The N-CoR/histone deacetylase 3 complex is required for repression by thyroid hormone receptor

Abstract

Nuclear receptor corepressors (N-CoR) and silencing mediator for retinoid and thyroid receptors (SMRT) have both been implicated in thyroid hormone receptor (TR)-mediated repression. Here we show that endogenous N-CoR, TBL1, and histone deacetylase 3 (HDAC3), but not HDAC1, -2, or -4, are recruited to a stably integrated reporter gene repressed by unliganded TR as well as the orphan receptor RevErb. Unliganded TR also recruits this complex to a transiently transfected reporter, and transcriptional repression is associated with local histone deacetylation that is reversed by the presence of thyroid hormone. Knockdown of N-CoR using small interfering RNAs markedly reduces repression by the TR ligand binding domain in human 293T cells, whereas knockdown of SMRT has little effect. RevErb repression appears to involve both corepressors in this system. Knockdown of HDAC3 markedly reduces repression by both TR and RevErb, while knockdown of HDAC1 or 2 has more modest, partly nonspecific effects. Thus, HDAC3 is critical for repression by multiple nuclear receptors and the N-CoR HDAC3 complex plays a unique and necessary role in TR-mediated gene repression in human 293T cells.

FIG. 1.

Unliganded TR specifically recruits N-CoR, HDAC3, and TBL1 to a repressed, stably integrated target gene. (a) Transcription assay. A (Gal4 × 5)-TK-luciferase plasmid was stably transfected into 293T cells as described in Materials and Methods. Effects of transfected Gal4 DBD or DBD fusion to the TRβ LBD (in the presence or absence of T3) are shown. Results are plotted as repression levels. (b) ChIP analysis of the experiment shown in panel a. (c) ChIP analysis of cells with stably integrated reporters transfected with a Gal4 DBD alone or fused to HDAC1 or HDAC3. Control IgG, control immunoglobulin G.

FIG. 2.

RevErb specifically recruits N-CoR, HDAC3, and TBL1 to a repressed, stably integrated target gene. (a) Transcription assay. The stable 293T cell line containing the (Gal4 × 5)-TK-luciferase reporter was transfected with Gal4 DBDs either alone or fused to RevErb LBD. Results are plotted as repression levels. (b) ChIP analysis of the experiment shown in panel a.

FIG. 3.

Unliganded TR specifically recruits N-CoR and HDAC3 to a repressed, transiently transfected reporter gene. (a) Transcription assay. The (Gal4 × 5)-TK-luciferase reporter was transiently transfected with pCMX or pCMX-Gal4-TR LBDs into 293T cells. (b) ChIP analysis of the experiment shown in panel a. IgG, immunoglobulin G. (c) T3 dose response.

FIG. 4.

Knockdown of N-CoR but not SMRT reduces repression by TR. (a) Knockdown using SiRNA for N-CoR and SMRT. Immunoblot for N-CoR, SMRT, and HDAC2 (Control) after transfecting 293T cells with SiRNA for N-CoR or SMRT. (b) Transcription assay. The (Gal4 × 5)-SV40-luciferase reporter was transiently transfected along with the Gal4 DBD, Gal4-TRα, Gal4-TRβ, or Gal4-Mad into 293T cells treated with SiRNA for N-CoR or SMRT as in panel a.

FIG. 5.

SMRT contributes to repression by RevErb. (a) The (Gal4 × 5)-SV40-luciferase reporter was transiently transfected along with the Gal4 DBD, Gal4-TRα, Gal4-TRβ, or Gal4-RevErb into 293T cells treated with SiRNA for N-CoR or SMRT or both. (b) Knockdown of SMRT. (c) SMRT-B SiRNA attenuates repression by RevErb but not TR.

FIG. 6.

Knockdown of HDAC3 markedly reduces repression by TR and RevErb. (a) Knockdown using SiRNA for HDAC1, -2, and -3. Shown are immunoblots for HDAC1 to -3 and HDAC4 (Control) after transfecting 293T cells with SiRNA for HDAC1, -2, and -3. (b) Transcription assay. The (Gal4 × 5)-TK-luciferase reporter was transiently transfected along with Gal4 DBD, Gal4-TRβ, or Gal4-RevErb into 293T cells treated with SiRNA for HDAC1 to -3 as described for panel a.