Ligand-dependent Transcriptional Regulators Research Articles

8464 A Unique SERM Reveals An Estrogen Receptor-Dependent Secretion Of DKK1 In Breast Cancer Cells

Abstract Disclosure: K.S. Young: None. G. Hancock: None. S. Kregel: None. S.W. Fanning: Grant Recipient; Self; Olema Oncology. Although Estrogen Receptor positive (ER+) breast cancer has been successfully targeted in primary tumors, the critical limitations of hormone therapies are ultimately revealed as many women often experience disease recurrence. A subset of these patients harbor a distinctive tyrosine to serine missense mutation at position 537 (Y537S) in ER leading to allele-specific transcriptional programs that enhance metastasis. ER is a ligand-dependent master transcriptional regulator. Probing unique ER structural features can reveal new transcriptional activities. We developed a structurally unconventional Selective Estrogen Receptor Modulator (SERM), T6I-29, to better understand these structure-transcriptional relationships. T6I-29 shows effective anti-tumoral activities in Y537S ESR1 breast cancer cells. To examine the anti-tumoral mechanism of T6I-29 action, we performed RNA-sequencing on ESR1 Y537S mutant breast cancer cells. Compared to other clinically relevant compounds, our drug uniquely and significantly downregulated DKK1, a tumor secreted protein known to modulate the Wnt/β-Catenin and PI3K/Akt pathways. Upregulation of DKK1 correlates with metastatic burden across multiple cancers but has not been evaluated in ESR1 mutant breast cancers. We find that DKK1 levels are significantly elevated in the plasma of 108 ER+ breast cancer patients compared to 100 matched healthy donors. Furthermore, in wild-type (WT) PIK3CA breast cancer cell lines, those with Y537S ESR1 mutation show constitutive expression and secretion of DKK1, while DKK1 expression and secretion is estrogen-dependent in WT breast cancer cells. Intriguingly, WT cells show significant surface bound DKK1, which is secreted into the media upon hormone stimulation and blocked by T6I-29. Understanding the ER-dependence and functional importance of DKK1 secretion offers a novel approach to potentially reduce breast cancer metastatic burden. Presentation: 6/3/2024

7993 Thienopyrimidine-Based Estrogen Receptor Modulators Adopt Unique Ligand Binding Poses to Elicit Anti-Proliferative Activities in ER+ Breast Cancer Cells

Abstract Disclosure: E.C. Fink: None. S.L. Mateus: None. N. Meganathan: None. V.K. Sammeta: None. J.D. Norris: None. D.P. McDonnell: None. T. Willson: None. S.W. Fanning: None. One in eight women will be diagnosed with breast cancer in their lifetimes. Estrogen receptor alpha (ER) drives breast cancer pathology and is expressed in approximately seventy percent of tumors. Hormone therapies are used to treat and prevent the metastasis of ER+ breast cancers and these patients have favorable 5-year survivals. However, most breast cancer patient deaths are ER+. While the next generation of antiestrogens have shown promise in the advanced setting, more work needs to be done to fully address these mortalities. ER is a ligand-dependent master transcriptional regulator, whereby ligand-specific ER conformational ensembles redirect multiple programs to produce oncogenic or therapeutic endpoints. To better understand ER structure-activity relationships, we have developed new antiestrogenic small molecules based on a thienopyrimidine scaffold. Comprehensive structural, ER activity, and cancer endpoint profiling shows that these new molecules adopt a unique ligand binding pose, which perturbs new structural elements within the orthosteric hormone binding pocket. These molecules downregulate ER target genes and halt the proliferation of ER+ breast cancer cells. Presentation: 6/3/2024

Unconventional isoquinoline-based SERMs elicit fulvestrant-like transcriptional programs in ER+ breast cancer cells

