Chromatin remodeling mediated by liganded and unliganded nuclear receptors

Transcriptional silencing mediated by nuclear receptors is important in development, differentiation and oncogenesis. The mechanism underlying this effect is unknown but is one key to understanding the molecular basis of hormone action. A receptor-interacting factor, SMRT, has been identified as a silencing mediator (co-repressor) for retinoid and thyroid-hormone receptors. SMRT is a previously undiscovered protein whose association with receptors both in solution and bound to DNA-response elements is destabilized by ligand. The interaction with mutant receptors correlates with their transcriptional silencing activities. In vivo, SMRT functions as a potent co-repressor, and a GAL4 DNA-binding domain fusion of SMRT behaves as a frank repressor of a GAL4-dependent reporter. Together, these results identify a new class of cofactors that may be important mediators of hormone action (Chen, 1995).

Transcriptional repression represents an important component in the regulation of cell differentiation and oncogenesis mediated by nuclear hormone receptors. Hormones act to relieve repression, thus allowing receptors to function as transcriptional activators. The transcriptional corepressor SMRT was identified as a silencing mediator for retinoid and thyroid hormone receptors. SMRT is highly related to another corepressor, N-CoR, suggesting the existence of a new family of receptor-interacting proteins. SMRT is a ubiquitous nuclear protein that interacts with unliganded receptor heterodimers in mammalian cells. Furthermore, expression of the receptor-interacting domain of SMRT acts as an antirepressor, suggesting the potential importance of splicing variants as modulators of thyroid hormone and retinoic acid signaling (Chen, 1996).

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, and repression is mediated by corepressors, proteins that associate with unliganded nuclear receptors that assemble 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 (see Drosophila Sin3A). 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).

Histone acetylation is thought to have a role in transcription. To gain insight into the role of histone acetylation in retinoid-dependent transcription, a study was performed of the effects of trichostatin A (TSA), a specific inhibitor of histone deacetylase, on P19 embryonal carcinoma cells. Coaddition of TSA and retinoic acid (RA) markedly enhances neuronal differentiation in these cells, although TSA alone does not induce differentiation but causes extensive apoptosis. Consistent with the cooperative effect of TSA and RA, coaddition of the two agents synergistically enhances transcription from stably integrated RA-responsive promoters. The transcriptional synergy by TSA and RA requires the RA-responsive element and a functional retinoid X receptor (RXR)/retinoic acid receptor (RAR) heterodimer, both obligatory for RA-dependent transcription. TSA leads to promoter activation by an RXR-selective ligand, which otherwise is inactive in transcription. In addition, TSA enhances transcription from a minimum basal promoter, independently of the RA-responsive element. TSA alone or in combination with RA increases in vivo endonuclease sensitivity within the RA-responsive promoter, suggesting that TSA treatment might alter a local chromatin environment to enhance RXR/RAR heterodimer action. Thus, these results indicate that histone acetylation influences activity of the heterodimer, which is in line with the observed interaction between the RXR/RAR heterodimer and a histone acetylase (Minucci, 1997).

A novel cofactor, ACTR, directly binds nuclear receptors and stimulates their transcriptional activities in a hormone-dependent fashion. ACTR has sequence homology to SRC-1 and TIF2, factors that associate with members of the nuclear receptor family and augment the transcription activity of steroid receptors such as glucocorticoid receptor and estrogen receptor. ACTR possesses an N-terminal PAS/bHLH domain, a central receptor-interaction domain able to interact with RAR, RXR (Drosophila homolog: Ultraspiracle) and TR (See Ecdysone Receptor for more information) in a hormone dependent fashion, and a C-terminal histone acetyl transferase domain. ACTR also recruits two other nuclear factors, CBP and P/CAF, and thus plays a central role in creating a multisubunit coactivator complex. In addition, and unexpectedly, purified ACTR is a potent histone acetyltransferase and appears to define a distinct evolutionary branch to this recently described family. ACTR is able to recruit P/CAF. Thus, hormonal activation by nuclear receptors involves the mutual recruitment of at least three classes of histone acetyltransferases that may act cooperatively as an enzymatic unit to reverse the effects of histone deacetylase shown to be part of the nuclear receptor corepressor complex. Interestingly, while all currently identified histone acetyltransferases (including ACTR) are capable of acetylating core histones H3 and H4, ACTR displays an unusual additional activity as an effective acetylator of mononucleosomes, similar only to that observed in CBP/p300 (Chen, 1997).

