Corepressor SMRT Research Articles

Nuclear Receptor Corepressors NCOR1 and SMRT Regulate Metabolism via Intestinal Regulation of Carbohydrate Transport.

Nuclear receptor action is mediated in part by the nuclear receptor corepressor 1 (NCOR1) and the silencing mediator of retinoic acid and thyroid hormone receptor (SMRT). NCOR1 and SMRT regulate metabolic pathways that govern body mass, insulin sensitivity, and energy expenditure, representing an understudied area in the realm of metabolic health and disease. Previously, we found that NCOR1 and SMRT are essential for maintaining metabolic homeostasis and their knockout (KO) leads to rapid weight loss and hypoglycemia, which is not survivable. Because of a potential defect in glucose absorption, we sought to determine the role of NCOR1 and SMRT specifically in intestinal epithelial cells (IECs). We used a postnatal strategy to disrupt NCOR1 and SMRT throughout IECs in adult mice. These mice were characterized metabolically and underwent metabolic phenotyping, body composition analysis, and glucose tolerance testing. Jejunal IECs were isolated and profiled by bulk RNA sequencing. We found that the postnatal KO of NCOR1 and SMRT from IECs leads to rapid weight loss and hypoglycemia with a significant reduction in survival. This was accompanied by alterations in glucose metabolism and activation of fatty acid oxidation in IECs. Metabolic phenotyping confirmed a reduction in body mass driven by a loss of body fat without altered food intake. This appeared to be mediated by a reduction of key intestinal carbohydrate transporters, including SGLT1, GLUT2, and GLUT5. Intestinal NCOR1 and SMRT act in tandem to regulate glucose levels and body weight. This in part may be mediated by regulation of intestinal carbohydrate transporters.

Mutant Tbl1x male mice have a short life span and do not breed: unexpected findings.

Humans with the mutation Y509C in transducin beta like 1 X-linked (TBL1X HGNC ID HGNC:11585) have been reported to present with the combination of central congenital hypothyroidism and impaired hearing. TBL1X belongs to the WD40 repeat-containing protein family, is part of NCoR and SMRT corepressor complexes, and thereby involved in thyroid hormone signaling. In order to investigate the effects of the Y509C mutation in TBL1X on cellular thyroid hormone action, we aimed to generate a hemizygous male mouse cohort carrying the Tbl1x Y459C mutation which is equivalent to the human TBL1X Y509C mutation using CRISPR/Cas9 technology. Hemizygous male mice were small at birth and inactive. Their life span (median life span 93 days) was very short compared with heterozygous female mice (survived to >200 days with no welfare issues). About 52% of mice did not survive to weaning (133 mice). Of the remaining 118 mice, only 8 were hemizygous males who were unable to mate whereby it was impossible to generate homozygous female mice. In conclusion, the Tbl1x Y459C mutation in male mice has a marked negative effect on birth weight, survival, and fertility of male mice. The present findings are unexpected as they are in contrast to the mild phenotype in human males carrying the equivalent TBL1X Y509C mutation.

Modulation of TCDD induced Cyp1a1 mRNA by the Co-repressor SMRT

Modulation of TCDD induced Cyp1a1 mRNA by the Co-repressor SMRT

Mutation of two residues converts the ligand-binding domain of RXRα into a uniform monomer without impairing the binding of retinoic acid and cofactors

