Differential Recruitment Of Coregulators Research Articles

Selective Modulation of the Human Glucocorticoid Receptor Compromises GR Chromatin Occupancy and Recruitment of p300/CBP and the Mediator Complex

Exogenous glucocorticoids are frequently used to treat inflammatory disorders and as adjuncts for the treatment of solid cancers. However, their use is associated with severe side effects and therapy resistance. Novel glucocorticoid receptor (GR) ligands with a patient-validated reduced side effect profile have not yet reached the clinic. GR is a member of the nuclear receptor family of transcription factors and heavily relies on interactions with coregulator proteins for its transcriptional activity. To elucidate the role of the GR interactome in the differential transcriptional activity of GR following treatment with the selective GR agonist and modulator dagrocorat compared to classic (ant)agonists, we generated comprehensive interactome maps by high-confidence proximity proteomics in lung epithelial carcinoma cells. We found that dagrocorat and the antagonist RU486 both reduced GR interaction with CREB-binding protein/p300 and the mediator complex compared to the full GR agonist dexamethasone. Chromatin immunoprecipitation assays revealed that these changes in GR interactome were accompanied by reduced GR chromatin occupancy with dagrocorat and RU486. Our data offer new insights into the role of differential coregulator recruitment in shaping ligand-specific GR-mediated transcriptional responses.

Confirmation of high-throughput screening data and novel mechanistic insights into FXR-xenobiotic interactions by orthogonal assays

Toxicology in the 21st Century (Tox21) is a federal collaboration employing a high-throughput robotic screening system to test 10,000 environmental chemicals. One of the primary goals of the program is prioritizing toxicity evaluations through in vitro high-throughput screening (HTS) assays for large numbers of chemicals already in commercial use for which little or no toxicity data is available. Within the Tox21 screening program, disruption in nuclear receptor (NR) signaling represents a particular area of interest. Given the role of NR’s in modulating a wide range of biological processes, alterations of their activity can have profound biological impacts. Farnesoid X receptor (FXR) is a member of the nuclear receptor superfamily that has demonstrated importance in bile acid homeostasis, glucose metabolism, lipid homeostasis and hepatic regeneration. In this study, we re-evaluated 24 FXR agonists and antagonists identified through Tox21 using select orthogonal assays. In transient transactivation assays, 7/8 putative agonists and 4/4 putative inactive compounds were confirmed. Likewise, we confirmed 9/12 antagonists tested. Using a mammalian two hybrid approach we demonstrate that both FXR agonists and antagonists facilitate FXRα-coregulator interactions suggesting that differential coregulator recruitment may mediate activation/repression of FXRα mediated transcription. Additionally, we tested the ability of select FXR agonists and antagonists to facilitate hepatic transcription of FXR gene targets Shp and Bsep in a teleost (Medaka) model. Through application of in vitro cell-based assays, in silico modeling and in vivo gene expressions, we demonstrated the molecular complexity of FXR:ligand interactions and confirmed the ability of diverse ligands to modulate FXRα, facilitate differential coregulator recruitment and activate/repress receptor-mediated transcription. Overall, we suggest a multiplicative approach to assessment of nuclear receptor function may facilitate a greater understanding of the biological and mechanistic complexities of nuclear receptor activities and further our ability to interpret broad HTS outcomes.

The Structure-Function Relationship of Angular Estrogens and Estrogen Receptor Alpha to Initiate Estrogen-Induced Apoptosis in Breast Cancer Cells

High-dose synthetic estrogen therapy was the standard treatment of advanced breast cancer for three decades until the discovery of tamoxifen. A range of substituted triphenylethylene synthetic estrogens and diethylstilbestrol were used. It is now known that low doses of estrogens can cause apoptosis in long-term estrogen deprived (LTED) breast cancer cells resistant to antiestrogens. This action of estrogen can explain the reduced breast cancer incidence in postmenopausal women over 60 who are taking conjugated equine estrogens and the beneficial effect of low-dose estrogen treatment of patients with acquired aromatase inhibitor resistance in clinical trials. To decipher the molecular mechanism of estrogens at the estrogen receptor (ER) complex by different types of estrogens—planar [17β-estradiol (E2)] and angular triphenylethylene (TPE) derivatives—we have synthesized a small series of compounds with either no substitutions on the TPE phenyl ring containing the antiestrogenic side chain of endoxifen or a free hydroxyl. In the first week of treatment with E2 the LTED cells undergo apoptosis completely. By contrast, the test TPE derivatives act as antiestrogens with a free para-hydroxyl on the phenyl ring that contains an antiestrogenic side chain in endoxifen. This inhibits early E2-induced apoptosis if a free hydroxyl is present. No substitution at the site occupied by the antiestrogenic side chain of endoxifen results in early apoptosis similar to planar E2. The TPE compounds recruit coregulators to the ER differentially and predictably, leading to delayed apoptosis in these cells.SIGNIFICANCE STATEMENTIn this paper we investigate the role of the structure-function relationship of a panel of synthetic triphenylethylene (TPE) derivatives and a novel mechanism of estrogen-induced cell death in breast cancer, which is now clinically relevant. Our study indicates that these TPE derivatives, depending on the positioning of the hydroxyl groups, induce various conformations of the estrogen receptor’s ligand-binding domain, which in turn produces differential recruitment of coregulators and subsequently different apoptotic effects on the antiestrogen-resistant breast cancer cells.

