CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (ChIP-Seq)

CWO reinforces PER-TIM repression of core clock gene transcription by antagonizing CLK-CYC binding to E-boxes, but also functions to promote CLK-CYC transcription of core clock genes. To determine the relationship between CWO and CLK-CYC binding across the genome, we identified CWO and CLK binding sites via ChIP-seq. ChIP-seq: to identify CWO target genes, HA antibody was used to IP CWO-HA from heads of cwo-HA; cwo5073 flies collected during transcriptional repression at Zeitgeber Time 2 (ZT2, where ZT0 is lights-on and ZT12 is lights-off during an LD cycle) and transcription activation at ZT14 for ChIP-seq analysis. CLK was IPed with anti-CLK antibody in parallel

CWO通过拮抗CLK-CYC结合E-box元件，增强PER-TIM对核心生物钟基因的转录抑制作用，同时亦可促进CLK-CYC介导的核心生物钟基因转录。为明确CWO与全基因组范围内CLK-CYC结合的关联，我们通过染色质免疫共沉淀测序（ChIP-seq）鉴定了CWO与CLK的结合位点。为鉴定CWO的靶基因，我们分别于转录抑制期（授时因子时间2，Zeitgeber Time 2，ZT2，其中ZT0对应光照开启、ZT12对应光照关闭的光暗LD循环条件下）与转录激活期（ZT14时刻）收集cwo-HA; cwo5073果蝇的头部样本，通过HA抗体免疫沉淀CWO-HA融合蛋白以进行ChIP-seq分析；同时并行使用抗CLK抗体免疫沉淀CLK蛋白，开展平行实验。