Ecdysone receptor

Evolutionary homologs

Table of contents

Chromatin mediated activation and silencing of retinoic acid responsive genes: SMRT and N-CoR

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; 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 (see Drosophila Sin3A), a corepressor for the Mad-Max heterodimer (See Drosophila Myc) 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).

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).

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 is 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 is shown to result 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).

Transcriptional repression plays crucial roles in diverse aspects of metazoan development, implying critical regulatory roles for corepressors such as N-CoR and SMRT. Altered patterns of transcription in tissues and cells derived from N-CoR gene-deleted mice and the resulting block at specific points in CNS, erythrocyte, and thymocyte development indicate that N-CoR is a required component of short-term active repression by nuclear receptors and MAD and of a subset of long-term repression events mediated by REST/NRSF. Unexpectedly, N-CoR and a specific deacetylase are also required for transcriptional activation of one class of retinoic acid response element. Together, these findings suggest that specific combinations of corepressors and histone deacetylases mediate the gene-specific actions of DNA-bound repressors in development of multiple organ systems (Jepsen, 2000).

In vivo evidence has been provided that the corepressor N-CoR is required for mediation of active repression by specific nuclear receptors and several additional classes of transcription factors on a subset of gene targets, thereby acting as a critical factor for specific developmental events in erythrocytic, thymic, and neural systems. This data has also validated the hypothesis that antagonist activity of ER-mediated transcription conferred by 4-Hydroxy-Tamoxifen requires N-CoR. This has clear implications in acquired drug resistance to antagonists that are used in the treatment of breast cancer. Evidence is also provided, through several independent types of analyses, that N-CoR can act as a required component of long-term gene repression events mediated by the DNA binding repressor REST/NRSF. CoREST (see Drosophila CoREST) is required for the repression of other REST/NRSF response elements, but interestingly, while N-CoR is present on the promoter of the SCG10 gene, CoREST is not. Thus there is likely to be gene specificity in cofactor recruitment to REST/NRSF response elements. These data indicate that the REST/NRSF-dependent restriction of genes out of specific cell types may involve independent mechanisms and that for at least a subset of transcription units, N-CoR is both recruited to a REST/NRSF-dependent repression complex and is required for its effective long-term repression (Jepsen, 2000).

The unexpected requirement for N-CoR in RA-dependent activation of a DR+1 element suggests that, directly or indirectly, N-CoR can repress or activate gene expression, analogous to roles of Sin3 and rpd3 in yeast. This activation also requires enzymatically active HDAC3, which has been recently seen to be in stable association with an N-CoR/SMRT complex. Based on the complexity of the events on the DR+1 site, activation by N-CoR/HDAC3 could be mediated through effects on receptors, coactivators, or components of chromatin. Indeed, corepressor/HDAC complexes may serve as positive coregulators for other classes of transcription factors. For example, HDAC-containing complexes could antagonize the proposed CBP-mediated repression of Drosophila TCF (Pangolin) through acetylation and subsequent abolishment of TCF interaction with the coactivator ß-catenin/Armadillo (Jepsen, 2000 and references therein).

N-CoR appears to exert critical roles at specific steps in both erythroid and thymocyte development, linking a specific corepressor to discrete cell-type specification events. Studies in primary avian erythroblasts show that overexpression of T₃R in the absence of T₃ results in sustained proliferation and tightly arrested differentiation of erythroblasts, while addition of T₃ causes loss of self-renewal capacity and induced terminal differentiation. Since N-CoR^-/- erythroblasts have enhanced levels of the CA II, unliganded T₃R appears to require N-CoR for repression events critical to expansion of erythroblast progenitors. Proliferation in the absence of T₃ appears to require cooperation with c-kit, because in the absence of the c-kit ligand SCF, differentiation occurs. This suggests that stages of development that require SCF may also involve T₃R. It is then interesting to note that CFU-E colony formation, which does not require SCF, is normal in N-CoR^-/- embryos at the ages tested, while SCF-dependent BFU-E colony formation is impaired (Jepsen, 2000).

