About 200 coactivators play a central role in promoting gene expression mediated by nuclear receptors. This diverse group of proteins are key integrators of signals from steroid hormones and have been implicated in cancer and other diseases.

Nuclear receptor coactivators (NCOAs) function as coactivators for nuclear receptors as well as several other transcription factors and potentiate their transcriptional activity. NCOAs play an important role in biology of hormone-dependent and -independent cancers. MCB-613 is a recently described, small molecule stimulator of NCOAs and anti-neoplastic compound that leads to the death of tumour cells due to increased cellular stress. In the present study we investigated the molecular mechanism of MCB-613-induced cell death. We report that absence of NCOA3 leads to compromised activation of PERK signalling pathway during unfolded protein response (UPR). We found that chemical and genetic inhibition of NCOA3 attenuated the expression of PERK at mRNA and protein level. We show that loss of NCOA3 renders cells hypersensitive to UPR induced cell death. Our results show that MCB-613 induced cell death is attenuated in NCOA3 knockout HeLa cells and MCB-613 leads to enhanced PERK signalling in wild-type HeLa cells. The knockdown of PERK provides resistance to MCB-613 mediated cell death while knockdown of XBP1 and ATF6 have no such effect. Our results suggest that hyperstimulation of NCOA3 by MCB-613 induces cell death by evoking constitutive PERK signalling. Taken together our results point to NCOA3 as an important determinant in regulating cell fate during ER stress, with too little and too much NCOA3 both producing deleterious effects.

Adult rat lumbar motoneurons in the spinal nucleus of the bulbocavernosus (SNB) respond to androgens with an increase in soma size. This response is mediated by the androgen receptor (AR) in these motoneurons. Interestingly, other lumbar motoneurons in the rat possess the AR, yet do not respond to androgens in this fashion. This paradox suggests the existence and participation of nuclear receptor coregulators in conferring direct androgen-responsiveness to select motoneurons in the adult rat spinal cord. Nuclear receptor coregulators have received much attention recently for their proposed role in enhancing or repressing the transcriptional activity of steroid hormone receptors. The present study used immunocytochemistry to identify a number of nuclear receptor coactivators that are expressed by adult lumbar motoneurons: SRC-1, SRC-2, CBP, p300, and cJUN. Results of this study indicate that all five of these coactivators are abundantly expressed in the androgen-responsive SNB, and in two adjacent motor pools, the androgen-responsive dorsolateral nucleus (DLN), and the androgen-unresponsive retrodorsolateral nucleus (RDLN). While we detected significant regional differences for only SRC-1 and cJUN, the SNB consistently contained the highest percentage of immunoreactive motoneurons for all five cofactors examined. Our results indicate five different putative cofactors have the potential to participate in motoneuronal responses to androgens, since their distribution overlaps well with the distribution of ARs in these motoneurons.

The ovarian hormones, estradiol (E) and progesterone (P) facilitate the expression of sexual behavior in female rats. E and P mediate many of these behavioral effects by binding to their respective intracellular receptors in specific brain regions. Nuclear receptor coactivators, including Steroid Receptor Coactivator-1 (SRC-1) and CREB Binding Protein (CBP), dramatically enhance ligand-dependent steroid receptor transcriptional activity in vitro. Previously, our lab has shown that SRC-1 and CBP modulate estrogen receptor (ER)-mediated induction of progestin receptor (PR) gene expression in the ventromedial nucleus of the hypothalamus (VMN) and hormone-dependent sexual receptivity in female rats. Female sexual behaviors can be activated by high doses of E alone in ovariectomized rats, and thus are believed to be ER-dependent. However, the full repertoire of female sexual behavior, in particular, proceptive behaviors such as hopping, darting and ear wiggling, are considered to be PR-dependent. In the present experiments, the function of SRC-1 and CBP in distinct ER- (Exp. 1) and PR- (Exp. 2) dependent aspects of female sexual behavior was investigated. In Exp. 1, infusion of antisense oligodeoxynucleotides to SRC-1 and CBP mRNA into the VMN decreased lordosis intensity in rats treated with E alone, suggesting that these coactivators modulate ER-mediated female sexual behavior. In Exp. 2, antisense to SRC-1 and CBP mRNA around the time of P administration reduced PR-dependent ear wiggling and hopping and darting. Taken together, these data suggest that SRC-1 and CBP modulate ER and PR action in brain and influence distinct aspects of hormone-dependent sexual behaviors. These findings support our previous studies and provide further evidence that SRC-1 and CBP function together to regulate ovarian hormone action in behaviorally-relevant brain regions.

