Smrter/SANT domain protein
SMRT and N-CoR interactions with transcription factors
SMRT contains within its C-terminal region at least two subdomains, denoted RID-1 and RID-2, that are independently able to confer physical and functional interactions with a defined subset of the nuclear hormone receptor family. Intriguingly, there is no extensive amino acid relatedness between RID-1 and RID-2, and different receptors display different abilities to interact with these two SMRT subdomains. T3Ralpha interacts with both SMRT RID-1 and RID-2 in vitro and in two-hybrid assays in vivo in both yeast and mammalian cells. T3Ralpha also interacts with both of the analogous RIDs of N-CoR; these interaction domains of N-CoR are related but are not identical in sequence to the corresponding interaction domains of SMRT. Perhaps reflecting this nonidentity of the SMRT and N-CoR RIDs, RARalpha interacts almost exclusively with RID-1 of SMRT but interacts moderately well with both RID-1 and RID-2 of N-CoR. Thus, different receptors make different patterns of contact with the SMRT and N-CoR corepressors, and these distinct patterns of contact may potentially be manifested as differences in transcriptional regulation (Wong, 1998a).
Unexpectedly, not all isoforms within a given receptor family interact equally well with a corepressor; specifically, RARbeta interacts very poorly with SMRT and N-CoR, whereas RARalpha and RARgamma interact quite well with both corepressors; these different RAR isoforms are thought to perform distinct functions in development and differentiation; the fact that they possess distinct corepressor interaction properties suggests at least one biochemical basis for their nonidentical physiological roles. The divergent corepressor association properties of the RARbeta isoform map to a small cluster of amino acids within the D domain of the receptor; these differ from the equivalent sequences in RARalpha and RARgamma. Preliminary analysis suggests that changing individual nonconserved amino acids from the RARbeta sequence to that of RARalpha (such as an A175P or a T181I substitution) fails to confer strong corepressor association; apparently more subtle, or multiple, amino acid divergences within this small cluster contribute to the isoform specificity. Notably, this amino acid cluster is proximal to the N-CoR box, a domain previously implicated in corepressor binding by RARs and T3Rs. Recently, it was proposed that the N-CoR box may itself play only an indirect role in the receptor-corepressor interaction, perhaps by stabilizing the conformation of the receptor rather than by providing the actual amino acid contacts involved in the binding of the corepressor. Consistent with this view, conservation of the N-CoR box itself is not necessary for corepressor binding; COUP-TF, RXRs, and PPARs, for example, all lack a detectable N-CoR box but, nonetheless, tether SMRT and N-CoR. However, whether by direct or indirect means, the amino acids within and immediately flanking the N-CoR box play a critical role in defining the ability of RARs and T3Rs to associate with corepressors (Wong, 1998).
The nuclear corepressor (NCoR) binds to the thyroid hormone receptor (TR) in the absence of ligand. NCoR-TR interactions are mediated by two interaction domains in the C-terminal portion of NCoR. Binding of NCoR to TR results in ligand-independent repression on positive thyroid hormone response elements. However, the interactions between NCoR interaction domains and TR on DNA response elements have not been well characterized. Both interaction domains are capable of binding TR on thyroid hormone response elements. In addition, the NCoR interaction domains interact much more strongly with the TR than those present in the silencing mediator of retinoic acid and TRs (SMRT). Furthermore, deletion of either NCoR interaction domain does not significantly impair ligand-independent effects on positive or negative thyroid hormone response elements. Finally, both NCoR interaction domains appear to preferentially bind TR homodimer over TR-retinoid X receptor heterodimer in electrophoretic mobility shift assays. These data suggest that either NCoR interaction domain is capable of mediating the ligand-independent effects of TR on positive and negative thyroid hormone response elements (Cohen, 1998).
