SMRT Regulates Metabolic Homeostasis and Adipose Tissue Macrophage Phenotypes in Tandem.

The Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) is a nuclear corepressor, regulating the transcriptional activity of many transcription factors critical for metabolic processes. While the importance of the role of SMRT in the adipocyte has been well-established, our comprehensive understanding of its in vivo function in the context of homeostatic maintenance is limited due to contradictory phenotypes yielded by prior generalized knockout mouse models. Multiple such models agree that SMRT deficiency leads to increased adiposity, although the effects of SMRT loss on glucose tolerance and insulin sensitivity have been variable. We therefore generated an adipocyte-specific SMRT knockout (adSMRT-/-) mouse to more clearly define the metabolic contributions of SMRT. In doing so, we found that SMRT deletion in the adipocyte does not cause obesity-even when mice are challenged with a high-fat diet. This suggests that adiposity phenotypes of previously described models were due to effects of SMRT loss beyond the adipocyte. However, an adipocyte-specific SMRT deficiency still led to dramatic effects on systemic glucose tolerance and adipocyte insulin sensitivity, impairing both. This metabolically deleterious outcome was coupled with a surprising immune phenotype, wherein most genes differentially expressed in the adipose tissue of adSMRT-/- mice were upregulated in pro-inflammatory pathways. Flow cytometry and conditioned media experiments demonstrated that secreted factors from knockout adipose tissue strongly informed resident macrophages to develop a pro-inflammatory, MMe (metabolically activated) phenotype. Together, these studies suggest a novel role for SMRT as an integrator of metabolic and inflammatory signals to maintain physiological homeostasis.

Nuclear corepressor SMRT is a strong regulator of body weight independently of its ability to regulate thyroid hormone action.

Silencing Mediator of Retinoid and Thyroid Hormone Receptors (SMRT) and the nuclear receptor co-repressor1 (NCoR1) are paralogs and regulate nuclear receptor (NR) function through the recruitment of a multiprotein complex that includes histone deacetylase activity. Previous genetic strategies which deleted SMRT in a specific tissue or which altered the interaction between SMRT and NRs have suggested that it may regulate adiposity and insulin sensitivity. However, the full role of SMRT in adult mice has been difficult to establish because its complete deletion during embryogenesis is lethal. To elucidate the specific roles of SMRT in mouse target tissues especially in the context of thyroid hormone (TH) signaling, we used a tamoxifen-inducible post-natal disruption strategy. We found that global SMRT deletion causes dramatic obesity even though mice were fed a standard chow diet and exhibited normal food intake. This weight gain was associated with a decrease in energy expenditure. Interestingly, the deletion of SMRT had no effect on TH action in any tissue but did regulate retinoic acid receptor (RAR) function in the liver. We also demonstrate that the deletion of SMRT leads to profound hepatic steatosis in the setting of obesity. This is unlike NCoR1 deletion, which results in hepatic steatosis due to the upregulation of lipogenic gene expression. Taken together, our data demonstrate that SMRT plays a unique and CoR specific role in the regulation of body weight and has no role in TH action. This raises the possibility that additional role of CoRs besides NCoR1 and SMRT may exist to regulate TH action.

Corepressor SMRT is required to maintain Hox transcriptional memory during somitogenesis.

Nuclear hormone receptors (NRs), such as retinoic acid receptors (RARs), play critical roles in vertebrate development and homeostasis by regulating target gene transcription. Their activity is controlled by ligand-dependent release of corepressors and subsequent recruitment of coactivators, but how these individual receptor modes contribute to development are unknown. Here, we show that mice carrying targeted knockin mutations in the corepressor Silencing Mediator of Retinoid and Thyroid hormone receptor (SMRT) that specifically disable SMRT function in NR signaling (SMRTmRID), display defects in cranial neural crest cell-derived structures and posterior homeotic transformations of axial vertebrae. SMRTmRID embryos show enhanced transcription of RAR targets including Hox loci, resulting in respecification of vertebral identities. Up-regulated histone acetylation and decreased H3K27 methylation are evident in the Hox loci whose somitic expression boundaries are rostrally shifted. Furthermore, enhanced recruitment of super elongation complex is evident in rapidly induced non-Pol II-paused targets in SMRTmRID embryonic stem cells. These results demonstrate that SMRT-dependent repression of RAR is critical to establish and maintain the somitic Hox code and segmental identity during fetal development via epigenetic marking of target loci.

