The retinoid X receptor has a critical role in synthetic rexinoid-induced increase in cellular all-trans-retinoic acid.

Rexinoids are agonists of nuclear rexinoid X receptors (RXR) that heterodimerize with other nuclear receptors to regulate gene transcription. A number of selective RXR agonists have been developed for clinical use but their application has been hampered by the unwanted side effects associated with the use of rexinoids and a limited understanding of their mechanisms of action across different cell types. Our previous studies showed that treatment of organotypic human epidermis with the low toxicity UAB30 and UAB110 rexinoids resulted in increased steady-state levels of all-trans-retinoic acid (ATRA), the obligatory ligand of the RXR-RAR heterodimers. Here, we investigated the molecular mechanism underlying the increase in ATRA levels using a dominant negative RXRα that lacks the activation function 2 (AF-2) domain. The results demonstrated that overexpression of dnRXRα in human organotypic epidermis markedly reduced signaling by resident ATRA, suggesting the existence of endogenous RXR ligand, diminished the biological effects of UAB30 and UAB110 on epidermis morphology and gene expression, and nearly abolished the rexinoid-induced increase in ATRA levels. Global transcriptome analysis of dnRXRα-rafts in comparison to empty vector-transduced rafts showed that over 95% of the differentially expressed genes in rexinoid-treated rafts constitute direct or indirect ATRA-regulated genes. Thus, the biological effects of UAB30 and UAB110 are mediated through the AF-2 domain of RXRα with minimal side effects in human epidermis. As ATRA levels are known to be reduced in certain epithelial pathologies, treatment with UAB30 and UAB110 may represent a promising therapy for normalizing the endogenous ATRA concentration and signaling in epithelial tissues.

Molecular interplay between ecdysone receptor and retinoid X receptor in regulating the molting of the Chinese mitten crab, Eriocheir sinensis.

Purpose: Molting is a pivotal biological process regulated by the ecdysteroid signaling pathway that requires molecular coordination of two transcription factors, Ecdysone receptor (EcR) and ultraspiracle (USP) in arthropods. However, the molecular interplay of EcR and Retinoid X receptor (RXR), the crustacean homolog of USP in the ecdysteroid signaling pathway, is not well understood. Methods: In this study, we conducted temporal and spatial expression, co-immunoprecipitation (CO-IP), and luciferase reporter assay experiments to investigate the molecular function and interplay of EcR and RXR during the molting process of the Chinese mitten crab, Eriocheir sinensis. Results: The results showed that the expression level of RXR was more stable and significantly higher than EcR during the entire molting process. However, the expression level of EcR fluctuated dynamically and increased sharply at the premolt stage. The CO-IP and luciferase reporter assay results confirmed the molecular interplay of EcR and RXR. The heterodimer complex formed by the two transcription factors significantly induced the transcription of E75, an essential gene in the ecdysteroid signaling pathway. Conclusions: Our study unveiled the diverse molecular function and molecular interplay of EcR and RXR; RXR is possibly a "constitutive-type" gene, and EcR is possibly a vital speed-limiting gene while both EcR and RXR are required to initiate the ecdysteroid signaling cascade, which may be indispensable for molting regulation in E. sinensis. The results provide a theoretical basis for the endocrine control of molting in E. sinensis and novel insights into the molecular mechanism of molting mediated by the ecdysteroid signaling pathway in crustaceans.

Development of a yeast reporter gene assay to detect ligands of freshwater cladoceran Daphnia magna ultraspiracle, a homolog of vertebrate retinoid X receptors.

