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The Hypoxia Inducible Factor (HIF) transcription factors are imperative for cell adaption to low oxygen conditions and development; however, they also contribute to ischaemic disease and cancer. To identify novel genetic regulators which target the HIF pathway or small molecules for therapeutic use, cell-based reporter systems are commonly used. Here, we present a new, highly sensitive and versatile reporter system, NanoFIRE: a NanoLuciferase and Fluorescent Integrated Reporter Element. Under the control of a Hypoxic Response Element (HRE-NanoFIRE), this system is a robust sensor of HIF activity within cells and potently responds to both hypoxia and chemical inducers of the HIF pathway in a highly reproducible and sensitive manner, consistently achieving 20 to 150-fold induction across different cell types and a Z' score > 0.5. We demonstrate that the NanoFIRE system is adaptable via substitution of the response element controlling NanoLuciferase and show that it can report on the activity of the transcriptional regulator Factor Inhibiting HIF, and an unrelated transcription factor, the Progesterone Receptor. Furthermore, the lentivirus-mediated stable integration of NanoFIRE highlights the versatility of this system across a wide range of cell types, including primary cells. Together, these findings demonstrate that NanoFIRE is a robust reporter system for the investigation of HIF and other transcription factor-mediated signalling pathways in cells, with applications in high throughput screening for the identification of novel small molecule and genetic regulators.

Human aquaporin 1 (hAQP1) forms homotetrameric channels that facilitate fluxes of water and small solutes across cell membranes. In addition to water channel activity, hAQP1 displays non-selective monovalent cation-channel activity gated by intracellular cyclic GMP. Dual water and ion-channel activity of hAQP1, thought to regulate cell shape and volume, could offer a target for novel therapeutics relevant to controlling cancer cell invasiveness. This study probed properties of hAQP1 ion channels using proteoliposomes, which, unlike conventional cell-based systems such as Xenopus laevis oocytes, are relatively free of background ion channels. Histidine-tagged recombinant hAQP1 protein was synthesized and purified from the methylotrophic yeast, Pichia pastoris , and reconstituted into proteoliposomes for biophysical analyses. Osmotic water channel activity confirmed correct folding and channel assembly. Ion-channel activity of hAQP1-Myc-His 6 was recorded by patch-clamp electrophysiology with excised patches. In symmetrical potassium, the hAQP1-Myc-His 6 channels displayed coordinated gating, a single-channel conductance of approximately 75 pS, and multiple subconductance states. Applicability of this method for structure-function analyses was tested using hAQP1-Myc-His 6 D48A/D185A channels modified by site-directed mutations of charged Asp residues estimated to be adjacent to the central ion-conducting pore of the tetramer. No differences in conductance were detected between mutant and wild-type constructs, suggesting the open-state conformation could differ substantially from expectations based on crystal structures. Nonetheless, the method pioneered here for AQP1 demonstrates feasibility for future work defining structure-function relationships, screening pharmacological inhibitors, and testing other classes in the broad family of aquaporins for previously undiscovered ion-conducting capabilities., Competing Interests: The authors declare no competing interests., (© 2023 The Author(s).)

Single-minded 2 (SIM2) is a neuron-enriched basic Helix-Loop-Helix/PER-ARNT-SIM (bHLH/PAS) transcription factor essential for mammalian survival. SIM2 is located within the Down syndrome critical region (DSCR) of chromosome 21, and manipulation in mouse models suggests Sim2 may play a role in brain development and function. During the screening of a clinical exome sequencing database, nine SIM2 non-synonymous mutations were found which were subsequently investigated for impaired function using cell-based reporter gene assays. Many of these human variants attenuated abilities to activate transcription and were further characterized to determine the mechanisms underpinning their deficiencies. These included impaired partner protein dimerization, reduced DNA binding, and reduced expression and nuclear localization. This study highlighted several SIM2 variants found in patients with disabilities and validated a candidate set as potentially contributing to pathology., (© 2022 The Author(s).)

Aberrations in the excitatory/inhibitory balance within the brain have been associated with both intellectual disability (ID) and schizophrenia (SZ). The bHLH-PAS transcription factors NPAS3 and NPAS4 have been implicated in controlling the excitatory/inhibitory balance, and targeted disruption of either gene in mice results in a phenotype resembling ID and SZ. However, there are few human variants in NPAS3 and none in NPAS4 that have been associated with schizophrenia or neurodevelopmental disorders. From a clinical exome sequencing database we identified three NPAS3 variants and four NPAS4 variants that could potentially disrupt protein function in individuals with either developmental delay or ID. The transcriptional activity of the variants when partnered with either ARNT or ARNT2 was assessed by reporter gene activity and it was found that variants which truncated the NPAS3/4 protein resulted in a complete loss of transcriptional activity. The ability of loss-of-function variants to heterodimerise with neuronally enriched partner protein ARNT2 was then determined by co-immunoprecipitation experiments. It was determined that the mechanism for the observed loss of function was the inability of the truncated NPAS3/4 protein to heterodimerise with ARNT2. This further establishes NPAS3 and NPAS4 as candidate neurodevelopmental disorder genes.

Single-minded homologue 1 (SIM1) is a transcription factor with numerous different physiological and developmental functions. SIM1 is a member of the class I basic helix-loop-helix-PER-ARNT-SIM (bHLH-PAS) transcription factor family, that includes several other conserved proteins, including the hypoxia-inducible factors, aryl hydrocarbon receptor, neuronal PAS proteins, and the CLOCK circadian regulator. Recent studies of HIF-a-ARNT and CLOCK-BMAL1 protein complexes have revealed the organization of their bHLH, PASA, and PASB domains and provided insight into how these heterodimeric protein complexes form; however, experimental structures for SIM1 have been lacking. Here, we describe the first full-length atomic structural model for human SIM1 with its binding partner ARNT in a heterodimeric complex and analyze several pathogenic variants utilizing state-of-the-art simulations and algorithms. Using local and global positional deviation metrics, deductions to the structural basis for the individual mutants are addressed in terms of the deleterious structural reorganizations that could alter protein function. We propose new experiments to probe these hypotheses and examine an interesting SIM1 dynamic behavior. The conformational dynamics demonstrates conformational changes on local and global regions that represent a mechanism for dysfunction in variants presented. In addition, we used our ab initio hybrid model for further prediction of variant hotspots that can be engineered to test for counter variant (restoration of wild-type function) or basic research probe.