Perspective on unraveling the versatility of 'co-repressor' complexes.

A multitude of post-translational modifications take place on histones, one of the best studied being acetylation on lysine residues, which is generally associated with gene activation. During the last decades, several so-called co-repressor protein complexes that carry out the reverse process, histone deacetylation, have been identified and characterized, such as the Sin3, N-CoR/SMRT and NuRD complexes. Although a repressive role for these complexes in regulating gene expression is well established, accumulating evidence also points to a role in gene activation. Here, we argue that integration of various state-of-the-art technologies, addressing different aspects of transcriptional regulation, is essential to unravel this apparent biological versatility of 'co-repressor' complexes.

AT1R blockade in adverse milieus: role of SMRT and corepressor complexes.

ANG II type 1 receptor blockade (AT1R-BLK) is used extensively to slow down the progression of proteinuric kidney diseases. We hypothesized that AT1R-BLK provides podocyte protection through regulation of silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) and vitamin D receptor (VDR) expression under adverse milieus such as high glucose and human immunodeficiency virus infection. Both AT1R-BLK and VDR agonists (VDAs) stimulated VDR complex formation that differed not only in their composition but also in their functionality. AT1R-BLK-induced VDR complexes contained predominantly unliganded VDR, SMRT, and phosphorylated histone deacetylase 3, whereas VDA-VDR complexes were constituted by liganded VDR and CREB-binding protein/p300. AT1R-BLK-induced complexes attenuated podocyte acetyl-histone 3 levels as well as cytochrome P-450 family 24A1 expression, thus indicating their deacetylating and repressive properties. On the other hand, VDA-VDR complexes not only increased podocyte acetyl-histone 3 levels but also enhanced cytochrome P-450 family 24A1 expression, thus suggesting their acetylating and gene activation properties. AT1R-BLK- induced podocyte SMRT inhibited expression of the proapoptotic gene BAX through downregulation of Wip1 and phosphorylation of checkpoint kinase 2 in high-glucose milieu. Since SMRT-depleted podocytes lacked AT1R-BLK-mediated protection against DNA damage, it appears that SMRT is necessary for DNA repairs during AT1R-BLK. We conclude that AT1R-BLK provides podocyte protection in adverse milieus predominantly through SMRT expression and partly through unliganded VDR expression in 1,25(OH)2D-deficient states; on the other hand, AT1R-BLK contributes to liganded VDR expression in 1,25(OH)2D-sufficient states.

Growth factor receptor/steroid receptor cross talk in trastuzumab-treated breast cancer.

Treatment with tyrosine kinase inhibitors (TKIs) including trastuzumab has revolutionized the management of HER2-positive breast cancer. Recent evaluation of clinical trial data suggests that a subset of HER2/ER double-positive cancers may not receive significant benefit from the TKI therapy. Here we investigate the cross talk between HER2 and ER in breast cancer and monitor the effect of trastuzumab on the tyrosine kinase effector transcription factor Myc. In HER2-positive breast cancer patients treated with neoadjuvant trastuzumab, steroid receptor-negative status (ER and PR negative) of pre-treatment biopsies predicted pathological complete response (pCR) (n=31 patients, P=0.0486), whereas elevated Myc protein inversely associated with pCR (P=0.0446). Liquid chromatography mass spectrometry identified the corepressor SMRT as a novel Myc-interacting protein. Trastuzumab treatment enhanced Myc-SMRT interactions in HER2-overexpressing breast cancer cells (LCC1) and inhibited expression of the Myc target gene survivin. In HER2-low, ER-positive steroid-dominant cells (MCF7), trastuzumab therapy repressed Myc-SMRT interactions and upregulated survivin expression. Trastuzumab treatment induced ER-CBP interactions, enhanced ER transcriptional activity and upregulated expression of the ER target gene pS2. The absence of pS2 expression in pre-treatment biopsies predicted pCR to neoadjuvant trastuzumab in breast cancer patients (n=25, P=0.0089) and pS2 expression associated with residual cancer burden (P=0.0196). Furthermore, metastatic tissues from patients who had failed trastuzumab therapy were pS2 positive. In HER2-overexpressing cells, trastuzumab treatment can repress Myc transcriptional activity and clinical response is favorable. However, with co-expression of the steroid pathway, this inhibition is lost and response to treatment is often poor.