Estrogen receptor alpha (ERα) is a ligand-dependent master transcriptional regulator and key driver of breast cancer pathology. Small molecule hormones and competitive antagonists favor unique ERα conformational ensembles that elicit ligand-specific transcriptional programs in breast cancer and other hormone-responsive tissues. By affecting disparate ligand binding domain structural features, unconventional ligand scaffolds can redirect ERα genomic binding patterns to engage novel therapeutic transcriptional programs. To improve our understanding of these ERα structure-transcriptional relationships, we develop a series of chemically unconventional antagonists based on the antiestrogens elacestrant and lasofoxifene. High-resolution x-ray co-crystal structures show that these molecules affect both classical and unique structural motifs within the ERα ligand binding pocket. They show moderately reduced antagonistic potencies on ERα genomic activities but are effective anti-proliferative agents in luminal breast cancer cells. Interestingly, they favor a 4-hydroxytamoxifen-like accumulation of ERα in breast cancer cells but lack uterotrophic activities in an endometrial cell line. Importantly, RNA sequencing shows that the lead molecules engage transcriptional pathways similar to the selective estrogen receptor degrader fulvestrant. This advance shows that fulvestrant-like genomic activities can be achieved without affecting ERα accumulation in breast cancer cells.

Design and in vitro characterization of RXR variants as tools to investigate the biological role of endogenous rexinoids

Retinoid X receptors (RXRα, β, and γ) are essential members of the nuclear receptor (NR) superfamily of ligand-dependent transcriptional regulators that bind DNA response elements and control the expression of large gene networks. As obligate heterodimerization partners of many NRs, RXRs are involved in a variety of pathophysiological processes. However, despite this central role in NR signaling, there is still no consensus regarding the precise biological functions of RXRs and the putative role of the endogenous ligands (rexinoids) previously proposed for these receptors. Based on available crystal structures, we introduced a series of amino acid substitutions into the ligand-binding pocket of all three RXR subtypes in order to alter their binding properties. Subsequent characterization using a battery of cell-based and in vitro assays led to the identification of a double mutation abolishing the binding of any ligand while keeping the other receptor functions intact and a triple mutation that selectively impairs interaction with natural rexinoids but not with some synthetic ligands. We also report crystal structures that help understand the specific ligand-binding capabilities of both variants. These RXR variants, either fully disabled for ligand binding or retaining the property of being activated by synthetic compounds, represent unique tools that could be used in future studies to probe the presence of active endogenous rexinoids in tissues/organs and to investigate their role in vivo. Last, we provide data suggesting a possible involvement of fatty acids in the weak interaction of RXRs with corepressors.

The genomic regulatory elements for estrogen receptor alpha transactivation-function-1 regulated genes.

Estrogen receptor alpha (ERα) is a ligand-dependent transcription regulator, containing two transactivation functional domains, AF-1 and AF-2. The selective estrogen receptor modulators (SERMs), including 4-hydroxytamoxifen (4OHT), activate AF-1 preferentially rather than AF-2. However, it is unclear whether this specific function is related to the tissue-selective functionality of SERMs. Moreover, there is no information determining AF-1-dependent estrogenic-genes existing in tissues. We sought to identify AF-1-dependent estrogenic-genes using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an AF-2 disrupted estradiol (E2)-insensitive mutant ERα, but AF-1-dependent transcription can be activated by the estrogen-antagonists, fulvestrant (ICI) and 4OHT. Gene profiling and ChIP-Seq analysis identified Klk1b21 as an ICI-inducible gene in AF2ERKI uterus. The regulatory activity was analyzed further using a cell-based reporter assay. The 5'-flanking 0.4kbp region of Klk1b21 gene responded as an ERα AF-1-dependent estrogen-responsive promoter. The 150bp minimum ERα binding element (EBE) consists of three direct repeats. These three half-site sequences were essential for the ERα-dependent transactivation and were differentially recognized by E2 and 4OHT for the gene activation. This response was impaired when the minimum EBE was fused with a thymidine-kinase promoter but could be restored by fusion with the 100bp minimum transcription initiation element (TIE) of Klk1b21, suggesting that the cooperative function of EBE and TIE is essential for mediating AF-1-dependent transactivation. These findings provide the first in vivo evidence that endogenous ERα AF-1 dominant estrogenic-genes exist in estrogen-responsive organs. Such findings will aid in understanding the mechanism of ERα-dependent tissue-selective activity of SERMs.

