Function of SMRT and N-CoR: role of nuclear receptor ligands
Nuclear hormone receptors are ligand-regulated transcription factors that modulate gene expression in response to small, hydrophobic hormones, such as retinoic acid and thyroid hormone. The thyroid hormone and retinoic acid receptors typically repress transcription in the absence of hormone and activate it in the presence of hormone. Transcriptional repression is mediated, in part, through the ability of these receptors to physically associate with ancillary polypeptides called corepressors. It is of interest to understand the mechanism by which corepressors are recruited to unliganded nuclear hormone receptors and are released on the binding of hormone. An alpha-helical domain located at the thyroid hormone receptor C terminus appears to undergo a hormone-induced conformational change required for release of corepressor; amino acid substitutions that abrogate this conformational change can impair or prevent corepressor release. In contrast, retinoid X receptors appear neither to undergo an equivalent conformational alteration in their C termini nor to release corepressor in response to cognate hormone, consistent with the distinct transcriptional regulatory properties displayed by this class of receptors (Lin, 1997).
Nuclear receptors are structurally related, ligand-activated regulators of a complex array of genes involved in cell proliferation, differentiation, morphogenesis, and homeostasis. In the absence of ligand, several nuclear receptors associate with a nuclear receptor corepressor (N-CoR) or the related factor SMRT (silencing mediator of retinoid and thyroid receptors) to mediate repression. Their regulatory function is further modulated by both physiologic and pharmacologic ligands and by the actions of various signal transduction pathways that result in ligand-independent gene activation of diverse nuclear receptor family members. N-CoR and SMRT appear to be components of cellular complexes containing histone deacetylases (HDACs) and homologs of the yeast repressor Sin3, which are recruited to DNA via targeting by diverse DNA-binding, site-specific transcription factors. Conversely, transcriptional activation by nuclear hormone receptors requires the ligand-dependent association of a coactivator complex that includes a family of nuclear receptor coactivators (NCoAs) and also includes the histone acetylases Creb-binding protein (CBP)/p300 and P/CAF. Several lines of evidence indicate that the nuclear receptor corepressor (N-CoR) complex imposes ligand dependence on transcriptional activation by the retinoic acid receptor and mediates the inhibitory effects of estrogen receptor antagonists, such as tamoxifen, suppressing a constitutive N-terminal, Creb-binding protein/coactivator complex-dependent activation domain. Functional interactions between specific receptors and N-CoR or SMRT corepressor complexes are regulated, positively or negatively, by diverse signal transduction pathways (Lavinsky, 1998).
The ER/N-CoR interactions are decreased by brief exposure of either MCF7 or HeLa cells to forskolin or epidermal growth factor (EGF), agents that can switch the mixed anti-estrogen trans-hydroxytamoxifen (TOT) from antagonist to an agonist function. It is observed that a nonphosphorylatable mutant of ER (S118A) proves resistant to the effect of EGF on ER/N-CoR interaction. This resistence is consistent with the observation that EGF-induced activation of the ER depends on direct phosphorylation of serine 118. Microinjection of purified IgG against N-CoR, mSin3 A/B, or HDAC2 converts TOT into an anagonist in MCF-7 and Rat-1 cells, while exerting little effect on activity of the unliganded ER. In the microinjection assay, treatment with forskolin or EGF also prevents the inhibitory effects of TOT. Decreased levels of N-CoR correlate with the acquisition of tamoxifen resistance in a mouse model system for human breast cancer. These data suggest that N-CoR- and SMRT-containing complexes act as rate-limiting components in the actions of specific nuclear receptors, and that their actions are regulated by multiple signal transduction pathways (Lavinsky, 1998).