Endocrine-disrupting chemicals (EDCs) often affect homeostatic regulation in living organisms by directly acting on nuclear receptors (NRs). Retinoid X receptors (RXRs), the most highly conserved members of the NR superfamily during evolution, function as partners to form heterodimers with other NRs, such as retinoic acid, thyroid hormone, and vitamin D3 receptors. RXRs also homodimerize and induce the expression of target genes upon binding with their natural ligand, 9-cis-retinoic acid (9cRA), and typical EDCs organotin compounds, such as tributyltin and triphenyltin. In the present study, we established a new yeast reporter gene assay (RGA) to detect the ligands of freshwater cladoceran Daphnia magna ultraspiracle (Dapma-USP), a homolog of vertebrate RXRs. D. magna has been used as a representative crustacean species for aquatic EDC assessments in the Organization for Economic Corporation and Development test guidelines. Dapma-USP was expressed along with the Drosophila melanogaster steroid receptor coactivator Taiman in yeast cells carrying the lacZ reporter plasmid. The RGA for detecting agonist activity of organotins and o-butylphenol was improved by use of mutant yeast strains lacking genes encoding cell wall mannoproteins and/or plasma membrane drug efflux pumps as hosts. We also showed that a number of other human RXR ligands, phenol and bisphenol A derivatives, and terpenoid compounds such as 9c-RA exhibited antagonist activity on Dapma-USP. Our newly established yeast-based RGA system is valuable as the first screening tool to detect ligand substances for Dapma-USP and for evaluating the evolutionary divergence of the ligand responses of RXR homologs between humans and D. magna.

FOXA1 Reprogramming Dictates Retinoid X Receptor Response in ESR1-Mutant Breast Cancer.

Estrogen receptor alpha (ER/ESR1) mutations occur in 30% to 40% of endocrine resistant ER-positive (ER+) breast cancer. Forkhead box A1 (FOXA1) is a key pioneer factor mediating ER-chromatin interactions and endocrine response in ER+ breast cancer, but its role in ESR1-mutant breast cancer remains unclear. Our previous FOXA1 chromatin immunoprecipitation sequencing (ChIP-seq) identified a large portion of redistributed binding sites in T47D genome-edited Y537S and D538G ESR1-mutant cells. Here, we further integrated FOXA1 genomic binding profile with the isogenic ER cistrome, accessible genome, and transcriptome data of T47D cell model. FOXA1 redistribution was significantly associated with transcriptomic alterations caused by ESR1 mutations. Furthermore, in ESR1-mutant cells, FOXA1-binding sites less frequently overlapped with ER, and differential gene expression was less associated with the canonical FOXA1-ER axis. Motif analysis revealed a unique enrichment of retinoid X receptor (RXR) motifs in FOXA1-binding sites of ESR1-mutant cells. Consistently, ESR1-mutant cells were more sensitive to growth stimulation with the RXR agonist LG268. The mutant-specific response was dependent on two RXR isoforms, RXR-α and RXR-ß, with a stronger dependency on the latter. In addition, T3, the agonist of thyroid receptor (TR) also showed a similar growth-promoting effect in ESR1-mutant cells. Importantly, RXR antagonist HX531 blocked growth of ESR1-mutant cells and a patient-derived xenograft (PDX)-derived organoid with an ESR1 D538G mutation. Collectively, our data support the evidence for a stronger RXR response associated with FOXA1 reprograming in ESR1-mutant cells, suggesting development of therapeutic strategies targeting RXR pathways in breast tumors with ESR1 mutation. IMPLICATIONS: It provides comprehensive characterization of the role of FOXA1 in ESR1-mutant breast cancer and potential therapeutic strategy through blocking RXR activation.

Rxrs and their partner receptor genes inducing masculinization plausibly mediated by endocrine disruption in Paralichthys olivaceus.