Retinoid X receptor (RXRα) is a nuclear receptor (NR) for retinoic acid (RA) and regulates various NR signaling pathways. Ligand-binding domain (LBD) of RXRα can bind with its ligand 9-cis-RA and cofactors, and mediate the forming of homodimer and homotetramer of RXRα and its heterodimer with other NRs, conferring RXRα the ability to play complicated roles in development and diseases. Due to the coexistence of monomer, dimer and tetramer, there are difficulties to study the structure and interaction of RXRα-LBD with its ligands and cofactors in solution and to distinguish the roles of different forms of RXRα in cell. Here, through analyzing available structures of RXRα-LBD, we selected two residues, D379 and L420, in the homodimer interface to design three mutants of RXRα-LBD. Recombinant proteins of the three mutants showed decreased proportions of dimer and tetramer but unchanged overall structure and binding affinities to 9-cis-RA, corepressor SMRT, and coactivator SRC2. Especially, the double-site mutant RXRα-LBDD379A−L420G existed as a uniform monomer. Furthermore, L420 was found to play a similar role in forming RXRα-LBD homodimer and its heterodimer with various NRs, while the role of D379 varies a lot, as it shows almost no interaction with RARα/β, LXRα/β, and THRα/β. This study provides a new insight into the mechanism for forming RXRα-LBD homodimer and its heterodimer with other NRs, and will facilitate the studies on the structure and interaction of RXRα-LBD with ligands, cofactors and drugs in solution, and the broad physiological functions of RXRα cooperating with various NRs in cell.

Nuclear corepressor SMRT acts as a strong regulator of both β-oxidation and suppressor of fibrosis in the differentiation process of mouse skeletal muscle cells.

Silencing Mediator of Retinoid and Thyroid hormone receptors (SMRT; NCoR2) is a transcriptional corepressor (CoR) which has been recognized as an important player in the regulation of hepatic lipogenesis and in somatic development in mouse embryo. SMRT protein is also widely expressed in mouse connective tissues, for example adipocytes and muscle. We recently reported that mice with global deletion of SMRT develop significant obesity and muscle wasting which are independent from thyroid hormone (TH) signaling and thermogenesis. However, the tissue specific role of SMRT in skeletal muscle is still not clear. To clarify role of SMRT in muscle differentiation, we made myogenic C2C12 clones which lack SMRT protein (C2C12-SKO) by using CRISPR-Cas9. Wild-type C2C12 (C2C12-WT) and C2C12-SKO cells were cultured in differentiation medium, and the resulting gene and protein profiles were compared between the two cell lines both before and after differentiation. We also analyzed muscle tissues which were dissected from whole body SMRT knockout (KO) mice and their controls. We found significant up-regulation of muscle specific β-oxidation markers; Peroxisome proliferator-activated receptor δ (PPARδ) and PPARγ coactivator-1α (PGC-1α) in the C2C12-SKO cells, suggesting that the cells had a similar gene profile to what is found in exercised rodent skeletal muscle. On the other hand, confocal microscopic analysis showed the significant loss of myotubes in C2C12-SKO cells similar to the morphology found in immature myoblasts. Proteomics analysis also confirmed that the C2C12-SKO cells had higher expression of markers of fibrosis (ex. Collagen1A1; COL1A1 and Fibroblast growth factor-2; FGF-2), indicating the up-regulation of Transforming growth factor-β (TGF-β) receptor signaling. Consistent with this, treatment with a specific TGF-β receptor inhibitor ameliorated both the defects in myotube differentiation and fibrosis. Taken together, we demonstrate that SMRT functions as a pivotal transcriptional mediator for both β-oxidation and the prevention for the fibrosis via TGF-β receptor signaling in the differentiation of C2C12 myoblasts. In contrast to the results from C2C12 cells, SMRT does not appear to play a role in adult skeletal muscle of whole body SMRT KO mice. Thus, SMRT plays a significant role in the differentiation of myoblasts.

Abstract 2607: Inhibition of HDAC3 induces BRCAness and potent synergy with PARP inhibition in neuroendocrine prostate and small cell lung cancers