Ligand-dependent transcriptional regulation by nuclear receptors is believed to be mediated by intermediary factors (TIFs) acting on remodeling of the chromatin structure and/or the activity of the transcriptional machinery. The putative transcriptional mediator TIF1alpha is a nuclear protein kinase that has been identified via its interaction with liganded nuclear receptors, including retinoic acid (RAR), retinoid X (RXR) and estrogen (ER) receptors. TIF1alpha is a non-histone chromosomal protein tightly associated with highly accessible euchromatic regions of the genome. Immunofluorescence confocal microscopy reveals that TIF1alpha exhibits a finely granular distribution in euchromatin of interphase nuclei, while it is mostly excluded from condensed chromatin and metaphase chromosomes. Immunoelectron microscopy shows that, in contrast to the heterochromatin protein HP1alpha, most of TIF1alpha is associated with euchromatin, where it is preferentially localized on regions known to be sites for RNA polymerase II (perichromatin fibrils and borders between euchromatin and heterochromatin). Early mouse embryos as well as embryonal carcinoma (EC) and embryonic stem (ES) cells express high levels of TIF1alpha. These levels dramatically decrease during organogenesis and upon differentiation of P19 EC cells, indicating that TIF1alpha is preferentially expressed in undifferentiated pluripotent cells in the course of development. Therefore, TIF1alpha could belong to a novel class of chromatin-associated TIFs that facilitate the access of transregulators (e.g. liganded nuclear receptors) to their cognate sites in target genes, thereby participitating in the epigenetic control of transcription during embryonic development and cell differentiation (Remboutsika, 1999).

The CREB-binding protein (CBP) and its homolog P300 (See Drosophila dCREB2) act as cofactors mediating nuclear-receptor-activated gene transcription. The role of CBP/P300 in the transcriptional response to cyclic AMP, phorbol esters, serum, the lipophilic hormones and as the target of the E1A oncoprotein suggests they may serve as integrators of extracellular and intracellular signaling pathways leading to gene activation. Since CBP is known to be a histone acetyltransferase gene activation carried out by nuclear receptors is likely to involve chromatin modification (Chakravarti, 1996).

Whereas the histone acetylase PCAF has been suggested to be part of a coactivator complex mediating transcriptional activation by the nuclear hormone receptors, the physical and functional interactions between nuclear receptors and PCAF have remained unclear. Efforts to clarify these relationships have revealed two novel properties of nuclear receptors (Blanco, 1998).

(1) First, the RXR/RAR heterodimer directly recruits PCAF from mammalian cell extracts in a ligand-dependent manner and increased expression of PCAF leads to enhanced retinoid-responsive transcription. Of the two domains present in PCAF, the carboxy-terminal domain (from amino acid position 352 to 832) represents the region homologous to the yeast GCN5 and contains histone acetylase activity. The amino-terminal region (composed of amino acids 1-351) shares little homology with known genes, and its function has not been fully elucidated. To determine a region of PCAF involved in binding to the heterodimer, truncated recombinant PCAFs lacking either the amino-terminal or carboxy-terminal domain were examined. Recombinant deltaN1 or deltaN2, lacking either the amino-terminal domain alone or the amino-terminal region plus the additional 113 amino acids of the carboxy-terminal region, binds to the heterodimer-RARE complex, although the binding of deltaN2 is slightly weaker than that of deltaN1 and full-length PCAF. In contrast to this, deltaC shows little binding to the complex. Ligand has no effect on the binding activity of the truncated PCAF. These results indicate that the conserved carboxy-terminal domain is required for binding to the heterodimer-RARE complex (Blanco, 1998).

(2) With respect to the second novel property of nuclear receptors, PCAF directly associates with the DNA-binding domain of nuclear receptors, independently of p300/CBP binding, and therefore defines a novel cofactor interaction surface. These results show that dissociation of corepressors enables ligand-dependent PCAF binding to the receptors. This observation illuminates how a ligand-dependent receptor function can be propagated to regions outside the ligand-binding domain itself. To evaluate whether PCAF enhances ligand-dependent promoter activity through its histone acetylase activity, two additional deletion constructs were examined in which the recently identified catalytic domain of PCAF was deleted (deltaHAT1 and deltaHAT2). Histone acetylase activity is found to be completely abrogated in these deletion constructs in vitro. deltaHAT1 and deltaHAT2, similar to deltaC and deltaN2, fails to give full enhancement in promoter activity attained by the intact PCAF. These results support the idea that PCAF potentiates retinoid-dependent transcription at least partly through its histone acetylase activity. Immunoblot analysis performed with transfected cells shows that exogenous PCAF is expressed in a dose-dependent manner, while the expression of the endogenous p300 remains unchanged. On the basis of these observations, it is suggested that PCAF may play a more central role in nuclear receptor function than previously anticipated (Blanco, 1998).