Ncor2 is required for hematopoietic stem cell emergence by inhibiting Fos signaling in zebrafish.

Nuclear receptor corepressors (Ncors) are important for developmental and homeostatic processes in vertebrates, which exert transcriptional repression by coordinating with histone deacetylases. However, little is known about their roles in definitive hematopoiesis. In this study, we show that in zebrafish, ncor2 is required for hematopoietic stem cell (HSC) development by repressing fos-vegfd signaling. ncor2 is specifically expressed in the aorta-gonad-mesonephros (AGM) region in zebrafish embryos. ncor2 deficiency reduced the population of HSCs in both the AGM region and T cells in the thymus. Mechanistically, ncor2 knockdown upregulated fos transcription by modulating the acetylation level in the fos promoter region, which then enhanced Vegfd signaling. Consequently, the augmented Vegfd signaling induced Notch signaling to promote the arterial endothelial fate, therefore, possibly repressing the hemogenic endothelial specification, which is a prerequisite for HSC emergence. Thus, our findings identify a novel regulatory mechanism for Ncor2 through Fos-Vegfd-Notch signaling cascade during HSC development in zebrafish embryos.

Nuclear translocation of MEK1 triggers a complex T cell response through the corepressor silencing mediator of retinoid and thyroid hormone receptor.

MEK1 phosphorylates ERK1/2 and regulates T cell generation, differentiation, and function. MEK1 has recently been shown to translocate to the nucleus. Its nuclear function is largely unknown. By studying human CD4 T cells, we demonstrate that a low level of MEK1 is present in the nucleus of CD4 T cells under basal conditions. T cell activation further increases the nuclear translocation of MEK1. MEK1 interacts with the nuclear receptor corepressor silencing mediator of retinoid and thyroid hormone receptor (SMRT). MEK1 reduces the nuclear level of SMRT in an activation-dependent manner. MEK1 is recruited to the promoter of c-Fos upon TCR stimulation. Conversely, SMRT is bound to the c-Fos promoter under basal conditions and is removed upon TCR stimulation. We examined the role of SMRT in regulation of T cell function. Small interfering RNA-mediated knockdown of SMRT results in a biphasic effect on cytokine production. The production of the cytokines IL-2, IL-4, IL-10, and IFN-γ increases in the early phase (8 h) and then decreases in the late phase (48 h). The late-phase decrease is associated with inhibition of T cell proliferation. The late-phase inhibition of T cell activation is, in part, mediated by IL-10 that is produced in the early phase and, in part, by ß-catenin signaling. Thus, we have identified a novel nuclear function of MEK1. MEK1 triggers a complex pattern of early T cell activation, followed by a late inhibition through its interaction with SMRT. This biphasic dual effect most likely reflects a homeostatic regulation of T cell function by MEK1.

Integrative genomics identifies the corepressor SMRT as a gatekeeper of adipogenesis through the transcription factors C/EBPß and KAISO.

The molecular role of corepressors is poorly understood. Here, we studied the transcriptional function of the corepressor SMRT during terminal adipogenesis. Genome-wide DNA-binding profiling revealed that this corepressor is predominantly located in active chromatin regions and that most distal SMRT binding events are lost after differentiation induction. Promoter-proximal tethering of SMRT in preadipocytes is primarily mediated by KAISO through the conserved TCTCGCGAGA motif. Further characterization revealed that KAISO, similar to SMRT, accelerates the cell cycle and increases fat accumulation upon knockdown, identifying KAISO as an adipogenic repressor that likely modulates the mitotic clonal expansion phase of this process. SMRT-bound promoter-distal sites tend to overlap with C/EBPß-bound regions, which become occupied by proadipogenic transcription factors after SMRT clearance. This reveals a role for SMRT in masking enhancers from proadipogenic factors in preadipocytes. Finally, we identified SMRT as an adipogenic gatekeeper as it directly fine-tunes transcription of pro- and antiadipogenic genes.