Retinoic acid receptors (RARs) are hormone-regulated transcription factors that control key aspects of normal differentiation. Aberrant RAR activity may be a causal factor in neoplasia. Human acute promyelocytic leukemia, for example, is tightly linked to chromosomal translocations that fuse novel amino acid sequences (denoted PML, PLZF, and NPM) to the DNA-binding and hormone-binding domains of RARalpha. The resulting chimeric receptors have unique transcriptional properties that may contribute to leukemogenesis. Normal RARs repress gene transcription by associating with ancillary factors denoted corepressors (also referred to as SMRT, N-CoR, TRAC, or RIP13). The PML-RARalpha and PLZF-RARalpha oncoproteins retain the ability of RARalpha to associate with corepressors, and this corepressor association correlates with certain aspects of the leukemic phenotype. Unexpectedly, the PLZF moiety itself can interact with SMRT corepressor. This interaction with corepressor is mediated, in part, by a POZ motif within PLZF. Given the presence of POZ motifs in a number of known transcriptional repressors, similar interactions with SMRT may play a role in transcriptional silencing by a variety of both receptor and nonreceptor transcription factors (Hong, 1997).
Chicken ovalbumin upstream promoter-transcription factors (COUP-TFs) are orphan receptors that belong to the steroid/thyroid hormone receptor (TR) superfamily and can repress the transcriptional activity of several target genes; however, the precise mechanism of this repression is unknown. Transfection of a Gal4 DNA-binding domain fused to the putative ligand-binding domain of COUP-TFI (Gal4-COUP-TFI) significantly represses the basal transcriptional activity of a reporter gene containing Gal4-binding sites. Cotransfection of COUP-TFI can relieve the Gal4-COUP-TFI repression in a dose-dependent manner. In contrast, COUP-TFI delta35, which lacks the repressor domain (the C-terminal 35 amino acids), fails to relieve this repression. This finding suggests that the repressor domain of COUP-TFI may squelch a limiting amount of corepressor in HeLa cells. In addition, increasing concentrations of TRbeta also can relieve the COUP-TFI repression in a hormone-sensitive manner. Similarly, overexpression of increasing concentration of COUP-TFI, but not COUP-TFI delta35, can squelch the silencing activity of the unliganded TRbeta. Collectively, these results indicate that COUP-TFI and TRbeta share a common corepressor(s) for their silencing activity. To determine which corepressor is involved in the COUP-TF-silencing activity, a yeast two-hybrid assay and in vitro GST pull-down assay were used to demonstrate that COUP-TFI can interact with the fragment of N-CoR (nuclear receptor-corepressor) encoding amino acids 921-2453 and the fragments of SMRT (silencing mediator for retinoic acid receptor and TR) encoding amino acids 29-564 and 565-1289, respectively. Interestingly, the fragment of SMRT encoding amino acids 1192-1495, which strongly interacts with TRbeta, interacts very weakly with COUP-TFI. Furthermore, overexpression of N-CoR or SMRT potentiates the silencing activity of COUP-TFI and can relieve the COUP-TFI-mediated squelching of Gal4-COUP-TFI activity. Therefore, these studies indicate that N-CoR and SMRT act as corepressors for the COUP-TFI silencing activity (Shibata, 1997).
Many transcription factors function by repressing gene transcription. For a variety of these transcription factors the ability to physically recruit auxiliary proteins, denoted corepressors, is crucial for the ability to silence gene expression. The SMRT corepressor have been implicated in the actions of the PLZF transcription factor and in the function of its oncogenic derivative, PLZF-retinoic acid receptor (RARalpha), in promyelocytic leukemia. PLZF, and a structurally similar transcriptional repressor, BCL-6, can interact with a variety of corepressor proteins in addition to SMRT, including the mSin3A protein and (for PLZF) histone deacetylase-1. Unexpectedly, these additional interactions with corepressor components are nonequivalent for these otherwise similar oncoproteins, suggesting that transcriptional repression by BCL-6 and by PLZF may differ in mechanism. Furthermore, the oncogenic PLZF-RARalpha chimera lacks several important corepressor interaction sites that are present in the native PLZF protein. Thus the t(11;17) translocation that creates the PLZF-RARalpha chimera generates an oncoprotein with potentially novel regulatory properties distinct from those of either parental protein. These results demonstrate that otherwise similar transcription factors can differ notably in their interactions with the corepressor machinery (Wong, 1998b).
Nuclear hormone receptors are ligand-regulated transcription factors that play critical roles in metazoan homeostasis, development, and reproduction. Many nuclear hormone receptors exhibit bimodal transcriptional properties and can either repress or activate the expression of a given target gene. Repression appears to require a physical interaction between a receptor and a corepressor complex containing either the SMRT/TRAC or N-CoR/RIP13 polypeptides. Different receptors are found to interact with different domains in the SMRT and N-CoR corepressors and these divergent interactions may therefore contribute to distinct repression phenotypes. Intriguingly, different isoforms of a single nuclear hormone receptor class also differ markedly in their interactions with corepressors, indicative of their nonidentical actions in cellular regulation. Evidence is presented that combinatorial interactions between different receptors can, through the formation of heterodimeric receptors, result in novel receptor-corepressor interactions not observed for homomeric receptors (Wong, 1998c).
