Showing papers on "Aryl hydrocarbon receptor nuclear translocator published in 2022"

Butyrate acts through HDAC inhibition to enhance aryl hydrocarbon receptor activation by gut microbiota-derived ligands

Abstract: ABSTRACT Aryl hydrocarbon receptor (AhR) is a critical player in the crosstalk between the gut microbiota and its host. However, factors regulating AhR within the gut, which is a complex metabolomic environment, are poorly understood. This study investigates the effect of a combination of metabolites on the activation mechanism of AhR. AhR activity was evaluated using both a luciferase reporter system and mRNA levels of AhR target genes on human cell lines and human colonic explants. AhR activation was studied by radioligand-binding assay, nuclear translocation of AhR by immuofluorescence and protein co-immunoprecipitation of AhR with ARNT. Indirect activation of AhR was evaluated using several tests and inhibitors. The promoter of the target gene CYP1A1 was studied both by chromatin immunoprecipitation and by using an histone deacetylase HDAC inhibitor (iHDAC). Short-chain fatty acids, and butyrate in particular, enhance AhR activity mediated by endogenous tryptophan metabolites without binding to the receptor. This effect was confirmed in human intestinal explants and did not rely on activation of receptors targeted by SCFAs, inhibition of AhR degradation or clearance of its ligands. Butyrate acted directly on AhR target gene promoter to reshape chromatin through iHDAC activity. Our findings revealed that butyrate is not an AhR ligand but acts as iHDAC leading to an increase recruitment of AhR to the target gene promoter in the presence of tryptophan-derived AhR agonists. These data contribute to a novel understanding of the complex regulation of AhR activation by gut microbiota-derived metabolites.

The aryl hydrocarbon receptor as a model PAS sensor

Abstract: Proteins containing PER-ARNT-SIM (PAS) domains are commonly associated with environmental adaptation in a variety of organisms. The PAS domain is found in proteins throughout Archaea, Bacteria, and Eukarya and often binds small-molecules, supports protein-protein interactions, and transduces input signals to mediate an adaptive physiological response. Signaling events mediated by PAS sensors can occur through induced phosphorelays or genomic events that are often dependent upon PAS domain interactions. In this perspective, we briefly discuss the diversity of PAS domain containing proteins, with particular emphasis on the prototype member, the aryl hydrocarbon receptor (AHR). This ligand-activated transcription factor acts as a sensor of the chemical environment in humans and many chordates. We conclude with the idea that since mammalian PAS proteins often act through PAS-PAS dimers, undocumented interactions of this type may link biological processes that we currently think of as independent. To support this idea, we present a framework to guide future experiments aimed at fully elucidating the spectrum of PAS-PAS interactions with an eye towards understanding how they might influence environmental sensing in human and wildlife populations.

Structural insight into the ligand binding mechanism of aryl hydrocarbon receptor

Abstract: Abstract The aryl hydrocarbon receptor (AHR), a member of the basic helix–loop–helix (bHLH) Per–Arnt–Sim (PAS) family of transcription factors, plays important roles in regulating xenobiotic metabolism, cellular differentiation, stem cell maintenance, as well as immunity. More recently, AHR has gained significant interest as a drug target for the development of novel cancer immunotherapy drugs. Detailed understanding of AHR-ligand binding has been hampered for decades by the lack of a three-dimensional structure of the AHR PAS-B domain. Here, we present multiple crystal structures of the Drosophila AHR PAS-B domain, including its apo, ligand-bound, and AHR nuclear translocator (ARNT) PAS-B-bound forms. Together with biochemical and cellular assays, our data reveal structural features of the AHR PAS-B domain, provide insights into the mechanism of AHR ligand binding, and provide the structural basis for the future development of AHR-targeted therapeutics.

AhR promotes phosphorylation of ARNT isoform 1 in human T cell malignancies as a switch for optimal AhR activity

Abstract: Significance Nontoxic agonists and antagonists of the aryl hydrocarbon receptor (AhR) hold high therapeutic potential for treating autoimmune disease and cancer. However, AhR activation by different ligands can lead to opposing phenotypical outcomes in a cell- and tissue-specific manner. In this study, we demonstrate that proportional flux in the levels of aryl hydrocarbon receptor nuclear translocator (ARNT) isoforms 1 and 3 modulates AhR signaling in terms of amplitude and expression of distinct gene programs. These results delineate a molecular mechanism of ARNT isoform–mediated AhR regulation, simplify our understanding of a complex AhR signaling pathway, and provide feasibility for ARNT-targeted therapies that could be used in conjunction with nontoxic AhR ligands for the purpose of immunomodulation.

