Retinoic Acid Receptor Retinoid X Receptor Research Articles

Identification of a candidate enhancer for DMRT3 involved in spastic cerebral palsy pathogenesis

Cerebral palsy (CP) is a major neuronal disease and the most common movement disorder in children. Although environmental factors leading to CP have been greatly investigated, the genetic mechanism underlying CP is not well understood. Here we focused on two clinical reports that characterized a deletion involving the KANK1 gene locus in the 9p24.3 region. One report shows spastic CP and the other shows no spastic CP phenotype. Based on the epigenetic status and evolutionary conservation, we first found a functional genomic element at the noncoding region that was deleted only in patients with spastic CP. This element contains the retinoic acid receptor/retinoid X receptor (RAR/RXR) complex-binding motif that is widely conserved among placental mammals. RAR/RXR ChIP-seq data from mouse F9 embryonal carcinoma cells that were treated with trans-retinoic acids showed that the element has a binding ability. In addition, data regarding chromosome conformation capture from mouse neural progenitor and ES cells suggested that the element spatially interacts with the Doublesex and mab-3 related transcription factor 3 (Dmrt3) gene promoter that is located approximately 120 kb downstream of the RAR/RXR-binding site. Dmrt3 is detected in the developing mouse forebrain and in some interneurons in the spinal cord, and it works as a locomotion coordinator in horses and mice. Thus, the deletion of the cis-regulatory element for DMRT3 in humans may cause impaired development of the forebrain and gait abnormalities, resulting in spastic CP. In conclusion, this study provides new mechanistic insights into the genetic basis of CP.

Carotenoids, Chronic Disease Prevention and Dietary Recommendations.

Carotenoids are C-30, C-40 or C-50 terpenoids produced by a number of bacteria, fungi, and plants. In addition to acting as vitamin A precursors such as β-carotene, their dietary intake and blood plasma/serum and tissue levels have been associated in several epidemiological studies to the reduced incidence of chronic diseases, including the reduction of type 2 diabetes and other cardiometabolic diseases, as well as some types of cancer. Lutein and zeaxanthin also appear to play a role in the amelioration of age-related macular degeneration (AMD), the main cause of blindness in the elderly, and may be regarded as conditionally essential nutrients for the elderly. Furthermore, some studies have proposed that carotenoids may improve cognitive functions. Though the underlying mechanisms remain to be fully elucidated, it is perceived that direct antioxidant effects and protection from UV-light, as well as rather indirect effects, acting on transcription factors such as NF-κB, Nrf-2, and nuclear receptors such as RAR/RXR (retinoic acid receptor/retinoid X receptor), altering gene expression, all can play a role. Despite individual intervention trials suggesting negative effects of high doses of β-carotene on smokers, perhaps due to effects related to cytochrome enzyme activation, there is accumulating evidence that these colourful pigments indeed contribute to a healthy life and well-being. However, further research is warranted to better understand factors influencing variable inter-individual responses following carotenoid consumption and to establish more detailed recommendations regarding their dietary intake and toward establishing health claims.

Monitoring of the retinoic acid receptor–retinoid X receptor dimerization upon DNA binding by native mass spectrometry

Identifying protein–DNA interactions is essential to understand the regulatory networks of cells and their influence on gene expression. In this study, we use native electrospray mass spectrometry (ESI-MS) to investigate how the heterodimerization of retinoic acid receptor–retinoid X receptor (RAR-RXR) is mediated by DNA sequence. In presence of various RAR response elements (RAREs), three oligomeric states of RAR–RXR DNA binding domains (DBDs) bound to RAREs (monomer, homo- or heterodimers) were detected and individually monitored to follow subunit assembly and disassembly upon RAREs' abundancy or sequence. In particular, a cooperative heterodimerization was shown with RARb2 DR5 (5 base pair spaced direct repeat) while a high heterogeneity reflecting random complex formation could be observed with the DR0 response elements, in agreement with native gel electrophoresis data or molecular modeling. Such MS information will help to identify the composition of species formed in solution and to define which DR sequence is specific for RAR–RXR heterodimerization.

The Corepressor CTBP2 Is a Coactivator of Retinoic Acid Receptor/Retinoid X Receptor in Retinoic Acid Signaling

Retinoids play key roles in development, differentiation, and homeostasis through regulation of specific target genes by the retinoic acid receptor/retinoid X receptor (RAR/RXR) nuclear receptor complex. Corepressors and coactivators contribute to its transcriptional control by creating the appropriate chromatin environment, but the precise composition of these nuclear receptor complexes remains to be elucidated. Using an RNA interference-based genetic screen in mouse F9 cells, we identified the transcriptional corepressor CTBP2 (C-terminal binding protein 2) as a coactivator critically required for retinoic acid (RA)-induced transcription. CTBP2 suppression by RNA interference confers resistance to RA-induced differentiation in diverse murine and human cells. Mechanistically, we find that CTBP2 associates with RAR/RXR at RA target gene promoters and is essential for their transactivation in response to RA. We show that CTBP2 is indispensable to create a chromatin environment conducive for RAR/RXR-mediated transcription by recruiting the histone acetyltransferase p300. Our data reveal an unexpected function of the corepressor CTBP2 as a coactivator for RAR/RXR in RA signaling.

