Correction: The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation.

Since the publication of the above article, the authors have noted that the input data in Fig. 6E is incorrect. The correct data are included in the below Fig. 6E. The mistake does not affect the conclusions of the paper as the levels of input proteins remain similar between samples. We apologise for any inconvenience caused by this error.

A prospero-related homeobox gene Prox-1 is expressed during postnatal brain development as well as in the adult rodent brain.

Prox-1, a prospero-related homeobox gene, is known to be an important transcription factor during embryogenesis. However, very little is known about Prox-1 expression and functions in the adult nervous system. Here we have investigated the expression pattern of Prox-1 mRNA and protein during postnatal brain development and in adult rat and mouse brains using in situ hybridization (ISH), immunohistochemistry (IHC) and Western blotting. In the developing and adult brain, we found prominent, but restricted Prox-1 mRNA expression in the dentate gyrus of the hippocampus, in some thalamic nuclei, notably in the anterior thalamus, and in the cerebellar cortex. Other brain regions, such as the hypothalamus and nuclei belonging to the midbrain, revealed a moderate level of Prox-1 mRNA expression. In developing cerebral cortex, Prox-1 mRNA was seen only in the thin layer under the pial surface postnatally, and the signal almost disappeared by the 28th postnatal day (PD). Using IHC and ISH approaches, we demonstrated rather restricted, but intense Prox-1 labeling in adult brain of both rat and mouse species. During postnatal brain development Prox-1 proteins by IHC, were below the detection limit at PD 14, while Prox-1 mRNA remained at a high level. Western blotting demonstrated the existence of two different variants of Prox-1 protein, one of which was about 20 kDa larger than ordinary size. During the first PDs, the larger variant predominated. At PD 14, neither protein variant could be detected. From PD 16 onwards the smaller variant started to predominate and by PD 30 the larger size protein had almost disappeared. The prominent but limited distribution of Prox-1 in the brain suggests its potentially important role during postnatal brain development and in adult CNS, which remains to be ascertained in future studies.

Mechanistic principles in NR box-dependent interaction between nuclear hormone receptors and the coactivator TIF2.

Nuclear hormone receptors exert transcriptional activation of target genes upon hormone induction via interactions with the basal transcription machinery. This interaction is mediated by cofactors which physically bind to receptors, thereby acting as coactivators or corepressors leading to activation or repression, respectively. Here we report the screening for and cloning of a peroxisome proliferator receptor-interacting protein, the rat homolog of TIF2. By sequence comparison with the related coactivator SRC-1, we identified three short conserved motifs (NR boxes) in both proteins which are the putative binding sites of TIF2 to nuclear hormone receptors. We demonstrate here by generation of amino acid exchanges within the NR boxes that all three boxes located in the receptor interaction domain of TIF2 are necessary and sufficient for interaction. The three boxes individually can bind to hormone receptors but display preferences in binding for certain receptors. In addition, we show that the interaction domain of TIF2 can compete with other AF-2-dependent cofactors for binding to receptors. Finally, we demonstrate cooperative binding of two TIF2 molecules to a heterodimeric nuclear receptor complex even in the presence of only one cognate ligand, indicating an allosteric effect on the heterodimeric partner upon coactivator binding.

The orphan nuclear receptor SHP utilizes conserved LXXLL-related motifs for interactions with ligand-activated estrogen receptors.

SHP (short heterodimer partner) is an unusual orphan nuclear receptor consisting only of a ligand-binding domain, and it exhibits unique features of interaction with conventional nuclear receptors. While the mechanistic basis of these interactions has remained enigmatic, SHP has been suggested to inhibit nuclear receptor activation by at least three alternatives; inhibition of DNA binding via dimerization, direct antagonism of coactivator function via competition, and possibly transrepression via recruitment of putative corepressors. We now show that SHP binds directly to estrogen receptors via LXXLL-related motifs. Similar motifs, referred to as NR (nuclear receptor) boxes, are usually critical for the binding of coactivators to the ligand-regulated activation domain AF-2 within nuclear receptors. In concordance with the NR box dependency, SHP requires the intact AF-2 domain of agonist-bound estrogen receptors for interaction. Mutations within the ligand-binding domain helix 12, or binding of antagonistic ligands, which are known to result in an incomplete AF-2 surface, abolish interactions with SHP. Supporting the idea that SHP directly antagonizes receptor activation via AF-2 binding, we demonstrate that SHP variants, carrying either interaction-defective NR box mutations or a deletion of the repressor domain, have lost the capacity to inhibit agonist-dependent transcriptional estrogen receptor activation. Furthermore, our studies indicate that SHP may function as a cofactor via the formation of ternary complexes with dimeric receptors on DNA. These novel insights provide a mechanistic explanation for the inhibitory role of SHP in nuclear receptor signaling, and they may explain how SHP functions as a negative coregulator or corepressor for ligand-activated receptors, a novel and unique function for an orphan nuclear receptor.

