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. 2001 Sep;21(18):6091-101.
doi: 10.1128/MCB.21.18.6091-6101.2001.

The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3

M G Guenther  1 , O Barak, M A Lazar
Affiliations

The SMRT and N-CoR corepressors are activating cofactors for histone deacetylase 3

M G Guenther et al. Mol Cell Biol. 2001 Sep.

Abstract

Repression of gene transcription is linked to regulation of chromatin structure through deacetylation of core histone amino-terminal tails. This action is mediated by histone deacetylases (HDACs) that function within active multiprotein complexes directed to the promoters of repressed genes. In vivo, HDAC3 forms a stable complex with the SMRT corepressor. The SMRT-HDAC3 complex exhibits histone deacetylase activity, whereas recombinant HDAC3 is an inactive enzyme. Here we report that SMRT functions as an activating cofactor of HDAC3. In contrast, SMRT does not activate the class II HDAC4, with which it also interacts. Activation of HDAC3 is mediated by a deacetylase activating domain (DAD) that includes one of two SANT motifs present in SMRT. A cognate DAD is present in the related corepressor N-CoR, which can also activate HDAC3. Mutations in the DAD that abolish HDAC3 interaction also eliminate reconstitution of HDAC activity. Using purified components, the SMRT DAD is shown to be necessary and sufficient for activation of HDAC3. Moreover, the DAD is required both for HDAC3 to function enzymatically and for the major repression function of SMRT. Thus, SMRT and N-CoR do not serve merely as platforms for HDAC recruitment but function as an integral component of an active cellular HDAC3 enzyme.