In N-CoR^-/- embryos, the block in thymocyte development at the CD25⁺/CD44^- stage during the DN to DP transition is similar to that seen in mice with deletion of particular transcription factors. Mice lacking the transcriptional repressor Hes1 exhibit blocks at both CD25^-/CD44⁺ and CD25⁺/CD44^- stages of development, but preliminary results with a Gal4-Hes1 fusion protein have suggested that in MEFs Hes1 maintains its repressive function in the absence of N-CoR. TCF/LEF double gene-deleted mice exhibit a block at the CD25⁺/CD44^- stage, as well as at the immature CD8 single-positive (CD8⁺ ISP) stage, raising the possibility that N-CoR-associated deacetylase activity may affect TCF/LEF-dependent activation events (Jepsen, 2000).

The early events of neural induction appear to be largely maintained as assessed by the normal onset of nestin expression in N-CoR^-/- embryos. While the mechanism of downregulation of nestin at midgestation remains to be elucidated, it is intriguing that the enhancer that drives nestin gene expression in the CNS harbors binding sites for both POU factors and nuclear receptors. The finding that MAP2, which can be induced by retinoic acid and harbors RAREs in its promoter, ias upregulated in the outer layers of neocortex of N-CoR^-/- embryos identifies MAP2 as a putative target gene for N-CoR-mediated repression through unliganded RAR. Experiments in cell lines have suggested that decreased levels of MAP2 inhibit neuronal differentiation and neurite formation (Jepsen, 2000).

In conclusion, N-CoR appears to exert corepressor roles for subsets of genes under control of specific DNA binding repressors and is involved in repression events mediated by specific nuclear receptors and several other classes of DNA binding transcription factors. Because of the linkage between N-CoR and REST/NRSF-dependent repression, the evidence suggests that N-CoR participates in both transient and long-term repression events. Additionally, N-CoR appears to serve as a cofactor required, directly or indirectly, for gene activation on certain nuclear receptor response elements. Interestingly, both short- and long-term repressor functions and specific activation functions appear to require the actions of distinct HDACs, suggesting that there may be DNA site-, promoter-specific usage of N-CoR-associated HDACs. These studies functionally link the corepressor N-CoR to repressor-mediated determination of lineage progression in distinct cell types in mammalian development (Jepsen, 2000).

Chromatin mediated activation and silencing of retinoic acid responsive genes: CBP

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).

CREB binding protein (CBP) functions as an essential coactivator of transcription factors that are inhibited by the adenovirus early gene product E1A. Transcriptional activation by the signal transducer and activator of transcription-1 (STAT1) protein requires the C/H3 domain in CBP, which is the primary target of E1A inhibition. The C/H3 domain is not required for retinoic acid receptor (RAR) function, nor is it involved in E1A inhibition. Instead, E1A inhibits RAR function by preventing the assembly of CBP-nuclear receptor coactivator complexes, revealing differences in required CBP domains for transcriptional activation by RAR and STAT1 (Kurokawa, 1998).

Thyroid hormone (T3) and retinoic acid (RA) play important roles in erythropoiesis. The hematopoietic cell-specific bZip protein p45/NF-E2 (Drosophila homolog: Cap'n'collar) interacts with T3 receptor (TR) and RA receptor (RAR) but not retinoid X receptor. The interaction is between the DNA-binding domain of the nuclear receptor and the leucine zipper region of p45/NF-E2 but is markedly enhanced by cognate ligand. Remarkably, ligand-dependent transactivation by TR and RAR is markedly potentiated by p45/NF-E2. This effect of p45/NF-E2 is prevented by maf-like protein p18, which functions positively as a heterodimer with p45/NF-E2 on DNA. Potentiation of hormone action by p45/NF-E2 requires its activation domain, which interacts strongly with the multifaceted coactivator cyclic AMP response element protein-binding protein (CBP). The region of CBP which interacts with p45/NF-E2 is the same interaction domain that mediates inhibition of hormone-stimulated transcription by AP1 transcription factors. Overexpression of the bZip interaction domain of CBP specifically abolishes the positive cross talk between TR and p45/NF-E2. Thus, positive cross talk between p45/NF-E2 and nuclear hormone receptors requires direct protein-protein interactions between these factors and with CBP, whose integration of positive signals from two transactivation domains provides a novel mechanism for potentiation of hormone action in hematopoietic cells (Cheng, 1997).