A regulatory role for RIP140 in nuclear receptor activation.

Transcriptional regulation of gene expression by nuclear receptors requires negatively and positively acting cofactors. Recent models for receptor activation propose that certain receptors in the absence of ligands can recruit corepressors while ligand binding results in conformational changes leading to the recruitment of coactivators. Previous work has established a coactivator role for the SRC-1 family members as well as an involvement of the coactivators CBP/p300 in nuclear receptor signaling. However, in addition to coactivators, ligand-activated nuclear receptors bind a number of different proteins that possibly serve other functions. Using peroxisome proliferator-activated receptor-alpha (PPAR alpha) as bait in a yeast two-hybrid screening, we have isolated nuclear factor RIP140 whose function in receptor activation is unclear. We now report a detailed characterization of RIP140 action with a focus on the retinoid X receptor (RXR) heterodimeric receptors PPAR and thyroid hormone receptor (TR). We show that putative PPAR ligands enhance the interaction of RIP140 with the rat PPAR subtypes alpha and gamma in solution but not with PPAR/RXR heterodimers on DNA. However, RIP140 forms ternary complexes in the presence of RXR ligands. Similar experiments with TR support the high affinity of RIP140 to the RXR subunit and also suggest that either partner in the TR/RXR heterodimer can independently respond to ligand. Coactivation experiments in yeast and mammalian cells confirm the coactivator role for SRC-1, but not for RIP140. We provide important evidence that the in vitro binding of RIP140 and SRC-1 to nuclear receptors is competitive. Since RIP140 generally down-regulates receptor activity in mammalian cells and specifically down-regulates coactivation mediated by SRC-1, we propose a model in which RIP140 indirectly regulates nuclear receptor AF-2 activity by competition for coactivators such as SRC-1.

Regulation of glucocorticoid receptor activity by 14--3-3-dependent intracellular relocalization of the corepressor RIP140.

Proteins belonging to the 14--3-3 family interact with various regulatory proteins involved in cellular signaling, cell cycle regulation, or apoptosis. 14--3-3 proteins have been suggested to act by regulating the cytoplasmic/nuclear localization of their target proteins or by acting as molecular scaffolds or chaperones. We have previously shown that overexpression of 14--3-3 enhances the transcriptional activity of the glucocorticoid receptor (GR), which is a member of the nuclear receptor family. In this study, we show that 14--3-3 interacts with the nuclear receptor corepressor RIP140. In transfection assays, RIP140 antagonizes 14--3-3- enhanced GR transactivation. Using colocalization studies we demonstrate that 14--3-3 can export RIP140 out of the nucleus and, interestingly, can also change its intranuclear localization. Moreover, we also observed that 14--3-3 can bind various other nuclear receptors and cofactors. In summary, our findings suggest that 14--3-3-mediated intracellular relocalization of the GR corepressor RIP140 might be a novel mechanism to enhance glucocorticoid responsiveness of target genes. They furthermore indicate a more general role for 14--3-3 protein by influencing the nuclear availability of nuclear receptor-associated cofactors.

Ligand-independent coregulator recruitment by the triply activatable OR1/retinoid X receptor-alpha nuclear receptor heterodimer.

