Silencing Mediator Of Retinoic Acid And Thyroid Research Articles

Emerging roles of the corepressors NCoR1 and SMRT in homeostasis

Epigenetic regulation of gene expression is strongly influenced by the accessibility of nucleosomal DNA or the state of chromatin compaction. In this context, coregulators, including both coactivators and corepressors, are pivotal intermediates that bridge chromatin-modifying enzymes and transcription factors. NCoR1 (nuclear receptor corepressor) and SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) are among the best-characterized corepressors from a molecular point of view. These coregulators have conserved orthologs in lower organisms, which underscores their functional importance. Here we summarize the results from recent in vivo studies that reveal the wide-ranging roles of NCoR1 and SMRT in developmental as well as homeostatic processes, including metabolism, inflammation, and circadian rhythms. We also discuss the potential implications of NCoR1 and SMRT regulation of pathways ranging from genomic stability and carcinogenesis to metabolic diseases such as type 2 diabetes.

High-Mobility Group A1 Protein

Rationale: The nuclear receptor peroxisome proliferator–activated receptor-γ (PPARγ) is an important regulator of gene transcription in vascular cells and mediates the vascular protection observed with antidiabetic glitazones. Objective: To determine the molecular mechanism of ligand-dependent transrepression in vascular smooth muscle cells and their impact on the vascular protective actions of PPARγ. Methods and Results: Here, we report a molecular pathway in vascular smooth muscle cells by which ligand-activated PPARγ represses transcriptional activation of the matrix-degrading matrix metalloproteinase-9 (MMP-9) gene, a crucial mediator of vascular injury. PPARγ-mediated transrepression of the MMP-9 gene was dependent on the presence of the high-mobility group A1 (HMGA1) protein, a gene highly expressed in vascular smooth muscle cells, newly identified by oligonucleotide array expression analysis. Transrepression of MMP-9 by PPARγ and regulation by HMGA1 required PPARγ SUMOylation at K367. This process was associated with formation of a complex between PPARγ, HMGA1, and the SUMO E2 ligase Ubc9 (ubiquitin-like protein SUMO-1 conjugating enzyme). After PPARγ ligand stimulation, HMGA1 and PPARγ were recruited to the MMP-9 promoter, which facilitated binding of SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), a nuclear corepressor involved in transrepression. The relevance of HMGA1 for vascular PPARγ signaling was underlined by the complete absence of vascular protection through a PPARγ ligand in HMGA1 −/− mice after arterial wire injury. Conclusions: The present data suggest that ligand-dependent formation of HMGA1-Ubc9-PPARγ complexes facilitates PPARγ SUMOylation, which results in the prevention of SMRT corepressor clearance and induction of MMP-9 transrepression. These data provide new information on PPARγ-dependent vascular transcriptional regulation and help us to understand the molecular consequences of therapeutic interventions with PPARγ ligands in the vasculature.

Response of SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) and N-CoR (Nuclear Receptor Corepressor) Corepressors to Mitogen-Activated Protein Kinase Kinase Kinase Cascades Is Determined by Alternative mRNA Splicing

The SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) corepressors are important mediators of transcriptional repression by nuclear hormone receptors. SMRT is regulated by MAPK kinase kinase (MAPKKK) cascades that induce its release from its receptor partners, its export from nucleus to cytoplasm, and derepression of target gene expression. Intriguingly, the otherwise closely related N-CoR is refractory to MAPKKK signaling under the same conditions. However, both SMRT and N-CoR are expressed as a series of alternatively spliced protein variants differing in structure and function. We have now characterized the impact of this alternative mRNA splicing on the corepressor response to MAPKKK signaling. Whereas the SMRTalpha, SMRTtau, and SMRTsp2 splice variants are released from their nuclear receptor partners in response to MAPKKK activation, the SMRTsp18 variant, which resembles N-CoR in its overall molecular architecture, is relatively refractory to this kinase-induced release. Alternative splicing of N-CoR, in contrast, had only minimal effects on the resistance of this corepressor to MAPKKK inhibition. Notably, all of the SMRT splice variants examined redistributed from nucleus to cytoplasm in response to MAPKKK cascade signaling, but none of the N-CoR splice variants did so. Different tiers of the MAPKKK cascade hierarchy contributed to these different aspects of corepressor regulation, with MAP/ERK kinase kinase 1 and MAP/ERK kinase 1 regulating subcellular redistribution and ERK2 regulating nuclear receptor-corepressor interaction. We conclude that cells can customize their transcriptional response to MAPKKK cascade signaling by selective expression of the SMRT or N-CoR locus, by selective utilization of a specific corepressor splice variant, and by selective exploitation of specific tiers of the MAPK cascade.