The science of steroids

Steroids are complex lipophilic molecules that have many actions in the body to regulate cellular, tissue and organ functions across the life-span. Steroid hormones such as cortisol, aldosterone, estradiol and testosterone are synthesised from cholesterol in specialised endocrine cells in the adrenal gland, ovary and testis, and released into the circulation when required. Steroid hormones move freely into cells to activate intracellular nuclear receptors that function as multi-domain ligand-dependent transcriptional regulators in the cell nucleus. Activated nuclear receptors modify expression of hundreds to thousands of specific target genes in the genome. Steroid hormone actions in the fetus include developmental roles in the respiratory system, brain, and cardiovascular system. The synthetic glucocorticoid steroid betamethasone is used antenatally to reduce the complications of preterm birth. Development of novel selective partial glucocorticoid receptor agonists may provide improved therapies to treat the respiratory complications of preterm birth and spare the deleterious effects of postnatal glucocorticoids in other organs.

SUN-013 Estrogen Receptor Alpha Transactivation Function 1 Dependent Uterotropic Estrogenic Genes

Estrogen receptor alpha (ERα) is a ligand dependent transcription regulator, which contains two transactivation domains, AF-1 and AF-2. The activities of these domains are differentially regulated by various estrogenic signals. The chemicals which control the activities of ERα AF-1 and AF-2 in a discriminatory manner in target tissues are named Selective Estrogen Receptor Modulators (SERMs). However, there is very little information on AF-1 or AF-2 predominantly regulated genes within tissues. Therefore, we sought out to determine which estrogenic genes are regulated by AF-1 using the AF-2 mutated knock-in (KI) mouse model, AF2ERKI. AF2ER is an estrogen-insensitive mutant ERα but unique to this mutant, AF-1 can be activated by estrogen-antagonists, such as fulvestrant (ICI) and 4-hydroxytamoxifen, in vitro and in vivo. Uterine microarray analysis revealed that the several genes showed higher fold activation level in the ICI-treated AF2ERKI compared to the estradiol (E2)-treated WT. The genes belong to such category were considered as potential AF-1 regulated genes. We analyzed the promotor activity of estrogen responsive uterine genes using an in vitro cell based luciferase reporter assay using the HepG2 cells. In order to define the promoter functionality, we selected Klk1b21, which fold activation level was higher in the ICI-treated AF2ERKI than E2-treated WT uterus, and Mad2l1, which fold activation level was higher in the E2-treated WT than ICI-treated AF2ERKI uterus. We generated the luciferase reporter plasmids which contain 1k bp 5’-flanking sequence of Klk1b21 or the 1k bp 5’-flanking sequence of Mad2l1. The promoter activity of Klk1b21 was enhanced by AF2ER with ICI and WT-ERα with E2 at the same level. On the other hand, Mad2l1 promoter was activated by WT-ERα with E2 but not AF2ER with ICI. These results suggest that Klk1b21 gene is likely to be regulated by AF-1 predominantly. Further analyses revealed that an 80 bp element of Klk1b21 gene, containing a transcription initiator element (Inr) and a TATA-like sequence contributes to the AF-1 preferential activity of the gene, rather than the sequence of the ERα binding element. These results suggest that the involvement of ERα AF-1 and AF-2 varies in the regulation of each estrogen responsive gene. The AF2ERKI mouse is a useful model to define the AF-1 predominantly regulated genes in the estrogen target tissues. The information would enhance the understanding of the mechanism of selective functionality of SERMs in target organs.

Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay.