Abstract HDAC3 is essential for transcriptional repression and is required for the activity of NCOR1 & SMRT co-repressors complexes. Inhibition of HDAC3 results in the suppression of genes responsible for DNA damage repair and maintenance of genomic stability. HDAC3/NCOR1 also play critical roles in tumor metabolism and immune cell biology. GB-3103 is a novel and potent inhibitor of HDAC3 with an IC50 of 0.56nM that was tested for activity against human neuroendocrine prostate (NEPC) and small cell lung cancers (SCLC). RNA-seq analysis of GB-3103 treated human tumor cells revealed induction of BRCAness, inhibition of genes from DNA repair pathways and inhibition of PI3K-AKT1, MYC and p53 signaling. GB-3103 showed potent in vitro anticancer activity against the human H660 NEPC line, with an IC50 of 28nM, and 21nM against the murine PNEC30 line and potent IC50 of 16nM against NCI-H209 human lung neuroendocrine cancer. Because of the potent induction of BRCAness we used zero interaction potency (ZIP) model to test the activity of the combination of GB-3103 with a PARP inhibitor against human H660 and murine PNEC30 cell lines. These experiments revealed synergy for the combination of PARPi with GB-3103 in NEPC. GB-3103 was subsequently tested alone and in combination with olaparib in a xenograft model of H660. GB3103 (TGI=3%) showed minimal activity at the selected dose against H660 compared to single agent olaparib (TGI=67%). However, when combined with olaparib, GB-3103 revealed potent synergy (TGI=96%, p<0.01). In the NCI-H209 human lung neuroendocrine xenograft model, GB-3103 showed robust activity as a single agent (TGI=83%) compared to single agent olaparib (TGI=33%) and single agent talazoparib (TGI=69%). When combined with olaparib, GB-3103 induced synergistic antitumor effect (TGI=99%, p<t0.001) when compared to olaparib treatment alone. When combined with talazoparib, GB-3103 induced synergistic antitumor effect (TGI >100%, p<0.001) when compared to talazoparib treatment alone. Taken together, these data confirm the important role of HDAC3 in neuroendocrine prostate and small cell lung cancers and suggest HDAC3 inhibition by GB-3103 could be an effective approach for patients with neuroendocrine cancers particularly when combined with inhibitors of PARP. Citation Format: Stephanie Nagl, Kendra Devore, Gregg Hirschfeld, Kari Kotlarczyk, Damaris Diaz, Tong Wang, Maggie Murray, Paul Gonzales, Stephen Thomas Gately. Inhibition of HDAC3 induces BRCAness and potent synergy with PARP inhibition in neuroendocrine prostate and small cell lung cancers [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 2607.

Progesterone receptor isoform ratio dictates antiprogestin/progestin effects on breast cancer growth and metastases: A role for NDRG1.

Progesterone receptors (PRs) ligands are being tested in luminal breast cancer. There are mainly two PR isoforms, PRA and PRB, and their ratio (PRA/PRB) may be predictive of antiprogestin response. Our aim was to investigate: the impact of the PR isoform ratio on metastatic behaviour, the PR isoform ratio in paired primary tumours and lymph node metastases (LNM) and, the effect of antiprogestin/progestins on metastatic growth. Using murine and human metastatic models, we demonstrated that tumours with PRB > PRA (PRB-H) have a higher proliferation index but less metastatic ability than those with PRA > PRB (PRA-H). Antiprogestins and progestins inhibited metastatic burden in PRA-H and PRB-H models, respectively. In breast cancer samples, LNM retained the same PRA/PRB ratio as their matched primary tumours. Moreover, PRA-H LNM expressed higher total PR levels than the primary tumours. The expression of NDRG1, a metastasis suppressor protein, was higher in PRB-H compared to PRA-H tumours and was inversely regulated by antiprogestins/progestins. The binding of the corepressor SMRT at the progesterone responsive elements of the NDRG1 regulatory sequences, together with PRA, impeded its expression in PRA-H cells. Antiprogestins modulate the interplay between SMRT and AIB1 recruitment in PRA-H or PRB-H contexts regulating NDRG1 expression and thus, metastasis. In conclusion, we provide a mechanistic interpretation to explain the differential role of PR isoforms in metastatic growth and highlight the therapeutic benefit of using antiprogestins in PRA-H tumours. The therapeutic effect of progestins in PRB-H tumours is suggested.