OR1 is a member of the superfamily of steroid/thyroid hormone nuclear receptors and recognizes DNA as a heterodimer with the 9-cis-retinoic acid receptor RXR (retinoid X receptor). The heterodimeric complex has been shown to be transcriptionally activatable by the RXR ligand as well as certain oxysterols via OR1, but to date uniquely also by heterodimerization itself. Recent studies on other members of the superfamily of nuclear receptors have led to the identification of a number of nuclear receptor-interacting proteins that mediate their regulatory effects on transcription. Here, we address the question of involvement of some of these cofactors in the three modes of activation by the OR1/RXRalpha complex. We show that in vitro the steroid receptor coactivator SRC-1 can be recruited by RXRalpha upon addition of its ligand, and to OR1 upon addition of 22(R)-OH-cholesterol, demonstrating that the latter can act as a direct ligand to OR1. Additionally, heterodimerization is sufficient to recruit SRC-1 to OR1/RXRalpha, indicating SRC-1 as a molecular mediator of dimerization-induced activation. In transfection experiments, coexpression of a nuclear receptor-interacting fragment of SRC-1 abolishes constitutive activation by OR1/RXRalpha, which can be restored by over-expression of full-length SRC-1. This constitutes evidence for an in vivo role of SRC-1 in dimerization-induced activation by OR1/RXRalpha. Additionally, we show that the nuclear receptor-interacting protein RIP140 binds in vitro to OR1 and RXRalpha with requirements distinct from those of SRC-1, and that binding of the two cofactors is competitive. Taken together, our results suggest a complex modulation of differentially induced transactivation by OR1/RXR coregulatory molecules.

Mechanism of oestrogen signalling with particular reference to the role of ER beta in the central nervous system.

The discovery of a second oestrogen receptor (ER), ER beta, has drastically changed our view of oestrogen action. Since the two ERs, ER alpha and ER beta, have somewhat different tissue and cellular distribution as well as ligand binding specificity the possibility exists that they have different biological roles. Indeed, several observations seem to indicate that they may even have opposite effects so that ER beta diminishes the activity of ER alpha. The CNS contains both ER alpha and ER beta and it is conceivable that they may have specific and individual roles in oestrogen signalling in the brain.

The nuclear-receptor interacting protein (RIP) 140 binds to the human glucocorticoid receptor and modulates hormone-dependent transactivation.

The glucocorticoid receptor (GR) regulates target gene expression in response to corticosteroid hormones. We have investigated the mechanism of transcriptional activation by the GR by studying the role of the receptor interacting protein RIP140. Both in vivo and in vitro protein-protein interaction assays revealed a ligand-dependent interaction between the GR and RIP140. The ligand binding domain of the GR was sufficient for this interaction, while both the N- and C-terminal regions of RIP140 bound to the receptor. In a yeast transactivation assay RIP140 and SRC-1, a member of the steroid receptor coactivator family of proteins, both enhanced the transactivation activity of a GR protein (GRA-1) in which the potent N-terminal tau1 transactivation domain has been deleted. In contrast, in COS-7 cells increasing amounts of RIP140 significantly inhibited GRdeltatau1 function. In cotransfection studies in COS-7 cells, RIP140 also inhibited receptor activity in presence of both SRC-1 and the coactivator protein CBP together. Thus, in yeast cells a stimulation of receptor activity was observed, while in mammalian cells RIP140 repressed GR function. Taken together, these data suggest that, (1) RIP140 is a target protein for the GR and (2) RIP140 can modulate the transactivation activity of the receptor.

The oxysterol receptor LXRß protects against DSS- and TNBS-induced colitis in mice.

We examined the function of the oxysterol receptors (LXRs) in inflammatory bowel disease (IBD) through studying dextran sodium sulfate (DSS)- and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced colitis in mice and by elucidating molecular mechanisms underlying their anti-inflammatory action. We observed that Lxr-deficient mice are more susceptible to colitis. Clinical indicators of colitis including weight loss, diarrhea and blood in feces appeared earlier and were more severe in Lxr-deficient mice and particularly LXRß protected against symptoms of colitis. Addition of an LXR agonist led to faster recovery and increased survival. In contrast, Lxr-deficient mice showed slower recovery and decreased survival. In Lxr-deficient mice, inflammatory cytokines and chemokines were increased together with increased infiltration of immune cells in the colon epithelium. Activation of LXRs strongly suppressed expression of inflammatory mediators including TNFα. While LXRα had anti-inflammatory effects in CD11b(+) immune cell populations, LXRß in addition had anti-inflammatory effects in colon epithelial cells. Lack of LXRß also induced CD4(+)/CD3(+) immune cell recruitment to the inflamed colon. Expression of both LXRA and LXRB was significantly suppressed in inflamed colon from subjects with IBD compared with non-inflamed colon. Taken together, our observations suggest that the LXRs could provide interesting targets to reduce the inflammatory responses in IBD.