Retinoid X receptors (RXRs) can form homo- or heterodimers with orphan receptors involved in multiple intertwined signaling pathways. However, there is limited study on the formation of sex phenotypes and the regulation of steroidogenesis by RXRs in fish. Here, in Paralichthys olivaceus, we first indicated that PPARγ::RXRα was predictably a transcription factor for steroidogenesis genes, and Foxl2 and Dmrt1 were also transcription factors for rxrs and their partner receptor genes. When the flounder fry were exposed to LG100268 (LG, RXRs agonist, 50 mg/kg diet), the percentage of males increased from 50% to 71.4%. Before histological differentiation of the flounder ovary (3 cm TL) and testis (6 cm TL), the transcripts of rar ß and rar γ (P < 0.05) were activated, and the steroidogenesis gene Hsd3b1 was down-regulated (P < 0.05). The ratios of testosterone (T)/17ß-estradiol (E2) were all greatly increased (P < 0.05), and the ratio of 11-ketotestosterone (11-KT)/E2 was elevated at 3 cm TL. Moreover, LG was used to treat the cultured gonads in vitro (10 µM) and the fish with intraperitoneal injection in vivo (12 mg/kg body weight), respectively. LG was able to up-regulate rxr γ, rar γ, and ppar δ, and Hsd3b1 was significantly up-regulated (P < 0.05). The ratios of 11-KT/E2 in the culture medium and in the ovaries of the fish were decreased. Furthermore, the recombinant flounder Foxl2 protein was able to significantly down-regulate ppar γ (P < 0.05) and tr ß (P < 0.01) in the ovaries in vitro, and the result of the Dmrt1 in the testes was opposite to that of the Foxl2, probably indicating a feedback loop between RXRs' partner receptors and Foxl2/Dmrt1. These findings introduce for the first time the mode of action of RXRs on the flounder steroidogenesis and provide important data to learn the potential function of RXRs in fish sex differentiation and the potential role of RXRs in aquatic animals in the presence of water pollutants.

A transgenic reporter mouse model for in vivo assessment of retinoic acid receptor transcriptional activation.

Background: Vitamin A is essential for a wide range of life processes throughout embryogenesis to adult life. With the aim of developing an in vivo model to monitor retinoic acid receptor (RAR) transactivation real-time in intact animals, we generated transgenic mice carrying a luciferase (luc) reporter gene under the control of retinoic acid response elements (RAREs) consisting of three copies of a direct repeat with five spacing nucleotides (DR5). Methods: Transgenic mice carrying a RARE dependent luciferase reporter flanked with insulator sequence were generated by pronuclear injection. RARE dependent luciferase activity was detected by in vivo imaging or in tissue extracts following manipulations with RAR/retinoid X receptor (RXR) agonists, RAR antagonists or in vitamin A deficient mice. Results: We found a strong induction of luciferase activity in a time and dose dependent manner by retinoic acid as well as RAR agonists, but not by the RXR agonist (using n=4-6 per group; 94 mice). In addition, luciferase activity was strongly reduced in vitamin A-deficient mice (n=6-9; 30 mice). These observations confirm that luciferase activity was controlled by RAR activation in the RARE-luc mouse. Luciferase activity was detectable in various organs, with high activity especially in brain and testis, indicating strong retinoid signalling in these tissues. Conclusion: The RARE-luc transgenic mice, which enabled real-time in vivo assessment of RAR activation, will be useful in understanding the normal physiology of vitamin A, the role of retinoid signalling in pathologies as well as to evaluate pharmacological ligands for RARs.

Computational refinement identifies functional destructive single nucleotide polymorphisms associated with human retinoid X receptor gene.

Alterations in the nuclear retinoid X receptor (RXRs) signalling have been implicated in neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, stroke, multiple sclerosis and glaucoma. Single nucleotide polymorphisms (SNPs) are the main cause underlying single nucleic acid variations which in turn determine heterogeneity within various populations. These genetic polymorphisms have been suggested to associate with various degenerative disorders in population-wide analysis. This bioinformatics study was designed to investigate, search, retrieve and identify deleterious SNPs which may affect the structure and function of various RXR isoforms through a computational and molecular modelling approach. Amongst the 1,813 retrieved SNPs several were found to be deleterious with rs140464195_G139R, rs368400425_R358W and rs368586400_L383F RXRα mutant variants being the most detrimental ones causing changes in the interatomic interactions and decreasing the flexibility of the mutant proteins. Molecular genetics analysis identified seven missense mutations in RXRα/ß/γ isoforms. Two novel mutations SNP IDs (rs1588299621 and rs1057519958) were identified in RXRα isoform. We used several in silico prediction tools such as SIFT, PolyPhen, I-Mutant, Protein Variation Effect Analyzer (PROVEAN), PANTHER, SNP&Go, PhD-SNP and SNPeffect to predict pathogenicity and protein stability associated with RXR mutations. The structural assessment by DynaMut tool revealed that hydrogen bonds were affected along with hydrophobic and carbonyl interactions resulting in reduced flexibility at the mutated residue positions but ultimately stabilizing the molecule as a whole. Summarizing, analysis of the missense mutations in RXR isoforms showed a mix of conclusive and inconclusive genotype-phenotype correlations suggesting the use of sophisticated computational analysis tools for studying RXR variants.Communicated by Ramaswamy H. Sarma.