First High-Resolution Crystal Structures of the Glucocorticoid Receptor Ligand-Binding Domain–Peroxisome Proliferator-Activated γ Coactivator 1-α Complex with Endogenous and Synthetic Glucocorticoids

Both synthetic and endogenous glucocorticoids are important pharmaceutic drugs known to bind to the ligand-binding domain (LBD) of glucocorticoid receptor (GR), a member of the nuclear receptor (NR) superfamily. Ligand binding induces conformational changes within GR, resulting in subsequent DNA binding and differential coregulator recruitment, ultimately activating or repressing target gene expression. One of the most crucial coregulators is peroxisome proliferator-activated γ coactivator 1-α (PGC1α), which acts to regulate energy metabolism by directly interacting with GR to modulate gene expression. However, the mechanisms through which PGC1α senses GR conformation to drive transcription are not completely known. Here, an ancestral variant of the GR (AncGR2) LBD was used as a tool to produce stable protein for biochemical and structural studies. PGC1α is found to interact more tightly and form a more stable complex with AncGR2 LBD than nuclear receptor coactivator 2. We report the first high-resolution X-ray crystal structures of AncGR2 LBD in complex with PGC1α and dexamethasone (DEX) or hydrocortisone (HCY). Structural analyses reveal how distinct steroid drugs bind to GR with different affinities by unique hydrogen bonds and hydrophobic interactions. Important charge clamps are formed between the activation function 2 and PGC1α to mediate their specific interactions. These interactions lead to a high level of protection from hydrogen-deuterium exchange at the coregulator interaction site and strong intramolecular allosteric communication to ligand binding site. This is the first structure detailing the GR-PGC1α interaction providing a foundation for future design of specific therapeutic agents targeting these critical metabolic regulators. SIGNIFICANCE STATEMENT: High-resolution structures of AncGR2 LBD bound to DEX and HCY in complex with PGC1α reveal the molecular mechanism of PGC1α binding to AncGR2 LBD as well as the distinct affinities between DEX and HCY binding. Identifying the structural mechanisms that drive drug affinity is of pharmacologic interest to the glucocorticoid receptor field as an avenue to guide future drug design targeting GR-PGC1α signaling, which plays a crucial role in controlling hepatic glucose output.

Identification of a Novel Coregulator, SH3YL1, That Interacts With the Androgen Receptor N-Terminus.

Nuclear receptor (NR)-mediated transcriptional activity is a dynamic process that is regulated by the binding of ligands that induce distinct conformational changes in the NR. These structural alterations lead to the differential recruitment of coregulators (coactivators or corepressors) that control the expression of NR-regulated genes. Here, we show that a stretch of proline residues located within the N-terminus of androgen receptor (AR) is a bona fide coregulator binding surface, the disruption of which reduces the androgen-dependent proliferation and migration of prostate cancer (PCa) cells. Using T7 phage display, we identified a novel AR-interacting protein, Src homology 3 (SH3)-domain containing, Ysc84-like 1 (SH3YL1), whose interaction with the receptor is dependent upon this polyproline domain. As with mutations within the AR polyproline domain, knockdown of SH3YL1 attenuated androgen-mediated cell growth and migration. RNA expression analysis revealed that SH3YL1 was required for the induction of a subset of AR-modulated genes. Notable was the observation that ubinuclein 1 (UBN1), a key member of a histone H3.3 chaperone complex, was a transcriptional target of the AR/SH3YL1 complex, correlated with aggressive PCa in patients, and was necessary for the maximal androgen-mediated proliferation and migration of PCa cells. Collectively, these data highlight the importance of an amino-terminal activation domain, its associated coregulator, and downstream transcriptional targets in regulating cellular processes of pathological importance in PCa.