The atypical ubiquitin ligase RNF31 stabilizes estrogen receptor α and modulates estrogen-stimulated breast cancer cell proliferation.

Estrogen receptor α (ERα) is initially expressed in the majority of breast cancers and promotes estrogen-dependent cancer progression by regulating the transcription of genes linked to cell proliferation. ERα status is of clinical importance, as ERα-positive breast cancers can be successfully treated by adjuvant therapy with antiestrogens or aromatase inhibitors. Complications arise from the frequent development of drug resistance that might be caused by multiple alterations, including components of ERα signaling, during tumor progression and metastasis. Therefore, insights into the molecular mechanisms that control ERα expression and stability are of utmost importance to improve breast cancer diagnostics and therapeutics. Here we report that the atypical E3 ubiquitin ligase RNF31 stabilizes ERα and facilitates ERα-stimulated proliferation in breast cancer cell lines. We show that depletion of RNF31 decreases the number of cells in the S phase and reduces the levels of ERα and its downstream target genes, including cyclin D1 and c-myc. Analysis of data from clinical samples confirms correlation between RNF31 expression and the expression of ERα target genes. Immunoprecipitation indicates that RNF31 associates with ERα and increases its stability and mono-ubiquitination, dependent on the ubiquitin ligase activity of RNF31. Our data suggest that association of RNF31 and ERα occurs mainly in the cytosol, consistent with the lack of RNF31 recruitment to ERα-occupied promoters. In conclusion, our study establishes a non-genomic mechanism by which RNF31 via stabilizing ERα levels controls the transcription of estrogen-dependent genes linked to breast cancer cell proliferation.

Receptor interacting protein RIP140 inhibits both positive and negative gene regulation by glucocorticoids.

Recent development in the field of gene regulation by nuclear receptors (NRs) have identified a role for cofactors in transcriptional control. While some of the NR-associated proteins serve as coactivators, the effect of the receptor interacting protein 140 (RIP140) on NR transcriptional responses is complex. In this report we have studied the effect of RIP140 on gene regulation by the glucocorticoid receptor (GR). We demonstrate that RIP140 antagonized all GR-mediated responses tested, which included activation through classical GRE, the synergistic effects of glucocorticoids on AP-1 and Pbx1/HOXB1 responsive elements, as well as gene repression through a negative GRE and cross-talk with NF-kappaB (RelA). This involved the ligand-binding domain of the GR and did not occur when the GR was bound to the antagonist RU486. The strong repressive effect of RIP140 was restricted to glucocorticoid-mediated responses in as much as it slightly increased signaling through the RelA and the Pit-1/Pbx proteins and only slightly repressed signaling through the Pbx1/HOXB1 and AP-1 proteins, excluding general squelching as a mechanism. Instead, this suggests that RIP140 acts as a direct inhibitor of GR function. In line with a direct effect of RIP140 on the GR, we demonstrate a GR-RIP140 interaction in vitro by a glutathione S-transferase-pull down assay. Furthermore, the repressive effect of RIP140 could partially be overcome by overexpression of the coactivator TIF2, which involved a competition between TIF2 and RIP140 for binding to the GR.

Promoter specificity and deletion analysis of three heat stress transcription factors of tomato.

Transient expression assays in transformed tobacco (Nicotiana plumbaginifolia) mesophyll protoplasts were used to test the activity of three tomato heat stress transcription factors, HSF24, HSF8 and HSF30, in a trans-activation and a trans-repression assay. The results document differences between the three HSFs with respect to their response to the configuration of heat stress promoter elements (HSEs) in the reporter construct (promoter specificity) and to the stress regime used for activation. Analysis of C-terminal deletions identified acidic sequence elements with a central tryptophan residue, which are important for HSF activity control. Surprisingly, heterologous HSFs from Drosophila and human cells, but not from yeast, were also functional as heat stress-induced transcription factors in this tobacco protoplast system.