Nuclear IKK activity leads to dysregulated Notch-dependent gene expression in colorectal cancer

Nuclear functions for IkappaB kinase (IKK), including phosphorylation of histone H3 and nuclear corepressors, have been recently described. Here, we show that IKK is activated in colorectal tumors concomitant with the presence of phosphorylated SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor that is aberrantly localized in the cytoplasm. In these tumors, IKKalpha associates to the chromatin of specific Notch targets, leading to the release of SMRT. Abrogation of IKK activity by BAY11-7082 or by expressing dominant negative IKKalpha restores the association of SMRT with Notch target genes, resulting in specific gene repression. Finally, BAY11-7082 significantly reduces tumor size in colorectal cancer xenografts (CRC-Xs) implanted in nude mice.

Alternative mRNA Splicing of SMRT Creates Functional Diversity by Generating Corepressor Isoforms with Different Affinities for Different Nuclear Receptors

Many eukaryotic transcription factors are bimodal in their regulatory properties and can both repress and activate expression of their target genes. These divergent transcriptional properties are conferred through recruitment of auxiliary proteins, denoted coactivators and corepressors. Repression plays a particularly critical role in the functions of the nuclear receptors, a large family of ligand-regulated transcription factors involved in metazoan development, differentiation, reproduction, and homeostasis. The SMRT corepressor interacts directly with nuclear receptors and serves, in turn, as a platform for the assembly of a larger corepressor complex. We report here that SMRT is expressed in cells by alternative mRNA splicing to yield two distinct variants or isoforms. We designate these isoforms SMRTalpha and SMRTtau and demonstrate that these isoforms have significantly different affinities for different nuclear receptors. These isoforms are evolutionarily conserved and are expressed in a tissue-specific manner. Our results suggest that differential mRNA splicing serves to customize corepressor function in different cells, allowing the transcriptional properties of nuclear receptors to be adapted to different contexts.

Retinoic acid receptors beta and gamma do not repress, but instead activate target gene transcription in both the absence and presence of hormone ligand.

Retinoic acid receptors (RARs) are important mediators of retinoid signaling in morphogenesis, development, and cell differentiation. Three major isotypes of RARs, denoted alpha, beta, and gamma, have been identified, each encoded by a distinct genetic locus. Although RARalpha, RARbeta, and RARgamma share many structural and functional features, these three isotypes are known to play unique, as well as overlapping, roles in physiology and development. We report here that the three RAR isotypes display different transcriptional properties in the absence of hormone ligand; under these conditions, RARalpha is a strong repressor of target gene expression, whereas both RARbeta and RARgamma fail to repress and instead are able to mediate substantial levels of hormone-independent transcriptional activation. These differing transcriptional properties appear to reflect the differing abilities of the three RAR isotypes to interact with the SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor protein: RARalpha binds to SMRT strongly both in vitro and in vivo, whereas RARbeta and RARgamma interact only weakly with SMRT. The ability to repress or to activate transcription in the absence of hormone maps predominantly to isotype-specific differences in the sequence of helix 3 within the hormone binding domain of the RARs, and the transcriptional properties of one isotype can be exchanged with that of another by exchanging portions of helix 3. The different transcriptional properties of RARalpha, RARbeta, and RARgamma in the absence of hormone contribute to the distinctive biological functions of these proteins and provide a rationale for the strong conservation of the three distinct isotypes during the vertebrate evolutionary radiation.