Human fetal membranes play an important role in term and preterm labor and are responsive to steroids. We examined the expression of steroid receptor coactivators in fetal membranes obtained prior to and following labor at term. Proteins were localized by immunohistochemistry, Western analysis was carried out in nuclear extracts, and mRNA levels were determined by real-time RT-PCR. SRC-1, SRC-2, p300, and PCAF proteins were present in all nuclear extracts. The amnion nuclei expressed higher levels of SRC-1, p300, and PCAF than nuclei from the chorion-decidua, whereas the reverse was true for SRC-2. Chorion-decidua from patients before labor expressed higher levels of SRC-1 than those from patients after labor. Also, the PCAF level was higher in the amnion obtained before labor than the same tissue obtained after labor. In contrast to the protein expression, mRNA levels of SRC-1 and p300 were higher in the chorion-decidua compared to the amnion, whereas there was no difference in levels of SRC-2 and PCAF mRNAs between these two tissues. These data underline that the regulation of the expression of the coactivators in these tissues occurs during labor and is complex and tissue specific.

Estrogens act in brain to profoundly influence neurogenesis, sexual differentiation, neuroprotection, cognition, energy homeostasis, and female reproductive behavior and physiology through a variety of mechanisms, including the induction of progestin receptors (PRs). PRs are expressed as two isoforms, PR-A and PR-B, that have distinct functions in physiology and behavior. Because these PR isoforms cannot be distinguished using cellular resolution techniques, the present study used isoform-specific null mutant mice that lack PR-A or PR-B for the first time to investigate whether 17β-estradiol benzoate (EB) regulates the differential expression of the PR isoforms in the ventromedial nucleus of the hypothalamus (VMN), arcuate nucleus, and medial preoptic area, brain regions that are rich in EB-induced PRs. Interestingly, EB induced more PR-A than PR-B in all three brain regions, suggesting that PR-A is the predominant isoform in these regions. Given that steroid receptor coactivator (SRC)-1 and SRC-2 are important in estrogen receptor (ER)-dependent transcription in brain, including PR induction, we tested whether the expression of these coactivators was correlated with PR isoform expression. The majority of EB-induced PR cells expressed both SRC-1 and SRC-2 in the three brain regions of all genotypes. Interestingly, the intensity of PR-A immunoreactivity correlated with SRC-2 expression in the VMN, providing a potential mechanism for selective ER-mediated transactivation of PR-A over PR-B in a brain region-specific manner. In summary, these novel findings indicate that estrogens differentially regulate PR-A and PR-B expression in the female hypothalamus, and provide a mechanism by which steroid action in brain can selectively modulate behavior and physiology.

Nuclear receptor coactivators (NCOAs), consisting of coactivators and corepressors, dramatically enhance the transcriptional activity of nuclear receptors. Hypoxia-inducible factor-1α (HIF-1α) is a transcription factor that plays a major role under hypoxic conditions. This study was performed with the focus on the association of NCOAs with HIF-1α in the serum of chronic kidney disease (CKD) patients. Sixty patients with stage 5 CKD and 30 healthy controls from The Second Affiliated Hospital of Shantou University Medical College, between March 21, 2019, and October 30, 2019, were recruited in this prospective cohort study. We analyzed the serum levels of NCOAs (NCOA1, NCOA2, and NCOA3), HIF-1α, vascular endothelial growth factor (VEGF), etc. and assessed whether there was any relationship between these parameters and CKD disease. We found that circulating NCOA1 was positively associated with circulating NCOA2, NCOA3, and HIF-1α. A positive correlation was also observed between NCOA2 and NCOA1, NCOA3, HIF-1α, and VEGF. Furthermore, statistically significant correlations between NCOA3 and NCOA1, NCOA2, and HIF-1α were observed. The serum levels of VEGF in the CKD group were higher than those of the healthy control group. Circulating NCOA1 and circulating NCOA2 were negatively associated with procalcitonin. In conclusion, there was an association between circulating NCOA1, NCOA2, NCOA3, and circulating HIF-1α, and circulating VEGF was a risk factor for CKD disease. However, more studies should be performed to confirm this hypothesis.

