title: Two distinct modes of PERIOD recruitment onto dCLOCK reveal a novel role for TIMELESS in circadian transcription.
abstract: Negative transcriptional feedback loops are a core feature of eukaryotic circadian clocks and are based on rhythmic interactions between clock-specific repressors and transcription factors. In Drosophila, the repression of dCLOCK (dCLK)-CYCLE (CYC) transcriptional activity by dPERIOD (dPER) is critical for driving circadian gene expression. Although growing lines of evidence indicate that circadian repressors such as dPER function, at least partly, as molecular bridges that facilitate timely interactions between other regulatory factors and core clock transcription factors, how dPER interacts with dCLK-CYC to promote repression is not known. Here, we identified a small conserved region on dPER required for binding to dCLK, termed CBD (for dCLK binding domain). In the absence of the CBD, dPER is unable to stably associate with dCLK and inhibit the transcriptional activity of dCLK-CYC in a simplified cell culture system. CBD is situated in close proximity to a region that interacts with other regulatory factors such as the DOUBLETIME kinase, suggesting that complex architectural constraints need to be met to assemble repressor complexes. Surprisingly, when dPER missing the CBD (dPER(ΔCBD)) was evaluated in flies the clock mechanism was operational, albeit with longer periods. Intriguingly, the interaction between dPER(ΔCBD) and dCLK is TIM-dependent and modulated by light, revealing a novel and unanticipated in vivo role for TIM in circadian transcription. Finally, dPER(ΔCBD) does not provoke the daily hyperphosphorylation of dCLK, indicating that direct interactions between dPER and dCLK are necessary for the dCLK phosphorylation program but are not required for other aspects of dCLK regulation.
authors.affiliation: Neuroscience Graduate Program, Institute for Medical Sciences, Ajou University School of Medicine, San5, Wonchon-dong, Suwon, Kyunggi-do, 443-721, Republic of Korea.
authors.forename: Woo Chul
authors.initials: WC
authors.lastname: Sun
authors.forename: Eun Hee
authors.initials: EH
authors.lastname: Jeong
authors.forename: Hyun-Jeong
authors.initials: HJ
authors.lastname: Jeong
authors.forename: Hyuk Wan
authors.initials: HW
authors.lastname: Ko
authors.forename: Isaac
authors.initials: I
authors.lastname: Edery
authors.forename: Eun Young
authors.initials: EY
authors.lastname: Kim
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D015153:Blotting, Western; D056926:CLOCK Proteins / Q000235:genetics / Q000502:physiology*; D002478:Cells, Cultured; D002940:Circadian Rhythm / Q000502:physiology*; D004330:Drosophila; D029721:Drosophila Proteins / Q000235:genetics / Q000502:physiology*; D007150:Immunohistochemistry; D008027:Light; D009043:Motor Activity / Q000502:physiology; D056950:Period Circadian Proteins / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D010957:Plasmids / Q000235:genetics; D011485:Protein Binding / Q000235:genetics; D012313:RNA / Q000096:biosynthesis / Q000235:genetics; D012097:Repressor Proteins / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000502:physiology
chemical_list: C120526:Clk protein, Drosophila; D029721:Drosophila Proteins; C539040:PER protein, Drosophila; D056950:Period Circadian Proteins; D012097:Repressor Proteins; C096445:tim protein, Drosophila; D012313:RNA; D056926:CLOCK Proteins