Context: Single-minded homologue 1 (SIM1) is a transcription factor with several physiological and developmental functions. Haploinsufficiency of SIM1 is associated with early-onset obesity with or without Prader-Willi-like (PWL) features and may exhibit incomplete penetrance., Case Description: Next-generation sequencing was performed for 2 male patients with obesity, including 1 man presenting with intellectual disability (ID), body mass index (BMI) of 47.4, and impulse-control disorder, and the other man with early obesity (BMI of 36); sequencing revealed a missense variant in SIM1 (c.2144G>T; p.G715V) in both individuals. Previous studies have identified several disease-associated variants that fall near the p.G715V variant within the C-terminal domain of SIM1. We examined p.G715V variant stability and activity in a doxycycline-inducible stable cell line transfected with an artificial reporter construct and either ARNT or ARNT2 as a partner protein., Conclusions: Functional testing of the p.G715V variant revealed a significant reduction in SIM1-mediated transcriptional activity. We also generated the first ab initio hybrid protein model for full-length SIM1 to show the predicted spatial relationship between p.G715V and other previously described variants in this region and identified a putative mutation hotspot within the C-terminus. Significant clinical heterogeneity has been observed in patients with SIM1 variants, particularly with regards to the PWL phenotype. In the patient with ID, a second variant of uncertain significance in CHD2 was identified that may contribute to his ID and behavioral disturbances, emphasizing the role of additional genetic modifiers., (© Endocrine Society 2019. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Objective: Genetic studies in obese rodents and humans can provide novel insights into the mechanisms involved in energy homeostasis., Methods: In this study, we genetically mapped the chromosomal region underlying the development of severe obesity in a mouse line identified as part of a dominant N-ethyl-N-nitrosourea (ENU) mutagenesis screen. We characterized the metabolic and behavioral phenotype of obese mutant mice and examined changes in hypothalamic gene expression. In humans, we examined genetic data from people with severe early onset obesity., Results: We identified an obese mouse heterozygous for a missense mutation (pR108W) in orthopedia homeobox (Otp), a homeodomain containing transcription factor required for the development of neuroendocrine cell lineages in the hypothalamus, a region of the brain important in the regulation of energy homeostasis. Otp R108W/+ mice exhibit increased food intake, weight gain, and anxiety when in novel environments or singly housed, phenotypes that may be partially explained by reduced hypothalamic expression of oxytocin and arginine vasopressin. R108W affects the highly conserved homeodomain, impairs DNA binding, and alters transcriptional activity in cells. We sequenced OTP in 2548 people with severe early-onset obesity and found a rare heterozygous loss of function variant in the homeodomain (Q153R) in a patient who also had features of attention deficit disorder., Conclusions: OTP is involved in mammalian energy homeostasis and behavior and appears to be necessary for the development of hypothalamic neural circuits. Further studies will be needed to investigate the contribution of rare variants in OTP to human energy homeostasis., (Copyright © 2017 The Authors. Published by Elsevier GmbH.. All rights reserved.)

Two decades of research into functions of the ubiquitous transcription factor HIF have revealed pervasive roles in development, oxygen homeostasis, metabolism, cancer and responses to ischemia. Unsurprisingly, HIF activities impinge on many pathologies, for which underlying molecular mechanisms are actively sought. HIF is a member of the heterodimeric bHLH/PAS family of transcription factors, a set of proteins that commonly function in developmental pathways and adaptive responses to environmental or physiological stress. Similarities in the mechanisms that regulate gene targeting by these transcription factors create opportunities for extensive crosstalk between family members. Data supporting pathway interactions between HIF1a and other bHLH/PAS factors, both collaborative and antagonistic, is beginning to surface in the areas of cancer, circadian rhythm, and immune responses. This review summarises the status of HIF1a-bHLH/PAS protein crosstalk and is dedicated to the memory of Lorenz Poellinger, a pioneer investigator into the molecular mechanisms of HIF, AHR, and ARNT bHLH/PAS factors., (Crown Copyright © 2017. Published by Elsevier Inc. All rights reserved.)

Transcription factors of the basic helix-loop-helix (bHLH) PER-ARNT-SIM (PAS) family generally have critical and nonredundant biological roles, but some bHLH PAS proteins compete for common cofactors or recognise similar DNA elements. Identifying factors that regulate function of bHLH PAS proteins, particularly in cells where multiple family members are coexpressed, is important for understanding bHLH PAS factor biology. This study identifies and characterises a novel interaction between melanoma-associated antigen D1 (MAGED1) and select members of the bHLH PAS transcription factor family. MAGED1 binds and positively regulates the transcriptional activity of family members SIM1, SIM2, NPAS4 and ARNT2, but does not interact with AhR, HIF1α and ARNT. This interaction is mediated by PAS repeat regions which also form the interface for bHLH PAS dimerisation, and accordingly MAGED1 is not found in complex with bHLH PAS dimers. We show that MAGED1 does not affect bHLH PAS protein levels and cannot be acting as a coactivator of transcriptionally active heterodimers, but rather appears to interact with nascent bHLH PAS proteins in the cytoplasm to enhance their function prior to nuclear import. As a selective regulator, MAGED1 may play an important role in the biology of these specific factors and in general bHLH PAS protein dynamics., (© 2016 Federation of European Biochemical Societies.)

Manipulation of gene expression to invoke loss of function (LoF) or gain of function (GoF) phenotypes is important for interrogating complex biological questions both in vitro and in vivo. Doxycycline (Dox)-inducible gene expression systems are commonly used although success is often limited by high background and insufficient sensitivity to Dox. Here we develop broadly applicable platforms for reliable, tightly controlled and reversible Dox-inducible systems for lentiviral mediated generation of cell lines or FLP Recombination-Mediated Cassette Exchange (RMCE) into the Collagen 1a1 (Col1a1) locus (FLP-In Col1a1) in mouse embryonic stem cells. We significantly improve the flexibility, usefulness and robustness of the Dox-inducible system by using Tetracycline (Tet) activator (Tet-On) variants which are more sensitive to Dox, have no background activity and are expressed from single Gateway-compatible constructs. We demonstrate the usefulness of these platforms in ectopic gene expression or gene knockdown in multiple cell lines, primary neurons and in FLP-In Col1a1 mouse embryonic stem cells. We also improve the flexibility of RMCE Dox-inducible systems by generating constructs that allow for tissue or cell type-specific Dox-inducible expression and generate a shRNA selection algorithm that can effectively predict potent shRNA sequences able to knockdown gene expression from single integrant constructs. These platforms provide flexible, reliable and broadly applicable inducible expression systems for studying gene function.

Factor inhibiting HIF (FIH, also known as HIF1AN) is an oxygen-dependent asparaginyl hydroxylase that regulates the hypoxia-inducible factors (HIFs). Several proteins containing ankyrin repeat domains (ARDs) have been characterised as substrates of FIH, although there is little evidence for a functional consequence of hydroxylation on these substrates. This study demonstrates that the transient receptor potential vanilloid 3 (TRPV3) channel is hydroxylated by FIH on asparagine 242 within the cytoplasmic ARD. Hypoxia, FIH inhibitors and mutation of asparagine 242 all potentiated TRPV3-mediated current, without altering TRPV3 protein levels, indicating that oxygen-dependent hydroxylation inhibits TRPV3 activity. This novel mechanism of channel regulation by oxygen-dependent asparaginyl hydroxylation is likely to extend to other ion channels., (© 2015. Published by The Company of Biologists Ltd.)

