Retinoic Acid Response Element Research Articles

AP-2α decreases TMZ resistance of recurrent GBM by downregulating MGMT expression and improving DNA damage

AimsThe incidence of recurrent gliomas is high, exerting low survival rates and poor prognoses. Transcription factor AP-2α has been reported to regulate the progression of primary glioblastoma (GBM). However, the function of AP-2α in recurrent gliomas is largely unclear. MethodsThe expression of AP-2α and O6-methylguanine DNA-methyltransferase (MGMT) was detected in recurrent glioma tissues and cell lines by Western blots, the regulation mechanisms between AP-2α/MGMT promoter and RA/AP-2α promoter were studied by luciferase reporter assays, EMSA, and chIP assays. The effects of AP-2α and TMZ/RA treatment on cell viability in vitro and in vivo were investigated by MTT assays, γH2AX staining, comet assays and intracranial injection. Key findingsAP-2α expression negatively correlates with the expression of MGMT in glioma samples. AP-2α could directly bind with the promoter of the MGMT gene, suppresses transcriptional levels of MGMT and downregulate MGMT expression in TMZ-resistant U87MG-R and T98G cells, but TMZ treatment decreases AP-2α expression and increases MGMT expression. The extended TMZ treatment and increased TMZ concentrations reversed these effects. Moreover, AP-2α overexpression combines with TMZ to decrease cell viability, concurrently with improved DNA damage marker γH2AX. Furthermore, retinoic acid (RA) activates RAR/RXR heterodimers, which bind to RA-responsive elements (RAREs) of the AP-2α promoter, and activates AP-2α expression in recurrent glioma cells. Finally, in intracranial relapsed glioma mouse model, both RA and TMZ could retard tumor development and prolong the mouse survival. SignificanceAP-2α activation by gene overexpression or RA treatment reveals the suppressive effects on glioma relapse, providing a novel therapeutic strategy against malignant refractory gliomas.

Retinoic Acid Regulates Spermiogenesis Via Hoxb1 and Shh Signaling in Testicular Germ Cells.

Retinoic acid (RA) regulates all four major events in spermatogenesis; spermatogonial differentiation, meiotic entry, spermiogenesis, and spermiation. For the pre-meiotic phase, Sertoli cells are the source of RA and for the post-meiotic phase, pachytene spermatocytes are the source of RA. While the entire spermatogenic process is regulated by RA, how each of these phases is regulated by RA remains completely unknown. Homeobox B1 (Hoxb1) has two retinoic acid response elements (RARE) upstream and downstream of the gene. In this study, we investigated if RA facilitates spermatogenesis by its action on Hoxb1. The expressions of the Hoxb1 and Sonic hedgehog (Shh) genes were analyzed in the post-natal mouse testes and the testicular localizations of Hoxb1, Shh and Gli1 were analyzed by immunohistochemistry in the adult rat testis. To delineate the signaling mechanisms, Hoxb1 expression was altered in vitro and in vivo using retinoic acid and miR-361-3p. Finally, the levels of miR-361-3p and HOXB1 were analyzed in infertile human sperm samples. Hoxb1 and Shh gene expressions were found to be low in the testis of post-natal Swiss mice of 7, 14, 28, 35, and 60days, after which the expressions of both spiked. Immunohistochemistry in the adult mouse testis showed the expressions of Hoxb1, Shh, and Gli1 in the elongating spermatids. Exposure of GC2 cells to RA and in vivoIP RA injection upregulated Hoxb1 andShh signaling in the testiswith increased expressions of Shh, Gli1, and Hdac1. Retinoic acid administration in Swiss mice compromised sperm production and reduced epididymal sperm count. The analysis of infertile human semen samples revealed an increased level of HOXB1 and a decreased level of miR-361-3p as compared to fertile controls. We conclude that retinoic acid regulates late stage of spermatogenesis (spermiogenesis) by affecting Hoxb1 and Shh signaling.

Exploring the role of trifarotene against RAR-α: an investigation of expression pattern and clinicopathological significance of RAR-α in breast cancer.