Structural basis of Apt48 inhibition of the BCL6 BTB domain

B cell lymphoma 6 (BCL6) is a transcriptional repressor that is deregulated in diffuse large B cell lymphoma, and the peptide aptamer, Apt48, inhibits BCL6 by an unknown mechanism. We report the crystal structure of BCL6 in complex with an Apt48 peptide, and show that Apt48 binds to a therapeutically uncharacterized region at the bottom of the BCL6 BTB domain. We show that the corepressor binding site of the BTB domain may be divided conceptually into two low-affinity, peptide-binding regions. An upper region, the lateral groove, binds peptides in robust three-dimensional conformations, whereas a lower binding site is permissive to less-specific interactions. We show that, even with little sequence specificity, the interactions of the lower region are required for the high-affinity binding of the SMRT corepressor and other peptides to the BTB domain. This has relevance for the design of new BCL6 inhibitors and for understanding the evolution of corepressor interactions with the BTB domain.

Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen by coordinating a crosstalk between Med1, miR205 and ErbB

Obese women with hormone receptor-positive breast cancer exhibit poor response to therapy and inferior outcomes. However, the underlying molecular mechanisms by which obesity/hyperleptinemia may reduce the efficacy of hormonal therapy remain elusive. Obese mice with hyperleptinemia exhibit increased tumor progression and respond poorly to tamoxifen compared to non-obese mice. Exogenous leptin abrogates tamoxifen-mediated growth inhibition and potentiates breast tumor growth even in the presence of tamoxifen. Mechanistically, leptin induces nuclear translocation of phosphorylated-ER and increases the expression of ER-responsive genes, while reducing tamoxifen-mediated gene repression by abrogating tamoxifen-induced recruitment of corepressors NCoR, SMRT, and Mi2 and potentiating coactivator binding. Furthermore, in silico analysis revealed that coactivator Med1 potentially associates with 48 (out of 74) obesity-signature genes. Interestingly, leptin upregulates Med1 expression by decreasing miR-205, and increases its functional activation via phosphorylation, which is mediated by activation of Her2 and EGFR. It is important to note that Med1 silencing abrogates the negative effects of leptin on tamoxifen efficacy. In addition, honokiol or adiponectin treatment effectively inhibits leptin-induced Med1 expression and improves tamoxifen efficacy in hyperleptinemic state. These studies uncover the mechanistic insights how obese/hyperleptinemic state may contribute to poor response to tamoxifen implicating leptin-miR205-Med1 and leptin-Her2-EGFR-Med1 axes, and present bioactive compound honokiol and adipocytokine adiponectin as agents that can block leptin’s negative effect on tamoxifen.

Comparative analysis of CSL-binding motifs of SMRT and KyoT2 corepressors

Comparative analysis of CSL-binding motifs of SMRT and KyoT2 corepressors

Hormone-dependent activation and repression of microRNAs by the ecdysone receptor in the dengue vector mosquito Aedes aegypti

Female mosquitoes transmit numerous devastating human diseases because they require vertebrate blood meal for egg development. MicroRNAs (miRNAs) play critical roles across multiple reproductive processes in female Aedes aegypti mosquitoes. However, how miRNAs are controlled to coordinate their activity with the demands of mosquito reproduction remains largely unknown. We report that the ecdysone receptor (EcR)-mediated 20-hydroxyecdysone (20E) signaling regulates miRNA expression in female mosquitoes. EcR RNA-interference silencing linked to small RNA-sequencing analysis reveals that EcR not only activates but also represses miRNA expression in the female mosquito fat body, a functional analog of the vertebrate liver. EcR directly represses the expression of clustered miR-275 and miR-305 before blood feeding when the 20E titer is low, whereas it activates their expression in response to the increased 20E titer after a blood meal. Furthermore, we find that SMRTER, an insect analog of the vertebrate nuclear receptor corepressors SMRT and N-CoR, interacts with EcR in a 20E-sensitive manner and is required for EcR-mediated repression of miRNA expression in Ae. aegypti mosquitoes. In addition, we demonstrate that miR-275 and miR-305 directly target glutamate semialdehyde dehydrogenase and AAEL009899, respectively, to facilitate egg development. This study reveals a mechanism for how miRNAs are controlled by the 20E signaling pathway to coordinate their activity with the demands of mosquito reproduction.