Corepressor SMRT promotes oxidative phosphorylation in adipose tissue and protects against diet-induced obesity and insulin resistance.

The ligand-dependent competing actions of nuclear receptor (NR)-associated transcriptional corepressor and coactivator complexes allow for the precise regulation of NR-dependent gene expression in response to both temporal and environmental cues. Here we report the mouse model termed silencing mediator of retinoid and thyroid hormone receptors (SMRT)(mRID1) in which targeted disruption of the first receptor interaction domain (RID) of the nuclear corepressor SMRT disrupts interactions with a subset of NRs and leads to diet-induced superobesity associated with a depressed respiratory exchange ratio, decreased ambulatory activity, and insulin resistance. Although apparently normal when chow fed, SMRT(mRID1) mice develop multiple metabolic dysfunctions when challenged by a high-fat diet, manifested by marked lipid accumulation in white and brown adipose tissue and the liver. The increased weight gain of SMRT(mRID1) mice on a high-fat diet occurs predominantly in fat with adipocyte hypertrophy evident in both visceral and s.c. depots. Importantly, increased inflammatory gene expression was detected only in the visceral depots. SMRT(mRID1) mice are both insulin-insensitive and refractory to the glucose-lowering effects of TZD and AICAR. Increased serum cholesterol and triglyceride levels were observed, accompanied by increased leptin and decreased adiponectin levels. Aberrant storage of lipids in the liver occurred as triglycerides and cholesterol significantly compromised hepatic function. Lipid accumulation in brown adipose tissue was associated with reduced thermogenic capacity and mitochondrial biogenesis. Collectively, these studies highlight the essential role of NR corepressors in maintaining metabolic homeostasis and describe an essential role for SMRT in regulating the progression, severity, and therapeutic outcome of metabolic diseases.

Differential recruitment of nuclear receptor coregulators in ligand-dependent transcriptional repression by estrogen receptor-α.

Estrogen receptors (ERs) are normally expressed in breast tissues and mediate hormonal functions during development and in female reproductive physiology. In the majority of breast cancers, ERs are involved in regulating tumor cell proliferation and serve as prognostic markers and therapeutic targets in the management of hormone-dependent tumors. At the molecular level, ERs function as ligand-dependent transcription factors and activate target-gene expression following hormone stimulation. Recent transcriptomic and whole-genome-binding studies suggest, however, that ligand-activated ERs can also repress the expression of a significant subset of target genes. To characterize the molecular mechanisms of transcriptional repression by ERs, we examined recruitment of nuclear receptor coregulators, histone modifications and RNA polymerase II docking at ER-binding sites and cis-regulatory regions adjacent to repressed target genes. Moreover, we utilized gene expression data from patient samples to determine potential roles of repressed target genes in breast cancer biology. Results from these studies indicate that nuclear receptor corepressor recruitment is a key feature of ligand-dependent transcriptional repression by Ers, and some repressed target genes are associated with disease progression and response to endocrine therapy. These findings provide preliminary insights into a novel aspect of the molecular mechanisms of ER functions and their potential roles in hormonal carcinogenesis and breast cancer biology.

Aberrant expression and modification of silencing mediator of retinoic acid and thyroid hormone receptors involved in the pathogenesis of tumoral cortisol resistance.