The members retinoic acid receptors (RARs) (α, β, and γ) and retinoid X receptors (RXRs) (α, β, and γ) belong to the retinoid receptor family. They regulate the biological action of classical retinoids through nuclear retinoid receptors, a transcription factor that is regulated by ligands. Through the binding of particular retinoic acid-responsive elements (RAREs) located in target gene promoters, RARs and members of the RXRs form heterodimers. By binding to its nuclear receptors and triggering the transcription of the target genes downstream, retinoic acid (RA) mediates the expression of certain genes. Retinoids so mainly control gene expression to carry out their biological actions. RARs are essential for many biological processes, such as development, immunity, reproduction, organogenesis, and homeostasis. Apart from their physiological functions, RARs are also linked to pathologies and tumors due to mutations, protein fusions, changes in expression levels, or abnormal post-translational changes that lead to aberrant functions and homeostasis breakdown. The oncogenic development of animal tissues or cultured cells is linked to altered expression of retinoid receptors. The RAR-α is over-expressed in several malignancies. Increased invasion and migration in several cancer forms, including HNSC carcinoma, pediatric low-grade gliomas, lung adenocarcinoma, and breast cancer, have been linked to its upregulated expression. Numerous approved therapeutic regimens targeting RAR-α have been developed, improving patient survival rates. This study's main objective was to identify novel RAR-α-targeting drugs and evaluate the expression patterns of RAR-α in breast cancer patients. In-silico investigation using a variety of bioinformatics tools like UALCAN, TISCH, TIMER 2.0, ENRICHR, and others were employed to examine the expression of RAR-α. Further we evaluated in-silico inhibition of RAR-α with trifarotene and also tested the cytotoxicity of trifarotene in breast cancer cells. Our research indicates that RAR-α is upregulated in several malignancies including Breast Cancer. It regulates granulocyte differentiation and has an association with the retinoic acid receptor signaling pathway and cellular response to estrogen stimulus. Furthermore, trifarotene was found as a potential synthetic compound that targets RAR-α through in silico and in-vitro study. Overall, this research indicates that elevated expression of RAR-α enhances the onset of breast cancer. Using trifarotene medication to target RAR-α will significantly boost the response of breast cancer individuals to treatment and delay the development of resistance to drugs.

Rewiring of the epigenome and chromatin architecture by exogenously induced retinoic acid signaling during zebrafish embryonic development.

Retinoic acid (RA) is the ligand of RA receptors (RARs), transcription factors that bind to RA response elements. RA signaling is required for multiple processes during embryonic development, including body axis extension, hindbrain antero-posterior patterning and forelimb bud initiation. Although some RA target genes have been identified, little is known about the genome-wide effects of RA signaling during in vivo embryonic development. Here, we stimulate the RA pathway by treating zebrafish embryos with all-trans-RA (atRA) and use a combination of RNA-seq, ATAC-seq, ChIP-seq and HiChIP to gain insight into the molecular mechanisms by which exogenously induced RA signaling controls gene expression. We find that RA signaling is involved in anterior/posterior patterning, central nervous system development, and the transition from pluripotency to differentiation. AtRA treatment also alters chromatin accessibility during early development and promotes chromatin binding of RARαa and the RA targets Hoxb1b, Meis2b and Sox3, which cooperate in central nervous system development. Finally, we show that exogenous RA induces a rewiring of chromatin architecture, with alterations in chromatin 3D interactions involving target genes. Altogether, our findings identify genome-wide targets of RA signaling and provide a molecular mechanism by which developmental signaling pathways regulate target gene expression by altering chromatin topology.

ALDH1A1 promotes PARP inhibitor resistance by enhancing retinoic acid receptor-mediated DNA polymerase θ expression

