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

The Drosophila circadian clock is driven by a transcriptional feedback loop in which the bHLH transcription factor CLOCK-CYCLE (CLK-CYC) binds E-boxes to transcribe genes encoding the PERIOD-TIMELESS (PER-TIM) repressor, which releases CLK-CYC from E-boxes to inhibit transcription. The bHLH-Orange transcription factor CLOCKWORK ORANGE (CWO) reinforces repression by binding E-boxes to displace CLK-CYC, but also acts through an unknown mechanism to promote CLK-CYC transcription. To determine how CWO activates CLK-CYC transcription, we identified CWO DNA binding targets that are upregulated in the absence of CWO repression, conserved in mammals and preferentially expressed in brain pacemaker neurons. Among the genes identified was the Drosophila ortholog of mammalian Clock Interacting Protein Circadian (Cipc) that acts to repress CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens circadian period while overexpressing Cipc lengthens circadian period in flies, consistent with previous analysis showing that Drosophila Cipc represses CLK-CYC transcription in S2 cell culture. Long period rhythms of cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates period lengthening of cwo mutants. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. Such a mechanism may be conserved given that orthologs of cwo and Cipc carry out analogous roles in the mammalian circadian clock. Overall design: RNA-seq: mRNA profiles of heads from Drosophila wild type (WT) and cwo5073 mutant at six different timepoints, ZT 02, ZT 06, ZT 10, ZT 14, ZT 18, ZT 22

果蝇昼夜节律时钟由转录反馈环路驱动，其中碱性螺旋-环-螺旋（basic helix-loop-helix，bHLH）转录因子CLOCK-CYCLE（CLK-CYC）可结合E-box元件，转录编码PERIOD-TIMELESS（PER-TIM）阻遏蛋白的基因，该阻遏蛋白可使CLK-CYC脱离E-box并抑制转录。碱性螺旋-环-螺旋-Orange（basic helix-loop-helix-Orange，bHLH-Orange）转录因子CLOCKWORK ORANGE（CWO）可通过结合E-box置换CLK-CYC来增强阻遏作用，同时还通过未知机制促进CLK-CYC的转录。为解析CWO激活CLK-CYC转录的具体机制，本研究鉴定了在CWO缺失阻遏时上调、在哺乳动物中保守且优先在脑起搏神经元中表达的CWO DNA结合靶标。其中鉴定到的基因包括哺乳动物节律性Clock相互作用蛋白（Clock Interacting Protein Circadian，Cipc）的果蝇同源物，该蛋白可抑制CLOCK-BMAL1的转录。在果蝇中降低或敲除Cipc表达会缩短昼夜节律周期，而过表达Cipc则会延长果蝇的昼夜节律周期，这与此前在S2细胞培养体系中证实果蝇Cipc抑制CLK-CYC转录的研究结果一致。当cwo5073突变型果蝇的Cipc表达被降低或敲除时，其长周期节律可得到大幅挽救，这表明Cipc表达上调介导了cwo突变体的周期延长表型。上述结果揭示了CWO依赖型CLK-CYC激活的机制：CWO对CIPC阻遏作用的抑制，可促进CLK-CYC的转录。鉴于cwo和Cipc的同源基因在哺乳动物昼夜节律时钟中发挥类似功能，这一机制可能具有进化保守性。实验整体设计：RNA测序：采集野生型（wild type，WT）与cwo5073突变型果蝇头部的mRNA表达谱，共设置6个采样时间点：ZT 02、ZT 06、ZT 10、ZT 14、ZT 18、ZT 22。