The SMRT Corepressor Is Regulated by a MEK-1 Kinase Pathway: Inhibition of Corepressor Function Is Associated with SMRT Phosphorylation and Nuclear Export

The SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor participates in the repression of target gene expression by a variety of transcription factors, including the nuclear hormone receptors, promyelocytic leukemia zinc finger protein, and B-cell leukemia protein 6. The ability of SMRT to associate with these transcription factors and thereby to mediate repression is strongly inhibited by activation of tyrosine kinase signaling pathways, such as that represented by the epidermal growth factor receptor. We report here that SMRT function is potently inhibited by a mitogen-activated protein kinase (MAPK) kinase kinase (MAPKKK) cascade that operates downstream of this growth factor receptor. Intriguingly, the SMRT protein is a substrate for phosphorylation by protein kinases operating at multiple levels in this MAPKKK pathway, including the MAPKs, MAPK-extracellular signal-regulated kinase 1 (MEK-1), and MEK-1 kinase (MEKK-1). Phosphorylation of SMRT by MEKK-1 and, to a lesser extent, MEK-1 inhibits the ability of SMRT to physically tether to its transcription factor partners. Notably, activation of MEKK-1 or MEK-1 signaling in transfected cells also leads to a redistribution of the SMRT protein from a nuclear compartment to a more perinuclear or cytoplasmic compartment. We suggest that SMRT-mediated repression is regulated by the MAPKKK cascade and that changes both in the affinity of SMRT for its transcription factors and in the subcellular distribution of SMRT contribute to the loss of SMRT function that is observed in response to kinase signal transduction.

Silencing Subdomains of v-ErbA Interact Cooperatively with Corepressors: Involvement of Helices 5/6

Members of the thyroid hormone receptor (TR) family act on vertebrate development and homeostasis by activating or repressing transcription of specific target genes in a ligand-dependent way. Repression by TR in the absence of ligand is mediated by an active silencing mechanism. The oncogene v-ErbA is a variant form of TR unable to bind hormone and thus acts as a constitutive repressor. Functional studies and mutation analysis revealed that the TR/v-ErbA silencing domain is composed of three silencing subdomains (SSD1-3) which, although nonfunctional individually, synergize such that silencing activity is restored when they are combined in a heteromeric complex. Here we demonstrate, using protein interaction assays in vitro and in vivo, that the inactive v-ErbA point mutant L489R within helix 5/6 in SSD2 fails to interact with the two corepressors N-CoR (nuclear receptor corepressor) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptor). Furthermore, mutants in SSD1 and SSD3 exhibit a reduced corepressor recruitment corresponding to their weak residual silencing activity. In mammalian two-hybrid assays, only the combination of all three silencing subdomains, SSD1-3, leads to a cooperative binding to the corepressors N-CoR or SMRT comparable to that of the full-length v-ErbA repression domain. In conclusion, full silencing activity requires corepressor interaction with all three silencing subdomains, SSD1-3. Among these, SSD2 is a new target for N-CoR and SMRT and is essential for corepressor binding and function.

The nuclear receptor corepressor N-CoR regulates differentiation: N-CoR directly interacts with MyoD.

Classical ligand-activated nuclear receptors (e.g. thyroid hormone receptor, retinoic acid receptor), orphan nuclear receptors (e.g. Rev-erbAalpha/beta), Mad/Max bHLH (basic helix loop helix)-LZ proteins, and oncoproteins, PLZF and LAZ3/BCL6, bind DNA and silence transcription by recruiting a repressor complex that contains N-CoR (nuclear receptor corepressor)/SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), Sin3A/B, and HDAc-1/-2 proteins. The function of the corepressor, N-CoR, in the process of cellular differentiation and coupled phenotypic acquisition, has not been investigated. We examined the functional role of N-CoR in myogenesis (muscle differentiation), an ideal paradigm for the analysis of the determinative events that govern the cell's decision to divide or differentiate. We observed that the mRNA encoding N-CoR was suppressed as proliferating myoblasts exited the cell cycle, and formed morphologically and biochemically differentiated myotubes. Exogenous expression of N-CoR (but not RIP13) in myogenic cells ablated 1) myogenic differentiation, 2) the expression of the myoD gene family that encode the myogenic specific bHLH proteins, and 3) the crucial cell cycle regulator, p21Waf-1/Cip-1 mRNA. Furthermore, N-CoR expression efficiently inhibits the myoD-mediated myogenic conversion of pluripotential C3H10T1/2 cells. We demonstrate that MyoD-mediated transactivation and activity are repressed by N-CoR. The mechanism involves direct interactions between MyoD and N-CoR; moreover, the interaction was dependent on the amino-terminal repression domain (RD1) of N-CoR and the bHLH region of MyoD. Trichostatin A treatment significantly stimulated the activity of MyoD by approximately 10-fold and inhibited the ability of N-CoR to repress MyoD-mediated transactivation, consistent with the involvement of the corepressor and the recruitment of a histone deacteylase activity in the process. This work demonstrates that the corepressor N-CoR is a key regulator of MyoD activity and mammalian differentiation, and that N-CoR has a multifaceted role in myogenesis.