PPARγ lipodystrophy mutants reveal intermolecular interactions required for enhancer activation.

Peroxisome proliferator-activated receptor γ (PPARγ) is the master regulator of adipocyte differentiation, and mutations that interfere with its function cause lipodystrophy. PPARγ is a highly modular protein, and structural studies indicate that PPARγ domains engage in several intra- and inter-molecular interactions. How these interactions modulate PPARγ's ability to activate target genes in a cellular context is currently poorly understood. Here we take advantage of two previously uncharacterized lipodystrophy mutations, R212Q and E379K, that are predicted to interfere with the interaction of the hinge of PPARγ with DNA and with the interaction of PPARγ ligand binding domain (LBD) with the DNA-binding domain (DBD) of the retinoid X receptor, respectively. Using biochemical and genome-wide approaches we show that these mutations impair PPARγ function on an overlapping subset of target enhancers. The hinge region-DNA interaction appears mostly important for binding and remodelling of target enhancers in inaccessible chromatin, whereas the PPARγ-LBD:RXR-DBD interface stabilizes the PPARγ:RXR:DNA ternary complex. Our data demonstrate how in-depth analyses of lipodystrophy mutants can unravel molecular mechanisms of PPARγ function.

Regulation of Tyrosinase Gene Expression by Retinoic Acid Pathway in the Pacific Oyster Crassostrea gigas.

Retinoic acid (RA) plays important roles in various biological processes in animals. RA signaling is mediated by two types of nuclear receptors, namely retinoic acid receptor (RAR) and retinoid x receptor (RXR), which regulate gene expression by binding to retinoic acid response elements (RAREs) in the promoters of target genes. Here, we explored the effect of all-trans retinoic acid (ATRA) on the Pacific oyster Crassostera gigas at the transcriptome level. A total of 586 differentially expressed genes (DEGs) were identified in C. gigas upon ATRA treatment, with 309 upregulated and 277 downregulated genes. Bioinformatic analysis revealed that ATRA affects the development, metabolism, reproduction, and immunity of C. gigas. Four tyrosinase genes, including Tyr-6 (LOC105331209), Tyr-9 (LOC105346503), Tyr-20 (LOC105330910), and Tyr-12 (LOC105320007), were upregulated by ATRA according to the transcriptome data and these results were verified by real-time quantitative polymerase chain reaction (RT-qPCR) analysis. In addition, increased expression of Tyr (a melanin-related TYR gene in C. gigas) and Tyr-2 were detected after ATRA treatment. The yeast one-hybrid assay revealed the DNA-binding activity of the RA receptors CgRAR and CgRXR, and the interaction of CgRAR with RARE present in the Tyr-2 promoter. These results provide evidence for the further studies on the role of ATRA and the mechanism of RA receptors in mollusks.

A novel nuclear receptor subfamily enlightens the origin of heterodimerization.