Objective: Several deletions of chromosome 6q, including SIM1, were reported in obese patients with developmental delay. Furthermore, rare loss-of-function SIM1 mutations were shown to contribute to severe obesity, yet the role of these mutations in developmental delay remained unclear. Here, SIM1 in children with neurodevelopmental abnormalities was screened and the functional effect of the identified mutations was investigated., Methods: SIM1 was sequenced in 283 children presenting with developmental delay and at least overweight. The effect of the identified mutations on SIM1 transcriptional activity in stable human cell lines was assessed using luciferase gene reporter assays., Results: Two novel mutations (c.886A>G/p.R296G and c.925A>G/p.S309G) in two boys with variable degrees of cognitive deficits and weight issues were identified. The child mutated for p.R296G presented with a generally more severe phenotype than the p.S309G carrier (obesity, compulsive eating, neonatal hypotonia versus overweight only), while both mutations had strong loss-of-function effects on SIM1 transcriptional activity., Conclusions: Severe loss-of-function SIM1 mutations can be associated with a spectrum of developmental delay phenotypes and obesity. Our data suggest that SIM1 sequencing should be performed more systematically in patients with developmental delay, even in the absence of severe obesity. These results deserve further SIM1 screening studies., (© 2014 The Obesity Society.)

The bHLH (basic helix-loop-helix) PAS (Per/Arnt/Sim) transcription factor SIM1 (single-minded 1) is important for development and function of regions of the hypothalamus that regulate energy homoeostasis and the feeding response. Low-activity SIM1 variants have been identified in individuals with severe early-onset obesity, but the underlying molecular causes of impaired function are unknown. In the present study we assess a number of human SIM1 variants with reduced activity and determine that impaired function is frequently due to defects in dimerization with the essential partner protein ARNT2 (aryl hydrocarbon nuclear translocator 2). Equivalent variants generated in the highly related protein SIM2 (single-minded 2) produce near-identical impaired function and dimerization defects, indicating that these effects are not unique to the structure of SIM1. On the basis of these data, we predict that other select SIM1 and SIM2 variants reported in human genomic databases will also be deficient in activity, and identify two new low-activity SIM1 variants (V290E and V326F) present in the population. The cumulative data is used in homology modelling to make novel observations about the dimerization interface between the PAS domains of SIM1 and ARNT2, and to define a mutational 'hot-spot' in SIM1 that is critical for protein function.

Neuronal Per-Arnt-Sim homology (PAS) Factor 4 (NPAS4) is a neuronal activity-dependent transcription factor which heterodimerises with ARNT2 to regulate genes involved in inhibitory synapse formation. NPAS4 functions to maintain excitatory/inhibitory balance in neurons, while mouse models have shown it to play roles in memory formation, social interaction and neurodegeneration. NPAS4 has therefore been implicated in a number of neuropsychiatric or neurodegenerative diseases which are underpinned by defects in excitatory/inhibitory balance. Here we have explored a broad set of non-synonymous human variants in NPAS4 and ARNT2 for disruption of NPAS4 function. We found two variants in NPAS4 (F147S and E257K) and two variants in ARNT2 (R46W and R107H) which significantly reduced transcriptional activity of the heterodimer on a luciferase reporter gene. Furthermore, we found that NPAS4.F147S was unable to activate expression of the NPAS4 target gene BDNF due to reduced dimerisation with ARNT2. Homology modelling predicts F147 in NPAS4 to lie at the dimer interface, where it appears to directly contribute to protein/protein interaction. We also found that reduced transcriptional activation by ARNT2 R46W was due to disruption of nuclear localisation. These results provide insight into the mechanisms of NPAS4/ARNT dimerisation and transcriptional activation and have potential implications for cognitive phenotypic variation and diseases such as autism, schizophrenia and dementia.

NPAS4 is a brain restricted, activity-induced transcription factor which regulates the expression of inhibitory synapse genes to control homeostatic excitatory/inhibitory balance in neurons. NPAS4 is required for normal social interaction and contextual memory formation in mice. Protein and mRNA expression of NPAS4 is tightly coupled to neuronal depolarization and most prevalent in the cortical and hippocampal regions in the brain, however the precise mechanisms by which the NPAS4 gene is controlled remain unexplored. Here we show that expression of NPAS4 mRNA is actively repressed by RE-1 silencing transcription factor/neuron-restrictive silencer factor (REST/NRSF) in embryonic stem cells and non-neuronal cells by binding multiple sites within the promoter and Intron I of NPAS4. Repression by REST also appears to correlate with the binding of the zinc finger DNA binding protein CTCF within Intron I of NPAS4. In addition, we show that the 3' untranslated region (3'UTR) of NPAS4 can be targeted by two microRNAs, miR-203 and miR-224 to further regulate its expression. miR-224 is a midbrain/hypothalamus enriched microRNA which is expressed from an intron within the GABAA receptor epsilon (GABRE) gene and may further regionalize NPAS4 expression. Our results reveal REST and microRNA dependent mechanisms that restrict NPAS4 expression to the brain., (Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.)

Mammalian basic HLH (helix-loop-helix)-PER-ARNT-SIM (bHLH-PAS) proteins are heterodimeric transcription factors that sense and respond to environmental signals (such as pollutants) or to physiological signals (for example, hypoxia and circadian rhythms) through their two PAS domains. PAS domains form a generic three-dimensional fold, which commonly contains an internal cavity capable of small-molecule binding and outer surfaces adept at protein-protein interactions. These proteins are important in several pro-tumour and antitumour pathways and their activities can be modulated by both natural metabolites and oncometabolites. Recently determined structures and successful small-molecule screening programmes are now providing new opportunities to discover selective agonists and antagonists directed against this multitasking family of transcription factors.

The aryl hydrocarbon receptor (AhR) is traditionally defined as a transcriptional regulator involved in adaptive xenobiotic response, however, emerging evidence supports physiological functions of AhR in normal cell development and immune response. The role of AhR in immunomodulation is multi-dimensional. On the one hand, activation of AhR by TCDD and other ligands leads to profound immunosuppression, potentially via skewed Th1/Th2 cell balance toward Th1 dominance, and boosted Treg cell differentiation. On the other hand, activation of AhR can also induce Th17 cell polarization and increase the severity of autoimmune disease. In addition to T lymphocytes, the AhR also appears to play a vital role in B cell maturation, and regulates the activity of macrophages, dendritic cells and neutrophils following lipopolysaccharide challenge or influenza virus infection. In these scenarios, activation of AhR is associated with decreased host response and reduced survival. Furthermore, gene knock out studies suggest that AhR is indispensable for the postnatal maintenance of intestinal intraepithelial lymphocytes and skin-resident dendritic epidermal gamma delta T cells, providing a potential link between AhR and gut immunity and wound healing. It is well accepted that the magnitude and the type of immune response is dependent on the local cytokine milieu and the AhR appears to be one of the key factors involved in the fine turning of this cytokine balance., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Single-minded 1 (SIM1) is a basic helix-loop-helix transcription factor involved in the development and function of the paraventricular nucleus of the hypothalamus. Obesity has been reported in Sim1 haploinsufficient mice and in a patient with a balanced translocation disrupting SIM1. We sequenced the coding region of SIM1 in 2,100 patients with severe, early onset obesity and in 1,680 controls. Thirteen different heterozygous variants in SIM1 were identified in 28 unrelated severely obese patients. Nine of the 13 variants significantly reduced the ability of SIM1 to activate a SIM1-responsive reporter gene when studied in stably transfected cells coexpressing the heterodimeric partners of SIM1 (ARNT or ARNT2). SIM1 variants with reduced activity cosegregated with obesity in extended family studies with variable penetrance. We studied the phenotype of patients carrying variants that exhibited reduced activity in vitro. Variant carriers exhibited increased ad libitum food intake at a test meal, normal basal metabolic rate, and evidence of autonomic dysfunction. Eleven of the 13 probands had evidence of a neurobehavioral phenotype. The phenotypic similarities between patients with SIM1 deficiency and melanocortin 4 receptor (MC4R) deficiency suggest that some of the effects of SIM1 deficiency on energy homeostasis are mediated by altered melanocortin signaling.