Retinoic-acid receptor-alpha (RAR-alpha) and peroxisome proliferator-activated receptor-gamma (PPAR-gamma) are members of the nuclear-receptor superfamily that bind to DNA as heterodimers with retinoid-X receptors (RXRs). PPAR-RXR heterodimers can be activated by PPAR or RXR ligands, whereas RAR-RXR heterodimers are selectively activated by RAR ligands only, because of allosteric inhibition of the binding of ligands to RXR by RAR. However, RXR ligands can potentiate the transcriptional effects of RAR ligands in cells. Transcriptional activation by nuclear receptors requires a carboxy-terminal helical region, termed activation function-2 (AF-2), that forms part of the ligand-binding pocket and undergoes a conformational change required for the recruitment of co-activator proteins, including NCoA-1/SRC-1. Allosteric inhibition of RXR results from a rotation of the RXR AF-2 helix that places it in contact with the RAR coactivator-binding site. Recruitment of an LXXLL motif of SRC-1 to RAR in response to ligand displaces the RXR AF-2 domain, allowing RXR ligands to bind and promote the binding of a second LXXLL motif from the same SRC-1 molecule. These results may partly explain the different responses of nuclear-receptor heterodimers to RXR-specific ligands (Westin, 1998).
Nuclear hormone receptors are ligand-regulated transcription factors that modulate the expression of specific target genes in response to the binding of small, hydrophobic hormone ligands. Many nuclear hormone receptors, such as the retinoic acid receptors, can both repress and activate target gene expression; these bimodal transcription properties are mediated by the ability of these receptors to tether auxiliary factors (termed corepressors and coactivators). Corepressors are typically bound by receptors in the absence of cognate hormone, whereas binding of an appropriate hormone agonist induces an allosteric alteration in the receptor resulting in release of the corepressor and recruitment of coactivator. Structural analysis indicates that there is a close induced fit between the hormone ligand and the receptor polypeptide chain. This observation suggests that different ligands, once bound, may confer distinct conformations on the receptor that may invoke, in turn, distinct functional consequences. Different retinoids do differ in the ability to release corepressor once bound to retinoic acid receptor; these differences in corepressor release may manifest as differences in transcriptional regulation (Hong, 1999).
Steroid receptor antagonists, such as the antiestrogen tamoxifen or the antiprogestin RU486, can have inappropriate agonist-like effects in tissues and tumors. To explain this paradox it was postulated that coactivators are inadvertently brought to the promoters of DNA-bound, antagonist-occupied receptors. The human (h) progesterone receptor (PR) hinge-hormone binding domain (H-HBD) was used as bait in a two-hybrid screen of a HeLa cDNA library, in which the yeast cells were treated with RU486. Two interesting steroid receptor-interacting proteins have been isolated and characterized that regulate transcription in opposite directions. The first is L7/SPA, a previously described 27-kDa protein containing a basic region leucine zipper domain, having no known nuclear function. When coexpressed with tamoxifen-occupied estrogen receptors (hER) or RU486-occupied hPR or glucocorticoid receptors (hGR), L7/SPA increases the partial agonist activity of the antagonists by 3- to 10-fold, but it has no effect on agonist-mediated transcription. The interaction of L7/SPA with hPR maps to the hinge region, and indeed, the hPR hinge region squelches L7/SPA-dependent induction of antagonist-mediated transcription. Interestingly, pure antagonists that lack partial agonist effects, such as the antiestrogen ICI164,384 or the antiprogestin ZK98299, cannot be up-regulated by L7/SPA. Also isolated, cloned, and sequenced was the human homolog (hN-CoR) of the 270-kDa mouse (m) thyroid/retinoic acid receptor corepressor. Binding of hN-CoR maps to the hPR-HBD. mN-CoR, and a related human corepressor, SMRT, suppress RU486 or tamoxifen-mediated partial agonist activity by more than 90%. This suppression is completely squelched by overexpression of the hPR H-HBD. Additionally, both corepressors reverse the antagonist-dependent transcriptional up-regulation produced by L7/SPA. These data suggest that the direction of transcription by antagonist-occupied steroid receptors can be controlled by the ratio of coactivators to corepressors recruited to the transcription complex by promoter-bound receptors. In normal tissues and in hormone-resistant breast cancers in which the agonist activity of mixed antagonists predominates, steroid receptors may be preferentially bound by coactivators. This suggests a strategy by which such partial agonist activity can be eliminated and by which candidate receptor ligands can be screened for this activity (Jackson, 1997).