Estrogen receptor alpha (ERα) is an estrogenic ligand-dependent transcription regulator. The sequence of ERα protein is highly conserved among species. It has been thought that the function of human and mouse ERαs is identical. We demonstrate the differential 4-hydroxy-tamoxifen (4OHT) effect on mouse and human ERα ligand-binding domain (LBD) dimerization activity using the mammalian two-hybrid (M2H) assay. The M2H assay can demonstrate the efficiency of LBD homodimerization activity in mammalian cells, utilizing the transfection of two protein expression plasmids (GAL4 DNA-binding domain [DBD] fusion LBD and VP16 transactivation domain [VP16AD] fusion LBD) and a GAL4-responsive element (GAL4RE) fused luciferase reporter plasmid. When the GAL4DBD fusion LBD and the VP16AD fusion LBD make a dimer in the cells, this protein complex binds to the GAL4RE and, then, activates a luciferase gene expression through the VP16AD dependent transcription activity. The 4OHT-mediated luciferase activation is higher in the HepG2 cells that were transfected with the mouse ERα LBD fusion protein expression plasmids than in the human ERα LBD fusion protein expression plasmid transfected cells. This result suggests that the efficacy of the 4OHT-dependent mouse ERα LBD homodimerization activity is higher than human ERα LBD. In general, the utilization of the M2H assay is not ideal for the evaluation of nuclear receptor LBD dimerization activity, because agonistic ligands enhance the basal level of the LBD activity and that impedes the detection of LBD dimerization activity. We found that 4OHT does not enhance ERα LBD basal activity. That is a key factor for being able to determine and detect the 4OHT-dependent LBD dimerization activity for successfully using the M2H assay. ERα LBD-based M2H assays may be applied to study the partial agonist activity of selective estrogen receptor modulators (e.g., 4OHT) in various mammalian cell types.

N-terminal transactivation function, AF-1, of estrogen receptor alpha controls obesity through enhancement of energy expenditure

ObjectiveStudies using the estrogen receptor alpha (ERα) knock-out (αERKO) mice have demonstrated that ERα plays a crucial role in various estrogen-mediated metabolic regulations. ERα is a ligand dependent transcription regulator and its activity is regulated by estrogenic compounds. ERα consists of two transcriptional activation domains, AF-1 and AF-2. The activities of these domains are regulated through different mechanisms; however, the specific physiological role in metabolic regulation by these domains is still unclear. MethodsWe utilized an ERα AF-2 mutant knock-in mouse (AF2ERKI) to evaluate the physiological functionality of ERα transactivation domains. Due to the estrogen insensitive AF-2 mutation, the phenotypes of AF2ERKI mice are seemingly identical to the global αERKO including obesity in the females. Distinct from the αERKO, the AF-1 function of AF2ERKI mice can be activated by tamoxifen (Tam). Ovariectomized (OVX) AF2ERKI and WT females were treated with Tam and fed a high-fat diet (HFD) for 10 weeks. Additionally, indirect calorimetric analysis was performed using metabolic chambers with food intake and locomotor activity recorded for Tam-treated AF2ERKI and αERKO females. ResultsObesity in HFD-fed AF2ERKI females was prevented by Tam treatment; particularly, inguinal fat accumulation was strongly blocked by Tam treatment. Alterations in fat metabolism genes, however, were not found in either inguinal fat nor visceral fat to be Tam-regulated, even though fat accumulation was strongly reduced by Tam treatment. Indirect calorimetric analysis revealed that without alteration of food intake and locomotor activity Tam treatment increased energy expenditure in AF2ERKI but not αERKO females. ConclusionsThese results suggest that the activation of ERα AF-1 prevents fat accumulation. The prevention of obesity through AF-1 is mediated by induction of energy expenditure rather than ERα AF-1 functionality of lipid metabolism gene regulation in fat tissues.

Structural basis for specific ligation of the peroxisome proliferator-activated receptor δ

The peroxisome proliferator-activated receptor (PPAR) family comprises three subtypes: PPARα, PPARγ, and PPARδ. PPARδ transcriptionally modulates lipid metabolism and the control of energy homeostasis; therefore, PPARδ agonists are promising agents for treating a variety of metabolic disorders. In the present study, we develop a panel of rationally designed PPARδ agonists. The modular motif affords efficient syntheses using building blocks optimized for interactions with subtype-specific residues in the PPARδ ligand-binding domain (LBD). A combination of atomic-resolution protein X-ray crystallographic structures, ligand-dependent LBD stabilization assays, and cell-based transactivation measurements delineate structure-activity relationships (SARs) for PPARδ-selective targeting and structural modulation. We identify key ligand-induced conformational transitions of a conserved tryptophan side chain in the LBD that trigger reorganization of the H2'-H3 surface segment of PPARδ. The subtype-specific conservation of H2'-H3 sequences suggests that this architectural remodeling constitutes a previously unrecognized conformational switch accompanying ligand-dependent PPARδ transcriptional regulation.