Sim1 haploinsufficiency in mice induces hyperphagic obesity and developmental abnormalities of the brain. In humans, abnormalities in chromosome 6q16, a region that includes SIM1, were reported in obese children with a Prader-Willi-like syndrome; however, SIM1 involvement in obesity has never been conclusively demonstrated. Here, SIM1 was sequenced in 44 children with Prader-Willi-like syndrome features, 198 children with severe early-onset obesity, 568 morbidly obese adults, and 383 controls. We identified 4 rare variants (p.I128T, p.Q152E, p.R581G, and p.T714A) in 4 children with Prader-Willi-like syndrome features (including severe obesity) and 4 other rare variants (p.T46R, p.E62K, p.H323Y, and p.D740H) in 7 morbidly obese adults. By assessing the carriers' relatives, we found a significant contribution of SIM1 rare variants to intra-family risk for obesity. We then assessed functional effects of the 8 substitutions on SIM1 transcriptional activities in stable cell lines using luciferase gene reporter assays. Three mutations showed strong loss-of-function effects (p.T46R, p.H323Y, and p.T714A) and were associated with high intra-family risk for obesity, while the variants with mild or no effects on SIM1 activity were not associated with obesity within families. Our genetic and functional studies demonstrate a firm link between SIM1 loss of function and severe obesity associated with, or independent of, Prader-Willi-like features.

Basic helix-loop-helix/Per-Arnt-Sim (bHLH/PAS) transcription factors function broadly in development, homeostasis and stress response. Active bHLH/PAS heterodimers consist of a ubiquitous signal-regulated subunit (e.g., hypoxia-inducible factors, HIF-1α/2α/3α; the aryl hydrocarbon receptor, AhR) or tissue-restricted subunit (e.g., NPAS1/3/4, Single Minded 1/2), paired with a general partner protein, aryl hydrocarbon receptor nuclear translocator (Arnt or Arnt2). We have investigated regulation of the neuron-enriched Arnt paralogue, Arnt2. We find high Arnt/Arnt2 ratios in P19 embryonic carcinoma cells and ES cells are dramatically reversed to high Arnt2/Arnt on neuronal differentiation. mRNA half-lives of Arnt and Arnt2 remain similar in both parent and neuronal differentiated cells. The GC-rich Arnt2 promoter, while heavily methylated in Arnt only expressing hepatoma cells, is methylation free in P19 and ES cells, where it is bivalent with respect to active H3K4me3 and repressive H3K27me3 histone marks. Typical of a 'transcription poised' developmental gene, H3K27me3 repressive marks are removed from Arnt2 during neuronal differentiation. Our data are consistent with a switch to predominant Arnt2 expression in neurons to allow specific functions of neuronal bHLH/PAS factors and/or to avoid neuronal bHLH/PAS factors from interfering with AhR/Arnt signalling.

The aryl hydrocarbon receptor (AhR) is a signal-regulated transcription factor, which is canonically activated by the direct binding of xenobiotics. In addition, switching cells from adherent to suspension culture also activates the AhR, representing a nonxenobiotic, physiological activation of AhR signaling. Here, we show that the AhR is recruited to target gene enhancers in both ligand [isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439)]-treated and suspension cells, suggesting a common mechanism of target gene induction between these two routes of AhR activation. However, gene expression profiles critically differ between xenobiotic- and suspension-activated AhR signaling. Por and Cldnd1 were regulated predominantly by ligand treatments, whereas, in contrast, ApoER2 and Ganc were regulated predominantly by the suspension condition. Classic xenobiotic-metabolizing AhR targets such as Cyp1a1, Cyp1b1, and Nqo1 were regulated by both ligand and suspension conditions. Temporal expression patterns of AhR target genes were also found to vary, with examples of transient activation, transient repression, or sustained alterations in expression. Furthermore, sequence analysis coupled with chromatin immunoprecipitation assays and reporter gene analysis identified a functional xenobiotic response element (XRE) in the intron 1 of the mouse Tiparp gene, which was also bound by hypoxia-inducible factor-1α during hypoxia and features a concatemer of four XRE cores (GCGTG). Our data suggest that this XRE concatemer site concurrently regulates the expression of both the Tiparp gene and its cis antisense noncoding RNA after ligand- or suspension-induced AhR activation. This work provides novel insights into how AhR signaling drives different transcriptional programs via the ligand versus suspension modes of activation.

The basic helix-loop-helix (bHLH).PAS dimeric transcription factors have crucial roles in development, stress response, oxygen homeostasis and neurogenesis. Their target gene specificity depends in part on partner protein choices, where dimerization with common partner Aryl hydrocarbon receptor nuclear translocator (Arnt) is an essential step towards forming active, DNA binding complexes. Using a new bacterial two-hybrid system that selects for loss of protein interactions, we have identified 22 amino acids in the N-terminal PAS domain of Arnt that are involved in heterodimerization with aryl hydrocarbon receptor (AhR). Of these, Arnt E163 and Arnt S190 were selective for the AhR/Arnt interaction, since mutations at these positions had little effect on Arnt dimerization with other bHLH.PAS partners, while substitution of Arnt D217 affected the interaction with both AhR and hypoxia inducible factor-1α but not with single minded 1 and 2 or neuronal PAS4. Arnt uses the same face of the N-terminal PAS domain for homo- and heterodimerization and mutational analysis of AhR demonstrated that the equivalent region is used by AhR when dimerizing with Arnt. These interfaces differ from the PAS β-scaffold surfaces used for dimerization between the C-terminal PAS domains of hypoxia inducible factor-2α and Arnt, commonly used for PAS domain interactions., (© The Author(s) 2011. Published by Oxford University Press.)

Background: Without appropriate cellular models the etiology of idiopathic Parkinson's disease remains unknown. We recently reported a novel patient-derived cellular model generated from biopsies of the olfactory mucosa (termed olfactory neurosphere-derived (hONS) cells) which express functional and genetic differences in a disease-specific manner. Transcriptomic analysis of Patient and Control hONS cells identified the NRF2 transcription factor signalling pathway as the most differentially expressed in Parkinson's disease., Results: We tested the robustness of our initial findings by including additional cell lines and confirmed that hONS cells from Patients had 20% reductions in reduced glutathione levels and MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium, inner salt] metabolism compared to cultures from healthy Control donors. We also confirmed that Patient hONS cells are in a state of oxidative stress due to higher production of H(2)O(2) than Control cultures. siRNA-mediated ablation of NRF2 in Control donor cells decreased both total glutathione content and MTS metabolism to levels detected in cells from Parkinson's Disease patients. Conversely, and more importantly, we showed that activation of the NRF2 pathway in Parkinson's disease hONS cultures restored glutathione levels and MTS metabolism to Control levels. Paradoxically, transcriptomic analysis after NRF2 pathway activation revealed an increased number of differentially expressed mRNAs within the NRF2 pathway in L-SUL treated Patient-derived hONS cells compared to L-SUL treated Controls, even though their metabolism was restored to normal. We also identified differential expression of the PI3K/AKT signalling pathway, but only post-treatment., Conclusions: Our results confirmed NRF2 as a potential therapeutic target for Parkinson's disease and provided the first demonstration that NRF2 function was inducible in Patient-derived cells from donors with uniquely varied genetic backgrounds. However, our results also demonstrated that the response of PD patient-derived cells was not co-ordinated in the same way as in Control cells. This may be an important factor when developing new therapeutics.