Aryl hydrocarbon receptor activation-mediated vascular toxicity of ambient fine particulate matter: contribution of polycyclic aromatic hydrocarbons and osteopontin as a biomarker

Abstract: Abstract Background Exposure to ambient fine particulate matter (PM 2.5 ) is associated with vascular diseases. Polycyclic aromatic hydrocarbons (PAHs) in PM 2.5 are highly hazardous; however, the contribution of PM 2.5 -bound PAHs to PM 2.5 -associated vascular diseases remains unclear. The ToxCast high-throughput in vitro screening database indicates that some PM 2.5 -bound PAHs activate the aryl hydrocarbon receptor (AhR). The present study investigated whether the AhR pathway is involved in the mechanism of PM 2.5 -induced vascular toxicity, identified the PAH in PM 2.5 that was the major contributor of AhR activation, and identified a biomarker for vascular toxicity of PM 2.5 -bound PAHs. Results Treatment of vascular smooth muscle cells (VMSCs) with an AhR antagonist inhibited the PM 2.5 -induced increase in the cell migration ability; NF-κB activity; and expression of cytochrome P450 1A1 (CYP1A1), 1B1 (CYP1B1), interleukin-6 (IL-6), and osteopontin (OPN). Most PM 2.5 -bound PAHs were extracted into the organic fraction, which drastically enhanced VSMC migration and increased mRNA levels of CYP1A1, CYP1B1, IL-6, and OPN. However, the inorganic fraction of PM 2.5 moderately enhanced VSMC migration and only increased IL-6 mRNA levels. PM 2.5 increased IL-6 secretion through NF-κB activation; however, PM 2.5 and its organic extract increased OPN secretion in a CYP1B1-dependent manner. Inhibiting CYP1B1 activity and silencing OPN expression prevented the increase in VSMC migration ability caused by PM 2.5 and its organic extract. The AhR activation potencies of seven PM 2.5 -bound PAHs, reported in the ToxCast database, were strongly correlated with their capabilities of enhancing the migration ability of VSMCs. Benzo(k)fluoranthene (BkF) contributed the most to the AhR agonistic activity of ambient PM 2.5 -bound PAHs. The association between PM 2.5 -induced vascular toxicity, AhR activity, and OPN secretion was further verified in mice; PM 2.5 -induced intimal hyperplasia in pulmonary small arteries and OPN secretion were alleviated in mice with low AhR affinity. Finally, urinary concentrations of 1-hydroxypyrene, a major PAH metabolite, were positively correlated with plasma OPN levels in healthy humans. Conclusions The present study offers in vitro, animal, and human evidences supporting the importance of AhR activation for PM 2.5 -induced vascular toxicities and that BkF was the major contributor of AhR activation. OPN is an AhR-dependent biomarker of PM 2.5 -induced vascular toxicity. The AhR activation potency may be applied in the risk assessment of vascular toxicity in PAH mixtures.

Aryl hydrocarbon receptor activation-mediated vascular toxicity of ambient fine particulate matter: contribution of polycyclic aromatic hydrocarbons and osteopontin as a biomarker