Mix ‘n’ match estrogens

Female sex hormones such as estrogen and progesterone are not only essential regulators of reproductive functions but play numerous physiological roles. Loss of endogenous hormone production after menopause can cause uncomfortable symptoms such as hot flashes and lead to the development of osteoporosis or cardiovascular disease. In order to maintain these beneficial anti-aging effects, various estrogen receptor ligands have been clinically tested for their use as postmenopausal hormone replacement therapy. To avoid potentially harmful stimulation of breast or endometrial cell proliferation, so-called selective estrogen receptor modulators or SERMs, have been developed. These compounds do not display the same profile as pure agonists or antagonists but can have differential effects in different tissues [1]. In this issue of Molecular Metabolism, Kim et al. investigate the use of the SERM bazedoxifene (BZA) in combination with estrogens to counter the weight gain and glucose intolerance associated with menopause [2]. The authors use ovariectomized mice fed a high fat diet and treated with various combinations of estrogen receptor (ER) ligands to study their effects on energy homeostasis. They demonstrate that a tissue-selective estrogen complex (TSEC) consisting of BZA plus conjugated equine estrogen, improves glucose homeostasis and insulin sensitivity without affecting the reproductive system. This therapy (BZA+CE) has been successful in the prevention of postmenopausal symptoms in phase III clinical trials, but its metabolic effects have not yet been studied [3,4]. Estrogen therapy for metabolic dysfunction is not a new concept [5,6], but, interestingly, the data presented by Kim et al. suggest that estrogens and BZA act via different pathways to increase hepatic lipid oxidation. As shown by ELISA and qRT-PCR assays on liver homogenates, only treatment with classical estrogens increases FGF21 expression levels, whereas BZA-containing complexes induce Sirtuin-1, PPARα and AMPK. FGF21 is known for its insulin-sensitizing potential and significant beneficial effects on several metabolic parameters [7]. Sirtuin-1 is the founder of the famous sirtuin deacetylase family, whose members play important roles in the regulation of lifespan and insulin sensitivity [8]. Likewise, the energy-sensing kinase adenosine monophosphate (AMP)-activated protein kinase (AMPK) has emerged as a central mediator of metabolism. AMPK responds to changes in the AMP:ATP ratio by promoting glucose and fatty acid catabolism or by inhibiting anabolic pathways. Linked to these pathways is the peroxisome proliferator-activated receptor PPARα, another nuclear receptor family member known to regulate fatty acid oxidation [9]. Activation of all of these pathways serves to enhance hepatic lipid oxidation and to elevate overall metabolic rate, and could explain the prevention of weight gain and insulin resistance observed during the experiments. How FGF21 activation in response to estrogens can achieve these effects to a similar extent as BZA-mediated SIRT1/PPARα/AMPK induction remains an open question. In the future, it will be revealing to determine the molecular mechanism for these observations. One might speculate how these ligands induce distinct target genes in the liver, for example by differential binding of ERα to target sites in the enhancer regions of Fgf21 versus Sirt1/PPARα. That would require BZA-bound ERα to bind to a different set of cis-regulatory elements than estrogen-ligand receptor. Another possibility would involve the activation of signaling pathways that indirectly influence hormone responses. It would also be interesting to test whether different receptor conformations lead to differential recruitment of coregulators. Finally, it is still unknown if these differential effects are only achieved via liver-specific pathways, or if systemic TSEC administration affects other systems such as adipose tissue, skeletal muscle or neuronal networks. Taken together, these findings provide mechanistic insight into ER-regulated metabolic gene programs and will hopefully provide a useful weapon in the battle against metabolic syndrome.

Differential recruitment of coregulators to the RORA promoter adds another layer of complexity to gene (dys) regulation by sex hormones in autism