BACKGROUND: Nuclear receptors are transcription factors of central importance in human biology and associated diseases. Much of the knowledge related to their major functions, such as ligand and DNA binding or dimerization, derives from functional studies undertaken in classical model animals. It has become evident, however, that a deeper understanding of these molecular functions requires uncovering how these characteristics originated and diversified during evolution, by looking at more species. In particular, the comprehension of how dimerization evolved from ancestral homodimers to a more sophisticated state of heterodimers has been missing, due to a too narrow phylogenetic sampling. Here, we experimentally and phylogenetically define the evolutionary trajectory of nuclear receptor dimerization by analyzing a novel NR7 subgroup, present in various metazoan groups, including cnidarians, annelids, mollusks, sea urchins, and amphioxus, but lost in vertebrates, arthropods, and nematodes. RESULTS: We focused on NR7 of the cephalochordate amphioxus B. lanceolatum. We present a complementary set of functional, structural, and evolutionary analyses that establish that NR7 lies at a pivotal point in the evolutionary trajectory from homodimerizing to heterodimerizing nuclear receptors. The crystal structure of the NR7 ligand-binding domain suggests that the isolated domain is not capable of dimerizing with the ubiquitous dimerization partner RXR. In contrast, the full-length NR7 dimerizes with RXR in a DNA-dependent manner and acts as a constitutively active receptor. The phylogenetic and sequence analyses position NR7 at a pivotal point, just between the basal class I nuclear receptors that form monomers or homodimers on DNA and the derived class II nuclear receptors that exhibit the classical DNA-independent RXR heterodimers. CONCLUSIONS: Our data suggest that NR7 represents the "missing link" in the transition between class I and class II nuclear receptors and that the DNA independency of heterodimer formation is a feature that was acquired during evolution. Our studies define a novel paradigm of nuclear receptor dimerization that evolved from DNA-dependent to DNA-independent requirements. This new concept emphasizes the importance of DNA in the dimerization of nuclear receptors, such as the glucocorticoid receptor and other members of this pharmacologically important oxosteroid receptor subfamily. Our studies further underline the importance of studying emerging model organisms for supporting cutting-edge research.

Adenosine N6-methylation upregulates the expression of human CYP2B6 by altering the chromatin status.

N6-Methyladenosine (m6A) modification is the most prevalent RNA modification in mammals. We have recently demonstrated that inhibition of m6A modification by 3-deazaadenosine results in an increase in the expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, and CYP2C8 in human liver-derived cells. In the present study, we aimed to clarify the mechanism of m6A-mediated regulation of CYP2B6 expression. RNA immunoprecipitation using an anti-m6A antibody revealed that CYP2B6 mRNA in human liver and hepatocarcinoma-derived HepaRG cells was m6A-modified around the stop codon. In contrast to the treatment with 3-deazaadenosine, double knockdown of methyltransferase like (METTL) 3 and METTL14 (METTL3/14) resulted in a decrease in the levels of CYP2B6 mRNA in Huh-7 and HepaRG cells and a decrease in bupropion hydroxylase activity, a marker activity of CYP2B6, in HepaRG cells. The stability of CYP2B6 mRNA was not influenced by siMETTL3/14. Reporter assays using the plasmids containing the last exon or 5'-flanking region of CYP2B6 indicated that reporter activities were not influenced by knockdown of METTL3/14. The expression levels of the constitutive androstane receptor, pregnane X receptor, and retinoid X receptor, which are the nuclear receptors regulating the transcription of CYP2B6, were not influenced by siMETTL3/14. The chromatin immunoprecipitation and formaldehyde-assisted enrichment of regulatory elements assays revealed that H3K9me2, a repressive histone marker, was enriched in the vicinity of the upstream region of CYP2B6, and knockdown of METTL3/14 induced the condensation of the chromatin structure in this region. In conclusion, we demonstrated that METTL3/14 upregulated CYP2B6 expression by altering the chromatin status.

Population-based meta-analysis and gene-set enrichment identifies FXR/RXR pathway as common to fatty liver disease and serum lipids.