Abstract: Abstract Background Exposure to ambient fine particulate matter (PM 2.5 ) is associated with vascular diseases. Polycyclic aromatic hydrocarbons (PAHs) in PM 2.5 are highly hazardous; however, the contribution of PM 2.5 -bound PAHs to PM 2.5 -associated vascular diseases remains unclear. The ToxCast high-throughput in vitro screening database indicates that some PM 2.5 -bound PAHs activate the aryl hydrocarbon receptor (AhR). The present study investigated whether the AhR pathway is involved in the mechanism of PM 2.5 -induced vascular toxicity, identified the PAH in PM 2.5 that was the major contributor of AhR activation, and identified a biomarker for vascular toxicity of PM 2.5 -bound PAHs. Results Treatment of vascular smooth muscle cells (VMSCs) with an AhR antagonist inhibited the PM 2.5 -induced increase in the cell migration ability; NF-κB activity; and expression of cytochrome P450 1A1 (CYP1A1), 1B1 (CYP1B1), interleukin-6 (IL-6), and osteopontin (OPN). Most PM 2.5 -bound PAHs were extracted into the organic fraction, which drastically enhanced VSMC migration and increased mRNA levels of CYP1A1, CYP1B1, IL-6, and OPN. However, the inorganic fraction of PM 2.5 moderately enhanced VSMC migration and only increased IL-6 mRNA levels. PM 2.5 increased IL-6 secretion through NF-κB activation; however, PM 2.5 and its organic extract increased OPN secretion in a CYP1B1-dependent manner. Inhibiting CYP1B1 activity and silencing OPN expression prevented the increase in VSMC migration ability caused by PM 2.5 and its organic extract. The AhR activation potencies of seven PM 2.5 -bound PAHs, reported in the ToxCast database, were strongly correlated with their capabilities of enhancing the migration ability of VSMCs. Benzo(k)fluoranthene (BkF) contributed the most to the AhR agonistic activity of ambient PM 2.5 -bound PAHs. The association between PM 2.5 -induced vascular toxicity, AhR activity, and OPN secretion was further verified in mice; PM 2.5 -induced intimal hyperplasia in pulmonary small arteries and OPN secretion were alleviated in mice with low AhR affinity. Finally, urinary concentrations of 1-hydroxypyrene, a major PAH metabolite, were positively correlated with plasma OPN levels in healthy humans. Conclusions The present study offers in vitro, animal, and human evidences supporting the importance of AhR activation for PM 2.5 -induced vascular toxicities and that BkF was the major contributor of AhR activation. OPN is an AhR-dependent biomarker of PM 2.5 -induced vascular toxicity. The AhR activation potency may be applied in the risk assessment of vascular toxicity in PAH mixtures.

The Contribution of Genetic Variation and Aberrant Methylation of Aryl Hydrocarbon Receptor Signaling Pathway Genes to Rheumatoid Arthritis

Abstract: The aryl hydrocarbon receptor (AHR) signaling pathway participates in immune regulation of multiple autoimmune diseases, including rheumatoid arthritis (RA). We conducted this study to investigate the association of AHR signaling pathway genes (AHR, ARNT, AHRR) single nucleotide polymorphisms (SNPs), as well as their methylation levels, with RA susceptibility. Nine SNPs (AHR gene rs2066853, rs2158041, rs2282885, ARNT gene rs10847, rs1889740, rs11204735, AHRR gene rs2292596, rs2672725, rs349583) were genotyped via improved multiple ligase detection reaction (iMLDR) in 479 RA patients and 496 healthy controls. We used the Illumina Hiseq platform to detect methylation levels of these genes in 122 RA patients and 123 healthy controls. A significant increase in rs11204735 C allele frequency was observed in RA patients when compared to controls. Further, rs11204735 polymorphism was associated with a decreased risk of RA under the dominant model. ARNT CCC haplotype frequency was significantly increased in RA patients in comparison to controls. In the AHRR gene, rs2672725 GG genotype, G allele frequencies were significantly related to an increased risk of RA and rs2292596, rs2672725 polymorphism were significantly associated with an increased risk of RA under the dominant model, recessive model, respectively. However, no significant association was identified between AHR gene polymorphism and RA susceptibility. The AHR methylation level in RA patients was significantly higher than the controls, while AHRR methylation level was abnormally reduced in RA patients. In addition, AHRR rs2672725 genotype distribution was significantly associated with the AHRR methylation level among RA patients. In summary, ARNT rs11204735, AHRR rs2292596, and rs2672725 polymorphisms were associated with RA susceptibility and altered AHR, AHRR methylation levels were related to the risk of RA.