BackgroundOur independent cohort studies have consistently shown the reduction of the nuclear receptor RORA (retinoic acid-related orphan receptor-alpha) in lymphoblasts as well as in brain tissues from individuals with autism spectrum disorder (ASD). Moreover, we have found that RORA regulates the gene for aromatase, which converts androgen to estrogen, and that male and female hormones regulate RORA in opposite directions, with androgen suppressing RORA, suggesting that the sexually dimorphic regulation of RORA may contribute to the male bias in ASD. However, the molecular mechanisms through which androgen and estrogen differentially regulate RORA are still unknown.MethodsHere we use functional knockdown of hormone receptors and coregulators with small interfering RNA (siRNA) to investigate their involvement in sex hormone regulation of RORA in human neuronal cells. Luciferase assays using a vector containing various RORA promoter constructs were first performed to identify the promoter regions required for inverse regulation of RORA by male and female hormones. Sequential chromatin immunoprecipitation methods followed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) analyses of RORA expression in hormone-treated SH-SY5Y cells were then utilized to identify coregulators that associate with hormone receptors on the RORA promoter. siRNA-mediated knockdown of interacting coregulators was performed followed by qRT-PCR analyses to confirm the functional requirement of each coregulator in hormone-regulated RORA expression.ResultsOur studies demonstrate the direct involvement of androgen receptor (AR) and estrogen receptor (ER) in the regulation of RORA by male and female hormones, respectively, and that the promoter region between −10055 bp and −2344 bp from the transcription start site of RORA is required for the inverse hormonal regulation. We further show that AR interacts with SUMO1, a reported suppressor of AR transcriptional activity, whereas ERα interacts with the coactivator NCOA5 on the RORA promoter. siRNA-mediated knockdown of SUMO1 and NCOA5 attenuate the sex hormone effects on RORA expression.ConclusionsAR and SUMO1 are involved in the suppression RORA expression by androgen, while ERα and NCOA5 collaborate in the up-regulation of RORA by estrogen. While this study offers a better understanding of molecular mechanisms involved in sex hormone regulation of RORA, it also reveals another layer of complexity with regard to gene regulation in ASD. Inasmuch as coregulators are capable of interacting with a multitude of transcription factors, aberrant expression of coregulator proteins, as we have seen previously in lymphoblasts from individuals with ASD, may contribute to the polygenic nature of gene dysregulation in ASD.

Identification of Ligand-Selective Peptide Antagonists of the Mineralocorticoid Receptor Using Phage Display

The mineralocorticoid receptor (MR) is a member of the nuclear receptor superfamily. Pathological activation of the MR causes cardiac fibrosis and heart failure, but clinical use of MR antagonists is limited by the renal side effect of hyperkalemia. The glucocorticoid cortisol binds the MR with equivalent affinity to that of the mineralocorticoids aldosterone and deoxycorticosterone. In nonepithelial tissues, including the myocardium, which do not express the cortisol-inactivating enzyme 11β hydroxysteroid dehydrogenase 2, cortisol has been implicated in the activation of MR. The mechanisms for ligand- and tissue-specific actions of the MR are undefined. Over the past decade, it has become clear that coregulator proteins are critical for nuclear receptor-mediated gene expression. A subset of these coregulators may confer specificity to MR-mediated responses. To evaluate whether different physiological ligands can induce distinct MR conformations that underlie differential coregulator recruitment and ligand-specific gene regulation, we utilized phage display technology to screen 10(8) 19mer peptides for their interaction with the MR in the presence of agonist ligands. We identified ligand-selective MR-interacting peptides that acted as potent antagonists of MR-mediated transactivation. This represents a novel mechanism of MR antagonism that may be manipulated in the rational design of a ligand- or tissue-selective MR modulator to treat diseases like heart failure without side effects such as hyperkalemia.

The mammalian mineralocorticoid receptor: tying down a promiscuous receptor.

The mineralocorticoid receptor (MR) has been called a promiscuous receptor because its intrinsic affinity for aldosterone, cortisol and corticosterone is similar. Since glucocorticoids circulate in concentrations 100- to 1000-fold those of aldosterone, stoichiometry dictates that MR should be activated by glucocorticoids, not aldosterone, yet MRs are expressed in many tissues and regulate diverse functions, many of them under the regulation of the renin-angiotensin-aldosterone system. A relatively small number of brain MRs are aldosterone selective and modulate blood pressure. Evidence for possible mechanisms conferring ligand specificity in the context of mineralocorticoid-induced hypertension and the brain are discussed. These include factors (or mechanisms) intrinsic to the receptor, such as alternative splice variants and translation start sites, and extrinsic to the MR, including differential access through the blood-brain barrier, differential recruitment of co-regulators and scaffolding proteins, 11beta-steroid dehydrogenase activity, synthesis of potent acylated aldosterone derivatives and the synthesis of relevant amounts of aldosterone in areas of the brain that modulate blood pressure.