Nuclear hormone receptors are ligand-activated transcription factors that regulate the expression of genes that are essential for development, reproduction and homeostasis. The hormone response is mediated through recruitment of p160 receptor coactivators and the general transcriptional coactivator CBP/p300, which function synergistically to activate transcription. These coactivators exhibit intrinsic histone acetyltransferase activity, function in the remodelling of chromatin, and facilitate the recruitment of RNA polymerase II and the basal transcription machinery. The activities of the p160 coactivators are dependent on CBP. Both coactivators are essential for proper cell-cycle control, differentiation and apoptosis, and are implicated in cancer and other diseases. To elucidate the molecular basis of assembling the multiprotein activation complex, a structural and thermodynamic analysis was undertaken of the interaction domains of CBP and the activator for thyroid hormone and retinoid receptors (ACTR). Although the isolated domains are intrinsically disordered, they combine with high affinity to form a cooperatively folded helical heterodimer. This study uncovers a unique mechanism, called 'synergistic folding', through which p160 coactivators recruit CBP/p300 to allow transmission of the hormonal signal to the transcriptional machinery (Demarest, 2002).

Extracellular signals and cell-intrinsic transcription factors cooperatively instruct generation of diverse neurons. However, little is known about how neural progenitors integrate both cues and orchestrate chromatin changes for neuronal specification. This paper reports that extrinsic signal retinoic acid (RA) and intrinsic transcription factor Neurogenin2 (Ngn2) collaboratively trigger transcriptionally active chromatin in spinal motor neuron genes during development. Retinoic acid receptor (RAR) binds Ngn2 and is thereby recruited to motor neuron genes targeted by Ngn2. RA then facilitates the recruitment of a histone acetyltransferase CBP to the Ngn2/RAR-complex, markedly inducing histone H3/H4-acetylation. Correspondingly, timely inactivation of CBP and its paralog p300 results in profound defects in motor neuron specification and motor axonal projection, accompanied by significantly reduced histone H3-acetylation of the motor neuron enhancer. This study uncovers the mechanism by which extrinsic RA-signal and intrinsic transcription factor Ngn2 cooperate for cell fate specification through their synergistic activity to trigger transcriptionally active chromatin (Lee, 2009).

Chromatin mediated activation and silencing of retinoic acid responsive genes: PCAF

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).

Chromatin mediated activation and silencing of retinoic acid responsive genes: NSD1

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).

Co-activator complexes: Transcriptional intermediary factor (TIF)and Thyroid hormone receptor-associated protein (TRAP)

The nuclear receptor (NR) coactivator TIF2 possesses a single NR interaction domain (NID) and two autonomous activation domains, AD1 and AD2. The TIF2 NID is composed of three NR-interacting modules each containing the NR box motif LxxLL. Mutation of boxes I, II and III abrogates TIF2-NR interaction and stimulation, in transfected cells, of the ligand-induced activation function-2 (AF-2) present in the ligand-binding domains (LBDs) of several NRs. The presence of an intact NR interaction module II in the NID is sufficient for both efficient interaction with NR holo-LBDs and stimulation of AF-2 activity. Modules I and III are poorly efficient on their own, but synergistically can promote interaction with NR holo-LBDs and AF-2 stimulation. TIF2 AD1 activity appears to be mediated through CBP, since AD1 could not be separated mutationally from the CBP interaction domain. In contrast, TIF2 AD2 activity apparently does not involve interaction with CBP. TIF2 exhibits the characteristics expected for a bona fide NR coactivator, in both mammalian and yeast cells. Moreover, in mammalian cells, a peptide encompassing the TIF2 NID inhibits the ligand-induced AF-2 activity of several NRs, indicating that NR AF-2 activity is either mediated by endogenous TIF2 or by coactivators recognizing a similar surface on NR holo-LBDs (Voegel, 1998).

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 majority of Nuclear hormone and orphan receptors (NRs) utilizes two distinct domains for transcription activation, located in the N and C termini, respectively: a constitutive AF-1 and a ligand-regulated AF-2 as part of the multifunctional ligand-binding domain (LBD). NRs function in concert with multiple transcriptional cofactors, including basal transcription factors, corepressors, and coactivators. Substantial progress in structural and functional analysis has allowed a more detailed understanding of interactions between the AF-2 domain and associated cofactors and further reveals a conserved mechanism for NR activation upon ligand binding. Briefly, ligand activation is associated with structural rearrangements within the LBD, causing the dissociation of corepressors and permitting the recruitment of coactivators or other cofactors with regulatory functions (e.g., RIP140). In agreement with the structural conservation of the coactivator interaction surface, most AF-2 domain-binding proteins contain short conserved LXXLL interaction motifs, referred to as the NR box (Treuter, 1999 and references therein).