The Role of Nuclear Receptor Corepressors NCoR1 and SMRT on Physiologic Function in the Adult Mouse

Thyroid hormone (TH) plays an essential role in maintaining homeostasis and regulating metabolism in all organ systems beginning with embryogenesis and continuing throughout life. TH action is mediated by the thyroid hormone receptor (TR), which is a nuclear receptor, and it’s coregulators. The nuclear receptor corepressor 1 (NCoR1) and the silencing mediator of retinoid and thyroid hormone receptors (SMRT) are two critical corepressors of the TR that inhibit gene transcription in the absence of TH. Repression is mediated by complexing with histone deacetylase 3 (HDAC3), which is stabilized by NCoR1 and SMRT. NCoR1 and SMRT are critical for maintaining metabolic homeostasis and act to mediate energy expenditure, insulin sensitivity, and body weight. We sought to elucidate the roles of NCoR1 and SMRT in maintaining global physiologic function in the adult mouse. In order to study the post-natal role of these corepressors, we used a tamoxifen-inducible Cre recombinase (UBC-Cre-ERT2) to knock-out (KO) NCoR1, SMRT, or NCoR1 and SMRT together in adult mice because global deletion of either corepressor during embryogenesis is lethal. Mice were injected with tamoxifen at 8 weeks of age to KO either NCoR1 (NCoR1-KO; NKO), SMRT (SMRT-KO; SKO), or both NCoR1 and SMRT (double KO; DKO) and metabolic parameters were analyzed. While postnatal deletion of either NCoR1 or SMRT did not impact mortality, KO of both NCoR1 and SMRT resulted in a rapidly lethal phenotype heralded by weight loss, hypoglycemia and hypothermia. Metabolic phenotyping confirmed a loss of body mass and in particular fat mass in addition to a reduction in energy expenditure and increase in fecal caloric density. Further analysis showed the rapid development of hepatosteatosis and disturbances in lipid metabolism with a profound increase in beta-oxidation. We also found a reduction in HDAC3 protein levels in the DKO mice but no rapidly lethal phenotype in HDAC3 KO mice. Overall, we show that NCoR1 and SMRT together are critical for life as their deletion results in a rapidly lethal phenotype. While NCoR1 and SMRT are required to stabilize the corepressor complex, including HDAC3, HDAC3 KO resulted in a distinct and separate phenotype.

Nuclear translocation of MRTFA in MCF7 breast cancer cells shifts ERα nuclear/genomic to extra-nuclear/non genomic actions

The Myocardin-related transcription factor A [MRTFA, also known as Megakaryoblastic Leukemia 1 (MKL1))] is a major actor in the epithelial to mesenchymal transition (EMT). We have previously shown that activation and nuclear accumulation of MRTFA mediate endocrine resistance of estrogen receptor alpha (ERα) positive breast cancers by initiating a partial transition from luminal to basal-like phenotype and impairing ERα cistrome and transcriptome. In the present study, we deepen our understanding of the mechanism by monitoring functional changes in the receptor's activity. We demonstrate that MRTFA nuclear accumulation down-regulates the expression of the unliganded (Apo-)ERα and causes a redistribution of the protein localization from its normal nuclear place to the entire cell volume. This phenomenon is accompanied by a shift in Apo-ERα monomer/dimer ratio towards the monomeric state, leading to significant functional consequences on ERα activities. In particular, the association of Apo-ERα with chromatin is drastically decreased, and the remaining ERα binding sites are substantially less enriched in ERE motifs than in control conditions. Monitored by proximity Ligation Assay, ERα interactions with P160 family coactivators are partly impacted when MRTFA accumulates in the nucleus, and those with SMRT and NCOR1 corepressors are abolished. Finally, ERα interactions with kinases such as c-src and PI3K are increased, thereby enhancing MAP Kinase and AKT activities. In conclusion, the activation and nuclear accumulation of MRTFA in ERα positive breast cancer cells remodels both ERα location and functions by shifting its activity from nuclear genome regulation to extra-nuclear non-genomic signaling.