Mutation of the orphan nuclear receptor RORalpha results in a severe impairment of cerebellar development by unknown mechanisms. RORalpha activates transcription from only a subset of sites to which it binds strongly as a monomer. RORalpha also selectively binds as a homodimer to a direct repeat of this monomer site with a 2-bp spacing between the AGGTCA sequences (Rev-DR2 site) and is a much more potent transcriptional activator on this site than on monomer sites or other direct repeats. To better understand the transcriptional regulatory functions of RORalpha, its C terminus was fused to a heterologous DNA-binding domain. Mutational analysis reveals that RORalpha contains both transcriptional activation and transcriptional repression domains, with the repression domain being more active in some cell types. The abilities of RORalpha polypeptides to repress transcription correlate with their abilities to interact with the nuclear receptor corepressors N-CoR and SMRT in vitro. However, the AF2 region of RORalpha inhibits corepressor interaction on DNA, consistent with the lack of repression by the full-length receptor. Thus, transcriptional regulation by RORalpha is complex and likely to be regulated in a cell type- and target gene-specific manner (Harding, 1997).
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. Tepression 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. It is concluded 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 (Hong, 1998).
A novel class of ligands for the human progesterone receptor (PR) have been defined that function as mixed agonists. These compounds induce a conformational change upon binding the receptor that is different from those changes induced by agonists and antagonists. This establishes a correlation between the structure of a ligand-receptor complex and its transcriptional activity. In an attempt to define the cellular components that distinguish between different ligand-induced PR conformations, it has been determined, by using a mammalian two-hybrid assay, that the nuclear receptor corepressor (NCoR) and the silencing mediator for retinoid and thyroid hormone receptor (SMRT) differentially associate with PR depending on the class of ligand bound to the receptor. Specifically, it has been observed that the corepressors preferentially associate with antagonist-occupied PR and that overexpression of these corepressors suppresses the partial agonist activity of antagonist-occupied PR. Binding studies performed in vitro, however, reveal that recombinant SMRT can interact with PR in a manner that is not influenced by the nature of the bound ligand. Thus, the inability of SMRT or NCoR to interact with agonist-activated PR when assayed in vivo may relate more to the increased affinity of PR for coactivators, with a subsequent displacement of corepressors, than to an inherent low affinity for the corepressor proteins. Previous work from other groups has shown that 8-bromo-cyclic AMP (8-bromo-cAMP) can convert the PR antagonist RU486 into an agonist and, additionally, can potentiate the transcriptional activity of agonist-bound PR. In this study, it is shown that exogenous expression of NCoR or SMRT suppresses all 8-bromo-cAMP-mediated potentiation of PR transcriptional activity. Further analysis reveals that 8-bromo-cAMP addition decreases the association of NCoR and SMRT with PR. Thus, it is proposed that 8-bromo-cAMP-mediated potentiation of PR transcriptional activity is due, at least in part, to a disruption of the interaction between PR and the corepressors NCoR and SMRT. Cumulatively, these results suggest that NCoR and SMRT expression may play a pivotal role in PR pharmacology (Wagner, 1998).
Two principles of corepressor function have been identified that account for differences in transcriptional repression by nuclear hormone receptors (NHRs): (1) it has been determined that receptor stoichiometry is a crucial determinant of transcriptional repression mediated by the corepressors N-CoR and SMRT. This provides a molecular explanation for the observation that NHRs repress transcription as dimers but not monomers. (2) Corepressor function is restricted by steric effects related to DNA binding in a receptor-specific manner. Thus, although N-CoR and SMRT are capable of binding to several NHRs in solution, they are highly selective about receptor binding on DNA, a context that reflects their in vivo function more accurately. These stoichiometric and steric principles govern specific interactions between corepressors and NHRs, thus providing evidence that N-CoR and SMRT do not serve redundant functions but rather contribute to receptor-specific transcriptional repression (Zamir, 1997a).