Phosphorylation Alters Oestrogen Receptor β-Mediated Transcription in Neurones.

Nuclear steroid hormone receptors are ubiquitously expressed transcription factors whose activity can be altered by post-translational modifications, such as phosphorylation. The consequences of post-translational modifications have been described for several members of the nuclear steroid hormone receptor superfamily; however, little is known about the effects of oestrogen receptor (ER)β phosphorylation in the brain. Moreover, to our knowledge, the presence of phosphorylated ERβ has not been detected in the brain of any species to date. Oestrogen receptor β is highly expressed in several regions of the brain and invitro studies have demonstrated that it can be phosphorylated at two serine residues (S87 and S105) in the N-terminal AF-1 region. The present study aimed to determine whether phosphorylated ERβ is detectable in the hippocampus of aged female rats, as well as the functional consequences of ERβ S87 and S105 phosphorylation on transcriptional activity in neuronal cells. First, we used a novel PhosTag(™) approach to detect phosphorylated forms of ERβ in the dorsal hippocampus of aged female rats. The data obtained demonstrated abundant forms of phosphorylated ERβ in the dorsal hippocampus, suggesting that this post-translational modification might be an important regulator of ERβ function. To assess the functional consequences of ERβ phosphorylation in neuronal cells, we created phospho-mimetic (S87E, S105E) and phospho-null (S87A, S105A) ERβ receptors that were transiently transfected in a hippocampal-derived cell line. Collectively, our results showed that phosphorylation of S87 and S105 altered both ligand-independent and ligand-dependent ERβ transcriptional regulation. Overall, these data demonstrate that phosphorylated forms of ERβ are present in the brain of aged female rats and that phosphorylation of ERβ could differentially alter ERβ-mediated gene expression.

Oridonin Upregulates All-Trans Retinoic Acid Receptor Alpha and Induces Differentiation Of NB4 Cells Though NF-Kb Pathway

Nuclear retinoic acid receptors (RARs) function as ligand-dependent transcriptional regulators, often heterodimerized with retinoid X receptors. They play critical roles in a variety of biological processes, including development, differentiation and homeostasis. However, it is still not very clear about the mechanism of RARα catabolism. Almost all cases of acute promyelocytic leukemia (APL) express oncogenic fusion protein involving the RARα due to chromosome translocations. Despite the great success of ATRA therapy, it is still important to search for a variety of promising compounds to enhance the pharmacologic activity of ATRA and alleviate its adverse effects. And some of these compounds may be useful as a probe to investigate the mechanism of RARα catabolism.Our previous studies showed that oridonin (Ori), a diterpenoid isolated from Rabdosia rubescens, could have an ability to accumulate RARα, enhance ATRA-induced differentiation in ATRA-sensitive as well as -resistant APL cells, and induce apoptosis which is initiated by reactive oxygen species (ROS) production. Based on those discoveries, we supposed that inhibition of RARα degradation may be responsible for these serial actions. Herein, we used Ori as a probe to explore how Ori stabilized RARα and its potent mechanisms. Firstly, our results showed that Ori upregulated RARa in a dose- and time- dependent manner in NB4 cells while anti-oxidant agent N-acetyl-L-cysteine (NAC) addition completely blocked this effect, indicating that RARa was stabilized via Ori-induced intracellular ROS formation. Secondly, we found H2O2, which could increase ROS level also, upregulated RARa in a dose- and time- dependent manner. More of interest, we found that both Ori and H2O2 upregulated RARa through NF-kB-signaling, proved by the accumulate evidence that either NF-κB pathway inhibitor or knockdown of P65 or inhibition IkBa phosphorylation could block the RARa stabilization. Whereas, P65 overexpression or knockdown of IkBa or some NF-κB pathway activating agents like tumor necrosis factor alpha (TNFa) and lipopolysaccharide ( LPS) could increase the RARa level.Collectively, these findings suggested that Ori is an extraordinary tool to enhance the pathophysiological and potential therapeutic roles of RARα expression though NF-kB pathway. Moreover, an approach combining ATRA with Ori warrants further investigation as a therapeutic strategy for APL patients. Disclosures:No relevant conflicts of interest to declare.