Abstract PS18-32: Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases

Abstract Background and Aim: Obese state is associated with increased breast cancer growth, metastasis, and poor overall survival. In this study, we seek to decipher the underlying molecular mechanisms by which obesity/hyperleptinemia reduces the efficacy of tamoxifen. Methods: The impact of obesity on tamoxifen was evaluated utilizing clonogenicity, high-fat-diet- induced obese mice and leptin-treated xenograft-models. Mechanistic studies involved immunoblotting, real-time PCR, immunocytochemistry, chromatin immunoprecipitation assay, phosphokinase array and in silico analysis. Results: Obese mice with hyperleptinemia exhibit increased tumor progression and respond poorly to tamoxifen compared to non-obese mice. Exogenous leptin abrogates tamoxifen-mediated growth inhibition and potentiates breast tumor growth even in the presence of tamoxifen. Mechanistically, leptin induces nuclear translocation of phosphorylated-ER and increases the expression of ER-responsive genes while reducing tamoxifen-mediated gene repression by abrogating tamoxifen-induced recruitment of corepressors NCoR, SMRT and Mi2. Further, we found that coactivator Med1 potentially associates with 48 (out of 75) obesity-signature genes. Interestingly, leptin upregulates Med1 expression by decreasing miR-205 and increases its functional activation via phosphorylation that is mediated by activation of Her2 and EGFR. It is important to note that Med1 silencing abrogates the negative effects of leptin on tamoxifen efficacy. Additionally, honokiol or adiponectin treatment effectively inhibits leptin-induced Med1 expression and improve tamoxifen efficacy in hyperleptinemic state.Conclusion: In conclusion, these studies show the molecular mechanisms by which obese/hyperleptinemic state may contribute to poor response to tamoxifen implicating leptin-miR205-Med1 and leptin-Her2-EGFR-Med1 axes and present bioactive compound honokiol and adipocytokine adiponectin as agents that can block leptin’s negative effect on tamoxifen. Citation Format: Arumugam Nagalingam, Nethaji Muniraj, Sumit Siddharth, Dimiter Avtanski, Sheetal Parida, Pajamurthy Kuppusamy, Neeraj K Saxena, Nethaji Muniraj. Hyperleptinemia in obese state renders luminal breast cancers refractory to tamoxifen coordinating a crosstalk between Med1, miR205 and Erb B kinases [abstract]. In: Proceedings of the 2020 San Antonio Breast Cancer Virtual Symposium; 2020 Dec 8-11; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2021;81(4 Suppl):Abstract nr PS18-32.

The corepressors GPS2 and SMRT control enhancer and silencer remodeling via eRNA transcription during inflammatory activation of macrophages

While the role of transcription factors and coactivators in controlling enhancer activity and chromatin structure linked to gene expression is well established, the involvement of corepressors is not. Using inflammatory macrophage activation as a model, we investigate here a corepressor complex containing GPS2 and SMRT both genome-wide and at the Ccl2 locus, encoding the chemokine CCL2 (MCP-1). We report that corepressors co-occupy candidate enhancers along with the coactivators CBP (H3K27 acetylase) and MED1 (mediator) but act antagonistically by repressing eRNA transcription-coupled H3K27 acetylation. Genome editing, transcriptional interference, and cistrome analysis reveals that apparently related enhancer and silencer elements control Ccl2 transcription in opposite ways. 4C-seq indicates that corepressor depletion or inflammatory signaling functions mechanistically similarly to trigger enhancer activation. In ob/ob mice, adipose tissue macrophage-selective depletion of the Ccl2 enhancer-transcribed eRNA reduces metaflammation. Thus, the identified corepressor-eRNA-chemokine pathway operates invivo and suggests therapeutic opportunities by targeting eRNAs in immuno-metabolic diseases.

