1: Cell Cycle. 2005 Jul;4(7):881-2. Epub 2005 Jul 11. Genetic instability: the dark side of the hypoxic response. To KK, Koshiji M, Hammer S, Huang LE. Laboratory of Human Carcinogenesis, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892-4255, USA. Under low oxygen tension, the activated transcription factor HIF-1alpha upregulates an array of hypoxia-inducible genes via heterodimerization with ARNT and binding to the hypoxia-responsive element in the promoter. Alternatively, HIF-1alpha regulates hypoxia-responsive genes by functionally antagonizing the oncoprotein Myc via protein-protein interactions. This so-called HIF-1alpha-Myc mechanism apparently not only accounts for the gene upregulation, but also for the gene downregulation during hypoxia, depending upon the activating and repressive nature of Myc in gene expression. Indeed, our recent study demonstrated that both mismatch repair genes, MSH2 and MSH6, are inhibited by this mechanism in a p53-dependent manner. In particular, the constitutively bound transcription factor Sp1 serves as a molecular switch by recruiting HIF-1alpha in hypoxia to displace the transcription activator Myc from the promoter. Therefore, our findings shed light on the mechanisms underlying hypoxia-induced genetic instability, an "adverse"effect of the hypoxic response, and yet a germane process to tumor survival and progression. PMID: 15970707 [PubMed - in process] --------------------------------------------------------------- 2: Exp Cell Res. 2003 Jul 15;287(2):237-48. Xenopus single-minded (xSim) is a nuclear factor allowing nuclear translocation of its cytoplasmic partner xArnt. Coumailleau P, Bollerot K, Lecomte C, Angelier N. Universite Pierre et Marie Curie, UMR7622-CNRS Biologie Moleculaire et Cellulaire du Developpement, 9 quai St Bernard, 75252 Paris Cedex 05, France. pascal.coumailleau@snv.jussieu.fr Transcription factors belonging to the basic helix-loop-helix Per-Arnt-Sim (bHLH/PAS) family control a wide variety of biological processes in mammalian and/or Drosophila. We have previously isolated bHLH/PAS Xenopus amphibian homologs of Single-minded (xSim) and aryl receptor nuclear translocator (xArnt) and characterized their expression pattern during embryogenesis. We show in this paper that xSim protein is a functional homolog of Drosophila or mammalian Sim(s). Biochemical analysis indicates that xSim forms a heterodimer with xArnt. Subcellular localization analysis of bHLH/PAS chimeric fluorescent versions in Xenopus or mammalian cell lines shows that xSim is constitutively localized in the nuclear compartment. On the opposite, xArnt appears to be predominantly expressed in the cytoplasm. In addition, we demonstrate that xArnt nuclear localization depends on the presence of xSim. Thus xSim appears to be an essential factor in the nuclear translocation of the xSim/xArnt complex. In perfect agreement, we show that the C-terminal half of xSim contains the information for this nuclear localization. PMID: 12837280 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 3: J Biol Chem. 2002 Mar 22;277(12):10236-43. Epub 2002 Jan 8. Differential activities of murine single minded 1 (SIM1) and SIM2 on a hypoxic response element. Cross-talk between basic helix-loop-helix/per-Arnt-Sim homology transcription factors. Woods SL, Whitelaw ML. Department of Molecular BioSciences, Center for the Molecular Genetics of Development, Adelaide University, South Australia 5005, Australia. 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. PMID: 11782478 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 4: Proc Natl Acad Sci U S A. 2000 Sep 12;97(19):10436-41. A genomewide survey of basic helix-loop-helix factors in Drosophila. Moore AW, Barbel S, Jan LY, Jan YN. Howard Hughes Medical Institute, Departments of Physiology and Biochemistry, University of California, San Francisco, CA 94143-0725, USA. The basic helix-loop-helix (bHLH) transcription factors play important roles in the specification of tissue type during the development of animals. We have used the information contained in the recently published genomic sequence of Drosophila melanogaster to identify 12 additional bHLH proteins. By sequence analysis we have assigned these proteins to families defined by Atonal, Hairy-Enhancer of Split, Hand, p48, Mesp, MYC/USF, and the bHLH-Per, Arnt, Sim (PAS) domain. In addition, one single protein represents a unique family of bHLH proteins. mRNA in situ analysis demonstrates that the genes encoding these proteins are expressed in several tissue types but are particularly concentrated in the developing nervous system and mesoderm. PMID: 10973473 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 5: Nucleic Acids Res. 1999 Aug 1;27(15):3205-12. Specificity of DNA binding of the c-Myc/Max and ARNT/ARNT dimers at the CACGTG recognition site. Swanson HI, Yang JH. Department of Pharmacology, University of Kentucky, Lexington, KY 40536, USA. hswan@pop.uky.edu Basic helix-loop-helix proteins that interact with the DNA recognition site CACGTG include the c-Myc/Max heterodimer and the ARNT (Ahreceptornucleartranslocator) homodimer. We have utilized a PCR-based protocol to identify high affinity binding sites of either the c-Myc/Max or ARNT/ARNT dimers and analyzed the ability of these dimers to interact with their derived consensus sequences and activate genes. chi(2)analysis of the selected DNA recognition sites revealed that DNA binding of the ARNT homodimer is symmetric, resulting in the consensus sequence RTCACGTGAY. Gel shift analysis demonstrated that the flanking nucleotides play an important role in dictating DNA binding affinity of the ARNT homodimer. These flanking sequences also regulate the ability of ARNT to competitively displace the c-Myc/Max heterodimer from a CACGTG-containing