Nonalcoholic fatty liver disease (NAFLD) is prevalent worldwide. NAFLD is associated with elevated serum triglycerides (TG), low-density lipoprotein cholesterol (LDL), and reduced high-density lipoprotein cholesterol (HDL). Both NAFLD and blood lipid levels are genetically influenced and may share a common genetic etiology. We used genome-wide association studies (GWAS)-ranked genes and gene-set enrichment analysis to identify pathways that affect serum lipids and NAFLD. We identified credible genes in these pathways and characterized missense variants in these for effects on serum traits. We used MAGENTA to identify 58 enriched pathways from publicly available TG, LDL, and HDL GWAS (n = 99,000). Three of these pathways were also enriched for associations with European-ancestry NAFLD GWAS (n = 7176). One pathway, farnesoid X receptor (FXR)/retinoid X receptor (RXR) activation, was replicated for association in an African-ancestry NAFLD GWAS (n = 3214) and plays a role in serum lipids and NAFLD. Credible genes (proteins) in FXR/RXR activation include those associated with cholesterol/bile/bilirubin transport/absorption (ABCC2 (MRP2) [ATP binding cassette subfamily C member (multidrug resistance-associated protein 2)], ABCG5, ABCG8 [ATP-binding cassette (ABC) transporters G5 and G8], APOB (APOB) [apolipoprotein B], FABP6 (ILBP) [fatty acid binding protein 6 (ileal lipid-binding protein)], MTTP (MTP) [microsomal triglyceride transfer protein], SLC4A2 (AE2) [solute carrier family 4 member 2 (anion exchange protein 2)]), nuclear hormone-mediated control of metabolism (NR0B2 (SHP) [nuclear receptor subfamily 0 group B member 2 (small heterodimer partner)], NR1H4 (FXR) [nuclear receptor subfamily 1 group H member 4 (FXR)], PPARA (PPAR) [peroxisome proliferator activated receptor alpha], FOXO1 (FOXO1A) [forkhead box O1]), or other pathways (FETUB (FETUB) [fetuin B]). Missense variants in ABCC2 (MRP2), ABCG5 (ABCG5), ABCG8 (ABCG8), APOB (APOB), MTTP (MTP), NR0B2 (SHP), NR1H4 (FXR), and PPARA (PPAR) that associate with serum LDL levels also associate with serum liver function tests in UK Biobank. Conclusion: Genetic variants in NR1H4 (FXR) that protect against liver steatosis increase serum LDL cholesterol while variants in other members of the family have congruent effects on these traits. Human genetic pathway enrichment analysis can help guide therapeutic development by identifying effective targets for NAFLD/serum lipid manipulation while minimizing side effects. In addition, missense variants could be used in companion diagnostics to determine their influence on drug effectiveness.

Molluscan RXR Transcriptional Regulation by Retinoids in a Drosophila CNS Organ Culture System.

Retinoic acid, the active metabolite of Vitamin A, is important for the appropriate development of the nervous system (e.g., neurite outgrowth) as well as for cognition (e.g., memory formation) in the adult brain. We have shown that many of the effects of retinoids are conserved in the CNS of the mollusc, Lymnaea stagnalis. RXRs are predominantly nuclear receptors, but the Lymnaea RXR (LymRXR) exhibits a non-nuclear distribution in the adult CNS, where it is also implicated in non-genomic retinoid functions. As such, we developed a CNS Drosophila organ culture-based system to examine the transcriptional activity and ligand-binding properties of LymRXR, in the context of a live invertebrate nervous system. The novel ligand sensor system was capable of reporting both the expression and transcriptional activity of the sensor. Our results indicate that the LymRXR ligand sensor mediated transcription following activation by both 9-cis RA (the high affinity ligand for vertebrate RXRs) as well as the vertebrate RXR synthetic agonist, SR11237. The LymRXR ligand sensor was also activated by all-trans RA, and to a much lesser extent by the vertebrate RAR synthetic agonist, EC23. This sensor also detected endogenous retinoid-like activity in the CNS of developing Drosophila larvae, primarily during the 3rd instar larval stage. These data indicate that the LymRXR sensor can be utilized not only for characterization of ligand activation for studies related to the Lymnaea CNS, but also for future studies of retinoids and their functions in Drosophila development.