NR coactivators are envisaged to function within larger multiprotein complexes. Multiple evidence suggests the existence of at least two distinct coactivator complexes for the ligand-regulated AF-2 domain referred to as the p160 complex and the thyroid hormone receptor-associated protein (TRAP) complex. The p160 complex is believed to integrate p160 family members (e.g. SRC-1, TIF2, and ACTR/p/CIP), CBP/p300, and PCAF. The composition of the complex has been proposed on the basis of the direct and functional interconnection of all subunits, although the existence in vivo has yet to be demonstrated. Because all these coactivators possess intrinsic histone acetyltransferase (HAT) activity and/or function in complex with other acetyltransferases, whereas corepressors apparently function in complex with deacetylases, functional connections between NR activation and the histone acetylation status have been proposed (Treuter, 1999 and references therein).

A different NR coactivator complex, the TRAP complex, has been biochemically identified as a TR-associated multiprotein complex and has been demonstrated to function as coactivator in in vitro transcription systems, suggesting a direct bridging function to the basal transcription machinery. This complex consists of at least nine different subunits and apparently does not contain p160/CBP coactivators. The AF-2 domain-binding 220-kDa subunit of the TRAP complex, referred to as TRAP220, was recently cloned and found to be identical to a putative NR coactivator named PBP/TRIP2, which has been isolated in two-hybrid screenings using TR and PPAR. To get more insights into the relationship between TRAP220 and p160 coactivators, this study has attempted to compare the interaction characteristics of TRAP220 and the p160 family member TIF2 in the context of the TR AF-2 domain, as well as in the context of the TR/RXR heterodimer. Novel features of TRAP220 are described and a competition model is suggested that may also have relevance for the recruitment of p160 and TRAP coactivator complexes to NRs and may further help to integrate these different complexes into current NR activation models (Treuter, 1999 and references therein).

The recent discovery that all components of the putative p160 complex exhibit HAT activity strongly suggests functional connections to chromatin by modification of histones. Intriguingly, it is now known that even non-histone proteins, such as the general transcription factors THIIEbeta and TFIIF or the tumor suppressor protein p53, can become acetylated. Thus, it is tempting to speculate that transcriptional activation mediated by p160 and associated coactivators is more than just a matter of chromatin modification. In case of the TRAP complex, its coactivator function in in vitro transcription systems strongly suggests direct links to the basal transcription machinery. In support of a direct bridging function, it has been demonstrated that TRAP220 exhibits intrinsic transcriptional activity and may contact putative targets, such as TBP, ADA2, or CBP. Intriguingly, each of these proteins is considered a putative target factor for 'classical' transcription activation domains, and TRAP220 may function in conjunction with other TRAPs as a true bridging factor between the unique AF-2 activation domain of NRs and the RNA polymerase II complex. Although no evidence has been provided yet for functional connections of the TRAP complex to chromatin, it is interesting to note that the TRAP-related DRIP complex apparently exhibits HAT activity and in vitro interactions have been observed of TRAP220 to CBP and ADA2, which exhibit intrinsic HAT activity and function within HAT complexes, respectively (Treuter, 1999 and references therein).

Under consideration of envisaged functional differences between p160 (HAT) complexes and the TRAP complex, a sequential multistep activation model for NRs has been proposed. Briefly, in the absence of activating ligands, NRs remain transcriptionally inactive or repress due to the binding of corepressors, which may connect them to histone deacetylase complexes and chromatin repression mechanisms. Upon ligand binding or other activating signals, structural changes within the LBD cause dissociation of corepressors and promote the association of NRs to chromatin-modifying HAT complexes containing p160 coactivators, CBP/p300, and/or PCAF/GCN5. Subsequently, HAT complexes are suggested to work either in concert with or (consistent with a competition model) to be replaced by the TRAP complex, which now directly connects NRs to the RNA polymerase II complex. Because these results indicate competitive interactions of TRAP220 and TIF2 with NRs, the proposed sequential activation model would require regulated changes in either the relative affinity or the relative availability of TIF2 and TRAP220 to their receptor targets (Treuter, 1999 and references therein).

