title: Regulation of CDKN2B expression by interaction of Arnt with Miz-1--a basis for functional integration between the HIF and Myc gene regulatory pathways.
abstract: BACKGROUND
  Hypoxia- and Myc-dependent transcriptional regulatory pathways are frequently deregulated in cancer cells. These pathways converge in many cellular responses, but the underlying molecular mechanisms are unclear.
  
  
  METHODS
  The ability of Miz-1 and Arnt to interact was identified in a yeast two-hybrid screen. The mode of interaction and the functional consequences of complex formation were analyzed by diverse molecular biology methods, in vitro. Statistical analyses were performed by Student's t-test and ANOVA.
  
  
  RESULTS
  In the present study we demonstrate that the aryl hydrocarbon receptor nuclear translocator (Arnt), which is central in hypoxia-induced signaling, forms a complex with Miz-1, an important transcriptional regulator in Myc-mediated transcriptional repression. Overexpression of Arnt induced reporter gene activity driven by the proximal promoter of the cyclin-dependent kinase inhibitor 2B gene (CDKN2B), which is an established target for the Myc/Miz-1 complex. In contrast, mutated forms of Arnt, that were unable to interact with Miz-1, had reduced capability to activate transcription. Moreover, repression of Arnt reduced endogenous CDKN2B expression, and chromatin immunoprecipitation demonstrated that Arnt interacts with the CDKN2B promoter. The transcriptional activity of Arnt was counteracted by Myc, but not by a mutated variant of Myc that is unable to interact with Miz-1, suggesting mutually exclusive interaction of Arnt and Myc with Miz-1. Our results also establish CDKN2B as a hypoxia regulated gene, as endogenous CDKN2B mRNA and protein levels were reduced by hypoxic treatment of U2OS cells.
  
  
  CONCLUSIONS
  Our data reveal a novel mode of regulation by protein-protein interaction that directly ties together, at the transcriptional level, the Myc- and hypoxia-dependent signaling pathways and expands our understanding of the roles of hypoxia and cell cycle alterations during tumorigenesis.
authors.affiliation: Department of Biomedicine, University of Bergen, Jonas Lies vei 91, N-5009 Bergen, Norway. Reidun.Asoy@biomed.uib.no.
authors.forename: Reidun
authors.initials: R
authors.lastname: Aesoy
authors.forename: Katarina
authors.initials: K
authors.lastname: Gradin
authors.forename: Kathrine S
authors.initials: KS
authors.lastname: Aasrud
authors.forename: Erling A
authors.initials: EA
authors.lastname: Hoivik
authors.forename: Jorge L
authors.initials: JL
authors.lastname: Ruas
authors.forename: Lorenz
authors.initials: L
authors.lastname: Poellinger
authors.forename: Marit
authors.initials: M
authors.lastname: Bakke
mesh_terms: D051784:Aryl Hydrocarbon Receptor Nuclear Translocator / Q000378:metabolism*; D063646:Carcinogenesis / Q000235:genetics*; D045744:Cell Line, Tumor; D047369:Chromatin Immunoprecipitation; D050762:Cyclin-Dependent Kinase Inhibitor p15 / Q000096:biosynthesis* / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic; D016259:Genes, myc / Q000235:genetics*; D006801:Humans; D051793:Hypoxia-Inducible Factor 1 / Q000235:genetics*; D015151:Immunoblotting; D047468:Immunoprecipitation; D051741:Kruppel-Like Transcription Factors / Q000378:metabolism*; D034622:RNA Interference; D020133:Reverse Transcriptase Polymerase Chain Reaction; D015398:Signal Transduction / Q000502:physiology; D014162:Transfection; D020798:Two-Hybrid System Techniques
chemical_list: C497396:ARNT protein, human; C493966:CDKN2B protein, human; D050762:Cyclin-Dependent Kinase Inhibitor p15; D051793:Hypoxia-Inducible Factor 1; D051741:Kruppel-Like Transcription Factors; C107961:ZBTB17 protein, human; D051784:Aryl Hydrocarbon Receptor Nuclear Translocator