Poly (ADP-ribose) Polymerase (PARP) inhibitors (PARPi) have been approved for both frontline and recurrent setting in ovarian cancer with homologous recombination (HR) repair deficiency. However, more than 40% of BRCA1/2-mutated ovarian cancer lack the initial response to PARPi treatment, and the majority of those that initially respond eventually develop resistance. Our previous study has demonstrated that increased expression of aldehyde dehydrogenase 1A1 (ALDH1A1) contributes to PARPi resistance in BRCA2-mutated ovarian cancer cells by enhancing microhomology-mediated end joining (MMEJ) but the mechanism remains unknown. Here, we find that ALDH1A1 enhances the expression of DNA polymerase θ (Polθ, encoded by the POLQ gene) in ovarian cancer cells. Furthermore, we demonstrate that the retinoic acid (RA) pathway is involved in the transcription activation of the POLQ gene. The RA receptor (RAR) can bind to the retinoic acid response element (RARE) located in the promoter of the POLQ gene, promoting transcription activation-related histone modification in the presence of RA. Given that ALDH1A1 catalyzes the biosynthesis of RA, we conclude that ALDH1A1 promotes POLQ expression via the activation of the RA signaling pathway. Finally, using a clinically-relevant patient-derived organoid (PDO) model, we find that ALDH1A1 inhibition by the pharmacological inhibitor NCT-505 in combination with the PARP inhibitor olaparib synergistically reduce the cell viability of PDOs carrying BRCA1/2 mutation and positive ALDH1A1 expression. In summary, our study elucidates a new mechanism contributing to PARPi resistance in HR-deficient ovarian cancer and shows the therapeutic potential of combining PARPi and ALDH1A1 inhibition in treating these patients.

The Endogenous Retinoic Acid Receptor Pathway Is Exploited by Mycobacterium tuberculosis during Infection, Both InVitro and InVivo.

Retinoic acid (RA) is a fundamental vitamin A metabolite involved in regulating immune responses through the nuclear RA receptor (RAR) and retinoid X receptor. While performing experiments using THP-1 cells as a model for Mycobacterium tuberculosis infection, we observed that serum-supplemented cultures displayed high levels of baseline RAR activation in the presence of live, but not heat-killed, bacteria, suggesting that M. tuberculosis robustly induces the endogenous RAR pathway. Using invitro and invivo models, we have further explored the role of endogenous RAR activity in M. tuberculosis infection through pharmacological inhibition of RARs. We found that M. tuberculosis induces classical RA response element genes such as CD38 and DHRS3 in both THP-1 cells and human primary CD14+ monocytes via a RAR-dependent pathway. M. tuberculosis-stimulated RAR activation was observed with conditioned media and required nonproteinaceous factor(s) present in FBS. Importantly, RAR blockade by (4-[(E)-2-[5,5-dimethyl-8-(2-phenylethynyl)-6H-naphthalen-2-yl]ethenyl]benzoic acid), a specific pan-RAR inverse agonist, in a low-dose murine model of tuberculosis significantly reduced SIGLEC-F+CD64+CD11c+high alveolar macrophages in the lungs, which correlated with 2× reduction in tissue mycobacterial burden. These results suggest that the endogenous RAR activation axis contributes to M. tuberculosis infection both invitro and invivo and reveal an opportunity for further investigation of new antituberculosis therapies.

Shared retinoic acid responsive enhancers coordinately regulate nascent transcription of Hoxb coding and non-coding RNAs in the developing mouse neural tube.

Signaling pathways regulate the patterns of Hox gene expression that underlie their functions in the specification of axial identity. Little is known about the properties of cis-regulatory elements and underlying transcriptional mechanisms that integrate graded signaling inputs to coordinately control Hox expression. Here, we optimized a single molecule fluorescent in situ hybridization (smFISH) technique with probes spanning introns to evaluate how three shared retinoic acid response element (RARE)-dependent enhancers in the Hoxb cluster regulate patterns of nascent transcription in vivo at the level of single cells in wild-type and mutant embryos. We predominately detect nascent transcription of only a single Hoxb gene in each cell, with no evidence for simultaneous co-transcriptional coupling of all or specific subsets of genes. Single and/or compound RARE mutations indicate that each enhancer differentially impacts global and local patterns of nascent transcription, suggesting that selectivity and competitive interactions between these enhancers is important to robustly maintain the proper levels and patterns of nascent Hoxb transcription. This implies that rapid and dynamic regulatory interactions potentiate transcription of genes through combined inputs from these enhancers in coordinating the retinoic acid response.