The aryl hydrocarbon receptor (AhR) is traditionally defined as a transcription factor activated by exogenous polyaromatic and halogenated aromatic hydrocarbon (PAH/HAH) ligands. Active AhR induces genes involved in xenobiotic metabolism, including cytochrome P4501A1, which function to metabolize activating ligands. However, recent studies implicate AhR in biological events that are apparently unrelated to the xenobiotic response, implying that endogenous activation mechanisms exist. Three AhR genes in zebrafish (Danio rerio) encode proteins that demonstrate differential activation in response to PAH/HAHs, with the nonresponsive drAhR1a having some sequence divergence from the PAH/HAH-responsive AhRs in the ligand binding domain (LBD). We used these differences to guide the mutagenesis of mouse AhR (mAhR), aiming to generate variants that functionally discriminate between activation mechanisms. We found substitution of histidine 285 in the LBD with tyrosine gave a receptor that could be activated by isopropyl-2-(1,3-dithietane-2-ylidene)-2-[N-(4-methylthiazol-2-yl)carbamoyl]acetate (YH439), a potential AhR ligand chemically distinct from classic PAH/HAH-type ligands, but prevented activation by both exogenous PAH/HAH ligands and the endogenous activation mimics of suspension culture and application of shear-stressed serum. The differential response of H285Y mAhR to YH439 suggests that this activator has a novel mode of interaction that tolerates tyrosine at position 285 in the LBD and is distinct from the binding mode of the well characterized PAH/HAH ligands. In support of this, the PAH-type antagonist 3',4'-dimethoxyflavone blocked mAhR activation by 2,3,7,8-tetrachlorodibenzo-p-dioxin but not YH439. Furthermore, the strict correlation between response to exogenous PAH/HAH ligands and mimics of endogenous activation suggests that a PAH-type ligand may underpin endogenous mechanisms of activation.

The short isoform of single-minded 2 (SIM2s), a basic helix-loop-helix/PAS (bHLH/PAS) transcription factor, is upregulated in pancreatic and prostate tumours; however, a mechanistic role for SIM2s in these cancers is unknown. Microarray studies in prostate DU145 cells identified the pro-cell death gene, BNIP3 (Bcl-2/adenovirus E1B 19 kDa interacting protein 3), as a novel putative target of SIM2s repression. Further validation showed BNIP3 repression in several prostate and pancreatic carcinoma-derived cell lines with ectopic expression of human SIM2s. BNIP3 levels are enhanced in prostate carcinoma cells upon short interfering (si)RNA-mediated knockdown of endogenous SIM2s. Chromatin immunoprecipitation and promoter studies show that SIM2s represses BNIP3 through its activities at the proximal promoter hypoxia response element (HRE), the site through which the bHLH/PAS family member, hypoxia-inducible factor 1alpha (HIF1alpha), induces BNIP3. SIM2s attenuates BNIP3 hypoxic induction via the HRE, and increased hypoxic induction of BNIP3 occurs with siRNA knockdown of endogenous SIM2s in prostate PC3AR+ cells. BNIP3 is implicated in hypoxia-induced cell death processes. Prolonged treatment of PC3AR+ cells with hypoxia mimetics, DP and DMOG, confers hypoxia-induced autophagy, measured by enhanced LC3-II levels and SQSTM1/p62 turnover. We show that PC3AR+ cells expressing ectopic SIM2s have enhanced survival in these conditions. Induction of LC3-II and turnover of SQSTM1/p62 are attenuated in PC3AR+/SIM2s DMOG and hypoxia-treated cells, suggesting that SIM2s may attenuate autophagic cell death processes, perhaps through BNIP3 repression. These data show, for the first time, SIM2s cross-talk on an endogenous HRE. SIM2s' functional interference with HIF1alpha activities on BNIP3 may indicate a novel role for SIM2s in promoting tumourigenesis.

Tagged murine dioxin receptor was purified from mammalian cells, digested with trypsin, and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. Several chromatographically distinct semitryptic peptides matching two regions spanning residues Glu(409)-Arg(424) and Ser(547)-Arg(555) of the dioxin receptor were revealed by de novo sequencing. Methionine residues at 418 and 548 were detected in these peptides as either unmodified or modified by moieties of 16 (oxidation) or 57 amu (S-carboxamidomethylation) or in a form corresponding to degradative removal of 105 amu from the S-carboxamidomethylated methionine. MS/MS spectra revealed that the peptides containing modified methionine residues also existed in forms with a modification of +80 amu on serine residues 411, 415, and 547. The MS/MS spectra of these peptide ions also revealed diagnostic neutral loss fragment ions of 64, 98, and/or 80 amu, and in some instances combinations of these neutral losses were apparent. Taken together, these data indicated that serines 411 and 547 of the dioxin receptor were sulfonated and serine 415 was phosphorylated. Separate digests of the dioxin receptor were prepared in H(2)(16)O and H(2)(18)O, and enzymatic dephosphorylation was subsequently performed on the H(2)(16)O digest only. The digests were mixed in equal proportions and analyzed by capillary HPLC-MALDI-TOF/TOF-MS and -MS/MS. This strategy confirmed assignment of sulfonation as the cause of the +80-amu modifications on serines 411 and 547 and phosphorylation as the predominant cause of the +80-amu modification of serine 415. The relative quantitation of phosphorylation and sulfonation enabled by this differential phosphatase strategy also suggested the presence of sulfonation on a serine other than residue 411 within the sequence spanning Glu(409)-Arg(424). This represents the first description of post-translational sulfonation sites and identification of a new phosphorylation site of the latent dioxin receptor. Furthermore this is only the second report of serine sulfonation of eukaryotic proteins. Mutagenesis studies are underway to assess the functional consequences of these modifications.

Despite a growing number of descriptive studies that show Single-minded 2 (Sim2) is not only essential for murine survival, but also upregulated in colon, prostate and pancreatic tumours, there is a lack of direct target genes identified for this basic helix-loop-helix/PAS transcription factor. We have performed a set of microarray experiments aimed at identifying genes that are differentially regulated by SIM2, and successfully verified that the Myomesin2 (Myom2) gene is SIM2-responsive. Although SIM2 has been reported to be a transcription repressor, we find that SIM2 induces transcription of Myom2 and activates the Myom2 promoter sequence when co-expressed with the heterodimeric partner protein, ARNT1, in human embryonic kidney cells. Truncation and mutation of the Myom2 promoter sequence, combined with chromatin immunoprecipitation studies in cells, has lead to the delineation of a non-canonical E-box sequence 5'-AACGTG-3' that is bound by SIM2/ARNT1 heterodimers. Interestingly, in immortalized human myoblasts knock down of Sim2 results in increased levels of Myom2 RNA, suggesting that SIM2 is acting as a repressor in these cells and so its activity is likely to be highly context dependent. This is the first report of a direct SIM2/ARNT1 target gene with accompanying analysis of a functional response element.