Upregulation of proto-oncogene ski by thyroid hormone in the intestine and tail during Xenopus metamorphosis.

Thyroid hormone (T3) is important for adult organ function and vertebrate development, particularly during the postembryonic period when many organs develop/mature into their adult forms. Amphibian metamorphosis is totally dependent on T3 and can be easily manipulated, thus offering a unique opportunity for studying how T3 controls postembryonic development in vertebrates. Numerous early studies have demonstrated that T3 affects frog metamorphosis through T3 receptor (TR)-mediated regulation of T3 response genes, where TR forms a heterodimer with RXR (9-cis retinoic acid receptor) and binds to T3 response elements (TREs) in T3 response genes to regulate their expression. We have previously identified many candidate direct T3 response genes in Xenopus tropicalis tadpole intestine. Among them is the proto-oncogene Ski, which encodes a nuclear protein with complex function in regulating cell fate. We show here that Ski is upregulated in the intestine and tail of premetamorphic tadpoles upon T3 treatment and its expression peaks at stage 62, the climax of metamorphosis. We have further discovered a putative TRE in the first exon that can bind to TR/RXR in vitro and mediate T3 regulation of the promoter in vivo. These data demonstrate that Ski is activated by T3 through TR binding to a TRE in the first exon during Xenopus tropicalis metamorphosis, implicating a role of Ski in regulating cell fate during metamorphosis.

T Cell Transcriptome in Chromosome 22q11.2 Deletion Syndrome.

Phenotypic variations of chromosome 22q11.2 deletion syndrome (22qDS) have unclear explanations. T cell lymphopenia in 22qDS related to varying degrees of thymic hypoplasia contributes to the phenotypic heterogeneity. No phenotype correlation with genotype or deletion size is known for lymphopenia. We investigated gene expression in human T cells of participants with and without 22qDS and T cells of participants with 22qDS with low or normal T cells. Peripheral blood was collected from participants aged 5-8 y. Immune function was checked. RNA sequencing was completed on isolated T cells, and differential gene expression profiles of T cells between 22qDS and healthy control subjects were established. A total of 360 genes were differentially expressed (q < 0.05) between T cells of patients with 22qDS (n = 13) and healthy control subjects (n = 6) (log2 fold change range, -2.0747, 15.6724). We compared gene expression between participants with 22qDS with low (n = 7) and normal T cell counts (n = 6), finding 94 genes that were differentially expressed (q < 0.05) (log2 fold change range, -4.5445, 5.1297). Twenty-nine genes correlated with T cell counts and markers CD3, CD4, CD8, and CD45RA+CD4 (R ≥ 0.8). We found significantly differentially expressed genes in participants with 22qDS compared with healthy control subjects and in participants with 22qDS with low T cell counts compared with those with normal T cell counts. Several enriched pathways suggest a role of T cells in defective communication between T cells and the innate immune system in 22qDS. Among these, the liver X receptor/retinoid X receptor pathway was noted to show several differentially expressed genes affecting participants with 22qDS compared with healthy control subjects and more so those with low T cell counts than in those with normal T cell counts.

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

Retinoid X receptors (RXRα, ß, 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.

Functional Definition of Thyroid Hormone Response Elements Based on a Synthetic STARR-seq Screen.

When bound to thyroid hormone, the nuclear receptor TRα1 activates the transcription of a number of genes in many cell types. It mainly acts by binding DNA as a heterodimer with retinoid X receptors at specific response elements related to the DR4 consensus sequence. However, the number of DR4-like elements in the genome exceed by far the number of occupied sites, indicating that minor variations in nucleotides composition deeply influence the DNA-binding capacity and transactivation activity of TRα1. An improved protocol of synthetic self-transcribing active regulatory region sequencing was used to quantitatively assess the transcriptional activity of thousands of synthetic sites in parallel. This functional screen highlights a strong correlation between the affinity of the heterodimers for DNA and their capacity to mediate the thyroid hormone response.

A nuclear receptor heterodimer, CgPPAR2-CgRXR, acts as a regulator of carotenoid metabolism in Crassostrea gigas.