Hepatocyte Nuclear Factor 4α (HNF4α) Plays a Controlling Role in Expression of the Retinoic Acid Receptor β (RARβ) Gene in Hepatocytes.

HNF4α, a member of the nuclear receptor superfamily, regulates the genes involved in lipid and glucose metabolism. The expression of the RARβ gene in the liver of HNF4α knock-out mice was higher versus wildtype controls, whereas oppositely, RARβ promoter activity was 50% reduced by the overexpression of HNF4α in HepG2 cells, and treatment with retinoic acid (RA), a major metabolite of vitamin A, increased RARβ promoter activity 15-fold. The human RARβ2 promoter contains two DR5 and one DR8 binding motifs, as RA response elements (RARE) proximal to the transcription start site. While DR5 RARE1 was previously reported to be responsive to RARs but not to other nuclear receptors, we show here that mutation in DR5 RARE2 suppresses the promoter response to HNF4α and RARα/RXRα. Mutational analysis of ligand-binding pocket amino acids shown to be critical for fatty acid (FA) binding indicated that RA may interfere with interactions of FA carboxylic acid headgroups with side chains of S190 and R235, and the aliphatic group with I355. These results could explain the partial suppression of HNF4α transcriptional activation toward gene promoters that lack RARE, including APOC3 and CYP2C9, while conversely, HNF4α may bind to RARE sequences in the promoter of the genes such as CYP26A1 and RARβ, activating these genes in the presence of RA. Thus, RA could act as either an antagonist towards HNF4α in genes lacking RAREs, or as an agonist for RARE-containing genes. Overall, RA may interfere with the function of HNF4α and deregulate HNF4α targets genes, including the genes important for lipid and glucose metabolism.

NF-κB represses retinoic acid receptor-mediated GPRC5A transactivation in lung epithelial cells to promote neoplasia.

Chronic inflammation is associated with lung tumorigenesis, in which NF-κB-mediated epigenetic regulation plays a critical role. Lung tumor suppressor G protein-coupled receptor, family C, member 5A (GPRC5A), is repressed in most non-small cell lung cancer (NSCLC); however, the mechanisms remain unclear. Here, we show that NF-κB acts as a transcriptional repressor in suppression of GPRC5A. NF-κB induced GPRC5A repression both in vitro and in vivo. Intriguingly, transactivation of NF-κB downstream targets was not required, but the transactivation domain of RelA/p65 was required for GPRC5A repression. NF-κB did not bind to any potential cis-element in the GPRC5A promoter. Instead, p65 was complexed with retinoic acid receptor α/β (RARα/β) and recruited to the RA response element site at the GPRC5A promoter, resulting in disrupted RNA polymerase II complexing and suppressed transcription. Notably, phosphorylation on serine 276 of p65 was required for interaction with RARα/β and repression of GPRC5A. Moreover, NF-κB-mediated epigenetic repression was through suppression of acetylated histone H3K9 (H3K9ac), but not DNA methylation of the CpG islands, at the GPRC5A promoter. Consistently, a histone deacetylase inhibitor, but not DNA methylation inhibitor, restored GPRC5A expression in NSCLC cells. Thus, NF-κB induces transcriptional repression of GPRC5A via a complex with RARα/β and mediates epigenetic repression via suppression of H3K9ac.

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.

The antileukemic activity of decitabine upon PML/RARA-negative AML blasts is supported by all-trans retinoic acid: in vitro and in vivo evidence for cooperation

The prognosis of AML patients with adverse genetics, such as a complex, monosomal karyotype and TP53 lesions, is still dismal even with standard chemotherapy. DNA-hypomethylating agent monotherapy induces an encouraging response rate in these patients. When combined with decitabine (DAC), all-trans retinoic acid (ATRA) resulted in an improved response rate and longer overall survival in a randomized phase II trial (DECIDER; NCT00867672). The molecular mechanisms governing this in vivo synergism are unclear. We now demonstrate cooperative antileukemic effects of DAC and ATRA on AML cell lines U937 and MOLM-13. By RNA-sequencing, derepression of >1200 commonly regulated transcripts following the dual treatment was observed. Overall chromatin accessibility (interrogated by ATAC-seq) and, in particular, at motifs of retinoic acid response elements were affected by both single-agent DAC and ATRA, and enhanced by the dual treatment. Cooperativity regarding transcriptional induction and chromatin remodeling was demonstrated by interrogating the HIC1, CYP26A1, GBP4, and LYZ genes, in vivo gene derepression by expression studies on peripheral blood blasts from AML patients receiving DAC + ATRA. The two drugs also cooperated in derepression of transposable elements, more effectively in U937 (mutated TP53) than MOLM-13 (intact TP53), resulting in a “viral mimicry” response. In conclusion, we demonstrate that in vitro and in vivo, the antileukemic and gene-derepressive epigenetic activity of DAC is enhanced by ATRA.