The role of AHA motifs in the activator function of tomato heat stress transcription factors HsfA1 and HsfA2.

Using reporter assays in tobacco protoplasts and yeast, we investigated the function of the acidic C-terminal activation domains of tomato heat stress transcription factors HsfA1 and HsfA2. Both transcription factors contain short, essential peptide motifs with a characteristic pattern of aromatic and large hydrophobic amino acid residues embedded in an acidic context (AHA motifs). The prototype is the AHA1 motif of HsfA2, which has the sequence DDIWEELL. Our mutational analysis supports the important role of the aromatic and large hydrophobic amino acid residues in the core positions of the AHA motifs. The pattern suggests the formation of an amphipathic, negatively charged helix as the putative contact region with components of the basal transcription complex. In support of this concept, proline or positively charged residues in or adjacent to the AHA motifs markedly reduce or abolish their activity. Both AHA motifs of HsfA1 and HsfA2 contribute to activator potential, and they can substitute for each other; however, there is evidence for sequence and positional specificity.

Cloning and characterization of RAP250, a novel nuclear receptor coactivator.

Ligand-induced transcriptional activation of gene expression by nuclear receptors is dependent on recruitment of coactivators as intermediary factors. The present work describes the cloning and characterization of RAP250, a novel human nuclear receptor coactivator. The results of in vitro and in vivo experiments indicate that the interaction of RAP250 with nuclear receptors is ligand-dependent or ligand-enhanced depending on the nuclear receptor and involves only one short LXXLL motif called nuclear receptor box. Transient transfection assays further demonstrate that RAP250 has a large intrinsic glutamine-rich activation domain and can significantly enhance the transcriptional activity of several nuclear receptors, acting as a coactivator. Interestingly, Northern blot and in situ hybridization analyses reveal that RAP250 is widely expressed with the highest expression in reproductive organs (testis, prostate and ovary) and brain. Together, our data suggest that RAP250 may play an important role in mammalian gene expression mediated by nuclear receptor.

Heat stress transcription factors from tomato can functionally replace HSF1 in the yeast Saccharomyces cerevisiae.

The fact that yeast HSF1 is essential for survival under nonstress conditions can be used to test heterologous Hsfs for the ability to substitute for the endogenous protein. Our results demonstrate that like Hsf of Drosophila, tomato Hsfs A1 and A2 can functionally replace the corresponding yeast protein, but Hsf B1 cannot. In addition to survival at 28 degrees C, we checked the transformed yeast strains for temperature sensitivity of growth, induced thermotolerance and activator function using two different lacZ reporter constructs. Tests with full-length Hsfs were supplemented by assays using mutant Hsfs lacking parts of their C-terminal activator region or oligomerization domain, or containing amino acid substitutions in the DNA-binding domain. Remarkably, results with the yeast system are basically similar to those obtained by the analysis of the same Hsfs as transcriptional activators in a tobacco protoplast assay. Most surprising is the failure of HsfB1 to substitute for the yeast Hsf. The defect can be overcome by addition to HsfB1 of a short C-terminal peptide motif from HsfA2 (34 amino acid residues), which represents a type of minimal activator necessary for interaction with the yeast transcription apparatus. Deletion of the oligomerization domain (HR-A/B) does not interfere with Hsf function for survival or growth at higher temperatures. But monomeric Hsf has a markedly reduced affinity for DNA, as shown by lacZ reporter and band-shift assays.

Intracellular distribution and identification of the nuclear localization signals of two plant heat-stress transcription factors.

