHRs. GST pull-down assays were performed with bacterially expressed GST, GST–d-Alien, or GST–h-Alien incubated with various in vitro-translated, ³⁵S-labeled NHRs from mammals or Drosophila. The input lane shows 10% of total input. Luciferase served as a negative control. GST–h-Alien was used for the human receptors, and GST–d-Alien was used for the Drosophila receptors. (E) Schematic presentation of the Alien-receptor interaction of the GST pull-down experiments. Results obtained in the experiment in Fig. 3D were plotted as the percentage of bound receptor compared to the input of each nuclear receptor. Interaction was observed with TR, EcR, and FTZ-F1, weak interaction was observed with COUP-TF1 and SVP, and no significant interaction was observed with RXR, USP, DHR 3, DHR 38, DHR 78, and DHR 96. (F) Alien is complexed with TR in the absence of hormone in vivo. Coimmunoprecipitations were performed with HA-tagged TRα c.t. (top) expressed in COS1 cells and endogenous Alien, using anti-HA-antibody for coimmunoprecipitation and anti-Alien antibody for Western analysis. Transfected COS1 cells were treated with or without thyroid hormone (10⁻⁶ M) for one day prior to harvest. Nonspecific bands with a lower migration rate appear in both lanes with similar intensity, while Alien (arrow) is complexed only in the absence of ligand with TR. TRα f.l. complexed with Alien was used in coimmunoprecipitation experiments (bottom). HA-tagged full-length TRα from transfected COS cells coprecipitates Alien only in the absence of ligand. Extracts from HA-tag-transfected cells, untransfected cells, and added hormone in extracts transfected with full-length TRα did not coprecipitate Alien. In vitro-translated [³⁵S]methionine-labeled h-Alien is shown as a migration control.

FIG. 3

(A) Alien interacts specifically with TR but not with RAR, RXR, or GR. h-Alien was tested for interaction with TRα f.l., the C termini of the TRα, TRβ, the oncogene v-erbA, RARα, RXRα, and GRα in the presence or absence of the cognate hormones. Yeast two-hybrid experiments were performed as described for Fig. 1. As controls, the parental expression vectors were used. The hormones used were TRIAC (10⁻⁶ M) for TR and v-erbA; retinoic acids (10⁻⁵ M) for RAR and RXR; and triamcinolon diacetate (10⁻⁶ M) for GR. (B) Alien interacts with the silencing domains of both RAR (RARΔC60) and EcR. Yeast two-hybrid experiments were performed as described in the legend to Fig. 1 in the absence of hormone, with or without h-Alien, with the C terminus of EcR, with hRARα, and with C-terminal deletions of hRARα: a 43-aa deletion (RARΔC43), lacking the RAR F-region, and a 60-aa deletion (RARΔC60), lacking both the receptor F-region and the AF2-AD/τ4/τc. (C) Interaction of Alien with TR is hormone sensitive in vitro. GST pull-down experiments were performed with bacterially expressed GST or GST-hTRβ fusion and in vitro-translated, ³⁵S-labeled h-Alien. The ligand TRIAC (5 × 10⁻⁸ M) was added to the reaction mixture in the indicated lanes. (D) Alien interacts with a subset of Drosophila NHRs. GST pull-down assays were performed with bacterially expressed GST, GST–d-Alien, or GST–h-Alien incubated with various in vitro-translated, ³⁵S-labeled NHRs from mammals or Drosophila. The input lane shows 10% of total input. Luciferase served as a negative control. GST–h-Alien was used for the human receptors, and GST–d-Alien was used for the Drosophila receptors. (E) Schematic presentation of the Alien-receptor interaction of the GST pull-down experiments. Results obtained in the experiment in Fig. 3D were plotted as the percentage of bound receptor compared to the input of each nuclear receptor. Interaction was observed with TR, EcR, and FTZ-F1, weak interaction was observed with COUP-TF1 and SVP, and no significant interaction was observed with RXR, USP, DHR 3, DHR 38, DHR 78, and DHR 96. (F) Alien is complexed with TR in the absence of hormone in vivo. Coimmunoprecipitations were performed with HA-tagged TRα c.t. (top) expressed in COS1 cells and endogenous Alien, using anti-HA-antibody for coimmunoprecipitation and anti-Alien antibody for Western analysis. Transfected COS1 cells were treated with or without thyroid hormone (10⁻⁶ M) for one day prior to harvest. Nonspecific bands with a lower migration rate appear in both lanes with similar intensity, while Alien (arrow) is complexed only in the absence of ligand with TR. TRα f.l. complexed with Alien was used in coimmunoprecipitation experiments (bottom). HA-tagged full-length TRα from transfected COS cells coprecipitates Alien only in the absence of ligand. Extracts from HA-tag-transfected cells, untransfected cells, and added hormone in extracts transfected with full-length TRα did not coprecipitate Alien. In vitro-translated [³⁵S]methionine-labeled h-Alien is shown as a migration control.

