Chromatin immunoprecipitation (ChIP) assay of CWO protein with Drosophila genome tiling array

CWO binding sites were genome-widely searched with Drosophila genome tiling array. 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 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 tilling 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. Keywords: ChIP-chip 4 Drosophila genome tiling arrays were used as follows: Array1 Immunoprecipetated with anti-Flag antibody (Biological replicate no.1) for CWO binding signal Array2 Immunoprecipetated with anti-Flag antibody (Biological replicate no.2) for CWO binding signal Array3 Immunoprecipetated with anti-V5 antiboy (Biological replicate no.1) for background signal Array4 Immunoprecipetated with anti-V5 antiboy (Biological replicate no.2) for background signal