Association of Retinoic Acid Receptors with Extracellular Matrix Accumulation in Rats with Renal Interstitial Fibrosis Disease

The nuclear retinoic acid receptors (RARs) function as ligand-dependent transcriptional regulators and include three subtypes (RARα, RARβ and RARγ), which control the expression of specific gene subsets subsequent to ligand binding and to strictly controlled phosphorylation processes. Extracellular matrix (ECM) accumulation is the most important characteristic of renal interstitial fibrosis (RIF). This study was performed to investigate whether RARs were associated with ECM accumulation in the progression of RIF in rats. Eighty Wistar male rats were divided into a sham operation group (SHO) and a model group subjected to unilateral ureteral obstruction (GU) at random; n = 40, respectively. The RIF disease in GU group was established by left ureteral ligation. The renal tissues were collected at two weeks and four weeks after surgery. Protein expressions of RARα, RARβ, RARγ, transforming growth factor-βl (TGF-β1), collagen-IV (Col-IV) and fibronectin (FN) were detected using immunohistochemical analysis, and mRNA expressions of RARα, RARβ, RARγ and TGF-β1 in renal tissue were detected by real time reverse transcription polymerase chain reaction. RIF index in renal interstitium was also calculated. When compared with those in SHO group, expressions of RARα and RARβ (protein and mRNA) were markedly reduced in the GU group (each p < 0.01). There was no marked difference for the expression of RARγ (protein and mRNA) between the SHO group and the GU group. The expressions of TGF-β1, Col-IV, FN and the RIF index in the GU group were markedly increased when compared with those in the SHO group (each p < 0.01). The protein expression of RARα/RARβ was negatively correlated with protein expression of TGF-β1, Col-IV or FN and the RIF index (all p < 0.01). In conclusion, the low expression of RARα/RARβ is associated with ECM accumulation in the progression of RIF in rats, suggesting that RARα/RARβ is a potentially therapeutic target for prevention of RIF.

1α,25(OH)2-3-Epi-Vitamin D3, a Natural Physiological Metabolite of Vitamin D3: Its Synthesis, Biological Activity and Crystal Structure with Its Receptor

BackgroundThe 1α,25-dihydroxy-3-epi-vitamin-D3 (1α,25(OH)2-3-epi-D3), a natural metabolite of the seco-steroid vitamin D3, exerts its biological activity through binding to its cognate vitamin D nuclear receptor (VDR), a ligand dependent transcription regulator. In vivo action of 1α,25(OH)2-3-epi-D3 is tissue-specific and exhibits lowest calcemic effect compared to that induced by 1α,25(OH)2D3. To further unveil the structural mechanism and structure-activity relationships of 1α,25(OH)2-3-epi-D3 and its receptor complex, we characterized some of its in vitro biological properties and solved its crystal structure complexed with human VDR ligand-binding domain (LBD).Methodology/Principal FindingsIn the present study, we report the more effective synthesis with fewer steps that provides higher yield of the 3-epimer of the 1α,25(OH)2D3. We solved the crystal structure of its complex with the human VDR-LBD and found that this natural metabolite displays specific adaptation of the ligand-binding pocket, as the 3-epimer maintains the number of hydrogen bonds by an alternative water-mediated interaction to compensate the abolished interaction with Ser278. In addition, the biological activity of the 1α,25(OH)2-3-epi-D3 in primary human keratinocytes and biochemical properties are comparable to 1α,25(OH)2D3.Conclusions/SignificanceThe physiological role of this pathway as the specific biological action of the 3-epimer remains unclear. However, its high metabolic stability together with its significant biologic activity makes this natural metabolite an interesting ligand for clinical applications. Our new findings contribute to a better understanding at molecular level how natural metabolites of 1α,25(OH)2D3 lead to significant activity in biological systems and we conclude that the C3-epimerization pathway produces an active metabolite with similar biochemical and biological properties to those of the 1α,25(OH)2D3.