Complexes of SMRT and N-CoR: interactions with Sin3, histone deacetylases and other members of a repression complex
Whereas liganded nuclear receptors serve as transcriptional activators, unliganded nuclear receptors serve as repressors. How does the unliganded nuclear receptor transmit a repressive signal to the transcriptional apparatus and what is the nature of this signal? In fact, the target of the unliganded nuclear receptor is not RNA polymerase but chromatin; repression is mediated by corepressors. These are proteins that associate with unliganded nuclear receptors assembling a macromolecular complex that modifies chromatin so as to silence gene activity. The macromolecular complex acts to deacetylate histone. The transcriptional corepressors SMRT and N-CoR function as silencing mediators for retinoid and thyroid hormone receptors. SMRT and N-CoR directly interact with unliganded nuclear receptors, and these corepressors in turn directly interact with mSin3A, a corepressor for the Mad-Max heterodimer and a homolog of the yeast global-transcriptional repressor Sin3p. The recently characterized histone deacetylase 1 (HDAC1) interacts with Sin3A and SMRT to form a multisubunit, ternary repressor complex. Histone deacetylase in turn targets chromatin, converting it into a form that is unaccessable to the transcriptional apparatus. Consistent with this model, it is found that HDAC inhibitors synergize with retinoic acid to stimulate hormone-responsive genes and the differentiation of myeloid leukemia (HL-60) cells. Addition of a deacetylase inhibitor such as Trichostatin A relieves transcriptional repression resulting in a promoter that is sensitive to the addition of activating hormone. This work establishes a convergence of repression pathways for bHLH-Zip proteins and nuclear receptors and suggests that this type of regulation may be more widely conserved than previously suspected (Nagy, 1997).
Evidence is presented that both corepressors SMRT and N-CoR exist in large protein complexes with estimated sizes of 1.5-2 MDa in HeLa nuclear extracts. Using a combination of conventional and immunoaffinity chromatography, a SMRT complex has been isolated and histone deacetylase 3 (HDAC3) and transducin (beta)-like I (TBL1), a WD-40 repeat-containing protein, have been identified as the subunits of the purified SMRT complex. The HDAC3-containing SMRT and N-CoR complexes can bind to unliganded thyroid hormone receptors (TRs) in vitro. In Xenopus oocytes, both SMRT and N-CoR also associate with HDAC3 in large protein complexes; injection of antibodies against HDAC3 or SMRT/N-CoR leads to a partial relief of repression by unliganded TR/RXR. These findings thus establish both SMRT and N-CoR complexes as bona fide HDAC-containing complexes and shed new light on the molecular pathways by which N-CoR and SMRT function in transcriptional repression (Li, 2000).
Recent works demonstrate that some transcriptional repressors recruit histone deacetylases (HDACs) either through direct interaction, or as a member of a multisubunit repressing complex containing other components, referred to as corepressors. For instance, the bHLH-Zip transcriptional repressors MAD/MXI recruit HDACs, together with the mSIN3 corepressors, whereas unliganded nuclear receptors contact another corepressor, SMRT (or its relative N-CoR), which, in turn, associates with both mSIN3 and HDACs to form the repressor complex. Recently, SMRT also directly associates with LAZ3(BCL-6), a POZ/Zn finger transcriptional repressor involved in the pathogenesis of non-Hodgkin lymphomas. However, whether LAZ3 recruits the HDACs-containing repression complex is currently unknown. LAZ3 is shown to associate with corepressor mSIN3A both in vivo and in vitro , and a central region, which harbours autonomous repression activity, is mainly responsible for this interaction. Conversely, the N-terminal half of mSIN3A is both necessary and sufficient to bind LAZ3. Moreover, LAZ3 also interacts with an HDAC (HDAC-1) through its POZ domain in vitro while the immunoprecipitation of LAZ3 results in the coretention of an endogenous HDAC activity in vivo . Finally, inhibitors of HDACs significantly reduce the LAZ3-mediated repression. Taken together, it is concluded that LAZ3 recruits a repressing complex containing SMRT, mSIN3A and a HDAC, and that LAZ3's full repressing potential on transcription requires HDACs activity. These results identify HDACs as molecular targets of LAZ3 oncogene and further strengthen the connection between aberrant chromatin acetylation and human cancers (Dhordain, 1998).