Live-cell fluorescence correlation spectroscopy dissects the role of coregulator exchange and chromatin binding in retinoic acid receptor mobility

The retinoic acid receptor (RAR) is a member of the nuclear receptor superfamily. This ligand-inducible transcription factor binds to DNA as a heterodimer with the retinoid X receptor (RXR) in the nucleus. The nucleus is a dynamic compartment and live-cell imaging techniques make it possible to investigate transcription factor action in real-time. We studied the diffusion of EGFP-RAR by fluorescence correlation spectroscopy (FCS) to uncover the molecular interactions determining receptor mobility. In the absence of ligand, we identified two distinct species with different mobilities. The fast component has a diffusion coefficient of D(1)=1.8-6.0 μm(2)/second corresponding to small oligomeric forms, whereas the slow component with D(2)=0.05-0.10 μm(2)/second corresponds to interactions of RAR with the chromatin or other large structures. The RAR ligand-binding-domain fragment also has a slow component, probably as a result of indirect DNA-binding through RXR, with lower affinity than the intact RAR-RXR complex. Importantly, RAR-agonist treatment shifts the equilibrium towards the slow population of the wild-type receptor, but without significantly changing the mobility of either the fast or the slow population. By using a series of mutant forms of the receptor with altered DNA- or coregulator-binding capacity we found that the slow component is probably related to chromatin binding, and that coregulator exchange, specifically the binding of the coactivator complex, is the main determinant contributing to the redistribution of RAR during ligand activation.

β-Klotho and FGF-15/19 inhibit the apical sodium-dependent bile acid transporter in enterocytes and cholangiocytes

beta-Klotho, a newly described membrane protein, regulates bile acid synthesis. Fibroblast growth factor-15 (FGF-15) and FGF receptor-4 (FGFR4) knockout mice share a similar phenotype with beta-Klotho-deficient mice. FGF-15 secretion by the intestine regulates hepatic bile acid biosynthesis. The effects of beta-Klotho and FGF-15 on the ileal apical sodium bile transporter (ASBT) are unknown. beta-Klotho siRNA treatment of the mouse colon cancer cell line, CT-26, and the human intrahepatic biliary epithelial cells (HIBEC) resulted in upregulation of endogenous ASBT expression that was associated with reduced expression of the farnesoid X receptor (FXR) and the short heterodimer partner (SHP). Silencing beta-Klotho activated the ASBT promoter in CT-26, Mz-ChA-1 (human cholangiocarcinoma), and HIBEC cells. Site-directed mutagenesis of liver receptor homolog-1 (mouse) or retinoic acid receptor/retinoid X receptor (RAR/RXR) (human) cis-elements attenuated the basal activity of the ASBT promoter and abrogated its response to beta-Klotho silencing. siSHP, siFXR, or dominant-negative FXR treatment also eliminated the beta-Klotho response. FGF-15 secretion into cell culture media by CT-26 cells was diminished after siFGF-15 or sibeta-Klotho treatment and enhanced by chenodeoxycholic acid. Exogenous FGF-19 repressed ASBT protein expression in mouse ileum, gallbladder, and in HIBEC and repressed ASBT promoter activity in Caco-2, HIBEC, and Mz-ChA-1 cells. Promoter repression was dependent on the expression of FGFR4. These results indicate that both beta-Klotho and FGF-15/19 repress ASBT in enterocytes and cholangiocytes. These novel signaling pathways need to be considered in analyzing bile acid homeostasis.

Retinoid Regulated Association of Transcriptional Co-regulators and the Polycomb Group Protein SUZ12 with the Retinoic Acid Response Elements of Hoxa1, RARβ2, and Cyp26A1 in F9 Embryonal Carcinoma Cells

Hox gene expression is activated by all- trans retinoic acid (RA), through binding to retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimers bound at RA response elements (RAREs) of target genes. The RARs and RXRs each have three isotypes (α, β, and γ), which are encoded by distinct genes. Hox genes are also repressed by polycomb group proteins (PcG), though how these proteins are targeted is unclear. We used chromatin immunoprecipitation assays to investigate the association of RXRα, RARγ, cofactors, and the PcG protein SUZ12 with the Hoxa1, RARβ2, and Cyp26A1 RAREs in F9 embryonal carcinoma cells (teratocarcinoma stem cells) during RA treatment. We demonstrate that RARγ and RXRα are associated with RAREs prior to and during RA treatment. pCIP, p300, and RNA polymerase II levels increased at target RAREs upon exposure to RA. Conversely, SUZ12 was found associated with all RAREs studied and these associations were attenuated by treatment with RA. Upon RA removal, SUZ12 re-associated with RAREs. H3ac, H3K4me2, and H3K27me3 marks were simultaneously detected at target loci, indicative of a bivalent domain chromatin structure. During RA mediated differentiation, H3K27me3 levels decreased at target RAREs whereas H3ac and H3K4me2 levels remained constant. These studies provide insight into the dynamics of association of co-regulators with RAREs and demonstrate a novel link between RA signaling and PcG repression.