RETRACTED ARTICLE: Transcriptional Controls by Nuclear Fat-Soluble Vitamin Receptors through Chromatin Reorganization

Fat-soluble ligands like vitamin A/D and steroid hormones activate their cognate nuclear receptors for ligand-dependent transcriptional regulation. Nuclear receptors constitute a gene superfamily with 48 members in higher mammals, and act as ligand-dependent transcription factors to bind stably to specific DNA elements in ligand/NR target gene promoters. Hence, most of biological actions of fat-soluble ligands are generally thought to mediate NR-mediated gene regulation. Starting in early 1990, transcriptional co-regulators supporting ligand-dependent transcriptional controls by NRs have been characterized. Initially, the transcriptional co-regulators were believed to couple with histone acethylation/deacethylation, and thereby histone acethyltransferases (HATs) and histone deacethylases (HDACs) were characterized as NR co-activators and co-repressor, respectively. However, recent progress in chromatin biology and epigenome have revealed that other histone modifying enzymes and chromatin remodelers are potential co-regulators for NRs. In this review, these cuttingedge aspects are discussed together with our recent findings on NR co-regulators.

Evolution of Nuclear Retinoic Acid Receptor Alpha (RAR ) Phosphorylation Sites. Serine Gain Provides Fine-Tuned Regulation

The human nuclear retinoic acid (RA) receptor alpha (hRARα) is a ligand-dependent transcriptional regulator, which is controlled by a phosphorylation cascade. The cascade starts with the RA-induced phosphorylation of a serine residue located in the ligand-binding domain, S(LBD), allowing the recruitment of the cdk7/cyclin H/MAT1 subcomplex of TFIIH through the docking of cyclin H. It ends by the subsequent phosphorylation by cdk7 of an other serine located in the N-terminal domain, S(NTD). Here, we show that this cascade relies on an increase in the flexibility of the domain involved in cyclin H binding, subsequently to the phosphorylation of S(LBD). Owing to the functional importance of RARα in several vertebrate species, we investigated whether the phosphorylation cascade was conserved in zebrafish (Danio rerio), which expresses two RARα genes: RARα-A and RARα-B. We found that in zebrafish RARαs, S(LBD) is absent, whereas S(NTD) is conserved and phosphorylated. Therefore, we analyzed the pattern of conservation of the phosphorylation sites and traced back their evolution. We found that S(LBD) is most often absent outside mammalian RARα and appears late during vertebrate evolution. In contrast, S(NTD) is conserved, indicating that the phosphorylation of this functional site has been under ancient high selection constraint. This suggests that, during evolution, different regulatory circuits control RARα activity.

Aurora Kinase B Activity Is Modulated by Thyroid Hormone during Transcriptional Activation of Pituitary Genes

Covalent histone modifications clearly play an essential role in ligand-dependent transcriptional regulation by nuclear receptors. One of the predominant mechanisms used by nuclear receptors to activate or repress target-gene transcription is the recruitment of coregulatory factors capable of covalently modify the amino terminal ends of histones. Here we show that the thyroid hormone (T3) produces a rapid increase in histone H3Ser10 phosphorylation (H3Ser10ph) concomitant to the rapid displacement of the heterochromatin protein 1β (HP1β) to the nuclear periphery. Moreover, we found that T3-mediated pituitary gene transcription is associated with an increase in H3Ser10ph. Interestingly, the Aurora kinase B inhibitor ZM443979 abolishes the effect of T3 on H3Ser10ph, blocks HP1β delocalization, and significantly reduces ligand-dependent transactivation. Similar effects were shown when Aurora kinase B expression was abrogated in small interfering RNA assays. In an effort to understand the underlying mechanism by which T3 increases H3Ser10ph, we demonstrate that liganded thyroid hormone receptor directly interacts with Aurora kinase B, increasing its kinase activity. Moreover, using chromatin immunoprecipitation assays, we have shown that Aurora kinase B participates of a mechanism that displaces HP1β from promoter region, thus preparing the chromatin for the transcriptional activation of T3 regulated genes. Our findings reveal a novel role for Aurora kinase B during transcriptional initiation in GO/G1, apart from its well-known mitotic activity.