Rev-erbA alpha and RVR are orphan nuclear receptors that function as dominant transcriptional silencers. Ligand-independent repression of transcription by Rev-erbA alpha and RVR is mediated by the nuclear receptor corepressors, N-CoR and its variants RIP (RXR interacting protein) 13a and RIP13 delta 1. The physical association between the corepressors and Rev-erbA alpha and RVR is dependent on the presence of a receptor interaction domain (RID) in the N-CoR family. The E region of RVR and Rev-erbA alpha is necessary and sufficient for the in vivo interaction with the nuclear receptor corepressor, RIP13 delta 1. Two corepressor interaction regions, CIR-1 and CIR-2, separated by approximately 150 amino acids in the E region of RVR, are required for the interaction with N-CoR, RIP13a, and RIP13 delta A. The D region is not required for the physical interaction. In contrast, the D and E regions of Rev-erbA alpha are necessary for the interaction with the N-CoR and RIP13a-RIDs in vivo, suggesting that RIP13 delta 1 and N-CoR/RIP13a differentially interact with Rev-erbA alpha. Mutagenesis of CIR-1, a novel domain that is highly conserved between RVR and Rev-erbA alpha, demonstrates that the N-terminal portion of helix 3 plays a key role and is absolutely necessary for the interaction with RIP13 delta 1, RIP13a, and N-CoR. The phenylalanine residues, F402 and F441, in RVR and Rev-erbA alpha, respectively, are critical residues in supporting corepressor interaction. Cotransfection studies demonstrate that repression of a physiological target, the human Rev-erbA alpha promoter, by RVR is significantly impaired by mutation of CIR-1 or deletion of CIR-2. Furthermore, overexpression of either the N-CoR/RIP13a or RIP13 delta 1-RIDs alleviates RVR-mediated repression of the Rev-erbA alpha promoter, demonstrating that corepressor binding mediates the repression of a native target gene by RVR. A minimal region containing juxtapositioned CIR-1 and CIR-2 is sufficient for corepressor binding and transcriptional repression. In conclusion, this study has identified a new corepressor interaction region, CIR-1, in the N terminus of helix 3 in the E region of RVR and Rev-erbA alpha, that is required for transcriptional silencing. Furthermore, evidence is provided that CIR-1 and CIR-2 may form a single corepressor interaction interface (Burke, 1998).
The E-26 transforming specific (ETS)-related gene TEL (Drosophila homolog: Yan), also known as ETV6, encodes a strong transcription repressor that is rearranged in several recurring chromosomal rearrangements associated with leukemia and congenital fibrosarcoma. The TEL protein contains two functional domains that have been partially characterized: a helix-loop-helix (HLH) domain (also known as a pointed domain) at the N-terminus, which physically interacts with itself, with the SUMO-conjugating enzyme UBC9, and with FLI1; and, at the C-terminus, an ETS domain with DNA-binding properties. Little is known about the function of the central region of TEL. The HLH domain and the central region of TEL are consistently maintained in the t(12;21), which is the most frequent chromosomal translocation involving TEL. The HLH domain and the central region of TEL mediate transcription repression by two distinct mechanisms. The central region involves the recruitment of a repression complex, including SMRT and mSin3A. The HLH domain represses gene transcription through a mechanism that is independent of known corepressors. Thus, TEL belongs to a growing number of transcription factors rearranged by chromosomal translocations that are associated with the corepressor complexes (Chakrabarti, 1999).
SMRT and N-CoR interactions with transcription factors: mutationally altered nuclear receptors
Nuclear receptor corepressor (CoR)-histone deacetylase (HDAC) complex recruitment is indispensable for the biological activities of the retinoic acid receptor fusion proteins of acute promyelocytic leukemias. ETO (eight-twenty-one or MTG8), which is fused to the acute myelogenous leukemia 1 (AML1) transcription factor in t(8;21) AML, interacts via its zinc finger region with a conserved domain of the corepressors N-CoR and SMRT and recruits HDAC in vivo. The fusion protein AML1-ETO retains the ability of ETO to form stable complexes with N-CoR/SMRT and HDAC. Deletion of the ETO C terminus abolishes CoR binding and HDAC recruitment and severely impairs the ability of AML1-ETO to inhibit differentiation of hematopoietic precursors. These data indicate that formation of a stable complex with CoR-HDAC is crucial to the activation of the leukemogenic potential of AML1 by ETO and suggest that aberrant recruitment of corepressor complexes is a general mechanism of leukemogenesis (Gelmetti, 1998).