Characterization of the Interaction between Retinoic Acid Receptor/Retinoid X Receptor (RAR/RXR) Heterodimers and Transcriptional Coactivators through Structural and Fluorescence Anisotropy Studies

Retinoid receptors (RARs and RXRs) are ligand-activated transcription factors that regulate the transcription of target genes by recruiting coregulator complexes at cognate promoters. To understand the effects of heterodimerization and ligand binding on coactivator recruitment, we solved the crystal structure of the complex between the RARbeta/RXRalpha ligand-binding domain heterodimer, its 9-cis retinoic acid ligand, and an LXXLL-containing peptide (termed NR box 2) derived from the nuclear receptor interaction domain (NID) of the TRAP220 coactivator. In parallel, we measured the binding affinities of the isolated NR box 2 peptide or the full-length NID of the coactivator SRC-1 for retinoid receptors in the presence of various types of ligands. Our correlative analysis of three-dimensional structures and fluorescence data reveals that heterodimerization does not significantly alter the structure of individual subunits or their intrinsic capacity to interact with NR box 2. Similarly, we show that the ability of a protomer to recruit NR box 2 does not vary as a function of the ligand binding status of the partner receptor. In contrast, the strength of the overall association between the heterodimer and the full-length SRC-1 NID is dictated by the combinatorial action of RAR and RXR ligands, the simultaneous presence of the two receptor agonists being required for highest binding affinity. We identified an LXXLL peptide-driven mechanism by which the concerted reorientation of three phenylalanine side chains generates an "aromatic clamp" that locks the RXR activation helix H12 in the transcriptionally active conformation. Finally, we show how variations of helix H11-ligand interactions can alter the communication pathway linking helices H11, H12, and the connecting loop L11-12 to the coactivator-binding site. Together, our results reveal molecular and structural features that impact on the ligand-dependent interaction of the RAR/RXR heterodimer with nuclear receptor coactivators.

Bile acid-induced negative feedback regulation of the human ileal bile acid transporter.

Ileal expression of the apical sodium-dependent bile acid transporter (ASBT) in the rat is unaffected by bile salts, yet in the mouse it is under negative-feedback regulation. The bile acid responsiveness of human ASBT is unknown. The human ASBT promoter linked to a luciferase reporter was studied in Caco-2 cells treated with chenodeoxycholic acid (CDCA) and transfected with expression plasmids for farnesoid X-receptor (FXR), short heterodimer partner (SHP), and retinoic acid receptor/retinoid X receptor (RAR/RXR). CDCA treatment of Caco-2 cells led to a 75% reduction in steady-state ASBT messenger RNA levels and a 78% reduction in human ASBT promoter activity. A dominant negative FXR abrogated the response to CDCA. Site-directed mutagenesis of an RAR/RXR cis element in the human ASBT promoter reduced its activity by 50% and eliminated the bile acid response. Retinoic acid activated the human ASBT promoter fourfold. SHP repressed the activity of the ASBT promoter and reduced activation by retinoic acid. Antisense mediated knock-down of SHP in Caco-2 cells partially offset the bile acid mediated repression of ASBT promoter activity. In conclusion, the human ASBT is positively regulated by retinoic acid. Bile acids induce a negative feedback regulation of human ASBT via an FXR-mediated, SHP-dependent effect upon RAR/RXR activation of ASBT.

The receptor-DNA complex determines the retinoid response: a mechanism for the diversification of the ligand signal.

To obtain insights into the principles governing the complex biological responses to retinoids, we have analyzed the ligand sensitivities of various retinoid receptor-DNA complexes. We find that different retinoid receptor heterodimers show distinct activation patterns with various response elements while a given heterodimer can be activated at different retinoic acid concentrations on different response elements. In vitro binding experiments suggest that the same retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimer can have different ligand affinities, depending on the response element it is bound to. The differential responses of a particular receptor heterodimer with various retinoic acid responsive elements can be enhanced through the use of conformationally restricted retinoids. RAR- and RXR-selective retinoids can also synergistically activate the receptor heterodimers, indicating that both partners in the heterodimer can contribute to ligand-induced transcriptional activation. However, the relative influence of the RAR or RXR partner is specific for each response element. Together, our data demonstrate that it is the receptor-DNA complex and not the receptor alone that determines the ligand response. This flexibility allows for a highly pleiotropic retinoid response. Furthermore, conformationally restricted retinoids can accentuate the differential responses and exhibit a certain degree of gene selectivity by differentially activating the RAR or RXR component in the context of a given response element.