Alternative Splicing of the Aryl Hydrocarbon Receptor Nuclear Translocator (ARNT) Is Regulated by RBFOX2 in Lymphoid Malignancies

Abstract: Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ABSTRACT Aberrant alternative splicing (AS) of pre-mRNAs promotes the development and proliferation of cancerous cells. Accordingly, we had previously observed higher levels of the aryl hydrocarbon receptor nuclear translocator (ARNT) spliced variant isoform 1 in human lymphoid malignancies compared to that in normal lymphoid cells, which is a consequence of increased inclusion of alternative exon 5. ARNT is a transcription factor that has been implicated in the survival of various cancers. Notably, we found that ARNT isoform 1 promoted the growth and survival of lymphoid malignancies, but the regulatory mechanism controlling ARNT AS is unclear. Here, we report cis- and trans-regulatory elements which are important for the inclusion of ARNT exon 5. Specifically, we identified recognition motifs for the RNA-binding protein RBFOX2, which are required for RBFOX2-mediated exon 5 inclusion. RBFOX2 upregulation was observed in lymphoid malignancies, correlating with the observed increase in ARNT exon 5 inclusion. Moreover, suppression of RBFOX2 significantly reduced ARNT isoform 1 levels and cell growth. These observations reveal RBFOX2 as a critical regulator of ARNT AS in lymphoid malignancies and suggest that blocking the ARNT-specific RBFOX2 motifs to decrease ARNT isoform 1 levels is a viable option for targeting the growth of lymphoid malignancies.

LncRNA MALAT1 Promotes PPARα/CD36-Mediated Hepatic Lipogenesis in Nonalcoholic Fatty Liver Disease by Modulating miR-206/ARNT Axis

Abstract: Long non-coding RNAs (lncRNAs) are known to play crucial roles in nonalcoholic fatty liver disease (NAFLD). This research sought to explore mechanisms by which lncRNA MALAT1 regulates the progression of NAFLD. Thus, in order to detect the function of MALAT1 in NAFLD, in vitro and in vivo model of NAFLD were established. Then, fatty acid uptake and triglyceride level were investigated by BODIPY labeled-fatty acid uptake assay and Oil red O staining, respectively. The expressions of MALAT1, miR-206, ARNT, PPARα and CD36 were detected by western blotting and qPCR. Dual luciferase, RIP and ChIP assay were used to validate the relation among MALAT1, miR-206, ARNT and PPARα. The data revealed expression of MALAT1 was up-regulated in vitro and in vivo in NAFLD, and knockdown of MALAT1 suppressed FFA-induced lipid accumulation in hepatocytes. Meanwhile, MALAT1 upregulated the expression of ARNT through binding with miR-206. Moreover, miR-206 inhibitor reversed MALAT1 knockdown effects in decreased lipid accumulation in FFA-treated hepatocytes. Furthermore, ARNT could inhibit the expression of PPARα via binding with PPARα promoter. Knockdown of MALAT1 significantly upregulated the level of PPARα and downregulated the expression of CD36, while PPARα knockdown reversed these phenomena. MALAT1 regulated PPARα/CD36 -mediated hepatic lipid accumulation in NAFLD through regulation of miR-206/ARNT axis. Thus, MALAT1/miR-206/ARNT might serve as a therapeutic target against NAFLD.

Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1β

Abstract: The main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer.This project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed.BaP can enhance the binding ability of HIF-1α protein to HIF-1β/ARNT in a dose-dependent manner without CoCl2. However, the binding ability of AhR protein to HIF-1β/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl2.It is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.

Structures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers reveal new dimerization modalities in the bHLH-PAS transcription factor family

Abstract: Significance Neuronal activities can regulate gene expression through the action of certain transcription factors (TFs). One such TF is the neuronal PER-ARNT-SIM (PAS) domain protein 4 (NPAS4), believed to modulate the excitatory-inhibitory balance of neural circuits. NPAS4 dysfunction has been linked to a variety of human neuropsychiatric and metabolic diseases. In a stimulus-dependent manner, NPAS4 forms distinct heterodimers with aryl hydrocarbon receptor nuclear translocator (ARNT) or ARNT2. Here, we visualized the molecular architectures of NPAS4-ARNT and NPAS4-ARNT2 heterodimers, finding unique architectural features and dimerization patterns relative to other members of their family. The visualization of three proteins, especially their PAS domains harboring ligand-binding pockets, and their unique inter-connections in each heterodimer will guide future therapeutic drug discovery directed against them.