Although a sequential NR activation model is very attractive, the possibility that NRs may use p160 or TRAP complexes as alternative coactivator complexes cannot be excluded. In such a model, both complexes would independently fulfill all functions required for transcriptional activation. In view of the coexpression of p160 and TRAP proteins in many tissues, it could be relevant for receptors that display different affinities to different coactivators. Furthermore, in light of the envisaged complex mode of interactions of NR box domains with NR dimers, dimer-specific affinity differences and additional influences of ligands, binding sites, or competitive coregulatory proteins have to be considered. Additional support for the alternative model comes from the possibility that TRAP220 contacts CBP/p300 and ADA2. As suggested earlier, p160 coactivators might be required for the recruitment of CBP/p300 to NRs. Because TRAP220 interacts with CBP in vitro, it may be able not only to displace p160s from NR heterodimers, but it may also substitute for these coactivators in fulfilling a bridging function between NRs and CBP/p300. Moreover, because ADA2 is part of the PCAF/GCN5 complex, and because interactions of TRAP220 to ADA2 via its putative activation domain(s) are observed, it is possible that TRAP220 may function even without the TRAP complex. This is consistent with the functionality of TRAP220 in yeast (Treuter, 1999 and references therein).

Finally, it is interesting to note that the TRAP220 interaction domain of CBP (aa 1678-1868) contains docking sites for other cofactors implicated to be directly or indirectly involved in certain aspects of NR signaling: PCAF, RNA helicase A, which may serve as direct bridging molecule to the RNA polymerase II complex, and the adenovirus E1A protein, which competes with PCAF for binding to CBP. E1A might inhibit NR activation in part through competition with p160/SRC-1 coactivators for CBP binding. The new findings imply that E1A may even compete for binding of TRAP220 to CBP. However, because CBP was not found within the TRAP complex, future studies have to investigate the relevance of the in vitro data for the function of TRAP220 in vivo, including the consequences of E1A expression on TRAP220-dependent coactivator function. Additionally, the functional interconnection or independence of both TRAP and p160 coactivator complexes has to be addressed experimentally by directly comparing their functionality in, for example, in vitro transcription systems utilizing chromatin and non-chromatin templates, or by performing comparable in vivo inhibition studies, for example by microinjection of antibodies and by generation of knockout animals for individual complex subunits (Treuter, 1999 and references therein).

Using a 'crude' chromatin-based transcription system that mimics transactivation by RAR/RXR heterodimers in vivo, it was not possible to demonstrate that chromatin remodeling is required to relieve nucleosomal repression. Using 'purified' chromatin templates, it has been shown that, irrespective of the presence of histone H1, both ATP-driven chromatin remodeling activities and histone acetyltransferase (HAT) activities of coactivators recruited by liganded receptors are required to achieve transactivation. DNA footprinting, ChIP analysis, and order of addition experiments indicate that coactivator HAT activities and two ATP-driven remodeling activities are sequentially involved at distinct steps preceding initiation of transcription. Thus, both ATP-driven chromatin remodeling and HAT activities act in a temporally ordered and interdependent manner to alleviate the repressive effects of nucleosomal histones on transcription by RARalpha/RXRalpha heterodimers (Dilworth, 2000).

The ATP requirement during the preincubation period preceding HeLa nuclear extract (NE) addition indicates the involvement of ATP-driven chromatin remodeling activity(ies) at an early stage in the process, leading to RAR/RXR-triggered transcriptional initiation. This involvement is strongly supported by the ATP requirement for heterodimers 'tight' binding to their chromatin cognate REs in the absence of any of the other components required to achieve efficient initiation of transcription. This tighter binding is indeed associated with a marked and selective disruption of the nucleosomal structure in the region encompassing the DR5 RE (direct repeat 5 response elements) to which RAR/RXR heterodimers are bound. It appears therefore that RAR/RXR heterodimers, although able to 'recognize' REs within a nucleosomal structure, cannot tightly bind them unless that structure is disrupted. Importantly, this 'tight' binding is ligand independent and does not require histone acetylation by p300/TIF2 coactivators (Dilworth, 2000).

