title: Twist1 Is a TNF-Inducible Inhibitor of Clock Mediated Activation of Period Genes.
abstract: BACKGROUND
  Activation of the immune system affects the circadian clock. Tumor necrosis factor (TNF) and Interleukin (IL)-1β inhibit the expression of clock genes including Period (Per) genes and the PAR-bZip clock-controlled gene D-site albumin promoter-binding protein (Dbp). These effects are due to cytokine-induced interference of E-box mediated transcription of clock genes. In the present study we have assessed the two E-box binding transcriptional regulators Twist1 and Twist2 for their role in cytokine induced inhibition of clock genes.
  
  
  METHODS
  The expression of the clock genes Per1, Per2, Per3 and of Dbp was assessed in NIH-3T3 mouse fibroblasts and the mouse hippocampal neuronal cell line HT22. Cells were treated for 4h with TNF and IL-1β. The functional role of Twist1 and Twist2 was assessed by siRNAs against the Twist genes and by overexpression of TWIST proteins. In luciferase (luc) assays NIH-3T3 cells were transfected with reporter gene constructs, which contain a 3xPer1 E-box or a Dbp E-box. Quantitative chromatin immunoprecipitation (ChIP) was performed using antibodies to TWIST1 and CLOCK, and the E-box consensus sequences of Dbp (CATGTG) and Per1 E-box (CACGTG).
  
  
  RESULTS
  We report here that siRNA against Twist1 protects NIH-3T3 cells and HT22 cells from down-regulation of Period and Dbp by TNF and IL-1β. Overexpression of Twist1, but not of Twist2, mimics the effect of the cytokines. TNF down-regulates the activation of Per1-3xE-box-luc, the effect being prevented by siRNA against Twist1. Overexpression of Twist1, but not of Twist2, inhibits Per1-3xE-box-luc or Dbp-E-Box-luc activity. ChIP experiments show TWIST1 induction by TNF to compete with CLOCK binding to the E-box of Period genes and Dbp.
  
  
  CONCLUSIONS
  Twist1 plays a pivotal role in the TNF mediated suppression of E-box dependent transactivation of Period genes and Dbp. Thereby Twist1 may provide a link between the immune system and the circadian timing system.
authors.affiliation: Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland.
authors.forename: Daniel
authors.initials: D
authors.lastname: Meier
authors.affiliation: Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland.
authors.forename: Martin
authors.initials: M
authors.lastname: Lopez
authors.affiliation: Center for Integrative Genomics, University of Lausanne, Lausanne, Switzerland.
authors.forename: Paul
authors.initials: P
authors.lastname: Franken
authors.affiliation: Institute of Experimental Immunology, University of Zurich, Zurich, Switzerland.
authors.forename: Adriano
authors.initials: A
authors.lastname: Fontana
mesh_terms: D000818:Animals; D002460:Cell Line; D016207:Cytokines / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism*; D005786:Gene Expression Regulation / Q000187:drug effects; D053583:Interleukin-1beta / Q000494:pharmacology; D051379:Mice; D041681:NIH 3T3 Cells; D009687:Nuclear Proteins / Q000378:metabolism*; D056950:Period Circadian Proteins / Q000378:metabolism*; D012097:Repressor Proteins / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*; D048069:Tumor Necrosis Factors / Q000494:pharmacology*; D051879:Twist-Related Protein 1 / Q000378:metabolism*
chemical_list: D016207:Cytokines; D004268:DNA-Binding Proteins; C498260:Dbp protein, mouse; D053583:Interleukin-1beta; D009687:Nuclear Proteins; D056950:Period Circadian Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; D048069:Tumor Necrosis Factors; D051879:Twist-Related Protein 1; C096852:Twist2 protein, mouse; C067250:Twist1 protein, mouse