Identification and Characterization of a Versatile Retinoid Response Element (Retinoic Acid Receptor Response Element-Retinoid X Receptor Response Element) in the Mouse Tissue Transglutaminase Gene Promoter

Tissue transglutaminase (transglutaminase type II) is an intracellular protein cross-linking enzyme that accumulates in connective tissue and in cells undergoing apoptosis. Retinoids regulate the transcription of the mouse tissue transglutaminase gene via activation of regulatory elements contained within 4 kilobases of the 5'-end of the gene. Co-transfection studies with retinoid receptor expression vectors in CV-1 cells demonstrated that the mouse tissue transglutaminase promoter is activated by ligand activation of either retinoic acid receptor-retinoid X receptor (RAR.RXR) heterodimers or RXR homodimers. Optimal induction is achieved with retinoid receptor panagonists; partial activation can also be achieved with either RAR-specific or RXR-specific retinoids. Retinoid-dependent activation of the tissue transglutaminase promoter depends on both a proximal regulatory region containing sequences highly conserved between the human and the mouse tissue transglutaminase promoters and a distal region that includes a 30-base pair retinoid response element (mTGRRE1). mTGRRE1 contains three hexanucleotide half-sites (two canonical and one non-canonical) in a DR7/DR5 motif that bind both RAR*RXR heterodimers and RXR homodimers. These studies suggest that retinoid-dependent expression of the mouse tissue transglutaminase gene is mediated by a versatile tripartite retinoid response element located 1.7 kilobases upstream of the transcription start site.

Adenovirus E1A functions as a cofactor for retinoic acid receptor beta (RAR beta) through direct interaction with RAR beta.

Transcription regulation by DNA-bound activators is thought to be mediated by a direct interaction between these proteins and TATA-binding protein (TBP), TFIIB, or TBP-associated factors, although occasionally cofactors or adapters are required. For ligand-induced activation by the retinoic acid receptor-retinoid X receptor (RAR-RXR) heterodimer, the RAR beta 2 promoter is dependent on the presence of E1A or E1A-like activity, since this promoter is activated by retinoic acid only in cells expressing such proteins. The mechanism underlying this E1A requirement is largely unknown. We now show that direct interaction between RAR and E1A is a requirement for retinoic acid-induced RAR beta 2 activation. The activity of the hormone-dependent activation function 2 (AF-2) of RAR beta is upregulated by E1A, and an interaction between this region and E1A was observed, but not with AF-1 or AF-2 of RXR alpha. This interaction is dependent on conserved region III (CRIII), the 13S mRNA-specific region of E1A. Deletion analysis within this region indicated that the complete CRIII is needed for activation. The putative zinc finger region is crucial, probably as a consequence of interaction with TBP. Furthermore, the region surrounding amino acid 178, partially overlapping with the TBP binding region, is involved in both binding to and activation by AF-2. We propose that E1A functions as a cofactor by interacting with both TBP and RAR, thereby stabilizing the preinitiation complex.

Retinoid receptors cause distortion of the retinoic acid response element in the phosphoenolpyruvate carboxykinase gene promoter.

Functional retinoic acid response elements (RAREs) have been described wherein the direct repeats are separated by 1, 2 or 5 bp (termed DR1, DR2 and DR5 respectively). We have previously shown that retinoic acid receptor/retinoid X receptor (RAR/RXR) binds a DR1 RARE within the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter and is the trans-acting complex that mediates the retinoic acid (RA) response. However, the mechanism of trans-activation is unknown. The consequences of RAR/RXR binding to the PEPCK RARE were examined using a circular permutation analysis as a first step to explore the possible role of DNA conformational changes in the RA response. The RAR/RXR heterodimer produced a distortion angle of 78 degrees. The DNA distortion was shown to be at the centre of the PEPCK RARE; RA did not affect the severity of the distortion angle or the location of the distortion centre. Monomers and homodimers of RAR also distorted the DNA, but to a lesser extent than did RAR/RXR. The results of a phasing analysis demonstrated that RAR/RXR heterodimers did not induce a static DNA bend, in either the presence or the absence of RA. A cyclization kinetics assay was employed to show that RAR/RXR binding affected DNA ring closure in a phase-sensitive, RA-insensitive, manner. Taken together, these observations support the idea that RAR/RXR heterodimers distort the structure of the PEPCK RARE, at least in part, by altering DNA flexibility. The conformational change in the PEPCK RARE upon RAR/RXR binding has implications for how RAR/RXR heterodimers recognize various RARE structures.