Purification, identification, and verification of SMRT complex components. (a) Pooled SMRT carboxy-terminal monoclonal antibodies are not cross-reactive with N-CoR. In vitro-translated SMRT, N-CoR, or unprogrammed rabbit reticulocyte lysate (RRL) control were subjected to immunoblot analysis with five pooled SMRT monoclonal antibodies or an anti-N-CoR monoclonal antibody. (b) Strategy to obtain SMRT-associated polypeptides. (c) SDS-PAGE and silver-staining analysis of SMRT complex purified as in b from HeLa nuclear extract. SMRT complex components are indicated by arrows. Asterisk denotes a band that was not reproducibly observed in eluates from the SMRT column. (d) Identity of SMRT complex subunits. Peptide sequences obtained by microsequencing are underlined. (e) Immunoblot analysis of SMRT complexes purified independently. Components isolated from nuclear extract or as in b were verified by immunoblot with anti-SMRT, mouse anti-TBL1, anti-HDAC3, anti-Sin3A, and anti-HDAC1. (f) HDAC assay of SMRT purified as in b.

Copurification of SMRT, HDAC3, and TBL1. (a) Multistep purification scheme. (b) Protein elution from Superose 6 column. (c) Immunoblot analysis of Superose 6 column fractions. Fractions were assayed by immunoblot with anti-SMRT, rabbit anti-TBL1, and anti-HDAC3.

In vivo characterization of the SMRT–TBL1–HDAC3 complex. (a) TBL1 and HDAC3 can each associate with SMRT. Extracts of 293T cells transfected with either Myc–TBL1 or Flag–HDAC3 were immunoprecipitated with anti-myc or anti-flag prior to immunoblot with SMRT monoclonal antibody. All inputs represent 1% of each total immunoprecipitation. (b) HDAC3 interacts with both TBL1 and SMRT in vivo. Extracts of 293T cells transfected with Myc–TBL1 and Flag–HDAC3 were immunoprecipitated with anti-flag prior to immunoblot with SMRT or myc polyclonal antibody. (c) TBL1 associates with histone deacetylase enzymatic activity. 293T cells were transfected with either myc-TBL1 or empty vector alone and subjected to immunoprecipitation with anti-myc followed by assay for deacetylase activity.

SMRT–TBL1–HDAC3 interactions in vitro. (a) Schematic representation of the SMRT corepressor indicating repression domains RD1, RD2, and RD3 and nuclear hormone receptor interaction domains ID1 and ID2. (b) GST-pulldown assay using GST fusions of SMRT RD1 [amino acids 1–303 of the full-length SMRT protein (Ordentlich et al. 1999)], SMRT RD2 (amino acids 763–1028), and SMRT RD3 (amino acids 1043–1514) with ³⁵S-labeled HDAC3 and TBL1. (c) Full-length SMRT interacts with HDAC3 in vitro. ³⁵S-labeled HDAC3 was incubated with control (unprogrammed) RRL (lanes 2,3) or unlabeled full-length SMRT translated in RRL (lanes 4,5) and subjected to immunoprecipitation with control IgG or SMRT monoclonal antibody pool. Input lane shows 1% of total. (d) The amino terminus of TBL1 interacts with N-CoR and SMRT. GST-pulldown assay using GST fusions of TBL1 full-length(1–577), amino-terminus (1–211), and carboxy-terminal WD40-repeat domain (211–577) with ³⁵S-labeled SMRT and N-CoR. (e) The SMRT–TBL1–HDAC3 interaction can be recapitulated in vitro. GST-pulldown assay using GST fusion to full-length TBL1 with ³⁵S-labeled HDAC3 in the presence of control RRL (lanes 2,3) or unlabeled SMRT translated in RRL (lanes 4,5).

Role of TBL1 in repression. (a) TBL1 potentiates repression by thyroid hormone receptor. A Gal4–TK-luciferase reporter was cotransfected with either Gal4 DBD, Gal4–TR, or Gal4–TR(V230R) and increasing amounts (black ramps representing 0.25–1.5 μg of DNA) of pCMX–HA–TBL1 into HeLa cells. Fold repression was measured as relative to Gal4 DBD alone and the results of duplicate samples are shown. (b) SMRT recruits TBL1 to TR. GST-pulldown assay using GST fusion to TRβ ligand-binding domain with ³⁵S-labeled TBL1 in the presence of control RRL (lanes 2,3) or unlabeled SMRT translated in RRL (lanes 4,5). (c) TBL1 contains an autonomous repression domain. Gal4–TBL1(1–577), Gal4–TBL1(1–211), and Gal4–TBL1(211–577) were transfected with the Gal4–TK-luciferase reporter and fold repression was measured relative to Gal4 DBD alone. (d) GST–TBL1 interacts with histone H3. HeLa Nuclear extract was incubated with either GST alone or GST–TBL1 and interacting histone H3 visualized by immunoblot.

Role of the core SMRT complex in repression. A model for SMRT-mediated repression indicating the core SMRT–HDAC3–TBL1 complex, with potential repression mechanisms including (1) HDAC activity via HDAC3, (2) Tup1/Groucho-like functions mediated by TBL1, and (3) interactions between SMRT and general transcription factors. These repression functions may be augmented by additional interactions with the mSin3/HDAC1 complex, as well as HDAC4, HDAC5, and HDAC7.