Signaling by Tyrosine Kinases Negatively Regulates the Interaction between Transcription Factors and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) Corepressor

Nuclear hormone receptors are hormone-regulated transcription factors that bind to specific sites on DNA and modulate the expression of adjacent target genes. Many nuclear hormone receptors display bimodal transcriptional properties; thyroid hormone receptors, for example, typically repress target gene expression in the absence of hormone, but activate target gene expression in the presence of hormone. The ability to repress is closely linked to the ability of the apo-receptor to physically bind to auxiliary corepressor proteins denoted SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor), which, in turn, help mediate the actual molecular events involved in transcriptional silencing. We report here that repression by thyroid hormone receptors can be regulated not only by cognate hormone, but also by certain tyrosine kinase signal transduction pathways, such as that represented by the epidermal growth factor-receptor. Activation of tyrosine kinase signaling leads to inhibition of T3R-mediated repression with relatively little effect on activation. These effects appear to be mediated by a kinase-initiated disruption of the ability of T3R to interact with SMRT corepressor. Intriguingly, tyrosine kinase signaling similarly disrupted the interactions of SMRT with v-Erb A, with retinoic acid receptors, and with PLZF, a nonreceptor transcriptional repressor. We conclude that tyrosine kinase signaling exerts potentially important regulatory effects on transcriptional silencing mediated by a variety of transcription factors that operate through the SMRT corepressor complex.

Signaling by Tyrosine Kinases Negatively Regulates the Interaction between Transcription Factors and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) Corepressor

Signaling by Tyrosine Kinases Negatively Regulates the Interaction between Transcription Factors and SMRT (Silencing Mediator of Retinoic Acid and Thyroid Hormone Receptor) Corepressor

Cell-specific inhibition of retinoic acid receptor-alpha silencing by the AF2/tau c activation domain can be overcome by the corepressor SMRT, but not by N-CoR.

The human retinoic acid receptor alpha (hRAR alpha) exhibits cell-specific transcriptional activity. Previously, it was shown that in the absence of hormone the wild-type receptor is a transcriptional silencer in L cells, whereas it lacks silencing function and is a weak activator in CV1 cells. Addition of hormone leads to a further increase in transactivation in CV1 cells. Thus, the retinoic acid response mediated by RAR alpha is weak in these cells. It was shown that the CV1-specific effect is due to the receptor C terminus. We show, that the failure of silencing by RAR is not due to a general lack of corepressors in CV1 cells, since the silencing domain of RAR is functionally active and exhibits active repression in these cells. Furthermore, we show that the conserved AF2/tau c activation function of RAR is responsible for the cell-specific inhibition of silencing. Thereby, the CV1 cell specificity was abolished by replacing AF2/tau c of RAR with the corresponding sequence of the thyroid hormone receptor. Thus, we find a new role of the C-terminal conserved activation function AF2/tau c in that, specifically, the RAR AF2/tau c-sequence is able to prevent silencing of RAR in a cell-specific manner. In addition, we show that the inhibitory effect of AF2/tau c in CV1 cells can be overcome by expression of the corepressor SMRT (silencing mediator of retinoic acid and thyroid hormone receptor), but not by that of N-CoR (nuclear receptor corepressor). The expression of these two corepressors, however, had no measurable effect on RAR-mediated silencing in L cells. Thus, the expression of a corepressor can lead to a dramatic increase of hormonal response in a cell-specific manner.

Characterization of receptor interaction and transcriptional repression by the corepressor SMRT.

SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.