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Figures

FIG. 1
FIG. 1
SMRT activates HDAC3 deacetylase activity in vitro. (a) SMRT core complex components were produced by T7-coupled transcription-translation. Myc-HDAC3 alone or mixed together with Gal4-TBL1 and/or Flag-SMRT was immunoprecipitated with anti-Myc agarose beads to isolate HDAC complexes, and the beads were assayed for HDAC activity. Immunoprecipitation of unprogrammed transcription-translation mix was used as a control. (b) Western blot analysis of the complexes in panel a. The upper panel was probed for SMRT (anti-Flag), the middle panel was probed for TBL1 (anti-Gal4), and the lower panel was probed for HDAC3 (anti-Myc). IP, immunoprecipitation.
FIG. 2
FIG. 2
SMRT binds but does not activate HDAC4. (a) Western blot analysis of HDAC complexes formed in the presence or absence of SMRT. Proteins produced by T7-coupled transcription-translation were mixed and immunoprecipitated (IP) with anti-Myc agarose beads. All samples were resoved by SDS-PAGE and subjected to immunoblotting. Flag-SMRT (top) was probed with anti-Flag. Myc-HDAC4 and Myc-HDAC3 (middle and bottom) were probed with anti-Myc. (b) HDAC activity of the complexes in panel a. Note that 50 μl of translated HDAC3 and 100 μl of HDAC4 were used.
FIG. 3
FIG. 3
SMRT contains a DAD. (a) Schematic representation of the SMRT corepressor and deletion mutants used to reconstitute HDAC3 activity. Gal4 DBD fusions to SMRT contained SMRT amino acids 1 to 2473 (full-length SMRT), SMRT amino acids 1961 to 2473 (fragment A), SMRT amino 1531 to 1959 (fragment B), SMRT amino acids 1068 to 1530 (fragment C), SMRT amino acids 1 to 1028 (fragment D), SMRT amino acids 1 to 763 (fragment E), SMRT amino acids 305 to 763 (fragment F), and SMRT amino acids 427 to 559 (fragment G). (b) HDAC activities of HDAC3-containing complexes. Myc-HDAC3 was incubated with equal amounts of Gal4-SMRT fusion proteins, immunoprecipitated with anti-Myc, and subjected to HDAC assay. (c) Immunoblot (IB) of anti-Myc agarose immunoprecipitated (IP) complexes. The upper panel was probed for SMRT polypeptides (anti-Gal4); the lower panel was probed for HDAC3 (anti-Myc).
FIG. 4
FIG. 4
The SMRT DAD is comprised of SANT1 and its amino-terminal flank. (a) Schematic representation of SMRT amino acids 305 to 763 containing SANT1 and SANT2. Gal4 DBD–SMRT fusions contained amino acids 305 to 763 (F), amino acids 427 to 669 (F1), amino acids 305 to 559 (F2), amino acids 305 to 489 (F3), amino acids 305 to 427 (F4), amino acids 305 to 455 (F5), amino acids 395 to 489 (F6 [DAD]), and amino acids 427 to 489 (F7). (b) HDAC activities of HDAC3-containing complexes. Myc-HDAC3 was incubated with equal amounts of Gal4-SMRT fusions, immunoprecipitated with anti-Myc, and subjected to HDAC assay. (c) Immunoblot (IB) of anti-Myc-precipitated complexes. The upper panel was probed for SMRT polypeptides (anti-Gal4), and the lower panel was probed for HDAC3 (anti-Myc). IP, immunoprecipitation.
FIG. 5
FIG. 5
N-CoR corepressor binds and activates HDAC3. (a) Alignment of the SMRT DAD with homologous regions of the corepressors N-CoR and SMRTER. Residues conserved in at least two of the three proteins are shown on black background. (b) Myc-HDAC3 alone or mixed with either Flag-SMRT or Flag–N-CoR was immunoprecipitated with anti-Myc and subjected to HDAC assay. Immunoprecipitation of an unprogrammed transcription-translation mix was used as a control. (c) Immunoblot (IB) of HDAC3 complexes immunoprecipitated (IP) by anti-Myc. The upper panel was probed for SMRT or N-CoR (anti-Flag); the lower panel was probed for HDAC3 (anti-Myc). (d) Myc-HDAC3 alone or mixed with either MTA1 or Flag-SMRT was immunoprecipitated with anti-Myc and subjected to HDAC assay. (e) Immunoblot analysis of the Myc-HDAC3 immunoprecipitates in panel d. The upper panel was probed with anti-MTA1, the middle panel was probed for SMRT (anti-Flag), and the lower panel was probed for HDAC3 (anti-Myc). Five-percent inputs are shown on the left, and all proteins were produced by T7-coupled transcription-translation.
FIG. 6
FIG. 6
HDAC3 activation correlates with SMRT DAD binding. Myc-HDAC3 was mixed with wild-type (WT) Gal4-DAD (Gal4 DBD fusion to SMRT amino acids 395 to 489) or the point mutant W432A, F440A, F451A, I454A, L458A, V463A, or Y470A. HDAC3 complexes were immunoprecipitated by anti-Myc agarose beads. (a) Immunoblot analysis of anti-Myc-precipitated complexes. The upper panel was probed for HDAC3 (anti-Myc), the middle panel was probed for associated SMRT DAD proteins (anti-Gal4), and the lower panel shows 5% inputs of SMRT DAD proteins. (b) HDAC-activating abilities of anti-Myc-precipitated complexes.
FIG. 7
FIG. 7
HDAC3 interaction with SMRT. SMRT 1-763 fused to Gal4 DBD was mixed with wild-type (WT) or mutant Myc-HDAC3 (Myc-HDAC3 amino acids 24 to 428, 78 to 428, or 1 to 390), and HDAC3 complexes were immunoprecipitated (IP) by anti-Myc agarose beads. (a) Immunoblot analysis of HDAC3 complexes. HDAC3 deletion mutants were visualized by anti-Myc, and SMRT polypeptides were probed with anti-Gal4. Inputs are shown on the left. (b) HDAC assay of immunocomplexes. Note that 50 μl of full-length HDAC3 translation mix and 100 μl of all translated HDAC3 mutants were used to normalize protein levels.
FIG. 8
FIG. 8
Purification of an enzymatically active HDAC3-SMRT complex. (a) Coomassie stain (top) of purified HDAC3 complex. Sf9 cells were mock infected (control) or infected with baculovirus expressing Flag-HDAC3, Myc-SMRT amino acids 1 to 587, or both. Sf9 lysates were purified by anti-Flag affinity chromatography and resolved by SDS-PAGE. Molecular weight markers (MW) and major polypeptides are indicated. Identities of complex components were verified by immunoblotting with anti-Myc and Flag-HDAC3 (below the Coomassie stain). (b) HDAC activities of the proteins in panel a.
FIG. 9
FIG. 9
The SMRT DAD is required for repression and deacetylase activity. (a) Repression of a Gal4-SV40-luciferase reporter by Gal4-SMRT 1-763 or Gal4-SMRT 1-763Δ DAD. Fold repression is expressed relative to Gal4 DBD. (b) In vitro HDAC activity of Myc-HDAC3 incubated with either Gal4 DBD, Gal4–SMRT 1-763, or Gal4–SMRT 1-763Δ DAD. HDAC3 complexes were immunoprecipitated (IP) with anti-Myc, and HDAC activity was expressed as the amount of acetate released. (c) HDAC activity of Gal4 DBD, Gal4–SMRT 1-763, or Gal4–SMRT 1-763Δ DAD immunoprecipitated by anti-Gal4 agarose after transfection into 293T cells. (d) Immunoblot (IB) of anti-Gal4 immunoprecipitates from 293T cells transfected with Gal4 DBD, Gal4–SMRT 1-763, or Gal4–SMRT 1-763Δ DAD. Inputs (2%) are shown on the left for comparison. The upper panels were probed for endogenous HDAC3 (anti-HDAC3), and the lower panels were probed for Gal4 DBD and Gal4-SMRT polypeptides (anti-Gal4).
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