Nuclear receptors (NRs) are central regulators of pathophysiological processes; however, how their responses intertwine is still not fully understood. The aim of this study was to determine whether and how steroid NRs can influence each other's activity under co-agonist treatment. We used a unique system consisting of a multicopy integration of an estrogen receptor responsive unit that allows direct visualization and quantification of estrogen receptor alpha (ERα) DNA binding, co-regulator recruitment and transcriptional readout. We find that ERα DNA loading is required for other type I nuclear receptors to be co-recruited after dual agonist treatment. We focused on ERα/glucocorticoid receptor interplay and demonstrated that it requires steroid receptor coactivators (SRC-2, SRC-3) and the mediator component MED14. We then validated this cooperative interplay on endogenous target genes in breast cancer cells. Taken together, this work highlights another layer of mechanistic complexity through which NRs cross-talk with each other on chromatin under multiple hormonal stimuli.

Pluripotency of embryonic stem cells (ESC) is tightly regulated by a network of transcription factors among which the estrogen-related receptor β (Esrrb). Esrrb contributes to the relaxation of the G1 to S-phase (G1/S) checkpoint in mouse ESCs by transcriptional control of the deubiquitylase Dub3 gene, contributing to Cdc25A persistence after DNA damage. We show that in mESCs, Dub3 gene expression is cell cycle regulated and is maximal prior G1/S transition. In addition, following UV-induced DNA damage in G1, Dub3 expression markedly increases in S-phase also suggesting a role in checkpoint recovery. Unexpectedly, we also observed cell cycle-regulation of Nanog expression, and not Oct4, reaching high levels prior to G1/S transition, finely mirroring Cyclin E1 fluctuations. Curiously, while Esrrb showed only limited cell-cycle oscillations, transcript levels of the p160 family of nuclear receptor coactivators (NCoAs) displayed strong cell cycle-dependent fluctuations. Since NCoAs function in concert with Esrrb in transcriptional activation, we focussed on NCoA1 whose levels specifically increase prior onset of Dub3 transcription. Using a reporter assay, we show that NCoA1 potentiates Esrrb-mediated transcription of Dub3 and we present evidence of protein interaction between the SRC1 splice variant NCoA1 and Esrrb. Finally, we show a differential developmental regulation of all members of the p160 family during neural conversion of mESCs. These findings suggest that in mouse ESCs, changes in the relative concentration of a coactivator at a given cell cycle phase, may contribute to modulation of the transcriptional activity of the core transcription factors of the pluripotent network and be implicated in cell fate decisions upon onset of differentiation.

Glucocorticoid receptor (GR) is a ligand-activated nuclear receptor which is widely distributed in the brain. Many types of neurons and glial cells are known to express GR, but the expression of GR in ependymal cells has yet to be identified. The present study therefore was undertaken to determine whether ependymal cells express GR and coactivators of GR, such as steroid receptor coactivator 1 (SRC-1) and p300. GR immunoreactivity was found in cells immunopositive to vimentin, a marker of ependymal cells, around the third ventricle (3V), the lateral ventricle (LV), the cerebral aqueduct and the fourth ventricle (4V), whereas the expression of GR in vimentin-immunoreactive (ir) cells was significantly reduced by adrenalectomy (ADX) in male rats. Vimentin-ir cells also expressed both SRC-1 and p300 at around 3V, LV, the cerebral aqueduct and 4V. ADX had no effect on the expression of SRC-1 or p300 in vimentin-ir cells. These results suggest that glucocorticoid may exert effects on ependymal cells through binding to GR followed by association with SRC-1 and p300 to maintain brain environment under stressful conditions.

Steroid receptors such as estrogen and androgen receptors are nuclear receptors involved in the transcriptional regulation of a large number of target genes. Steroid-dependent protein expression in the brain controls a large array of biological processes including spatial cognition, copulatory behavior and neuroprotection. The discovery of a competition, or squelching, between two different nuclear receptors introduced the notion that common cofactors may be involved in the modulation of transcriptional activity of nuclear receptors. These cofactors or coregulatory proteins are functionally divided into coactivators and corepressors and are involved in chromatin remodeling and stabilization of the general transcription machinery. Although a large amount of information has been collected about the in vitro function of these coregulatory proteins, relatively little is known regarding their physiological role in vivo, particularly in the brain. Our laboratory and others have demonstrated the importance of SRC-1 in the differentiation and activation of steroid-dependent sexual behaviors and the related neural genes. For example, we report that the inhibition of SRC-1 expression blocks the activating effects of exogenous testosterone on male sexual behaviors and increases the volume of the median preoptic area. Other coactivators are likely to be involved in the modulation in vivo of steroid receptor activity and it seems that the presence of a precise subset of coactivators could help define the phenotype of the cell by modulating a specific downstream pathway after steroid receptor activation. The very large number of coactivators and their association into preformed complexes potentially allows the determination of hundreds of different phenotypes. The study of the expression of the coactivator and their function in vivo is required to fully understand steroid action and specificity in the brain.