Genome-Wide ChIPseq Analysis of AhR, COUP-TF, and HNF4 Enrichment in TCDD-Treated Mouse Liver

Abstract: The aryl hydrocarbon receptor (AhR) is a ligand-activated transcription factor known for mediating the toxicity of 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and related compounds. Although the canonical mechanism of AhR activation involves heterodimerization with the aryl hydrocarbon receptor nuclear translocator, other transcriptional regulators that interact with AhR have been identified. Enrichment analysis of motifs in AhR-bound genomic regions implicated co-operation with COUP transcription factor (COUP-TF) and hepatocyte nuclear factor 4 (HNF4). The present study investigated AhR, HNF4α and COUP-TFII genomic binding and effects on gene expression associated with liver-specific function and cell differentiation in response to TCDD. Hepatic ChIPseq data from male C57BL/6 mice at 2 h after oral gavage with 30 µg/kg TCDD were integrated with bulk RNA-sequencing (RNAseq) time-course (2–72 h) and dose–response (0.01–30 µg/kg) datasets to assess putative AhR, HNF4α and COUP-TFII interactions associated with differential gene expression. Functional enrichment analysis of differentially expressed genes (DEGs) identified differential binding enrichment for AhR, COUP-TFII, and HNF4α to regions within liver-specific genes, suggesting intersections associated with the loss of liver-specific functions and hepatocyte differentiation. Analysis found that the repression of liver-specific, HNF4α target and hepatocyte differentiation genes, involved increased AhR and HNF4α binding with decreased COUP-TFII binding. Collectively, these results suggested TCDD-elicited loss of liver-specific functions and markers of hepatocyte differentiation involved interactions between AhR, COUP-TFII and HNF4α.

Interaction between AhR and HIF-1 signaling pathways mediated by ARNT/HIF-1β

Abstract: The main causes of lung cancer are smoking, environmental pollution and genetic susceptibility. It is an indisputable fact that PAHs are related to lung cancer, and benzo(a) pyrene is a representative of PAHs. The purpose of the current investigation was to investigate the interaction between AhR and HIF-1 signaling pathways in A549 cells, which provide some experimental basis for scientists to find drugs that block AhR and HIF-1 signaling pathway to prevent and treat cancer.This project adopts the CYP1A1 signaling pathways and the expression of CYP1B1 is expressed as a measure of AhR strength index. The expression of VEGF and CAIX volume as a measure of the strength of the signal path HIF-1 indicators. Through the construction of plasmid vector, fluorescence resonance energy transfer, real-time quantitative PCR, western blotting and immunoprecipitation, the interaction between AhR signaling pathway and HIF-1 signaling pathway was observed.BaP can enhance the binding ability of HIF-1α protein to HIF-1β/ARNT in a dose-dependent manner without CoCl2. However, the binding ability of AhR protein to HIF-1β/ARNT is inhibited by HIF-1α signaling pathway in a dose-dependent manner with CoCl2.It is shown that activation of the AhR signaling pathway does not inhibit the HIF-1α signaling pathway, but activation of the HIF-1α signaling pathway inhibits the AhR signaling pathway.

CYP1A1, VEGFA and Adipokine Responses of Human Adipocytes Co-exposed to PCB126 and Hypoxia

Abstract: It is increasingly recognized that hypoxia may develop in adipose tissue as its mass expands. Adipose tissue is also the main reservoir of lipophilic pollutants, including polychlorinated biphenyls (PCBs). Both hypoxia and PCBs have been shown to alter adipose tissue functions. The signaling pathways induced by hypoxia and pollutants may crosstalk, as they share a common transcription factor: aryl hydrocarbon receptor nuclear translocator (ARNT). Whether hypoxia and PCBs crosstalk and affect adipokine secretion in human adipocytes remains to be explored. Using primary human adipocytes acutely co-exposed to different levels of hypoxia (24 h) and PCB126 (48 h), we observed that hypoxia significantly inhibits the PCB126 induction of cytochrome P450 (CYP1A1) transcription in a dose-response manner, and that Acriflavine (ACF)—an HIF1α inhibitor—partially restores the PCB126 induction of CYP1A1 under hypoxia. On the other hand, exposure to PCB126 did not affect the transcription of the vascular endothelial growth factor-A (VEGFA) under hypoxia. Exposure to hypoxia increased leptin and interleukin-6 (IL-6), and decreased adiponectin levels dose-dependently, while PCB126 increased IL-6 and IL-8 secretion in a dose-dependent manner. Co-exposure to PCB126 and hypoxia did not alter the adipokine secretion pattern observed under hypoxia and PCB126 exposure alone. In conclusion, our results indicate that (1) hypoxia inhibits PCB126-induced CYP1A1 expression at least partly through ARNT-dependent means, suggesting that hypoxia could affect PCB metabolism and toxicity in adipose tissue, and (2) hypoxia and PCB126 affect leptin, adiponectin, IL-6 and IL-8 secretion differently, with no apparent crosstalk between the two factors.