Ectopic ACTH syndrome (EAS) accounts for 10-15% of cases of Cushing's syndrome and is mostly caused by small cell lung cancers or thymic carcinoids. EAS is characterized by tumoral cortisol resistance, whose underlying mechanism remains unknown. In this study, we reported that silencing mediator of retinoic acid and thyroid hormone receptors (SMRT), a major nuclear corepressor, was aberrantly expressed in ACTH-secreting thymic carcinoids. Overexpression and knockdown of SMRT in the ACTH-secreting AtT-20 cell line demonstrated that SMRT participated in the negative feedback of dexamethasone-mediated suppression of proopiomelanocortin. Posttranslational modification by the small ubiquitin-like modifiers (SUMO), i.e. SUMOylation plays an important role in fine-tuning transcriptional activities. SUMOylation of SMRT was observed in dexamethasone-resistant cell lines. Moreover, overexpression of the deSUMOylation enzyme enhanced the suppression of proopiomelanocortin by dexamethasone in AtT-20 cells. An evolutionarily conserved consensus SUMOylation site was identified close to the histone deacetylase 3 recruiting domain of SMRT, which might interfere with the recruiting process. These results suggested that aberrant expression and modification of SMRT might be involved in the pathogenesis of tumoral cortisol resistance. A therapeutic approach targeting SMRT SUMOylation might be developed for EAS patients.

Nuclear receptor transrepression pathways that regulate inflammation in macrophages and T cells.

Members of the nuclear receptor superfamily of ligand-dependent transcription factors regulate diverse aspects of immunity and inflammation by both positively and negatively regulating gene expression. Here, we review recent studies providing insights into the distinct mechanisms that enable nuclear receptors to antagonize pro-inflammatory programmes of gene expression in macrophages and T cells by altering the turnover or recruitment of co-repressors and co-activators in a gene-specific manner. These nuclear receptor-dependent transrepression pathways are proposed to have roles in controlling the initiation, magnitude and duration of pro-inflammatory gene expression and are amenable to pharmacological manipulation.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) regulates adipose tissue accumulation and adipocyte insulin sensitivity in vivo.

The silencing mediator of retinoid and thyroid hormone receptors (SMRT) serves as a corepressor for nuclear receptors and other factors. Recent evidence suggests that SMRT is an important regulator of metabolism, but its role in adipocyte function in vivo remains unclear. We generated heterozygous SMRT knock-out (SMRT(+/-)) mice to investigate the function of SMRT in the adipocyte and the regulation of adipocyte insulin sensitivity. We show that SMRT(+/-) mice are normal weight on a regular diet, but develop increased adiposity on a high-fat diet (HFD). The mechanisms underlying this phenotype are complex, but appear to be due to a combination of an increased number of smaller subcutaneous adipocytes as well as decreased leptin expression, resulting in greater caloric intake. In addition, adipogenesis of mouse embryonic fibroblasts (MEFs) derived from these mice was increased. However, adipocyte insulin sensitivity, measured by insulin-induced Akt phosphorylation and insulin-mediated suppression of lipolysis, was enhanced in SMRT(+/-) adipocytes. These finding suggest that SMRT regulates leptin expression and limits the ability of fat mass to expand with increased caloric intake, but that SMRT also negatively regulates adipocyte insulin sensitivity.

Anticancer therapy SMRT-ens up: targeting the BCL6-SMRT interaction in B cell lymphoma.

Transcription factors have proven to be difficult targets for the development of small-molecule drugs. In this issue of Cancer Cell, Cerchietti et al. identify and characterize a specific, small-molecule inhibitor of BCL6, an oncogenic transcriptional repressor, that has high clinical promise for treating diffuse large B cell lymphoma.

Hairless is a nuclear receptor corepressor essential for skin function.

The activity of nuclear receptors is modulated by numerous coregulatory factors. Corepressors can either mediate the ability of nuclear receptors to repress transcription, or can inhibit transactivation by nuclear receptors. As we learn more about the mechanisms of transcriptional repression, the importance of repression by nuclear receptors in development and disease has become clear. The protein encoded by the mammalian Hairless (Hr) gene was shown to be a corepressor by virtue of its functional similarity to the well-established corepressors N-CoR and SMRT. Mutation of the Hr gene results in congenital hair loss in both mice and men. Investigation of Hairless function both in vitro and in mouse models in vivo has revealed a critical role in maintaining skin and hair by regulating the differentiation of epithelial stem cells, as well as a putative role in regulating gene expression via chromatin remodeling.