title: A functional genomics strategy reveals clockwork orange as a transcriptional regulator in the Drosophila circadian clock.
abstract: The Drosophila circadian clock consists of integrated autoregulatory feedback loops, making the clock difficult to elucidate without comprehensively identifying the network components in vivo. Previous studies have adopted genome-wide screening for clock-controlled genes using high-density oligonucleotide arrays that identified hundreds of clock-controlled genes. In an attempt to identify the core clock genes among these candidates, we applied genome-wide functional screening using an RNA interference (RNAi) system in vivo. Here we report the identification of novel clock gene candidates including clockwork orange (cwo), a transcriptional repressor belonging to the basic helix-loop-helix ORANGE family. cwo is rhythmically expressed and directly regulated by CLK-CYC through canonical E-box sequences. A genome-wide search for its target genes using the Drosophila genome tiling array revealed that cwo forms its own negative feedback loop and directly suppresses the expression of other clock genes through the E-box sequence. Furthermore, this negative transcriptional feedback loop contributes to sustaining a high-amplitude circadian oscillation in vivo. Based on these results, we propose that the competition between cyclic CLK-CYC activity and the adjustable threshold imposed by CWO keeps E-box-mediated transcription within the controllable range of its activity, thereby rendering a Drosophila circadian clock capable of generating high-amplitude oscillation.
authors.affiliation: Laboratory for Systems Biology, Center for Developmental Biology, RIKEN, Kobe, Hyogo, Japan.
authors.forename: Akira
authors.initials: A
authors.lastname: Matsumoto
authors.forename: Maki
authors.initials: M
authors.lastname: Ukai-Tadenuma
authors.forename: Rikuhiro G
authors.initials: RG
authors.lastname: Yamada
authors.forename: Jerry
authors.initials: J
authors.lastname: Houl
authors.forename: Kenichiro D
authors.initials: KD
authors.lastname: Uno
authors.forename: Takeya
authors.initials: T
authors.lastname: Kasukawa
authors.forename: Brigitte
authors.initials: B
authors.lastname: Dauwalder
authors.forename: Taichi Q
authors.initials: TQ
authors.lastname: Itoh
authors.forename: Kuniaki
authors.initials: K
authors.lastname: Takahashi
authors.forename: Ryu
authors.initials: R
authors.lastname: Ueda
authors.forename: Paul E
authors.initials: PE
authors.lastname: Hardin
authors.forename: Teiichi
authors.initials: T
authors.lastname: Tanimura
authors.forename: Hiroki R
authors.initials: HR
authors.lastname: Ueda
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D030801:Animals, Genetically Modified; D001483:Base Sequence; D051792:Basic Helix-Loop-Helix Proteins / Q000235:genetics / Q000378:metabolism; D001683:Biological Clocks / Q000235:genetics*; D056926:CLOCK Proteins; D002478:Cells, Cultured; D002940:Circadian Rhythm / Q000235:genetics*; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D004331:Drosophila melanogaster / Q000235:genetics / Q000502:physiology*; D024721:E-Box Elements; D005786:Gene Expression Regulation; D049750:Genome, Insect; D023281:Genomics; D008954:Models, Biological; D008969:Molecular Sequence Data; D009474:Neurons / Q000378:metabolism; D012097:Repressor Proteins / Q000235:genetics / Q000502:physiology*; D017386:Sequence Homology, Amino Acid; D014157:Transcription Factors / Q000502:physiology; D014158:Transcription, Genetic
chemical_list: D051792:Basic Helix-Loop-Helix Proteins; D056926:CLOCK Proteins; D029721:Drosophila Proteins; D012097:Repressor Proteins; D014157:Transcription Factors; C120526:Clk protein, Drosophila; C521952:Cwo protein, Drosophila