A variety of eukaryotic transcription factors, including the nuclear hormone receptors, Max-Mad, BCL-6, and PLZF, appear to mediate transcriptional repression through the ability to recruit a multiprotein corepressor complex to the target promoter. This corepressor complex includes the SMRT/N-CoR polypeptides, mSin3A or -B, and histone deacetylase 1 or 2. The presence of a histone-modifying activity in the corepressor complex has led to the suggestion that gene silencing is mediated by modification of the chromatin template, perhaps rendering it less accessible to the transcriptional machinery. It is reported, however, that the corepressor complex actually appears to exhibit multiple mechanisms of transcriptional repression, only one of which corresponds with detectable recruitment of the histone deacetylase. Evidence is provided instead of an alternative pathway of repression that may be mediated by direct physical interactions between components of the corepressor complex and the general transcription factor TFIIB (Wong, 1998a).
Nuclear hormone receptors are potent repressors of transcription in the unliganded state. The cloning of a nuclear receptor corepressor that has been called SUN-CoR (Small Unique Nuclear receptor CoRepressor) is described that shows no homology to previously described nuclear hormone receptor corepressors, N-CoR, or SMRT. SUN-CoR is a highly basic, 16-kDa nuclear protein that is expressed at high levels in adult tissues and is induced during adipocyte and myogenic differentiation. SUN-CoR potentiates transcriptional repression by thyroid hormone receptor and RevErb in vivo, represses transcription when fused to a heterologous DNA binding domain, and interacts with RevErb as well as with thyroid hormone receptor in vitro. SUN-CoR also interacts with N-CoR and SMRT in vitro and with endogenous N-CoR in cells. It is concluded that SUN-CoR is a corepressor and may function as an additional component of the complex involved in transcriptional repression by unliganded and orphan nuclear hormone receptors (Zamir, 1997).
Normal mammalian growth and development are highly dependent on the regulation of the expression and activity of the Myc family of transcription factors. Mxi1-mediated inhibition of Myc activities requires interaction with mammalian Sin3A or Sin3B proteins, which are purported to act as scaffolds for additional co-repressor factors. The identification of two such Sin3-associated factors, the nuclear receptor co-repressor (N-CoR) and histone deacetylase (HD1), provides a basis for Mxi1/Sin3-induced transcriptional repression and tumor suppression (Alland, 1997).
Members of the Mad family of bHLH-Zip proteins heterodimerize with Max to repress transcription in a sequence-specific manner. Transcriptional repression by Mad:Max heterodimers is mediated by ternary complex formation with either of the corepressors mSin3A or mSin3B. mSin3A is an in vivo component of large, heterogeneous multiprotein complexes and is tightly and specifically associated with at least seven polypeptides. Two of the mSin3A-associated proteins, p50 and p55, are highly related to the histone deacetylase HDAC1. The mSin3A immunocomplexes possess histone deacetylase activity that is sensitive to the specific deacetylase inhibitor trapoxin. mSin3A-targeted repression of a reporter gene is reduced by trapoxin treatment, suggesting that histone deacetylation mediates transcriptional repression through Mad-Max-mSin3A multimeric complexes (Hassig, 1997).
Transcriptional repression by nuclear receptors has been correlated to binding of the putative co-repressor, N-CoR. A complex has been identified that contains N-CoR, the Mad presumptive co-repressor mSin3, and the histone deacetylase mRPD3; this complex is required for both nuclear receptor- and Mad-dependent repression, but not for repression by transcription factors of the ets-domain family. These data predict that the ligand-induced switch of heterodimeric nuclear receptors from repressor to activator functions involves the exchange of complexes containing histone deacetylases with those that have histone acetylase activity (Heinzel, 1997).