Nuclear receptor coregulators are the principal regulators of Estrogen Receptor (ER)-mediated transcription. ERβ, an ER subtype first identified in 1996, is associated with poor outcomes in breast cancer (BCa) subtypes, and the coexpression of the ERβ1 isoform and AIB-1 and TIF-2 coactivators in BCa-associated myofibroblasts is associated with high-grade BCa. We aimed to identify the specific coactivators that are involved in the progression of ERβ-expressing BCa. ERβ isoforms, coactivators, and prognostic markers were tested using standard immunohistochemistry. AIB-1, TIF-2, NF-kB, p-c-Jun, and/or cyclin D1 were differentially correlated with ERβ isoform expression in the BCa subtypes and subgroups. The coexpression of the ERβ5 and/or ERβ1 isoforms and the coactivators were found to be correlated with a high expression of P53, Ki-67, and Her2/neu and large-sized and/or high-grade tumors in BCa. Our study supports the notion that ERβ isoforms and coactivators seemingly coregulate the proliferation and progression of BCa and may provide insight into the potential therapeutic uses of the coactivators in BCa.

Peroxisome proliferator-activated receptor (PPAR)alpha, beta (also known as delta), and gamma function as sensors for fatty acids and fatty acid derivatives and control important metabolic pathways involved in the maintenance of energy balance. PPARs also regulate other diverse biological processes such as development, differentiation, inflammation, and neoplasia. In the nucleus, PPARs exist as heterodimers with retinoid X receptor-alpha bound to DNA with corepressor molecules. Upon ligand activation, PPARs undergo conformational changes that facilitate the dissociation of corepressor molecules and invoke a spatiotemporally orchestrated recruitment of transcription cofactors including coactivators and coactivator-associated proteins. While a given nuclear receptor regulates the expression of a prescribed set of target genes, coactivators are likely to influence the functioning of many regulators and thus affect the transcription of many genes. Evidence suggests that some of the coactivators such as PPAR-binding protein (PBP/PPARBP), thyroid hormone receptor-associated protein 220 (TRAP220), and mediator complex subunit 1 (MED1) may exert a broader influence on the functions of several nuclear receptors and their target genes. Investigations into the role of coactivators in the function of PPARs should strengthen our understanding of the complexities of metabolic diseases associated with energy metabolism.

Steroid hormones are pivotal modulators of pathophysiological processes in many organs, where they interact with nuclear receptors to regulate gene transcription. However, our understanding of hormone action at the single cell level remains incomplete. Here, we focused on estrogen stimulation of the well-characterized GREB1 and MYC target genes that revealed large differences in cell-by-cell responses, and, more interestingly, between alleles within the same cell, both over time and hormone concentration. We specifically analyzed the role of receptor level and activity state during allele-by-allele regulation and found that neither receptor level nor activation status are the determinant of maximal hormonal response, indicating that additional pathways are potentially in place to modulate cell- and allele-specific responses. Interestingly, we found that a small molecule inhibitor of the arginine methyltransferases CARM1 and PRMT6 was able to increase, in a gene specific manner, the number of active alleles/cell before and after hormonal stimulation, suggesting that mechanisms do indeed exist to modulate hormone receptor responses at the single cell and allele level.

How nuclear receptors (NRs) coordinate the sequential, ligand-dependent recruitment of multiple coactivator complexes (e.g., SRC complexes and Mediator) that share similar receptor binding determinants is unclear. We show that although the receptor binding subunits of these complexes (i.e., SRCs and Med220, respectively) share overlapping binding sites on estrogen receptor alpha (ERalpha), information contained in the receptor-coactivator interface allows the receptor to distinguish between them. In support of this conclusion, we have identified an ERalpha AF-2 point mutant (L540Q) that selectively binds and recruits Med220, but not SRCs, both in vitro and in vivo. In cells expressing this mutant, the recruitment of Med220 to the pS2 promoter is delayed, and the expression of the vast majority of estrogen target genes is impaired, suggesting a nearly global functional interdependence of these coactivators. Collectively, our results suggest that "facilitated recruitment," rather than competition, drives the sequential recruitment of SRC complexes and Mediator by NRs.