FIG. 4

Alien is localized predominantly in the cell nucleus. Rabbit anti-Alien peptide antibody (A to C) or preimmune antiserum (D to F) was used for indirect immunofluorescence analysis of HeLa cells with TRITC-conjugated anti-rabbit antibody. A triplicate picture was taken of the same cells: (A and D) immunofluorescence of immune and preimmune antisera, respectively; (B and E) DAPI staining; (C and F) phase-contrast pictures.

FIG. 5

Alien harbors an autonomous silencing function. Both d-Alien and h-Alien were fused as full-length proteins to the DBD of Gal4 (aa 1 to 94) and were tested with a UAS-tkCAT reporter in chicken HD3 cells for their ability to repress promoter activity. In addition, h-Alien was tested in HeLa, mouse Ltk−, and monkey CV1 cells. For Gal fusions, 3 pmol of expression vectors was transfected. Values obtained with Gal4 DBD were set arbitrarily to 1.

FIG. 6

Interaction of Alien with TR correlates with its silencing function. Yeast two-hybrid experiments with TR mutants as bait were tested for interaction with Alien as prey, as described in the legend to Fig. 1. This figure gives an overview of point mutants and deletions of TR used. Interaction with h-Alien is indicated as a plus, and the corresponding Miller units obtained are listed. The extent of the receptor-silencing domain and the silencing function of receptor mutants were described previously (4, 15, 51). Solid bars represent the single-amino-acid exchanges K419E, K415E, and N359S of TR and the naturally occurring P398R mutant of v-ErbA; numbers indicate the amino acid endpoints of the deletion of TR mutants. h: hormone-sensitive silencing function and interaction with Alien; const., constitutive silencing function and interaction with Alien.

FIG. 7

TR and Alien mutually enhance the ability to silence gene transcription. (A) The silencing function of TR is increased when Alien is coexpressed on a natural response element. The results of cotransfection of TRα with h-Alien and the reporter pTRElys_3× tkCAT (4.4 μg) bearing the natural lysozyme TRE in CV1 cells are shown. As a control (lane C), an empty expression vector was used. (B) The silencing mediated by the C terminus of TR is enhanced by coexpression of h-Alien. Gal-TRβ (1 μg) encompassing the Gal4 DBD fused to the TRβ c.t. was coexpressed with h-Alien (10 μg) and the reporter pUAS_6× tkCAT (3 μg) containing Gal4 binding sites. As controls, both the Gal4 DBD expression vector and an empty expression vector were used. (C) The silencing of Alien is enhanced by expression of TRα only in the absence of hormone. Expression vectors for Gal–h-Alien (1 pmol), a fusion of full-length h-Alien to the Gal4 DBD (aa 1 to 94), and TRα (2 pmol) were tested for hormone-dependent effects of the TR-Alien interaction with the reporter pUAS_6× tkCAT in CV1 cells (1 pmol) (mammalian one-hybrid). As controls, an empty expression vector (lane C), the Gal4 DBD alone, and RARα were used.

FIG. 8

Alien interacts with SIN3A but not with the SMRT/N-CoR class of corepressors. (A) Full-length h-Alien was tested for interaction with full-length SMRT (SMRT f.l.), the receptor interaction domain of N-CoR (N-CoR i.d.), or SIN3A. Yeast two-hybrid assays were done as in the experiment in Fig. 1. (B) Coimmunoprecipitation of endogenous h-Alien with SIN3A. HeLa cells were used for immunoprecipitation with the anti-Alien peptide antibody. Western blotting was performed with the anti-SIN3A antibody. Protein A-Sepharose alone and anti-GST antibody were used as negative controls. As a migration control, HeLa extract was loaded. (C) The histone deacetylase inhibitor TSA decreases the silencing activity of Alien. Cotransfection experiments were performed with the Gal-Alien expression vector and the UAS_6× tkCAT reporter in CV1 cells. TSA (100 ng/ml) was added 8 h prior to cell harvest. Gal-N-CoR was used as a positive control.