The corepressor SMRT mediates repression by thyroid hormone receptor (TR) as well as other nuclear hormone receptors and transcription factors. A novel SMRT-containing complex has been isolated from HeLa cells. This complex contains transducin beta-like protein 1 (TBL1: Drosophila homolog, Ebi), whose gene is mutated in human sensorineural deafness. It also contains HDAC3, a histone deacetylase not previously thought to interact with SMRT. TBL1 displays structural and functional similarities to Tup1 and Groucho corepressors, sharing their ability to interact with histone H3. In vivo, TBL1 is bridged to HDAC3 through SMRT and can potentiate repression by TR. Intriguingly, loss-of-function TRbeta mutations cause deafness in mice and humans. These results define a new TR corepressor complex with a physical link to histone structure and a potential biological link to deafness (Guenther, 2000).
Evidence is presented that both corepressors SMRT and N-CoR exist in large protein complexes with estimated sizes of 1.5-2 MDa in HeLa nuclear extracts. Using a combination of conventional and immunoaffinity chromatography, a SMRT complex has been successfully isolated and histone deacetylase 3 (HDAC3) and transducin (beta)-like I (TBL1), a WD-40 repeat-containing protein, have been identified as the subunits of the purified SMRT complex. The HDAC3-containing SMRT and N-CoR complexes can bind to unliganded thyroid hormone receptors (TRs) in vitro. In Xenopus oocytes, both SMRT and N-CoR associate with HDAC3 in large protein complexes. Injection of antibodies against HDAC3 or SMRT/N-CoR leads to a partial relief of repression by unliganded TR/RXR. These findings thus establish both SMRT and N-CoR complexes as bona fide HDAC-containing complexes and shed new light on the molecular pathways by which N-CoR and SMRT function in transcriptional repression (Li, 2002).
A yeast two-hybrid screen using the conserved carboxyl terminus of the nuclear receptor corepressor SMRT as a bait led to the isolation of a novel human gene termed SHARP (SMRT/HDAC1 Associated Repressor Protein), related to Drosophila Split ends. SHARP is a potent transcriptional repressor whose repression domain (RD) interacts directly with SMRT and at least five members of the NuRD complex including HDAC1 and HDAC2. In addition, SHARP binds to the steroid receptor RNA coactivator SRA via an intrinsic RNA binding domain and suppresses SRA-potentiated steroid receptor transcription activity. Accordingly, SHARP has the capacity to modulate both liganded and nonliganded nuclear receptors. Surprisingly, the expression of SHARP is itself steroid inducible, suggesting a simple feedback mechanism for attenuation of the hormonal response (Shi, 2001).
Spen proteins regulate the expression of key transcriptional effectors in diverse signaling pathways. They are large proteins characterized by N-terminal RNA-binding motifs and a highly conserved C-terminal SPOC domain. The specific biological role of the SPOC domain (Spen paralog and ortholog C-terminal domain), and hence, the common function of Spen proteins, has been unclear to date. The Spen protein, SHARP (SMRT/HDAC1-associated repressor protein), was identified as a component of transcriptional repression complexes in both nuclear receptor and Notch/RBP-J{kappa} signaling pathways. The 1.8 Å crystal structure of the SPOC domain from SHARP has been determined. This structure shows that essentially all of the conserved surface residues map to a positively charged patch. Structure-based mutational analysis indicates that this conserved region is responsible for the interaction between SHARP and the universal transcriptional corepressor SMRT/NCoR (silencing mediator for retinoid and thyroid receptors/nuclear receptor corepressor. This interaction involves a highly conserved acidic motif at the C terminus of SMRT/NCoR. These findings suggest that the conserved function of the SPOC domain is to mediate interaction with SMRT/NCoR corepressors, and that Spen proteins play an essential role in the repression complex (Ariyoshi, 2003).