Cells adapt to hypoxia by a cellular response, where hypoxia-inducible factor 1alpha (HIF-1alpha) becomes stabilized and directly activates transcription of downstream genes. In addition to this "canonical" response, certain aspects of the pathway require integration with Notch signaling, i.e., HIF-1alpha can interact with the Notch intracellular domain (ICD) to augment the Notch downstream response. In this work, we demonstrate an additional level of complexity in this cross-talk: factor-inhibiting HIF-1 (FIH-1) regulates not only HIF activity, but also the Notch signaling output and, in addition, plays a role in how Notch signaling modulates the hypoxic response. We show that FIH-1 hydroxylates Notch ICD at two residues (N(1945) and N(2012)) that are critical for the function of Notch ICD as a transactivator within cells and during neurogenesis and myogenesis in vivo. FIH-1 negatively regulates Notch activity and accelerates myogenic differentiation. In its modulation of the hypoxic response, Notch ICD enhances recruitment of HIF-1alpha to its target promoters and derepresses HIF-1alpha function. Addition of FIH-1, which has a higher affinity for Notch ICD than for HIF-1alpha, abrogates the derepression, suggesting that Notch ICD sequesters FIH-1 away from HIF-1alpha. In conclusion, the data reveal posttranslational modification of the activated form of the Notch receptor and an intricate mode of cross-coupling between the Notch and hypoxia signaling pathways.

This review examines the common theme of adaptive responses of bHLH/PAS proteins, using the dioxin receptor as a prototype. The bHLH/PAS family of transcriptional regulators are a group of key developmental and environmental stress sensing proteins. They employ a variety of post-translational control mechanisms to regulate their transcriptional output. Amongst this family, the dioxin receptor is best known for its ability to elicit toxic responses to dioxin and dioxin like chemicals even though it mediates more benign adaptive responses to non-toxic xenobiotics. We discuss what is known about dioxin receptor physiology, both adaptive and inherent, along with its molecular regulation and put this into the context of the wider bHLH/PAS family. We also raise the issue of its toxic responses, in particular the idea that it is the dysregulation of its poorly characterised housekeeping functions that leads to these outcomes.

The hypoxia-inducible factor (HIF)-1alpha and HIF-2alpha are closely related, key transcriptional regulators of the hypoxic response, countering a low oxygen situation with the up-regulation of target genes associated with numerous processes, including vascularization and glycolysis. This involves a dual mechanism of control through both stabilization and transactivation, regulated via prolyl and asparaginyl hydroxylation. Despite high similarity with respect to protein sequence and activation pathway, a growing number of physiological and mechanistic differences between HIF-1alpha and HIF-2alpha are being reported. To further characterize this nonredundancy, the stabilization of endogenous proteins and regulation of the transactivation domains were compared in a graded oxygen environment across a series of cell lines. Although generally similar results were found, interesting and specific differences between the HIF-alpha proteins were observed within certain cell lines, such as rat adrenal PC12s, emphasizing the cell-specific nature of HIF-alpha regulation. We characterize a conserved amino acid substitution between HIF-1alpha and HIF-2alpha that contributes to the intrinsically higher FIH-1-mediated asparaginyl hydroxylation of HIF-1alpha and, hence, lower HIF-1alpha activity. In addition, our data demonstrate that the different cell lines can be classified into two distinct groups: those in which stabilization and transactivation proceed in conjunction (HeLa, 293T, and COS-1) and those cells in which HIF-alpha is stabilized prior to transactivation (PC12, HepG2, and CACO2). Interestingly, the initial stabilization of HIF-alpha prior to transactivation up-regulation predicted from in vitro derived hydroxylation data is only true for a subset of cells.

Central issues surrounding the basic helix-loop-helix (bHLH) superfamily of dimeric transcription factors concern how specificity of partner selection and DNA binding are achieved. bHLH proteins bind DNA through the basic sequence that is contiguous with a helix-loop-helix dimerization domain. For the two subgroups within the family, dimerization is further regulated by an adjacent Per-Arnt-Sim homology (PAS) or leucine zipper (LZ) domain. We provide evidence that for the bHLH.PAS transcription factors Dioxin Receptor (DR) and Arnt, the DR PAS A domain has a unique interaction with the bHLH region that underpins both dimerization strength and affinity for an atypical E-box DNA sequence. A PAS swap heterodimer, where the DR bHLH domain was fused to Arnt PAS A and the Arnt bHLH fused to DR PAS A, gave strong DNA binding, but dimerization was only effective with the native arrangement, suggesting the PAS A domain is critical for each process via distinct mechanisms. LZ domains, which regulate heterodimerization for the bHLH.LZ family members Myc and Max, could not replace the PAS domains for either dimerization or DNA binding in the DR/Arnt heterodimer. In vitro footprinting revealed that the PAS domains influence the conformation of target DNA in a manner consistent with DNA bending. These results provide the first insights for understanding mechanisms of selective dimerization and DNA interaction that distinguish bHLH.PAS proteins from the broader bHLH superfamily.

The basic helix-loop-helix/PER-ARNT-SIM homology (bHLH/PAS) transcription factor ARNT (aryl hydrocarbon receptor nuclear translocator) is a key component of various pathways which induce the transcription of cytochrome P450 and hypoxia response genes. ARNT can be alternatively spliced to express Alt ARNT, containing an additional 15 amino acids immediately N-terminal to the DNA-binding basic region. Here, we show that ARNT and Alt ARNT proteins are differentially phosphorylated by protein kinase CKII in vitro. Phosphorylation had an inhibitory effect on DNA-binding to an E-box probe by Alt ARNT, but not ARNT, homodimers. This inhibitory phosphorylation occurs through Ser77. Moreover, a point mutant, Alt ARNT S77A, shows increased activity on an E-box reporter gene, consistent with Ser77 being a regulatory site in vivo. In contrast, DNA binding by an Alt ARNT/dioxin receptor heterodimer to the xenobiotic response element is not inhibited by phosphorylation with CKII, nor does Alt ARNT S77A behave differently from wild type Alt ARNT in the context of a dioxin receptor heterodimer.

The hypoxia-inducible factors 1alpha (HIF-1alpha) and 2alpha (HIF-2alpha) are key regulators of the transcriptional response to low oxygen and are closely related in domain architecture, DNA binding, and activation mechanisms. Despite these similarities, targeted disruption of the HIF-alpha genes in mice results in distinctly different phenotypes demonstrating nonredundancy of function, although the underlying mechanisms remain unclear. Here we report on the novel and specific interaction of HIF-2alpha, but not HIF-1alpha, with the NF-kappaB essential modulator (NEMO) using immunoprecipitation, mammalian two-hybrid, and in vitro protein interaction assays. Reporter gene assays demonstrate that this interaction specifically enhances normoxic HIF-2alpha transcriptional activity, independently of the HIF-2alpha transactivation domain, consistent with a model by which NEMO aids CBP/p300 recruitment to HIF-2alpha. In contrast, HIF-2alpha overexpression does not alter NF-kappaB signaling, suggesting that the functional consequence of the HIF-2alpha/NEMO interaction is limited to the HIF pathway. The specificity of NEMO for HIF-2alpha represents one of the few known differential protein-protein interactions between the HIF-alpha proteins, which has important implications for the activity of HIF-2alpha and is also the first postulated NF-kappaB-independent role for NEMO.