The immunomodulatory role of all-trans retinoic acid in tumor microenvironment.

Retinoids are essential nutrients for human beings. Among them, all-trans retinoic acid (ATRA), considered one of the most active metabolites, plays important roles in multiple biological processes. ATRA regulates the transcription of target genes by interacting with nuclear receptors bonded to retinoic acid response elements (RAREs). Besides its differentiation-inducing effect in the treatment of acute promyelocytic leukemia and some solid tumor types, its immunoregulatory role in tumor microenvironment (TME) has attracted considerable attention. ATRA not only substantially abrogates the immunosuppressive effect of tumor-infiltrating myeloid-derived suppressor cells but also activates the anti-tumor effect of CD8 + T cells. Notably, the combination of ATRA with other therapeutic approaches, including immune checkpoint inhibitors (ICIs), tumor vaccines, and chemotherapy, has been extensively investigated in a variety of tumor models and clinical trials. In this review, we summarize the current understanding of the role of ATRA in cancer immunology and immunotherapy, dissect the underlying mechanisms of ATRA-mediated activation or differentiation of different types of immune cells, and explore the potential clinical significance of ATRA-based cancer therapy.

Abstract 1519: ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression

Abstract Aldehyde dehydrogenase (ALDH) is a superfamily of 19 known enzymes that participated in the metabolism of endogenous and exogenous aldehydes. ALDH1A1 is a major subtype contributing to high ALDH activity out of all ALDH isoforms and plays a critical role in the maintenance of cancer stem cells. Our previous studies have shown that ALDH1A1 is able to reduce the sensitivity of BRCA2-/- ovarian cancer cells to PARP inhibitors by enhancing microhomology-mediated end joining (MMEJ). MMEJ uses 5-25 base pair micro homologous sequences to align the broken strands before joining, and this repair pathway requires DNA polymerase θ (Pol θ, encoded by POLQ). Here we show that ALDH1A1 can increase the expression of Pol θ, at both mRNA and protein levels, in ovarian cancer cell lines. ALDH1A1 is reported to catalyze the production of all-trans retinoic acid (ATRA), which can bind to the retinoic acid receptor (RAR), and promote transcription of the downstream target genes that possess retinoic acid response element (RARE) in their promoter regions. Interestingly we identified several RAREs in the promoter region of the POLQ gene. Thus, we hypothesized that ALDH1A1 promotes Pol θ expression via activation of the RAR signaling pathway. We found that ATRA and RAR agonist (CH55) increased but pan RAR antagonist (AGN193109) treatment decreased the expression of POLQ, indicating that the RAR pathway is involved in the transcription of POLQ. We also demonstrated that RARα can bind to the promoter region of Pol Q, activation of RAR signaling by treatment with ATRA or CH55 increased the enrichment of K27 acetylated histone H3 in this promoter region. Moreover, we showed that mutated ALDH1A1 lacking the enzyme activity was unable to enhance POLQ transcription. These data suggest that ALDH1A1 enhances the Pol Q expression by increasing the production of ATRA which further activates the RAR signaling pathway. Citation Format: Kousalya Lavudi, Ananya Banerjee, Shurui Cai, Na Li, Xuetao Bai, Wang Qi-En. ALDH1A1 enhances micro homology-mediated end joining by increasing POLQ expression [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1519.

A novel HNRNPH1::ERG rearrangement in aggressive acute myeloid leukemia.