It was found that corepressors from the N-CoR-SMRT family inhibit binding of recombinant PCAF to the nuclear receptor heterodimer in the absence of ligand, but that this binding is restored upon addition of ligand, concomitant with repressor release. These results suggest that corepressors, by virtue of their dissociation from the receptor, confer ligand dependence on PCAF binding. It has been shown that N-CoR and SMRT bind to the hinge region of receptors. Because the hinge region present in the ligand-binding domain is only ~30 amino acids away from the DNA-binding domain, corepressors could cause either a steric block of PCAF binding or induce a local conformational change that precludes PCAF binding. Adding to this passive regulation of activity exclusion, corepressors have recently been shown to be associated with the histone deacetylase HDAC-1 and mSin3, which are thought to establish transcriptional repression via modification of chromatin. A model depicting how ligand reverses this process in two steps is presented: first the ligand promotes the dissociation of the repressor complex, which in turn enables the second step of PCAF recruitment. Like the repressors, PCAF itself also functions in at least two ways: (1) as a histone acetylase it has the direct capacity to modify chromatin to reverse repression, and (2) via its p300/CBP- and SRC-interaction domains, it serves to recruit additional activators (Blanco, 1998).

NSD1, a novel 2588 amino acid mouse nuclear protein that interacts directly with the ligand-binding domain (LBD) of several nuclear receptors (NRs), has been identified and characterized. NSD1 contains a SET domain and multiple PHD fingers. The ~150-amino acid SET domain, located between residues 1834 and 1980, was first identified in three Drosophila chromosomal regulators: Suppressor of variegation 3-9, Enhancer of zeste [E(z)] and Trithorax (Trx). The SET domains most similar to that of NSD1 are encoded by the Drosophila trithorax-group gene Ash1 and the yeast ORF YJQ8 (>40% identity). As in Ash1 and YJQ8, the NSD1 SET domain is not C-terminal, unlike those of Su(var)3-9, E(z) and Trx. Immediately preceding the SET domain, NSD1 contains a Cys-rich domain (residues 1791-1833; herein referred to as the SAC domain), that is conserved at the same position in some, but not all, SET-domain-containing proteins. This SAC domain, originally noticed in E(z) and its murine homolog Enx-1, was also found adjacent to the SET domain of Ash1, YJQ8 and Su(var)3-9, but not Trx. As searches in protein databases have revealed, this Cys-rich domain occurs only in proteins containing the SET domain, hence the term SAC, for SET domain-associated cysteine-rich domain. In addition to the SAC and SET domains, NSD1 contains five zinc finger-like motifs that all match the consensus sequence of the PHD finger, also designated as the C4HC3 motif. There are also four PHD fingers located N-terminal to the SET domain of the Drosophila Trx protein and its human homolog (HRX/All-1/MLL). In contrast, Ash1 contains a single C-terminally located PHD finger, whereas Pcl, the product of the Drosophila polycomb-group gene Polycomb-like, has two PHD fingers, but no SET domain. In addition to these conserved domains found in both positive and negative Drosophila chromosomal regulators, NSD1 contains two distinct NR interaction domains, NID-L and NID+L, that exhibit binding properties of NIDs found in NR corepressors and coactivators, respectively. NID-L, but not NID+L, interacts with the unliganded LBDs of retinoic acid receptors (RAR) and thyroid hormone receptors (TR), and this interaction is severely impaired by mutations in the LBD alpha-helix 1 that prevent binding of corepressors and transcriptional silencing by apo-NRs. NID+L, but not NID-L, interacts with the liganded LBDs of RAR, TR, retinoid X receptor (RXR), and estrogen receptor (ER), and this interaction is abrogated by mutations in the LBD alpha-helix 12 that prevent binding of coactivators of the ligand-induced transcriptional activation function AF-2. A novel variant (FxxLL) of the NR box motif (LxxLL) is present in NID+L and is required for the binding of NSD1 to holo-LBDs. Interestingly, NSD1 contains separate repression and activation domains. Thus, NSD1 may define a novel class of bifunctional transcriptional intermediary factors playing distinct roles in both the presence and absence of ligand (Huang, 1998).

RXR proteins and cancer

All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies have demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, several synthetic retinoids were evaluated for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. RXR-selective retinoids, particularly in combination with RAR-selective retinoids, can significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but have low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. The effects of RXR-selective retinoids on MDA-MB-231 cells are most likely mediated by RXR-nur77 heterodimers that bind to the RA response element in the RARbeta promoter and activate the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurs through RXR-RAR alpha heterodimers that also bind to and activate the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids are suppressed, while the effects of RAR-selective retinoids are enhanced. Together, these results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells (Wu, 1997).

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