Nuclear receptors (NRs) are mostly ligand-activated transcription factors in animals and play essential roles in metabolism and homeostasis. The NR heterodimer composed of PPAR/RXR (peroxisome proliferator-activated receptor/retinoid X receptor) is considered a key regulator of lipid metabolism in vertebrate. However, in molluscs, how this heterodimer is involved in carotenoid metabolism remains unclear. To elucidate how this heterodimer regulates carotenoid metabolism, we identified a PPAR gene in C. gigas, designated as CgPPAR2 (LOC105323212), and functionally characterized it using two-hybrid and reporter systems. CgPPAR2 is a direct orthologue of vertebrate PPARs and the second PPAR gene identified in C. gigas genome in addition to CgPPAR1 (LOC105317849). The results demonstrated that CgPPAR2 protein can form heterodimer with C. gigas RXR (CgRXR), and then regulate carotenoid metabolism by controlling carotenoid cleavage oxygenases with different carotenoid cleavage efficiencies. This regulation can be affected by retinoid ligands, i.e., carotenoid derivatives, validating a negative feedback regulation mechanism of carotenoid cleavage for retinoid production. Besides, organotins may disrupt this regulatory process through the mediation of CgPPAR2/CgRXR heterodimer. This is the first report of PPAR/RXR heterodimer regulating carotenoid metabolism in mollusks, contributing to a better understanding of the evolution and conservation of this nuclear receptor heterodimer.

Retinoid-X receptor agonists increase thyroid hormone competence in lower jaw remodeling of pre-metamorphic Xenopus laevis tadpoles.

Thyroid hormone (TH) signaling plays critical roles during vertebrate development, including regulation of skeletal and cartilage growth. TH acts through its receptors (TRs), nuclear hormone receptors (NRs) that heterodimerize with Retinoid-X receptors (RXRs), to regulate gene expression. A defining difference between NR signaling during development compared to in adult tissues, is competence, the ability of the organism to respond to an endocrine signal. Amphibian metamorphosis, especially in Xenopus laevis, the African clawed frog, is a well-established in vivo model for studying the mechanisms of TH action during development. Previously, we've used one-week post-fertilization X. laevis tadpoles, which are only partially competent to TH, to show that in the tail, which is naturally refractive to exogenous T3 at this stage, RXR agonists increase TH competence, and that RXR antagonism inhibits the TH response. Here, we focused on the jaw that undergoes dramatic TH-mediated remodeling during metamorphosis in order to support new feeding and breathing styles. We used a battery of approaches in one-week-old tadpoles, including quantitative morphology, differential gene expression and whole mount cell proliferation assays, to show that both pharmacologic (bexarotene) and environmental (tributyltin) RXR agonists potentiated TH-induced responses but were inactive in the absence of TH; and the RXR antagonist UVI 3003 inhibited TH action. Bex and TBT significantly potentiated cellular proliferation and the TH induction of runx2, a transcription factor critical for developing cartilage and bone. Prominent targets of RXR-mediated TH potentiation were members of the matrix metalloprotease family, suggesting that RXR potentiation may emphasize pathways responsible for rapid changes during development.

TREM2 is thyroid hormone regulated making the TREM2 pathway druggable with ligands for thyroid hormone receptor.

Triggering receptor expressed on myeloid cells-2 (TREM2) is a cell surface receptor on macrophages and microglia that senses and responds to disease-associated signals to regulate the phenotype of these innate immune cells. The TREM2 signaling pathway has been implicated in a variety of diseases ranging from neurodegeneration in the central nervous system to metabolic disease in the periphery. Here, we report that TREM2 is a thyroid hormone-regulated gene and its expression in macrophages and microglia is stimulated by thyroid hormone and synthetic thyroid hormone agonists (thyromimetics). Our findings report the endocrine regulation of TREM2 by thyroid hormone, and provide a unique opportunity to drug the TREM2 signaling pathway with orally active small-molecule therapeutic agents.