FUS::ERG rearrangement is a recurrent abnormality seen in a subgroup of acute myeloid leukemia (AML) with a poor prognosis. We described here a novel HNRNPH1::ERG rearrangement in a de novo AML. The patient was unresponsive to routine chemotherapy and succumbed to the disease just 3 months after diagnosis. Two additional cases of AML with HNRNPH1::ERG rearrangement were discovered by searching a publicly available sequencing database. The three patients share several clinical phenotypes with the FUS::ERG rearranged AML, including high blast count at diagnosis, pediatric or young adult-onset, and poor overall survival. In addition, hnRNPH1 and FUS are both hnRNP family members, a group of RNA-binding proteins functioning in RNA metabolism and transport. Therefore, we suggest that patients with HNRNPH1::ERG or FUS::ERG rearrangement belong to the same distinct clinicopathologic subtype of AML, that is, AML with ERG rearrangement. Based on a previous study showing that FUS::ERG binds to the retinoic acid-responsive elements and that all-trans retinoic acid-induced cell differentiation of AML cells, we support the clinical evaluation of an APL-like therapeutic regimen for AML with ERG rearrangement.

Retinoic acid restores the levels of cellular cholesterol in Leishmania donovani infected macrophages by increasing npc1 and npc2 expressions

Visceral leishmaniasis (VL) is a fatal form among all forms of leishmaniasis and is caused by visceralization of the Leishmania donovani (Ld) parasite to the critical organs. Mild to severe malnutrition is common in VL patients and the deficiency of retinoic acid (RA), an important micronutrient, results in a compromised state of immune response in macrophages (mφ) leading to the increased parasite load. In the continuation of our earlier work, we observed loss of cellular cholesterol in infected mφ in the absence of RA i.e., upon inhibition of RALDH pathway. Moreover, the Leishmania utilizes host cholesterol for the establishment of infection and causes a decrease in the expressions of Niemann-Pick C2 (npc2) and Niemann-Pick C1 (npc1) genes involved in the uptake of extracellular cholesterol. This results in reduced levels of cellular cholesterol in infected mφ. Intrigued by this, as the first sign of our hypothesis, we investigated the presence of RA Response Element (RARE) sequences in the upstream of npc1 and npc2 genes. To functionally confirm this, we measured their expressions and the levels of cellular cholesterol in Ld infected mφ in the absence (i.e., using an inhibitor of RALDH pathway) and presence of RA. We found restoration of the levels of cellular cholesterol in infected mφ under the supplementation of RA resulting in the decreased parasite load. Hence, the supplementation of RA with the standard therapy and/or preventive use of RA could be potentially an advancement in the treatment and cure of VL patients.

Towards a Better Vision of Retinoic Acid Signaling during Eye Development.

Retinoic acid (RA) functions as an essential signal for development of the vertebrate eye by controlling the transcriptional regulatory activity of RA receptors (RARs). During eye development, the optic vesicles and later the retina generate RA as a metabolite of vitamin A (retinol). Retinol is first converted to retinaldehyde by retinol dehydrogenase 10 (RDH10) and then to RA by all three retinaldehyde dehydrogenases (ALDH1A1, ALDH1A2, and ALDH1A3). In early mouse embryos, RA diffuses to tissues throughout the optic placode, optic vesicle, and adjacent mesenchyme to stimulate folding of the optic vesicle to form the optic cup. RA later generated by the retina is needed for further morphogenesis of the optic cup and surrounding perioptic mesenchyme; loss of RA at this stage leads to microphthalmia and cornea plus eyelid defects. RA functions by binding to nuclear RARs at RA response elements (RAREs) that either activate or repress transcription of key genes. Binding of RA to RARs regulates recruitment of transcriptional coregulators such as nuclear receptor coactivator (NCOA) or nuclear receptor corepressor (NCOR), which in turn control binding of the generic coactivator p300 or the generic corepressor PRC2. No genes have been identified as direct targets of RA signaling during eye development, so future studies need to focus on identifying such genes and their RAREs. Studies designed to learn how RA normally controls eye development in vivo will provide basic knowledge valuable for determining how developmental eye defects occur and for improving strategies to treat eye defects.