The structure of the SPOC domain reveals a novel architecture for an independent protein domain. (The ß-barrel domain of Ku forms part of a larger structure.) It appears to be ideally suited to mediate interaction with other proteins through a number of deep grooves and clefts in the surface as well as two nonpolar loops. In addition, the N-terminal region seems to possess an intrinsic propensity to form a ß-sheet with partner proteins. Most significantly, the structure reveals a highly basic patch on the surface, which is absolutely conserved throughout the Spen protein family. It is likely, therefore, that the function of this patch is indicative of the conserved role of the Spen proteins (Ariyoshi, 2003).
Through a variety of interaction and mutagenesis experiments it has been shown that this basic patch mediates the tight and specific interaction of the Spen proteins with the conserved acidic C-terminal LSD peptide from the SMRT/NCoR corepressors. Remarkably, point mutations within the basic patch totally abolish interaction with the LSD peptide. This suggests that although complementary charges play an important role in the interaction, the precise positioning of side chains of the key basic residues is absolutely required for stereospecific recognition of the SMRT/NCoR LSD motif. Whereas the precise details of the interaction remain to be determined, some indication of a possible mode of interaction is seen within the crystal lattice. The N-terminal region of one molecule (Pro 3495-Gln 3500) makes a crystal packing interaction with the ß3 strand of an adjacent molecule (Arg 3548-Arg 3554). The interactions include backbone-backbone hydrogen bonds, as well as both electrostatic and hydrophobic interactions (Ariyoshi, 2003).
The conservation of the SPOC domain in Drosophila and Caenorhabditis elegans suggests that both these species should possess corepressor proteins with LSD motifs. It is clear that the rather divergent Drosophila corepressor SMRTER does have an almost identical LSD motif. The findings of this study suggest that a similar protein must also be present in the worm (Ariyoshi, 2003).
It remains to be seen how other proteins such as HDAC1 may interact with SHARP. It is striking however that the LSD peptide itself serves as a potent transcriptional repressor, suggesting that recruitment of SHARP to a promoter is sufficient to mediate strong repression of basal transcription (Ariyoshi, 2003).
In conclusion, the combination of structural and functional experiments with the SPOC domain of SHARP clearly demonstrate that the conserved function of the SPOC domain is to mediate interaction with corepressors and, therefore, that Spen proteins play an essential role in regulating transcriptional repression (Ariyoshi, 2003).
Nuclear hormone receptor independent functions of SMRT and N-CoR
Nuclear receptors inhibit synthesis of collagenase-1 (matrix metalloproteinase-1; MMP-1), an enzyme that degrades interstitial collagens and contributes to joint pathology in rheumatoid arthritis. SMRT (Silencing Mediator for Retinoid and Thyroid hormone receptors) mediates the repressive effect of nuclear receptors at hormone responsive elements (HREs), prompting an investigation to find out whether this co-repressor could also regulate transcription of MMP-1, which lacks any known HREs. Primary synovial fibroblasts express SMRT. When over-expressed by transient transfection, SMRT inhibits MMP-1 promoter activity induced by interleukin-1 (IL-1), phorbol myristate acetate (PMA) or v-Src. SMRT apparently inhibits MMP-1 gene expression by interfering with one or more transcriptional elements clustered in a region between -321 and +63. It is concluded that SMRT negatively regulates MMP-1 synthesis through a novel, HRE-independent mechanism that involves proximal regions of the MMP-1 promoter (Schroen, 1997).
The Delta-Notch signal transduction pathway has widespread roles in animal development in which it appears to control cell fate. CBF1/RBP-Jkappa, the mammalian homolog of Drosophila Suppressor of Hairless [Su(H)], switches from a transcriptional repressor to an activator upon Notch activation. The mechanism whereby Notch regulates this switch is not clear. Prior to induction CBF1/RBP-Jkappa interacts with a corepressor complex containing SMRT and the histone deacetylase HDAC-1. This complex binds via the CBF1 repression domain; mutants defective in repression fail to interact with the complex. Activation by Notch disrupts the formation of the repressor complex, thus establishing a molecular basis for the Notch switch. Finally, ESR-1, a Xenopus gene activated by Notch and X-Su(H), is induced in animal caps treated with TSA, an inhibitor of HDAC-1. The functional role for the SMRT/HDAC-1 complex in CBF1/RBP-Jkappa regulation reveals a novel genetic switch in which extracellular ligands control the status of critical nuclear cofactor complexes (Kao, 1998).