Similar to heat-stress transcription factors (HSFs) from non-plant sources, HSFA1 and HSFA2 from tomato (Lycopersicon esculentum Mill) contain two conserved clusters of basic amino acid residues (K/R1 and K/R2) which might serve as nuclear localization signal (NLS) motifs. Mutation of either one of them and functional testing of the corresponding proteins in a transient expression assay using tobacco (Nicotiana plumbaginifolia L:) protoplasts gave the following results. Whereas K/R1, positioned in all HSFs at the C-terminus of the DNA-binding domain, had no influence on nuclear import, the K/R1 mutants were impaired in their interaction with the DNA (band-shift assays). In contrast to this, mutants of the K/R2 motif, found 15-20 amino acid residues C-terminal of the oligomerization domain (HR-A/B region), had wild-type activity in DNA-binding but were defective in nuclear import. Thus, for the related tomato HSFA1 and HSFA2 the K/R2 cluster represents the only NLS motif, and in this function it cannot be replaced by K/R1.

Differential recruitment of the mammalian mediator subunit TRAP220 by estrogen receptors ERalpha and ERbeta.

Estrogen receptors (ERs) associate with distinct transcriptional coactivators to mediate activation of target genes in response to estrogens. Previous work has provided multiple evidence for a critical role of p160 coactivators and associated histone acetyltransferases in estrogen signaling. In contrast, the involvement of the mammalian mediator complex remains to be established. Further, although the two subtypes ERalpha and ERbeta appear to be similar in regard to principles of LXXLL-mediated coactivator binding to the AF-2 activation domain, there are indications that the context-dependent transcriptional activation profiles of the two ERs can be quite distinct. Potentially, this could be attributed to differences with regard to coregulator recruitment. We have here studied the interactions of the nuclear receptor-binding subunit of the mammalian mediator complex, referred to as TRAP220, with ERalpha and ERbeta. In comparison to the p160 coactivator TIF2, we find that TRAP220 displays ERbeta preference. Here, we show that this is a feature of the binding specificity of the TRAP220 LXXLL motifs and demonstrate that the ER subtype-specific F-domain influences TRAP220 interaction. Such differences with regard to coactivator recruitment indicate that the relative importance of individual coregulators in estrogen signaling could depend on the dominant ER subtype.

DAX-1 functions as an LXXLL-containing corepressor for activated estrogen receptors.

We have discovered that the orphan receptor DAX-1 (NROB1) interacts with the estrogen receptors ERalpha and ERbeta. Interaction occurs with ligand-activated ERs in solution and on DNA and is mediated by the unique DAX-1 N-terminal repeat domain. Each of the three repeats contains a leucine-rich receptor-binding motif, known as the LXXLL motif, which is usually found in nuclear receptor coactivators. We have demonstrated that DAX-1 functions as an inhibitor of ER activation in mammalian cells and suggest a mechanism involving two sequential events, occupation of the ligand-induced coactivator-binding surface and subsequent recruitment of corepressors. Accordingly, we propose that DAX-1 itself acts as a corepressor for ERs. Because DAX-1 is coexpressed with ERs in reproductive tissues, these interactions could play significant roles by influencing estrogen signaling pathways. Our results point at functional similarities between DAX-1 and the orphan receptor SHP (NROB2) in that they have acquired features of transcriptional coregulators that are unique for members of the nuclear receptor family.

The orphan nuclear receptor SHP inhibits agonist-dependent transcriptional activity of estrogen receptors ERalpha and ERbeta.

SHP (short heterodimer partner) is an unusual orphan nuclear receptor that contains a putative ligand-binding domain but lacks a conserved DNA-binding domain. Although no conventional receptor function has yet been identified, SHP has been proposed to act as a negative regulator of nuclear receptor signaling pathways, because it interacts with and inhibits DNA binding and transcriptional activity of various nonsteroid receptors, including thyroid hormone and retinoid receptors. We show here that SHP interacts directly with agonist-bound estrogen receptors, ERalpha and ERbeta, and inhibits ER-mediated transcriptional activation. SHP specifically targets the ligand-regulated activation domain AF-2 and competes for binding of coactivators such as TIF2. Thus, SHP may represent a new category of negative coregulators for ligand-activated nuclear receptors. SHP mRNA is widely expressed in rat tissues including certain estrogen target tissues, and subcellular localization studies demonstrate that SHP is a nuclear protein, suggesting a biological significance of the SHP interactions with ERs. Taken together, these results identify ERs as novel SHP targets and suggest that competition for coactivator-binding is a novel mechanism by which SHP may inhibit nuclear receptor activation.