Development of an adenovirus-mediated reporter assay system to detect a low concentration of retinoic acid in MCF-7 cells.

Retinoic acid, an active form of vitamin A, plays very important roles in mammalian embryogenesis. The concentration of retinoic acid is extremely low and strictly regulated by enzymes of cytochrome P450 (CYP) family, CYP26s (CYP26A1, CYP26B1 and CYP26C1) in the cells. Therefore, it is thought that changes in CYP26s activities due to exposure to a wide variety of drugs and chemicals exhibit teratogenicity. In this study, to easily detect the changes in retinoic acid level, we constructed an adenovirus-mediated reporter assay system using the promoter region of the CYP26A1 gene and inserting retinoic acid response element (RARE) and retinoid X response element (RXRE) into the downstream of the luciferase gene of reporter plasmid, which highly increased the response to retinoic acid. Reporter activity significantly increased in a concentration-dependent manner with retinoic acid; this increase was also observed at least after treatment with a very low concentration of 1 nM retinoic acid. This increase was suppressed by the accelerated metabolism of retinoic acid due to the overexpression of CYP26A1; however, this suppression was almost completely suspended by treatment with talarozole, a CYP26 inhibitor. In conclusion, the reporter assay system constructed using the induction of CYP26A1 expression is a risk assessment system that responds to extremely low concentrations of retinoic acid and is useful for assessing the excess vitamin A mediated teratogenicity caused by various chemicals at the cellular level.

Enhancer architecture-dependent multilayered transcriptional regulation orchestrates RA signaling-induced early lineage differentiation of ESCs.

Signaling pathway-driven target gene transcription is critical for fate determination of embryonic stem cells (ESCs), but enhancer-dependent transcriptional regulation in these processes remains poorly understood. Here, we report enhancer architecture-dependent multilayered transcriptional regulation at the Halr1–Hoxa1 locus that orchestrates retinoic acid (RA) signaling-induced early lineage differentiation of ESCs. We show that both homeobox A1 (Hoxa1) and Hoxa adjacent long non-coding RNA 1 (Halr1) are identified as direct downstream targets of RA signaling and regulated by RARA/RXRA via RA response elements (RAREs). Chromosome conformation capture-based screens indicate that RA signaling promotes enhancer interactions essential for Hoxa1 and Halr1 expression and mesendoderm differentiation of ESCs. Furthermore, the results also show that HOXA1 promotes expression of Halr1 through binding to enhancer; conversely, loss of Halr1 enhances interaction between Hoxa1 chromatin and four distal enhancers but weakens interaction with chromatin inside the HoxA cluster, leading to RA signaling-induced Hoxa1 overactivation and enhanced endoderm differentiation. These findings reveal complex transcriptional regulation involving synergistic regulation by enhancers, transcription factors and lncRNA. This work provides new insight into intrinsic molecular mechanisms underlying ESC fate determination during RA signaling-induced early differentiation.

Vitamin A and Vitamin E: Will the Real Antioxidant Please Stand Up?

Vitamin A, acting through its metabolite, all-trans-retinoic acid, is a potent transcriptional regulator affecting expression levels of hundreds of genes through retinoic acid response elements present within these genes. However, the literature is replete with claims that consider vitamin A to be an antioxidant vitamin, like vitamins C and E. This apparent contradiction in the understanding of how vitamin A acts mechanistically within the body is a major focus of this review. Vitamin E, which is generally understood to act as a lipophilic antioxidant protecting polyunsaturated fatty acids present in membranes, is often proposed to be a transcriptional regulator. The evaluation of this claim is another focus of the review. We conclude that vitamin A is an indirect antioxidant, whose indirect function is to transcriptionally regulate a number of genes involved in mediating the body's canonical antioxidant responses. Vitamin E, in addition to being a direct antioxidant, prevents the increase of peroxidized lipids that alter both metabolic pathways and gene expression profiles within tissues and cells. However, there is little compelling evidence that vitamin E has a direct transcriptional mechanism like that of vitamin A. Thus, we propose that the term antioxidant not be applied to vitamin A, and we discourage the use of the term transcriptional mediator when discussing vitamin E.

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.