Differential Requirements of Hsp90 and DNA for the Formation of Estrogen Receptor Homodimers and Heterodimers

The two estrogen receptor (ER) subforms, ERalpha and ERbeta, are capable of forming DNA-binding homodimers and heterodimers. Although binding to DNA is thought to stabilize ER dimers, how ERalpha/alpha, ERbeta/beta, and ERalpha/beta dimerization is regulated by DNA and the chaperone protein Hsp90 is poorly understood. Using our highly optimized bioluminescence resonance energy transfer assays in conjunction with assays for transcriptional activation of ERs, we determined that DNA binding appears to play a minor role in the stabilization of ER dimers, especially in the case of ERbeta/beta homodimers. These findings suggest that ER dimers form before they associate with chromatin and that DNA binding plays a minor role in stabilizing ER dimers. Additionally, although Hsp90 is essential for the proper dimerization of ERalpha/alpha and ERalpha/beta, it is not required for the proper dimerization of ERbeta/beta. Despite this, Hsp90 is critical for the estrogen-dependent transcriptional activity of the ERbeta/beta homodimer. Thus, Hsp90 is implicated as an important regulator of distinct aspects of ERalpha and ERbeta action.

Structure-Based Design of a Superagonist Ligand for the Vitamin D Nuclear Receptor

Vitamin D nuclear receptor (VDR), a ligand-dependent transcriptional regulator, is an important target for multiple clinical applications, such as osteoporosis and cancer. Since exacerbated increase of calcium serum level is currently associated with VDR ligands action, superagonists with low calcium serum levels have been developed. Based on the crystal structures of human VDR (hVDR) bound to 1alpha,25-dihydroxyvitamin D(3) and superagonists-notably, KH1060-we designed a superagonist ligand. In order to optimize the aliphatic side chain conformation with a subsequent entropy benefit, we incorporated an oxolane ring and generated two stereo diasteromers, AMCR277A and AMCR277B. Only AMCR277A exhibits superagonist activity in vitro, but is as calcemic in vivo as the natural ligand. The crystal structures of the complexes between the ligand binding domain of hVDR and these ligands provide a rational approach to the design of more potent superagonist ligands for potential clinical application.

Selective Glucocorticoid Receptor Ligands

Glucocorticoids are four-ring steroid compounds that regulate a wide range of physiological systems ranging from embryonic respiratory development, immune function and responses to acute or chronic stress. Glucocorticoids are taken up by many target cells where they bind and activate cytoplasmic glucocorticoid receptors (GRs), which then dimerize, translocate to the nucleus and function as ligand-dependent transcriptional regulators. Synthetic glucocorticoids such as dexamethasone and prednisolone have for decades been the cornerstone for the clinical treatment of inflammatory diseases, such as rheumatoid arthritis and asthma, yet prolonged use have undesirable side-effects such as persistent immune suppression, metabolic imbalance, obesity, diabetes, and osteoporosis. Detailed understanding of the cell signaling mechanisms of GR action has led to the development of novel selective glucocorticoid receptor ligands that appear to offer more efficient treatments for a number of diseases while eliciting fewer side-effects. Additionally, in cell-based and animal model systems a number of compounds such as the methane sulphonamides and a novel compound A-348441 have shown promise as GR antagonists. Other classes of ligands such as the benzopyranoquinolines and the arylpyrazoles have further been shown to selectively influence the transcriptional regulatory properties of GRs on different target gene in various cellular contexts. These selective GR modulators are believed to initiate transcriptional co-regulator recruitment that in turn promotes specific gene responses relevant to the more efficient and specific treatment of inflammatory conditions and metabolic diseases such as type-2 diabetes.