Innate immune responses to bacterial or viral infection require rapid transition of large cohorts of inflammatory response genes from poised/repressed to actively transcribed states, but the underlying repression/derepression mechanisms remain poorly understood. This study report that, while the nuclear receptor corepressor (NCoR) and silencing mediator of retinoic acid and thyroid hormone receptor (SMRT) corepressors establish repression checkpoints on broad sets of inflammatory response genes in macrophages and are required for nearly all of the transrepression activities of liver X receptors (LXRs), they can be selectively recruited via c-Jun or the Ets repressor Tel, respectively, establishing NCoR-specific, SMRT-specific, and NCoR/SMRT-dependent promoters. Unexpectedly, the binding of NCoR and SMRT to NCoR/SMRT-dependent promoters is frequently mutually dependent, establishing a requirement for both proteins for LXR transrepression and enabling inflammatory signaling pathways that selectively target NCoR or SMRT to also derepress/activate NCoR/SMRT-dependent genes. These findings reveal a combinatorial, corepressor-based strategy for integration of inflammatory and anti-inflammatory signals that play essential roles in immunity and homeostasis (Ghisletti, 2009).
A series of transcription factors critical for maintenance of the neural stem cell state have been identified, but the role of functionally important corepressors in maintenance of the neural stem cell state and early neurogenesis remains unclear. Previous studies have characterized the expression of both SMRT (also known as NCoR2, nuclear receptor co-repressor 2) and NCoR in a variety of developmental systems; however, the specific role of the SMRT corepressor in neurogenesis is still to be determined. This study reports a critical role for SMRT in forebrain development and in maintenance of the neural stem cell state. Analysis of a series of markers in SMRT-gene-deleted mice revealed the functional requirement of SMRT in the actions of both retinoic-acid-dependent and Notch-dependent forebrain development. In isolated cortical progenitor cells, SMRT is critical for preventing retinoic-acid-receptor-dependent induction of differentiation along a neuronal pathway in the absence of any ligand. These data reveal that SMRT represses expression of the jumonji-domain containing gene JMJD3, a direct retinoic-acid-receptor target that functions as a histone H3 trimethyl K27 demethylase and which is capable of activating specific components of the neurogenic program (Jepsen, 2007).
Many jumonji-C-domain containing proteins catalyse removal of methylated histone tails but nevertheless show distinct substrate specificity among various protein families. In vitro histone demethylase assays revealed selective removal of H3K27me3 and expression of full-length JMJD3, but not of jumonji-C-domain deleted protein, results in a dramatic decrease of global trimethyl histone H3K27 level, indicating that JMJD3 demethylates H3K27me3. Other histone modifications evaluated, including H3K27me2, were not diminished by overexpression of JMJD3. These data demonstrate that JMJD3 is a functional demethylase that specifically targets histone H3K27me3 and this function has also been reported for both JMJD3 and the highly homologous UTX protein (Jepsen, 2007).
Trimethyl K27 modification is important in transmitting epigenetic information during development, imprinting and X-chromosome inactivation and has limited reversibility. A global 'passive' decrease of K27me3 has been observed in embryonic stem cell differentiation, accompanied by concurrent loss of the Polycomb group protein Ezh2, the only known histone methyltransferase that targets K27. It was observed, however, that Ezh2 levels remain constant after RA-stimulated differentiation of neural stem cells, prompting the suggestion that epigenetic regulation via histone H3K27me3 modification by the histone demethylase JMJD3 is a contributory early event in neural differentiation. Indeed, RA-induced neuronal differentiation caused a decrease in histone H3K27me3 and recruitment of JMJD3 to the Dlx5 promoter. These results are consistent with several recent genome-wide location analyses that have suggested histone H3K27me3 to be a dynamic marker reflecting developmental potential (Jepsen, 2007).