Several ATP-driven remodeling complexes are present in 'crude' chromatin assembled in vitro using Drosophila extracts. NURF, CHRAC, and ACF complexes contain the dISWI ATPase subunit, while the Brahma ATPase subunit is present in dSWI/SNF. This latter complex was essentially removed through purification of 'crude' chromatin. Similar DNase I footprints were obtained in the presence of RARalpha/RXRalpha heterodimers with 'crude' (which contains ATP) and 'purified' (to which only ATP was added) chromatin preparations. The ability of purified hISWI-containing complexes, but not of purified hSWI/SNF complexes, to further enhance ATP-dependent footprints suggests that hISWI-containing complexes can mediate 'tight' binding of RAR/RXR heterodimers to their REs. This is consistent with studies showing a role for the dISWI-containing NURF complex in assisting binding of other transactivators to cognate REs. Thus, the 'weak' but clear footprint of heterodimers on their REs in the absence of ATP suggests that ISWI-catalyzed nucleosome remodeling activity(ies) results in a greater RE accessibility and therefore in 'tighter' binding. This initial 'weak' binding could be a limiting step, as maximal transcriptional initiation is achieved only upon heterodimer addition at the start of the preincubation period, whereas ATP can be added up to 20 min later with little decrease in transcription efficiency. Note in this respect that nucleosome remodeling by NURF is known to occur in vitro within minutes through short-range sliding, irrespective of histone H1 presence. Alternatively or concomitantly a limiting step may correspond to the possible recruitment/targeting of dISWI-containing complexes through interaction with RARalpha/RXRalpha heterodimers, as recently suggested in the case of progesterone receptor and demonstrated for SWI/SNF complexes recruited/targeted by a number of transactivators. It remains to be determined whether the hSNF2h(hISWI)-containing complexes, RSF and/or WCRF/hACF, could functionally substitute in this respect for the Drosophila ATP-driven remodeling activity that is responsible for 'tight' binding of RAR/RXR heterodimers. Human SWI/SNF chromatin remodeling complexes, which cannot be replaced by hSNF2h(hISWI)-containing complexes, are required at a later stage, as optimal transcription is still achieved upon hSWI/SNF addition at the same time as HeLa NE that provides the machinery required for Preinitiation complex (PIC) formation. In contrast, addition of hSWI/SNF just before NTPs is ineffective, indicating that some further chromatin remodeling is indeed required for PIC formation, but does not exclude additional effects on transcriptional elongation. Whether the effect of hSWI/SNF involves its targeting to the promoter region through direct or indirect recruitment by template-bound liganded heterodimers is unknown, but preliminary experiments have failed to reveal such interactions in immunoprecipitation assays using either purified components or F9 cell extracts. However, such a possibility is suggested by a number of in vivo and in vitro observations indicating that SWI/SNF complexes can be recruited through interaction with yeast or animal transactivators, including several NRs. Alternatively or concomitantly, hSWI/SNF may be preferentially recruited through the bromodomain of SNF2alpha/beta subunits by nucleosomes acetylated by coactivators, as bromodomains have been shown to exhibit a high affinity for acetylated lysine residues. This latter possibility may explain why in the absence of histone acetylation hSWI/SNF does not exert any stimulatory activity, unless it is added much before HeLa NE (Dilworth, 2000).

The second step in the process leading to transcriptional initiation triggered by RAR/RXR heterodimers corresponds to the ligand-dependent recruitment/targeting of coactivators that acetylates histones through intrinsic HAT activities. To be efficient, this step has to be preceded by the ATP-dependent ligand-independent receptor 'tight' binding step, and it appears to be a prerequisite for efficient PIC formation upon HeLa NE addition, since activation of transcription is strongly decreased when either Acetyl CoA, ligands, and/or p300/TIF2 is added at the same time as HeLa NE. This strongly suggests that the observed transcriptional activation can be attributed to histone acetylation but does not exclude that acetylation of either nonhistone chromatin proteins (components of the basal transcription machinery and/or activators) by coactivator acetyltransferases may play a role at a later stage(s) for enhanced PIC formation. In contrast, no stimulation of transcription by HeLa NE was observed on 'naked' cognate DNA templates in the presence of p300/TIF2, Acetyl CoA, and liganded heterodimers. Thus, nucleosomal histone acetylation by coactivators targeted to the promoter region through ligand-dependent recruitment by the receptors play a crucial role in transcriptional activation in vitro. Interestingly, both estrogens and RA induce histone hyperacetylation at target gene promoters in cultured cells, which may be mediated by receptors and their coactivators. Previous failures to reveal a role for nucleosomal acetylation in transcription activated in vitro by NRs in the presence of coactivators with known intrinsic HAT activities are most probably due to the use of 'crude' chromatin templates assembled in vitro with extracts that may contain Acetyl CoA and HAT activities. In fact, a recent study has demonstrated the importance of the p300 HAT domain in transcription activated by the estrogen receptor in vitro, although no Acetyl CoA requirement was reported. However, several yeast HAT activities recruited/targeted by transcriptional activators have been shown to facilitate transcription from nucleosomal templates in an Acetyl CoA-dependent fashion (Dilworth, 2000).