The uncharacterized SANT and BTB domain-containing protein SANBR inhibits class switch recombination

Class switch recombination (CSR) is the process by which B cells switch production from IgM/IgD to other immunoglobulin isotypes, enabling them to mount an effective immune response against pathogens. Timely resolution of CSR prevents damage due to an uncontrolled and prolonged immune response. While many positive regulators of CSR have been described, negative regulators of CSR are relatively unknown. Using an shRNA library screen targeting more than 28,000 genes in a mouse B cell line, we have identified a novel, uncharacterized protein of 82kD (KIAA1841, NM_027860), which we have named SANBR (SANT and BTB domain regulator of CSR), as a negative regulator of CSR. The purified, recombinant BTB domain of SANBR exhibited characteristic properties such as homodimerization and interaction with corepressor proteins, including HDAC and SMRT. Overexpression of SANBR inhibited CSR in primary mouse splenic B cells, and inhibition of CSR is dependent on the BTB domain while the SANT domain is largely dispensable. Thus, we have identified a new member of the BTB family that serves as a negative regulator of CSR. Future investigations to identify transcriptional targets of SANBR in B cells will reveal further insights into the specific mechanisms by which SANBR regulates CSR as well as fundamental gene regulatory activities of this protein.

PPARγ S273 Phosphorylation Modifies the Dynamics of Coregulator Proteins Recruitment.

The nuclear receptor PPARγ is essential to maintain whole-body glucose homeostasis and insulin sensitivity, acting as a master regulator of adipogenesis, lipid, and glucose metabolism. Its activation through natural or synthetic ligands induces the recruitment of coactivators, leading to transcription of target genes such as cytokines and hormones. More recently, post translational modifications, such as PPARγ phosphorylation at Ser273 by CDK5 in adipose tissue, have been linked to insulin resistance trough the dysregulation of expression of a specific subset of genes. Here, we investigate how this phosphorylation may disturb the interaction between PPARγ and some coregulator proteins as a new mechanism that may leads to insulin resistance. Through cellular and in vitro assays, we show that PPARγ phosphorylation inhibition increased the activation of the receptor, therefore the increased recruitment of PGC1-α and TIF2 coactivators, whilst decreases the interaction with SMRT and NCoR corepressors. Moreover, our results show a shift in the coregulators interaction domains preferences, suggesting additional interaction interfaces formed between the phosphorylated PPARγ and some coregulator proteins. Also, we observed that the CDK5 presence disturb the PPARγ-coregulator’s synergy, decreasing interaction with PGC1-α, TIF2, and NCoR, but increasing coupling of SMRT. Finally, we conclude that the insulin resistance provoked by PPARγ phosphorylation is linked to a differential coregulators recruitment, which may promote dysregulation in gene expression.

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

Proteasome inhibition creates a chromatin landscape favorable to RNA Pol II processivity

PPARβ/δ recruits NCOR and regulates transcription reinitiation of ANGPTL4.

In the absence of ligands, the nuclear receptor PPARβ/δ recruits the NCOR and SMRT corepressors, which form complexes with HDAC3, to canonical target genes. Agonistic ligands cause dissociation of corepressors and enable enhanced transcription. Vice versa, synthetic inverse agonists augment corepressor recruitment and repression. Both basal repression of the target gene ANGPTL4 and reinforced repression elicited by inverse agonists are partially insensitive to HDAC inhibition. This raises the question how PPARβ/δ represses transcription mechanistically. We show that the PPARβ/δ inverse agonist PT-S264 impairs transcription initiation by decreasing recruitment of activating Mediator subunits, RNA polymerase II, and TFIIB, but not of TFIIA, to the ANGPTL4 promoter. Mass spectrometry identifies NCOR as the main PT-S264-dependent interactor of PPARβ/δ. Reconstitution of knockout cells with PPARβ/δ mutants deficient in basal repression results in diminished recruitment of NCOR, SMRT, and HDAC3 to PPAR target genes, while occupancy by RNA polymerase II is increased. PT-S264 restores binding of NCOR, SMRT, and HDAC3 to the mutants, resulting in reduced polymerase II occupancy. Our findings corroborate deacetylase-dependent and -independent repressive functions of HDAC3-containing complexes, which act in parallel to downregulate transcription.

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.