Characterization of SMRT^mRID mice. (A) Schematic of SMRT functional domains. (B) Mammalian 2-hybrid experiment showing interaction of wt and SMRT^mRID Gal4-SMRT and indicated VP16-NHR-LBD fusion proteins in CV-1 cells. (C) Reduced body weights in SMRT^mRID mice compared with wt littermates are observed from 8 to 24 weeks of age. (D) H&E-stained sections of SMRT^mRID brains in P0 and adult mice reveal no apparent defects. Corpus callosum (cc) and anterior commissure (aca, acp) are indicated. (E) H&E-stained sections of SMRT^mRID heart reveal a thinner ventricular wall (boxed area shown magnified in lower left corner) at P0 compared with wt. By 4 months of age, SMRT^mRID hearts exhibit normal ventricular wall thickness.

SMRT^mRID mutant mice exhibit perturbed metabolic phenotype. (A) SMRT^mRID mice (10 weeks old, n = 6) display reduced heat production, O₂ consumption, CO₂ production, and respiratory exchange ratios (RERs). (B) Attenuated increase of serum cholesterol in SMRT^mRID mice [2–3 months old, n = 7 (wt) and 5 (SMRT^mRID)] in PTU/MMI-induced hypothyroidism. (C) Derepression of TR target genes in PTU/MMI-treated SMRT^mRID livers. Pituitary TSHα and TSHβ mRNA expression (D) and serum TSH levels (E) are normal in untreated and PTU/MMI-treated conditions. (F) Glucose tolerance test revealed increased fasting blood glucose and intolerance to i.p. glucose injection in SMRT^mRID mice compared with controls (8 weeks old, n = 12). (G) Hyperinsulinemic-euglycemic clamping on chow-fed 4-month-old, weight-matched littermates (n = 7) revealed hepatic glucose production (HGP) was higher at baseline in SMRT^mRID animals, but suppressed to similar levels after insulin injection. Insulin-stimulated glucose disposal was significantly diminished. Free fatty acid levels were not changed. (H) Increased epididymal fat pad (EFP) to body weight ratio in SMRT^mRID as compared with WT (n = 6) on high-fat diet (HFD). Body composition analysis (n = 10) confirms an increased percentage of SMRT^mRID dry weight is composed of lipids. (I) Serum adiponectin levels are elevated in SMRT^mRID mice at 2.5 (n = 13) and 3.5 months (n = 9) of age. *, significant differences with P values <0.05 by using the student t test. Standard error of the mean (SEM) is shown unless otherwise indicated.

SMRT^mRID MEFs exhibit enhanced adipogenic potential. (A) Postconfluent SMRT^mRID-derived MEFs after a 7-day exposure to DC. Oil Red O staining revealed nearly 100% differentiation of SMRT^mRID cells into mature adipocytes compared with wt (5%). Spontaneous adipocyte differentiation was seen in ≈10% of untreated SMRT^mRID cells. (B) Increased expression of PPARγ and PPARγ-target genes in SMRT^mRID MEFs during differentiation by using QPCR analysis. Expression levels under described experimental conditions are compared over time (indicated as days relative to addition of DC). (C) Lentiviral shRNA-knockdown of PPARγ expression abolished the adipogenic potential of SMRT^mRID MEFs as demonstrated by Oil Red O. (D) ChIP analysis of the aP2 promoter in the proximity of the PPRE revealed reduced SMRT occupancy and increased histone H3 acetylation. Immunoprecipitation was performed by using control IgG or antibodies against SMRT, PPARγ and acetyl-H3. (E) QPCR analysis of WAT from SMRT^mRID and wt mice that were treated with vehicle or Rosi for 5 days. Brackets with asterisks indicate P < 0.05.

Model for in vivo function of SMRT-mediated transcriptional repression. The corepressor SMRT is recruited by a wide variety of transcription factors, including NHRs, to mediate lineage- and signal-dependent transcriptional repression. In the standard model of NHR action, ligand binding is believed to actuate a bifunctional molecular switch, releasing the corepressor complex and recruiting a histone acetyltransferase coactivator complex, resulting in potent transcriptional activation of gene targets. Whole-body knockout of SMRT results in developmental defects and embryonic lethality. In contrast, mice with mutations that specifically disrupt SMRT interaction with NHRs are viable but display widespread metabolic defects, demonstrating that the lethality of SMRT knockout mice is mediated by non-NHR transcription factors. Derepression of SMRT enhances NHR sensitivity to ligand.