Interestingly, histone acetylation by p300/TIF2 is not restricted to nucleosomes surrounding REs to which heterodimers are bound. Whether this is related to the use of an artifactual circular template is unknown. Note that a similar 'wide' histone acetylation pattern has been observed for the yeast NuA4 HAT complex, whereas histone acetylation is restricted to the promoter proximal region in the case of the SAGA HAT complex. Consistent with recent yeast in vivo data showing an ordered involvement of ATP-driven chromatin remodeling and HAT activities, the present data unambiguously establish that the events leading to ligand-dependent enhancement of PIC formation mediated by retinoid receptors on chromatin templates in vitro are both temporally ordered and interdependent. Initially, cognate REs recognition by unliganded RAR/RXR heterodimers results in a 'weak' interaction. Next, receptor 'tight' binding is achieved through nucleosomal remodeling by an ATP-driven machinery, presumably hISWI-containing complexes. The third step is the ligand-dependent coactivator recruitment by heterodimers. The fourth step, occurring upon Acetyl CoA addition, corresponds to histone acetylation by coactivator intrinsic HAT activity. These first four steps take place in the absence of any of the transcription machinery components present in HeLa NE. Thus, the raison d'etre of these chromatin remodeling steps appears to be to create the proper environment for the next steps leading to PIC formation. In subsequent steps, RE-bound liganded receptors associated with p300/CBP and p160 coactivators (e.g., TIF2) may recruit the general transcription factors (GTFs), TFIID complexes, and RNA polymerase II (Pol II) to form PICs. Direct and indirect interactions between a number of GTFs, TFIID TAF subunits, and Pol II and either NR (including retinoid receptors) or p300/CBP have indeed been shown to occur in vitro, in transfected cells or in yeast two-hybrid assays, but their physiological relevance for PIC formation in vivo is unknown. Importantly, large multisubunit complexes that appear to be required for activator-enhanced initiation of transcription in vitro on both 'naked' and 'chromatin' templates have been recently characterized. These SMCC/TRAP, DRIP, and ARC complexes appear to be closely related. Most interesting is the fact that SMCC/TRAP/DRIP/ARC complexes may associate with several NRs through a direct interaction between one of their subunits (TRAP220/DRIP205) and liganded LBDs. Since other SMCC/DRIP/ARC subunits belong to human and mouse mediator complexes and are homologs of components of the yeast SRB/mediator complex that is found associated with Pol II holoenzyme, one of the function of the SMCC/DRIP/ARC complexes might be to recruit Pol II holoenzyme to promoters during PIC formation. Thus, the fifth step leading to ligand-dependent enhancement of PIC formation mediated by retinoid receptors on chromatin templates may correspond to the binding of SMCC/DRIP/ARC to liganded receptor LBDs. The next step would then be the recruitment of Pol II holoenzyme. Alternatively, the liganded receptor might directly recruit preformed SMCC/DRIP/ARC-Pol II holoenzyme complexes. Further in vitro studies using complexes and components from HeLa NE are required to discriminate between these possibilities and to reveal whether nucleosomal acetylation is a prerequisite for these recruitments. Such studies should also reveal when and how hSWI/SWF-mediated ATP-driven nucleosomal remodeling at the promoter region is involved in PIC formation. Furthermore, since TIF2, and presumably other p160 coactivators, as well as p300/CBP, are apparently bound to the same liganded-LBD surface as TRAP220, these studies will indicate whether the release of these coactivators from liganded LBDs is a prerequisite for SMCC/DRIP binding and whether it is achieved through their acetylation (Dilworth, 2000).

The TRAP220 component of the TRAP/SMCC complex, a mammalian homolog of the yeast Mediator that shows diverse coactivation functions, interacts directly with nuclear receptors. Ablation of the murine Trap220 gene reveals that null mutants die with heart failure during an early gestational stage and exhibit impaired neuronal development with extensive apoptosis. Primary embryonic fibroblasts derived from null mutants show an impaired cell cycle regulation and a prominent decrease of thyroid hormone receptor function that is restored by ectopic TRAP220 but no defect in activation by Gal4-RARalpha/RXRalpha, p53, or VP16. Moreover, haploinsufficient animals show growth retardation, pituitary hypothyroidism, and widely impaired transcription in certain organs. These results indicate that TRAP220 is essential for a wide range of physiological processes but also that it has gene- and activator-selective functions (Ito, 2000).