Adrenal corticosteroid effects in the central nervous system on long-term control of blood pressure

Adrenal corticosteroid effects in the central nervous system on long-term control of blood pressure

Identification of novel ER-α target genes in breast cancer cells: Gene- and cell-selective co-regulator recruitment at target promoters determines the response to 17β-estradiol and tamoxifen

Tamoxifen and 17β-estradiol are capable of up-regulating the expression of some genes and down-regulate the expression of others simultaneously in the same cell. In addition, tamoxifen shows distinct transcriptional activities in different target tissues. To elucidate whether these events are determined by differences in the recruitment of co-regulators by activated estrogen receptor-α (ER-α) at target promoters, we applied chromatin immunoprecipitation (ChIP) with promoter microarray hybridisation in breast cancer T47D cells and identified 904 ER-α targets genome-wide. On a selection of newly identified targets, we show that 17β-estradiol and tamoxifen stimulated up- or down-regulation of transcription correlates with the selective recruitment of co-activators or co-repressors, respectively. This is shown for both breast (T47D) and endometrial carcinoma cells (ECC1). Moreover, differential co-regulator recruitment also explains that tamoxifen regulates a number of genes in opposite direction in breast and endometrial cancer cells. Over-expression of co-activator SRC-1 or co-repressor SMRT is sufficient to alter the transcriptional action of tamoxifen on a number of targets. Our findings support the notion that recruitment of co-regulator at target gene promoters and their expression levels determine the effect of ER-α on gene expression to a large extent.

Retinoic Acid Receptor Isotype Specificity in F9 Teratocarcinoma Stem Cells Results from the Differential Recruitment of Coregulators to Retinoic Acid Response Elements

The retinoic acid receptor (RAR) alpha, beta(2), and gamma isotypes each regulate specific subsets of target genes in F9 teratocarcinoma stem cells. We used chromatin immunoprecipitation assays to monitor the association of RARgamma, retinoic X receptor (RXR) alpha, and coregulators with the RARbeta(2), Hoxa1, and Cyp26A1 retinoic acid response elements (RAREs) in F9 wild type and RARalpha, -beta(2), and -gamma null cells. Additionally we quantitatively monitored expression of the corresponding mRNAs. We demonstrated that the association of RARgamma and/or RXRalpha with a RARE was not sufficient for retinoic acid (RA)-mediated transcription of the corresponding target gene. However, the ability of RARgamma and/or RXRalpha to recruit pCIP (AIB1/ACTR/RAC-3/TRAM-1/SRC-3) and p300 to a RARE did correlate with RA-associated transcription of target mRNAs. Therefore, the specific functions of the RAR isotypes do not manifest at the level of their DNA binding but rather from a differential ability to recruit specific components of the transcriptional machinery. We also demonstrated that RA-mediated displacement of the polycomb group protein SUZ12 from a RARE was inhibited in the absence of RARgamma. Thus, transcriptional components of the RAR signaling pathway are specifically required for displacement of SUZ12 from RAREs during RA-mediated differentiation of F9 cells.

SUMOylation of Tr2 orphan receptor involves Pml and fine-tunes Oct4 expression in stem cells

The Tr2 orphan nuclear receptor can be SUMOylated, resulting in the replacement of coregulators recruited to the regulatory region of its endogenous target gene, Oct4. UnSUMOylated Tr2 activates Oct4, enhancing embryonal carcinoma-cell proliferation, and is localized to the promyelocytic leukemia (Pml) nuclear bodies. When its abundance is elevated, Tr2 is SUMOylated at Lys238 and seems to be released from the nuclear bodies to act as a repressor. SUMOylation of Tr2 induces an exchange of its coregulators: corepressor Rip140 replaces coactivator Pcaf, which switches Tr2 from an activator to a repressor. This involves dynamic partitioning of Tr2 into Pml-containing and Pml-free pools. These results support a model where SUMOylation-dependent partitioning and differential coregulator recruitment contribute to the maintenance of a homeostatic supply of activating, as opposed to repressive, Tr2, thus fine-tuning Oct4 expression and regulating stem-cell proliferation.

Yin Yang 1 Physically Interacts with Hoxa11 and Represses Hoxa11-dependent Transcription

Yin Yang 1 (YY1) plays an indispensable role in embryonic development. YY1 contains an evolutionarily conserved, 22-amino acid segment, the PHO homology region (PHR), which is located within its central domain (spacer) and has been shown previously to participate in the recruitment of Polycomb group of proteins and in YY1-mediated transcription. In this report, we show that the PHR physically interacts with several Abd-B-type Hox proteins. Although ectopic expression of Hoxa11 enhanced target promoter activity, overexpression of YY1 repressed this effect, which was abrogated by YY1 siRNA and the histone deacetylase inhibitor trichostatin A. We have further demonstrated that this suppression effect was the result of YY1-dependent recruitment of HDAC2 to the Hoxa11 target promoter. Taken together, our findings show that YY1 represses Hoxa11-dependent transcription via interactions with the Hox proteins and HDAC recruitment, providing a link between an Abd-type Hox protein and a Polycomb group protein at the level of direct protein-protein interactions. These findings not only provide a novel insight into YY1 function but also identify a new regulation of homeotic protein-mediated transcriptional regulation in general.