The hypoxia-inducible factor alpha subunits 1 and 2 (HIF-1alpha and HIF-2alpha) are subjected to oxygen-dependent asparaginyl hydroxylation, a modification that represses the carboxyl-terminal transactivation domain (CAD) at normoxia by preventing recruitment of the p300/cAMP-response element-binding protein coactivators. This hydroxylation is performed by the novel asparaginyl hydroxylase, factor-inhibiting HIF-1' (FIH-1), of which HIF-1alpha and HIF-2alpha are the only reported substrates. Here we investigated the substrate requirements of FIH-1 by characterizing its subcellular localization and by examining amino acids within the HIF-1alpha substrate for their importance in recognition and catalysis by FIH-1. Using immunohistochemistry, we showed that both endogenous and transfected FIH-1 are primarily confined to the cytoplasm and remain there under normoxia and following treatment with the hypoxia mimetic, dipyridyl. Individual alanine mutations of seven conserved amino acids flanking the hydroxylated asparagine in HIF-1alpha revealed the importance of the valine (Val-802) adjacent to the targeted asparagine. The HIF-1alpha CAD V802A mutant exhibited a 4-fold lower V(max) in enzyme assays, whereas all other mutants were hydroxylated as efficiently as the wild type HIF-1alpha CAD. Furthermore, in cell-based assays the transcriptional activity of V802A was constitutive, suggesting negligible normoxic hydroxylation in HEK293T cells, whereas the wild type and other mutants were repressed under normoxia. Molecular modeling of the HIF-1alpha CAD V802A in complex with FIH-1 predicted an alteration in asparagine positioning compared with the wild type HIF-1alpha CAD, providing an explanation for the impaired catalysis observed and confirming the importance of Val-802 in asparaginyl hydroxylation by FIH-1.

The basic helix-loop-helix (bHLH) PAS transcriptional regulators control critical developmental and metabolic processes, including transcriptional responses to stimuli such as hypoxia and environmental pollutants, mediated respectively by hypoxia inducible factors (HIF-alpha) and the dioxin (aryl hydrocarbon) receptor (DR). The bHLH proteins contain a basic DNA binding sequence adjacent to a helix-loop-helix dimerization domain. Dimerization among bHLH.PAS proteins is additionally regulated by the PAS region, which controls the specificity of partner choice such that HIF-alpha and DR must dimerize with the aryl hydrocarbon nuclear translocator (Arnt) to form functional DNA binding complexes. Here, we have analyzed purified bacterially expressed proteins encompassing the N-terminal bHLH and bHLH.PAS regions of Arnt, DR, and HIF-1alpha and evaluated the contribution of the PAS domains to DNA binding in vitro. Recovery of functional DNA binding proteins from bacteria was dramatically enhanced by coexpression of the bHLH.PAS regions of DR or HIF-1alpha with the corresponding region of Arnt. Formation of stable protein-DNA complexes by DR/Arnt and HIF-1alpha/Arnt heterodimers with their cognate DNA sequences required the PAS A domains and exhibited KD values of 0.4 nM and approximately 50 nM, respectively. In contrast, the presence of the PAS domains of Arnt had little effect on DNA binding by Arnt homodimers, and these bound DNA with a KD of 45 nM. In the case of the DR, both high affinity DNA binding and dimer stability were specific to its native PAS domain, since a chimera in which the PAS A domain was substituted with the equivalent domain of Arnt generated a destabilized protein that bound DNA poorly.

Basic helix-loop-helix (bHLH)/PAS proteins are critical regulators of gene expression networks underlying many essential physiological and developmental processes. These include transcriptional responses to environmental pollutants and low oxygen tension, mediated by the aryl hydrocarbon (Dioxin) receptor and hypoxia inducible factors (HIF), respectively, and controlling aspects of neural development, mediated by the single minded (SIM) proteins. bHLH proteins must dimerise to form functional DNA binding complexes and bHLH/PAS proteins are distinguished from other members of the broader bHLH superfamily by the dimerisation specificity conferred by their PAS homology domains. bHLH/PAS proteins tend to be ubiquitous, latent signal-regulated transcription factors that often recognise variant forms of the classic E-box enhancer sequence bound by other bHLH proteins. Two closely related forms of each of the hypoxia inducible factors alpha and single minded proteins and the general partner protein, aryl hydrocarbon receptor nuclear translocator (ARNT), are present in many cell types. Despite high sequence conservation within their DNA binding and dimerisation domains, and having very similar DNA recognition specificities, the homologues are functionally non-redundant and biologically essential. While the mechanisms controlling partner choice and target gene activation that determine this functional specificity are poorly understood, interactions mediated by the PAS domains are essential. Information on structures and protein/protein interactions for members of the steroid hormone/nuclear receptor superfamily has contributed to our understanding of the way these receptors function and assisted the development of highly specific agonists and antagonists. Similarly, it is anticipated that developing a detailed mechanistic and structural understanding of bHLH/PAS proteins will ultimately facilitate drug design.

The dioxin receptor (DR) is a ligand-activated transcription factor that is activated upon binding of dioxins or structurally related forms of xenobiotics. Upon binding ligand the DR translocates from the cytoplasm to the nucleus where it complexes with the partner protein Arnt to form a DNA binding heterodimer, which activates transcription of target genes involved in xenobiotic metabolism. Latency of the DR signaling pathway is maintained by association of the DR with a number of molecular chaperones including the 90-kDa heat shock protein (hsp90), the hepatitis B virus X-associated protein (XAP2), and the 23-kDa heat shock protein (p23). Here we investigated the role of XAP2 in DR signaling and demonstrated that reduced levels of XAP2 labilize the DR, arguing for a function of XAP2 beyond its reported role as a cytoplasmic retention factor. In addition, we showed that a constitutively nuclear DR is degraded in the nucleus and does not require nuclear export for efficient degradation. We also provided evidence implicating the ubiquitin ligase protein C-terminal hsp70-interacting protein (CHIP) in the degradation of the DR, and we demonstrated that this degradation can be overcome by overexpression of XAP2. XAP2 protection of CHIP-mediated degradation is dependent on the tetratricopeptide repeat domain of XAP2 and suggests a mechanism whereby competition for the C-terminal tetratricopeptide repeat acceptor site of hsp90 guides the protein triage decision, the point of determination for either maturation of DR folding or DR degradation.

Oxygen depravation in mammals leads to the transcriptional induction of a host of target genes to metabolically adapt to this deficiency, including erythropoietin and vascular endothelial growth factor. This response is primarily mediated by the hypoxia-inducible factors (HIFs) which are members of the basic-helix-loop-helix/Per-ARNT-Sim (bHLH/PAS) transcription factor family. The HIFs are primarily regulated via a two-step mechanism of HIF post-translational modification, increasing both protein stability and transactivation capacity. This review aims to summarise our current understanding of these processes, and discuss the important role of the HIFs in the pathophysiology of many human diseases.