The LAZ3/BCL6 (lymphoma-associated zinc finger 3/B cell lymphomas 6) gene frequently is altered in non-Hodgkin lymphomas. It encodes a sequence-specific DNA binding transcriptional repressor that contains a conserved N-terminal domain, termed BTB/POZ (bric-a-brac tramtrack broad complex/pox viruses and zinc fingers). The LAZ3/BCL6 BTB/POZ domain interacts with the SMRT (silencing mediator of retinoid and thyroid receptor) protein. SMRT originally was identified as a corepressor of unliganded retinoic acid and thyroid receptors and forms a repressive complex with a mammalian homolog of the yeast transcriptional repressor SIN3 and the HDAC-1 histone deacetylase. Protein binding assays demonstrate that the LAZ3/BCL6 BTB/POZ domain directly interacts with SMRT in vitro. DNA-bound LAZ3/BCL6 recruits SMRT in vivo, and both overexpressed proteins completely colocalize in nuclear dots. Overexpression of SMRT enhances the LAZ3/BCL6-mediated repression. These results define SMRT as a corepressor of LAZ3/BCL6 and suggest that LAZ3/BCL6 and nuclear hormone receptors repress transcription through shared mechanisms involving SMRT recruitment and histone deacetylation (Dhordain, 1997).
Aberrant nuclear hormone receptors have been implicated as causal agents in a number of endocrine and neoplastic diseases. The syndrome of Resistance to Thyroid Hormone (RTH) is a human genetic disease characterized by an impaired physiological response to thyroid hormone. RTH is associated with diverse mutations in the thyroid hormone receptor beta-gene. The resulting mutant receptors function as dominant negatives, interfering with the actions of normal thyroid hormone receptors coexpressed in the same cells. RTH receptors interact aberrantly with a newly recognized family of transcriptional corepressors variously denoted as nuclear receptor corepressor (N-CoR), retinoid X receptor interacting protein-13 (RIP-13), silencing mediator for retinoid and thyroid hormone receptors (SMRT), and thyroid hormone receptor-associating cofactor (TRAC). All RTH receptors tested exhibit an impaired ability to dissociate from corepressors in the presence of thyroid hormone. Two of the RTH mutations uncouple corepressor dissociation from hormone binding; two additional RTH mutants exhibit an unusually strong interaction with corepressor under all hormone conditions tested. Artificial mutants that abolish corepressor binding abrogate the dominant negative activity of RTH mutants. It is suggested that an altered corepressor interaction is likely to play a critical role in the dominant negative potency of RTH mutants and may contribute to the variable phenotype in this disorder (Yoh, 1997).
Thyroid hormone receptor (TR) functions as part of multiprotein complexes that also include retinoid X receptor (RXR) and transcriptional coregulators. Both the TR CoR box and ninth heptad are required for RXR interaction and in turn for interaction with corepressor proteins N-CoR and SMRT. Remarkably, the recruitment of RXR to repression-defective CoR box and ninth-heptad mutants via a heterologous dimerization interface restores both corepressor interaction and repression. The addition of thyroid hormone obviates the CoR box requirement for RXR interaction, provided that the AF2 activation helix at the C terminus of TR is intact. These results indicate that RXR differentially recognizes the unliganded and liganded conformations of TR and that these differences appear to play a major role in the recruitment of corepressors to TR-RXR heterodimers (Zhang, 1997).
The human retinoic acid receptor alpha (hRAR alpha) exhibits cell-specific transcriptional activity. 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. The CV1-specific effect is due to the receptor C terminus. 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, the conserved AF2/tau c activation function of RAR is responsible for the cell-specific inhibition of silencing. Thereby, the CV1 cell specificity is abolished by replacing AF2/tau c of RAR with the corresponding sequence of the thyroid hormone receptor. Thus, a new role of the C-terminal conserved activation function AF2/tau c is found; specifically, the RAR AF2/tau c-sequence is able to prevent silencing of RAR in a cell-specific manner. In addition, 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, has 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 (Baniahmad, 1998).
Function of SMRT and N-CoR: role of nuclear receptor ligands
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