sequence. However, transient transfection analyses in CV-1 cells revealed that ARNT and c-Myc/Max exhibited similar abilities to activate transcription through each other's consensus sequences. Taken together, these results indicate that although binding affinity of these dimers for the CACGTG core sequences may be differentially influenced by flanking nucleotides, transcriptional activity may also be determined by other factors, such as cellular concentrations of these proteins and their co-activators. PMID: 10454619 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 6: J Biol Chem. 1999 Apr 30;274(18):12391-400. Protein kinase C modulates aryl hydrocarbon receptor nuclear translocator protein-mediated transactivation potential in a dimer context. Long WP, Chen X, Perdew GH. Center for Molecular Toxicology, Pennsylvania State University, University Park, Pennsylvania 16802, USA. Protein kinase C (PKC)- and protein kinase A (PKA)-mediated modulation of the transactivation potential of human aryl hydrocarbon receptor nuclear translocator (hARNT), a basic helix-loop-helix (bHLH)-PAS transcription factor, and the bHLH-ZIP transcription factors USF-1 (for upstream regulatory factor 1) and c-Myc were examined. An 81 nM dose of the PKC activator phorbol-12-myristate-13-acetate (PMA), shown here to specifically activate PKC in COS-1 cells, or a 1 nM dose of the PKA activator 8-bromoadenosine-3',5'-cyclic monophosphate (8-Br-cAMP) results in 2. 6- and 1.9-fold enhancements, respectively, in hARNT-mediated transactivation of the class B, E-box-driven reporter pMyc3E1bLuc relative to identically transfected, carrier solvent-treated COS-1 cells. In contrast, 81 nM PMA and 1 nM 8-Br-cAMP did not enhance transactivation of pMyc3E1bLuc-driven by USF-1 and c-Myc expression relative to identically transfected, carrier-treated COS-1 cells. Co-transfection of pcDNA3/ARNT-474-Flag, expressing a hARNT carboxyl-terminal transactivation domain deletion, and pMyc3E1bLuc does not result in induction of reporter activity, suggesting PMA's effects do not involve formation of unknown hARNT-protein heterodimers. Additionally, PMA had no effect on hARNT expression relative to Me2SO-treated cells. Metabolic 32P labeling of hARNT in cells treated with carrier solvent or 81 nM PMA demonstrates that PMA does not increase the overall phosphorylation level of hARNT. These results demonstrate, for the first time, that the transactivation potential of ARNT in a dimer context can be specifically modulated by PKC or PKA stimulation and that the bHLH-PAS and bHLH-ZIP transcription factors are differentially regulated by these pathways in COS-1 cells. PMID: 10212212 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 7: Pharmacogenetics. 1993 Oct;3(5):213-30. The AH-receptor: genetics, structure and function. Swanson HI, Bradfield CA. Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611. The AH-receptor is a ligand-activated transcription factor that regulates a number of biological responses to planar aromatic hydrocarbons. Interest in this receptor is related to its role in the toxic action of a variety of environmental chemicals, the simplicity and elegance of the murine genetics that led to its characterization and the distinctive mechanism by which this receptor activates gene expression. Recent cloning experiments have demonstrated that the AH-receptor is structurally related to the Per, ARNT and Sim proteins. Members of this newly described gene family are characterized by two N-terminal domains, the most characteristic of which is a motif referred to as a PAS domain. In the AH-receptor, this domain harbours sequences involved in the formation of a hydrophobic pocket that bind receptor agonists. Adjacent to the PAS domain in the AH-receptor, ARNT and Sim proteins is a basic/helix-loop-helix (bHLH) domain that appears to mediate heterodimerization and sequence specific DNA binding properties. The observation that the bHLH domain is present in the AH-receptor and the ARNT protein, a factor required for proper AH-receptor function, suggests that these proteins are heterodimeric partners that activate gene expression in a manner similar to Myc/Max and MyoD/E2A. The objectives of this review are to describe recent experimental results in this field and to use this information to develop a molecular model of AH-receptor mediated signal transduction. Publication Types: Review PMID: 8287061 [PubMed - indexed for MEDLINE] --------------------------------------------------------------- 8: Proc Natl Acad Sci U S A. 1992 Sep 1;89(17):8185-9. Cloning of the Ah-receptor cDNA reveals a distinctive ligand-activated transcription factor. Burbach KM, Poland A, Bradfield CA. Department of Pharmacology, Northwestern University Medical School, Chicago, IL 60611. A cDNA encoding the murine Ah receptor (Ahb-1 allele for aromatic hydrocarbon responsiveness) has been isolated and characterized. Analysis of the deduced protein sequence revealed a region with similarity to the basic region/helix-loop-helix (BR/HLH) motif found in many transcription factors that undergo dimerization for function. In addition to the BR/HLH domain, the N-terminal domain of the Ah receptor has extensive sequence similarity to the human ARNT (aryl hydrocarbon receptor nuclear translocator) protein and two regulatory proteins of Drosophila, Sim and Per. Photoaffinity labeling and peptide mapping studies indicate that the Ah receptor binds agonist at a domain that lies within this conserved N-terminal domain. The Ah receptor appears to be a ligand-activated transcription factor with a helix-loop-helix motif similar to those found in a variety of DNA-binding proteins, including Myc and MyoD. PMID: 1325649 [PubMed - indexed for MEDLINE] ---------------------------------------------------------------