The results suggest that expression of JMJD3, a novel histone H3K27 demethylase, is regulated in neural stem cell differentiation in response to RA by SMRT-dependent, RA-receptor-mediated programs, and serves as a mechanistic component of this neuronal fate program. Together, these studies have uncovered a specific role of SMRT in maintaining the neural stem cell state, defending against an ability of unliganded RA receptor to initiate a differentiation program along a neuronal pathway based, at least in part, on the RA-dependent regulation of a component of the histone methyl ation/demethylation machinery (Jepsen, 2007).
SMRT and N-CoR expression in tumors
Recently, it has been shown that CYP17 gene transcription is activated by SF-1 (Steroidogenic Factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, whereas it is inhibited by COUP-TF (Chicken Ovalbumin Upstream Promoter-Transcription Factor) binding to the sequence. Transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor corepressor) and SMRT. Expression of COUP-TFI, N-CoR and SMRT in non-hyperfunctioning adrenocortical adenomas and normal adrenal glands have been compared. Significantly higher expression of COUP-TFI mRNA has been found in non-hyperfunctioning adenomas than in normal adrenals. Interestingly, the pattern of N-CoR and SMRT expression is different compared with COUP-TFI expression. These data suggest that COUP-TFI, N-CoR, and SMRT may play a differential role in steroid biosynthesis of non-hyperfunctioning adenomas (Shibata, 1998a).
Chicken ovalbumin upstream promoter-transcription factor I (COUP-TFI) is an orphan nuclear receptor essential for neurogenesis, organogenesis, and cell fate determination. CYP17 gene transcription has recently been shown to be activated by SF-1 (steroidogenic factor-1) binding to a cyclic AMP-responsive sequence within the promoter region of the gene, and inhibited by COUP-TF binding to the sequence. Thus, COUP-TF and SF-1 act as a transcriptional repressor and activator, respectively, of CYP17 gene expression. Transcriptional repression by COUP-TFI is mediated by corepressors, N-CoR (nuclear receptor-corepressor) and SMRT, whereas transcriptional activation by SF-1 is mediated by coactivator SRC-1 (steroid receptor coactivator-1). The expression of COUP-TFI, SF-1, SRC-1, N-CoR, and SMRT were examined in a variety of adrenocortical adenomas and the results were compared with CYP17 mRNA levels. Significantly high COUP-TFI mRNA expression is found in nonfunctional adenomas, a deoxycorticosterone-producing adenoma, and a pre-clinical Cushing's adenoma, intermediate COUP-TFI expression in cortisol-producing adenomas and low COUP-TFI expression in aldosterone-producing adenomas. In contrast to COUP-TFI, SF-1 mRNA expression does not vary significantly among adrenals. The expected negative correlation between COUP-TFI and CYP17 mRNA levels in adrenocortical adenomas is not detected. High COUP-TFI expression is associated with a nonfunctioning phenotype. Interestingly, the pattern of COUP-TFI expression is similar to the profile of N-CoR expression, but not of SMRT expression. These results indicate that COUP-TFI and N-CoR may play a role in steroidogenesis by human adrenocortical adenomas (Shibata, 1998b).
The corepressors N-CoR and SMRT partner with histone deacetylases (HDACs) in diverse repression pathways. GPS2, a protein involved in intracellular signaling, is an integral subunit of the N-CoR-HDAC3 complex. Structural motifs that direct the formation of a highly stable and active deacetylase complex have been determined. GPS2 and TBL1, another component of the N-CoR-HDAC3 complex, interact cooperatively with repression domain 1 of N-CoR to form a heterotrimeric structure and are indirectly linked to HDAC3 via an extended N-CoR SANT domain that also activates latent HDAC3 activity. More importantly, the N-CoR-HDAC3 complex inhibits JNK activation through the associated GPS2 subunit and thus could potentially provide an alternative mechanism for hormone-mediated antagonism of AP-1 function (Zhang, 2002).
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