Characterization of Receptor Interaction and Transcriptional Repression by the Corepressor SMRT

SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) and N-CoR (nuclear receptor corepressor) are two related transcriptional corepressors that contain separable domains capable of interacting with unliganded nuclear receptors and repressing basal transcription. To decipher the mechanisms of receptor interaction and transcriptional repression by SMRT/N-CoR, we have characterized protein-protein interacting surfaces between SMRT and nuclear receptors and defined transcriptional repression domains of both SMRT and N-CoR. Deletional analysis reveals two individual nuclear receptor domains necessary for stable association with SMRT and a C-terminal helix essential for corepressor dissociation. Coordinately, two SMRT domains are found to interact independently with the receptors. Functional analysis reveals that SMRT contains two distinct repression domains, and the corresponding regions in N-CoR also repress basal transcription. Both repression domains in SMRT and N-CoR interact weakly with mSin3A, which in turn associates with a histone deacetylase HDAC1 in a mammalian two-hybrid assay. Far-Western analysis demonstrates a direct protein-protein interaction between two N-CoR repression domains with mSin3A. Finally we demonstrate that overexpression of full-length SMRT further represses basal transcription from natural promoters. Together, these results support a role of SMRT/N-CoR in corepression through the utilization of multiple mechanisms for receptor interactions and transcriptional repression.

Coactivator and corepressor regulation of the agonist/antagonist activity of the mixed antiestrogen, 4-hydroxytamoxifen.

Mixed antiestrogens, such as 4-hydroxytamoxifen (4HT), act as either partial agonists or antagonists of estrogen receptor (ER) function in a tissue-, cell-, and promoter-specific manner, suggesting that intracellular factors modulate their ability to regulate transcription. To determine whether coactivators and corepressors have the capacity to modulate the relative agonist/antagonist activity of 4HT, ER-dependent gene expression was measured in the absence or presence of expression vectors for SRC-1 (steroid receptor coactivator-1) or SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). In Hep G2 cells in which 4HT is an agonist, exogenous SRC-1 enhanced estradiol (E2)- and 4HT-stimulated transcription in a dose-dependent manner, while SMRT overexpression strongly reduced basal and 4HT-stimulated gene expression with no effect on E2 activity. These observations were not cell- or promoter-specific inasmuch as similar results were obtained in HeLa cells under conditions in which 4HT is an antagonist. A protein-protein interaction assay indicated that the full-length ER binds to SMRT in vitro. To assess whether relative coactivator and corepressor expression within a given cell could modulate the balance of 4HT agonist/antagonist activity, SRC-1 and SMRT were coexpressed. SMRT overexpression blocked SRC-1 coactivation of 4HT-stimulated gene expression and preferentially inhibited 4HT agonist activity whether or not exogenous SRC-1 was present. The cumulative data in this model system indicate that the relative expression of coactivators and corepressors can modulate 4HT regulation of ER transcriptional activity and suggest they could contribute to the tissue-specific ability of mixed antiestrogens to activate or inhibit ER-mediated gene expression.

Differential effects of nuclear receptor corepressor (N-CoR) expression levels on retinoic acid receptor-mediated repression support the existence of dynamically regulated corepressor complexes.

Thyroid hormone and retinoic acid receptors are members of the nuclear receptor superfamily of ligand-dependent transcription factors that stimulate the transcription of target genes in the presence of activating ligands and repress transcription in their absence. Transcriptional repression by the thyroid hormone and retinoic acid receptors has been proposed to be mediated by the nuclear receptor corepressor, N-CoR, or the related factor, SMRT (silencing mediator of retinoic acid and thyroid hormone receptors). Recent studies have suggested that transcriptional repression by N-CoR involves a corepressor complex that also contains mSin3A/B and the histone deacetylase, RPD3. In this manuscript, we demonstrate that transcriptional repression by the retinoic acid receptor can be either positively or negatively regulated by changes in the levels of N-CoR expression, suggesting a relatively strict stoichiometric relationship between N-CoR and other components of the corepressor complex. Consistent with this interpretation, overexpression of several functionally defined domains of N-CoR also relieve repression by nuclear receptors. N-CoR is distributed throughout the nucleus in a nonuniform pattern, and a subpopulation becomes concentrated into several discrete dot structures when highly expressed. RPD3 is also widely distributed throughout the nucleus in a nonuniform pattern. Simultaneous imaging of RPD3 and N-CoR suggest that a subset of each of these proteins colocalize, consistent with the existence of coactivator complexes containing both proteins. In addition, a substantial fraction of both N-CoR and mSin3 A/B appear to be independently distributed. These observations suggest that interactions between RPD3 and Sin3/N-CoR complexes may be dynamically regulated.