Other co-activator complexes

PNRC (proline-rich nuclear receptor coregulatory protein) was identified using bovine SF1 (steroidogenic factor 1) as the bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC is unique in that it has a molecular mass of 35 kDa, significantly smaller than most of the coregulatory proteins reported so far, and it is proline-rich. PNRC's nuclear localization has been demonstrated. In the yeast two-hybrid assays, PNRC interacted with the orphan receptors SF1 and ERRalpha1 in a ligand-independent manner. PNRC was also found to interact with the ligand-binding domains of all the nuclear receptors tested in a ligand-dependent manner, including estrogen receptor (ER), androgen receptor (AR), glucocorticoid receptor (GR), progesterone receptor (PR), thyroid hormone receptor (TR), retinoic acid receptor (RAR), and retinoid X receptor (RXR). Functional AF2 domain is required for nuclear receptors to bind to PNRC. Furthermore, in vitro glutathione-S-transferase pull-down assay was performed to demonstrate a direct contact between PNRC and nuclear receptors such as SF1. A coimmunoprecipitation experiment using Hela cells that express PNRC and ER was performed to confirm the interaction of PNRC and nuclear receptors in vivo in a ligand-dependent manner. PNRC functions as a coactivator to enhance the transcriptional activation mediated by SF1, ERR1 (estrogen related receptor alpha-1), PR, and TR. A 23-amino acid sequence in the carboxy-terminal region, amino acids 278-300, is critical and sufficient for the interaction with nuclear receptors. This region is proline rich and contains a SH3-binding motif, S-D-P-P-S-P-S. The two conserved proline (P) residues in this motif are crucial for PNRC to interact with the nuclear receptors. The exact 23-amino acid sequence was also found in another protein isolated from the same yeast two-hybrid screening study. These two proteins belong to a new family of nuclear receptor coregulatory proteins (Zhou, 2000).

PNRC2 (proline-rich nuclear receptor co-regulatory protein 2) was identified using mouse steroidogenic factor 1 (SF1) as bait in a yeast two-hybrid screening of a human mammary gland cDNA expression library. PNRC2 is an unusual coactivator in that it is the smallest coactivator identified so far, with a molecular weight of 16 kDa, and interacts with nuclear receptors using a proline-rich sequence. In yeast two-hybrid assays PNRC2 interacted with orphan receptors SF1 and estrogen receptor-related receptor alpha1 in a ligand-independent manner. PNRC2 was also found to interact in a ligand-dependent manner with the ligand-binding domains of estrogen receptor, glucocorticoid receptor, progesterone receptor, thyroid receptor, retinoic acid receptor and retinoid X receptor. A functional activation function 2 domain is required for nuclear receptors to interact with PNRC2. Using the yeast two-hybrid assay, the region amino acids 85-139 were found to be responsible for the interaction with nuclear receptors. This region contains an SH3 domain-binding motif (SEPPSPS) and an NR box-like sequence (LKTLL). A mutagenesis study has shown that the SH3 domain-binding motif is important for PNRC2 to interact with all the nuclear receptors tested. These results reveal that PNRC2 has a structure and function similar to PNRC, a previously characterized coactivator. These two proteins represent a new type of nuclear receptor co-regulatory proteins (Zhou, 2001).

Retinoic acid receptor-α (RARα) is a known estrogen target gene in breast cancer cells. The consequence of RARα induction by estrogen was previously unknown. This study shows that RARα is required for efficient estrogen receptor-α (ER)-mediated transcription and cell proliferation. RARα can interact with ER-binding sites, but this occurs in an ER-dependent manner, providing a novel role for RARα that is independent of its classic role. This study shows, on a genome-wide scale, that RARα and ER can co-occupy regulatory regions together within the chromatin. This transcriptionally active co-occupancy and dependency occurs when exposed to the predominant breast cancer hormone, estrogen - an interaction that is promoted by the estrogen-ER induction of RARα. These findings implicate RARα as an essential component of the ER complex, potentially by maintaining ER-cofactor interactions, and suggest that different nuclear receptors can cooperate for effective transcriptional activity in breast cancer cells (Ross-Innes, 2010).

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