Cryo-EM structure of the agonist-bound Hsp90-XAP2-AHR cytosolic complex

Abstract: Summary Living organisms have developed protein sensors helping them to adapt to their environment 1 . The aryl hydrocarbon receptor (AHR) is an emblematic member of this class of proteins, and a ligand-dependent transcription factor that mediates a broad spectrum of (patho)physiological processes in response to numerous substances including pollutants, natural products and metabolites 2 . However, in the absence of high-resolution structural data, a molecular understanding of how AHR is activated by such diverse compounds is lacking. Here we present a 2.85 Å cryo-electron microscopy structure of the cytosolic complex comprising AHR bound to the ligand indirubin, the chaperone Hsp90 and the co-chaperone XAP2. The structure reveals a closed Hsp90 dimer with AHR threaded through its lumen. XAP2 directly interacts with Hsp90 and the AHR ligand-binding domain, thereby acting as a brace stabilizing the entire complex. Importantly, we provide the first experimental visualization of the AHR PAS-B domain bound to a ligand, revealing a unique organization of the ligand-binding pocket and the structural determinants of ligand-binding specificity and promiscuity of the receptor. By providing unprecedented structural details of the molecular initiating event leading to AHR activation, our study rationalizes prior biochemical data and provides a framework for future mechanistic studies and structure-guided drug design.

Core and Flanking bHLH-PAS:DNA interactions mediate specificity and drive obesity

Abstract: The basic-Helix-Loop-Helix Per-Arnt-Sim (PAS) homology domain (bHLH-PAS) transcription factor (TF) family comprises critical biological sensors of physiological (hypoxia, tryptophan metabolites, neuronal activity, and appetite) and environmental (diet derived metabolites and environmental pollutants) stimuli to regulate genes involved in signal adaptation and homeostasis1. bHLH TFs bind DNA as homo or heterodimers via E-box (CANNTG) response elements, however the DNA binding specificity of the PAS domain-containing bHLH subfamily remains unresolved1. We systematically analysed cognate DNA binding hierarchies of prototypical bHLH-PAS family members (ARNT, ARNT2, HIF1α, HIF2α, AhR, NPAS4, SIM1) and demonstrate distinct core (NNCGTG) specificities for different heterodimer classes. The results also show that bHLH-PAS TFs bind over a large footprint 12-15bp and recognise preferential DNA sequences flanking the core. For example, specificity beyond otherwise identical core binding by SIM1 and the HIFs is mediated through N-terminal HIFα-DNA interactions. We also reveal an intimate relationship between DNA shape and both core and flanking TF binding allowing motif sequence flexibility and underpinning TF binding specificity. Furthermore, DNA-shape affinity relationships revealed that novel downstream PAS-A-loop DNA interactions are associated with AT-rich sequences that lead to high-affinity binding, and that loss of this function underpins a monogenic cause of human hyperphagic obesity in a recapitulated SIM1.R171H knock-in mouse model. Importantly, models of protein-DNA binding accurately predict in vivo occupancy, while response element methylation blocks DNA binding and predicts cell type specific chromatin occupancy. These data provide a definitive and accurate map of bHLH-PAS TF specificity and target selectivity through novel flanking protein-DNA interactions that are crucial for in vivo biological function.

Why ARNT Prostate Tumors Responding to Enzalutamide?

Abstract: Prostate tumors can develop resistance to androgen receptor (AR)-targeted therapies through treatment-induced changes in transcription factor activity that promote transcriptional and morphologic features of a neuroendocrine lineage. This study identifies an unexpected role for the circadian protein ARNTL in resistance to enzalutamide, a second-generation AR-targeted therapy. See related article by Linder et al., p. 2074 (4).