The chicken ovalbumin upstream promoter-transcription factors (COUP-TFI and II) make up the most conserved subfamily of nuclear receptors that play key roles in angiogenesis, neuronal development, organogenesis, cell fate determination, and metabolic homeostasis. Although the biological functions of COUP-TFs have been studied extensively, little is known of their structural features or aspects of ligand regulation. Here we report the ligand-free 1.48 A crystal structure of the human COUP-TFII ligand-binding domain. The structure reveals an autorepressed conformation of the receptor, where helix alpha10 is bent into the ligand-binding pocket and the activation function-2 helix is folded into the cofactor binding site, thus preventing the recruitment of coactivators. In contrast, in multiple cell lines, COUP-TFII exhibits constitutive transcriptional activity, which can be further potentiated by nuclear receptor coactivators. Mutations designed to disrupt cofactor binding, dimerization, and ligand binding, substantially reduce the COUP-TFII transcriptional activity. Importantly, retinoid acids are able to promote COUP-TFII to recruit coactivators and activate a COUP-TF reporter construct. Although the concentration needed is higher than the physiological levels of retinoic acids, these findings demonstrate that COUP-TFII is a ligand-regulated nuclear receptor, in which ligands activate the receptor by releasing it from the autorepressed conformation.

We report our initial efforts in the analysis of endogenous nuclear receptor coactivator complexes as a research bridging strand of the Nuclear Receptor Signaling Atlas (NURSA) (www.NURSA.org). A proteomic approach is used to systematically isolate a variety of coactivator complexes using HeLa cells as a model cell line and to identify the coactivator-associated proteins with mass spectrometry. We have isolated and identified seven coactivator complexes including the p160 steroid receptor coactivator family, cAMP response element binding protein-binding protein, p300, coactivator of activating protein-1 and estrogen receptors, and E6 papillomavirus-associated protein. The newly identified coactivator-associated proteins provide unbiased clues and links for understanding of the endogenous hormone receptor coregulator network and its regulation. We hope that the electronic availability of these data to the general scientific community will facilitate generation and testing of new hypotheses to further our understanding of nuclear receptor signaling and coactivator functions.

In a little more than 10 years, nuclear receptor (NR) coregulators (coactivators and corepressors) have contributed to our present realization that a great level of sophistication exists in transcriptional regulation. Here, we discuss the implications of coregulators as versatile regulatory agents, influencing not only transcriptional initiation but also elongation, splicing, and translation. In addition to this, there is an increasing recognition that they also regulate a variety of biological processes outside of the nucleus. An important concept that we wish to emphasize is that coregulators are both targets and propagators of posttranslational modification (PTM) codes. This underlies a sophisticated epigenetic regulatory scheme from which a complex and dynamic mammalian phenotype emanates.

Proline-, glutamic acid-, and leucine-rich protein (PELP)1, also known as modulator of nongenomic actions of the estrogen receptor (MNAR), is a novel nuclear receptor coregulator with a multitude of functions. PELP1/MNAR serves as a scaffolding protein that couples various signaling complexes with nuclear receptors and participates in genomic and nongenomic functions. Recent data suggest that PELP1/MNAR expression is deregulated in several cancers, including breast, endometrial, prostate, and ovarian cancer, and that PELP1/MNAR interacts with several oncogenes. In this review, we summarize the emerging biological properties and functions of PELP1/MNAR.

We have systematically isolated a variety of coactivator complexes from HeLa S3 cells using proteomic approaches. In the present report, we have evaluated twelve coactivator complexes involved in nuclear receptor-dependent gene transcription that have been purified by using an immunoprecipitation method. The twelve purified coactivator complexes are SRC-1, SRC-2, SRC-3, CBP, p300, CAPER, E6-AP, ASC-1, CoREST, CRSP3, CRSP2, and CDK7 containing complexes. We have identified 153 protein components associated with these coactivator complexes using mass spectrometry. In order to systematically characterize the functional roles for these components in nuclear receptor-dependent gene transcription and their investigative potential, we have developed a scoring system. This scoring system is comprised of biological and experimental parameters. The biological evaluation considers aspects such as intrinsic enzymatic activity of a protein component, cellular signaling processes in which protein components may be involved, associations with human disease, specific protein motifs, and the known biological roles of other interacting partners of the identified protein. In the experimental evaluation, we include parameters, such as the availability of research materials for the functional study of the identified protein component; such as full-length cDNA clones, antibodies, and commercially available knock-out embryonic stem (ES) cells. Each scoring parameter has been assigned an arbitrary number of points according to perceived relative importance. On the basis of this scoring system, we prioritized each of the protein components in terms of the likelihood of their importance for coactivator complex networking in nuclear receptor-dependent gene transcription.