To sustain life mammals have an absolute and continual requirement for oxygen, which is necessary to produce energy for normal cell survival and growth. Hence, maintaining oxygen homeostasis is a critical requirement and mammals have evolved a wide range of cellular and physiological responses to adapt to changes in oxygen availability. In the past few years it has become evident that the transcriptional protein complex hypoxia-inducible factor (HIF) is a key regulator of these processes. In this review we will focus on the way oxygen availability regulates HIF proteins and in particular we will discuss the way oxygen-dependent hydroxylation of specific amino acid residues has been demonstrated to regulate HIF function at the level of both protein stability and transcriptional potency.

The dioxin (Aryl hydrocarbon) receptor (DR) is a unique bHLH transcription factor which is activated by binding of planar aromatic hydrocarbons typified by dioxin (TCDD). The active receptor is key to metabolism of aryl hydrocarbon xenobiotics by being a potent inducer of CYP1A1 gene activity. Chlorinated dioxins are inert to metabolism and initiate multifarious toxicities, including potent tumour promotion. These ill-effects are mediated by the activated DR and we are studying the mechanisms by which the ligand binding domain of the DR controls activity of the protein. The DR ligand binding domain resides within a PAS (Per/Arnt/Sim homology) region which is contiguous with the bHLH. The latent bHLH/PAS dioxin receptor (DR) is found in the cytoplasm of most mammalian cell types in a complex with heat shock protein 90, a novel immunophilin like protein termed ARA9/XAP2/AIP, and the co-chaperone p23. Here we use antisense ARA9 constructs to reveal that in the absence of ARA9, the DR is unable to form a transcriptionally active complex. Co-expression of antisense ARA9 with a form of the DR which is constitutively targeted to the nucleus leads to dramatically decreased levels of the nuclear DR protein, implying that ARA9 may function beyond its currently proposed role in cytoplasmic retention of the latent DR.

The dioxin/aryl hydrocarbon receptor (AhR) functions as a ligand-activated transcription factor regulating transcription of a battery of genes encoding xenobiotic metabolizing enzymes. Known receptor ligands are environmental pollutants including polycyclic aromatic hydrocarbons and polychlorinated dioxins. Loss-of-function (gene-disruption) studies in mice have demonstrated that the AhR is involved in toxic effects of dioxins but have not yielded unequivocal results concerning the physiological function of the receptor. Gain-of-function studies therefore were performed to unravel the biological functions of the AhR. A constitutively active AhR expressed in transgenic mice reduced the life span of the mice and induced tumors in the glandular part of the stomach, demonstrating the oncogenic potential of the AhR and implicating the receptor in regulation of cell proliferation.

Many molecular and physiological responses to hypoxia in mammals are controlled by the transcription factors Hypoxia-Inducible Factor-1alpha (HIF-1alpha) and HIF-2alpha. Their ability to promote the transcription of hypoxia-inducible genes is mediated by protein stability and regulation of a C-terminal transactivation domain. Oxygen-dependent hydroxylation of conserved proline and asparagine residues in HIF-alpha are required for targeting HIF-alpha to proteasomes for destruction, and for inhibiting its capacity for CBP/p300-dependent transactivation, respectively. In hypoxia, the O2 required for prolyl and asparaginyl hydroxylation is limiting, and HIF-alpha is thus stabilized and competent for transcription. Because these proteins participate in angiogenesis, glycolysis, programmed cell death, cancer, and ischemia, HIF-alpha and its mediators are attractive therapeutic targets.

Mammalian cells adapt to hypoxic conditions through a transcriptional response pathway mediated by the hypoxia-inducible factor, HIF. HIF transcriptional activity is suppressed under normoxic conditions by hydroxylation of an asparagine residue within its C-terminal transactivation domain, blocking association with coactivators. Here we show that the protein FIH-1, previously shown to interact with HIF, is an asparaginyl hydroxylase. Like known hydroxylase enzymes, FIH-1 is an Fe(II)-dependent enzyme that uses molecular O(2) to modify its substrate. Together with the recently discovered prolyl hydroxylases that regulate HIF stability, this class of oxygen-dependent enzymes comprises critical regulatory components of the hypoxic response pathway.

The basic helix-loop-helix/Per-Arnt-Sim homology (bHLH/PAS) protein family comprises a group of transcriptional regulators that often respond to a variety of developmental and environmental stimuli. Two murine members of this family, Single Minded 1 (SIM1) and Single Minded 2 (SIM2), are essential for postnatal survival but differ from other prototypical family members such as the dioxin receptor (DR) and hypoxia-inducible factors, in that they behave as transcriptional repressors in mammalian one-hybrid experiments and have yet to be ascribed a regulating signal. In cell lines engineered to stably express SIM1 and SIM2, we show that both are nuclear proteins that constitutively complex with the general bHLH/PAS partner factor, ARNT. We report that the murine SIM factors, in combination with ARNT, attenuate transcription from the hypoxia-inducible erythropoietin (EPO) enhancer during hypoxia. Such cross-talk between coexpressed bHLH/PAS factors can occur through competition for ARNT, which we find evident in SIM repression of DR-induced transcription from a xenobiotic response element reporter gene. However, SIM1/ARNT, but not SIM2/ARNT, can activate transcription from the EPO enhancer at normoxia, implying that the SIM proteins have the ability to bind hypoxia response elements and affect either activation or repression of transcription. This notion is supported by co-immunoprecipitation of EPO enhancer sequences with the SIM2 protein. SIM protein levels decrease with hypoxia treatment in our stable cell lines, although levels of the transcripts encoding SIM1 and SIM2 and the approximately 2-h half-lives of each protein are unchanged during hypoxia. Inhibition of protein synthesis, known to occur in cells during hypoxic stress in order to decrease ATP utilization, appears to account for the fall in SIM levels. Our data suggest the existence of a hypoxic switch mechanism in cells that coexpress hypoxia-inducible factor and SIM proteins, where up-regulation and activation of hypoxia-inducible factor-1alpha is concomitant with attenuation of SIM activities.

The hypoxia-inducible factors (HIFs) 1alpha and 2alpha are key mammalian transcription factors that exhibit dramatic increases in both protein stability and intrinsic transcriptional potency during low-oxygen stress. This increased stability is due to the absence of proline hydroxylation, which in normoxia promotes binding of HIF to the von Hippel-Lindau (VHL tumor suppressor) ubiquitin ligase. We now show that hypoxic induction of the COOH-terminal transactivation domain (CAD) of HIF occurs through abrogation of hydroxylation of a conserved asparagine in the CAD. Inhibitors of Fe(II)- and 2-oxoglutarate-dependent dioxygenases prevented hydroxylation of the Asn, thus allowing the CAD to interact with the p300 transcription coactivator. Replacement of the conserved Asn by Ala resulted in constitutive p300 interaction and strong transcriptional activity. Full induction of HIF-1alpha and -2alpha, therefore, relies on the abrogation of both Pro and Asn hydroxylation, which during normoxia occur at the degradation and COOH-terminal transactivation domains, respectively.