ARNT Inhibits H5N1 Influenza A Virus Replication by Interacting with the PA Protein

Abstract: Increasing evidence suggests that the polymerase acidic (PA) protein of influenza A viruses plays an important role in viral replication and pathogenicity. However, information regarding the interaction(s) of host factors with PA is scarce. By using a yeast two-hybrid screen, we identified a novel host factor, aryl hydrocarbon receptor nuclear translocator (ARNT), that interacts with the PA protein of the H5N1 virus. The interaction between PA and human ARNT was confirmed by co-immunoprecipitation and immunofluorescence microscopy. Moreover, overexpression of ARNT downregulated the polymerase activity and inhibited virus propagation, whereas knockdown of ARNT significantly increased the polymerase activity and virus replication. Mechanistically, overexpression of ARNT resulted in the accumulation of PA protein in the nucleus and inhibited both the replication and transcription of the viral genome. Interaction domain mapping revealed that the bHLH/PAS domain of ARNT mainly interacted with the C-terminal domain of PA. Together, our results demonstrate that ARNT inhibits the replication of the H5N1 virus and could be a target for the development of therapeutic strategies against H5N1 influenza viruses.

49. loss of hypoxia signaling limits physiologic and muscle adaptations to aerobic exercise in aging

Abstract: PURPOSE: This study investigates signaling pathways which contribute to aging associated loss of muscle adaptation to exercise. METHODS: Young (Y, 12 weeks) and Old (O, 25 months) mice were subjected to regimented treadmill running or no exercise for 8 weeks. Mice were then subjected to muscle physiology testing. The transcriptional response to exercise was evaluated using RNA Seq. A genetically modified mouse model, with a deletion of aryl hydrocarbon receptor nuclear translocator (ARNT) was then evaluated for response to exercise training as it mimics old mice with regards to transcription profile and exercise response. RESULTS: Y, trained mice experienced a significant increase in distance running, speed, and lean muscle mass in comparison to untrained controls. O mice did not improve significantly in these measures. Transcriptome analysis in muscle from Y mice demonstrated differential regulation of 96 genes with exercise. None of these genes were similarly regulated in the O group. Genes most upregulated in Y mice are targets of hypoxia signaling. ARNT, a regulator of hypoxia signaling, increased 3-fold with exercise in Y mice, but not in O mice following exercise. To assess whether loss of ARNT impairs the exercise response, we generated a mouse with muscle-specific knockout of ARNT (ARNT muscle (mKO). Following regimented exercise, ARNT mKO mice did not improve maximal distance running, maximal running speed, or lean muscle mass. Administration of an ARNT agonist restored the exercise response. CONCLUSION: Restoration of ARNT and hypoxia signaling may restore the physiologic response to exercise in aging.

Potential Molecular Mechanism of Upregulated Aryl Hydrocarbon Receptor Nuclear Translocator 2 in Nasopharyngeal Carcinoma

Abstract: Background Currently, the benefits of nasopharyngeal carcinoma (NPC) therapy are limited, and it is necessary to further explore possible therapeutic targets. Aryl hydrocarbon receptor nuclear translocator 2 (ARNT2) has been extensively studied in other cancer species, but little has been explored in NPC. The aim of this study was to verify the expression level of ARNT2 and its underlying mechanism in NPC. Methods Datasets containing ARNT2 mRNA expression levels were retrieved and collected from various databases to explore the expression status of ARNT2 in NPC. ARNT2-related coexpressed genes, differential expressed genes, and target genes were obtained for functional enrichment analysis. The potential target gene of ARNT2 and their regulatory relationship were studied through ChIP-seq data. CIBERSORTx was used to assess the immune infiltration of NPC, and the association with ARNT2 expression was calculated through correlation analysis. Results ARNT2 was upregulated and possessed an excellent discriminatory capability in NPC samples. ARNT2 positively correlated target genes were clustered in pathways in cancer, while negatively correlated target genes were enriched in immune-related pathway. The ChIP-seq information of ARNT2 and histone showed that prostaglandin-endoperoxide synthase 2 (PTGS2) was a potential target gene of ARNT2. CIBERSORTx revealed the immunity status in NPC, and ARNT2 expression was correlated with infiltration of five immune cells. Conclusions ARNT2 is overexpressed in NPC and may regulate PTGS2 to participate in the cancer process. ARNT2 serves as a key oncogenic target in NPC patients.