CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE165044 | GEO | 2021/07/23
REPOSITORIES: GEO
Similar Datasets
Project description:The Drosophila circadian clock is driven by a transcriptional feedback loop in which 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. CLOCKWORK ORANGE (CWO) reinforces PER-TIM repression by binding E-boxes to maintain PER-TIM bound CLK-CYC off DNA, but also promotes CLK-CYC transcription through an unknown mechanism. To determine how CWO activates CLK-CYC transcription, we identified CWO target genes that are upregulated in the absence of CWO repression, conserved in mammals, and preferentially expressed in brain pacemaker neurons. Among the genes identified was a putative ortholog of mouse Clock Interacting Protein Circadian (Cipc), which represses CLOCK-BMAL1 transcription. Reducing or eliminating Drosophila Cipc expression shortens period, while overexpressing Cipc lengthens period, which is consistent with previous work showing that Drosophila Cipc represses CLK-CYC transcription in S2 cells. Cipc represses CLK-CYC transcription in vivo, but not uniformly, as per is strongly repressed, tim less so, and vri hardly at all. Long period rhythms in cwo mutant flies are largely rescued when Cipc expression is reduced or eliminated, indicating that increased Cipc expression mediates the period lengthening of cwo mutants. Consistent with this behavioral rescue, eliminating Cipc rescues the decreased CLK-CYC transcription in cwo mutant flies, where per is strongly rescued, tim is moderately rescued, and vri shows little rescue. These results suggest a mechanism for CWO-dependent CLK-CYC activation: CWO inhibition of CIPC repression promotes CLK-CYC transcription. This mechanism may be conserved since cwo and Cipc perform analogous roles in the mammalian circadian clock.
| S-EPMC8511158 | biostudies-literature
Project description: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.
2021-07-23 | GSE165042 | GEO
Project description: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.
2021-07-23 | GSE165043 | GEO
Project description:CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the putative Drosophila ortholog of CLOCK INTERACTING PROTEIN CIRCADIAN
| PRJNA693022 | ENA
Project description:Gene transcription is a central timekeeping process in animal clocks. In Drosophila, the basic helix-loop helix (bHLH)-PAS transcription-factor heterodimer, CLOCK/CYCLE (CLK/CYC), transcriptionally activates the clock components period (per), timeless (tim), Par domain protein 1 (Pdp1), and vrille (vri), which feed back and regulate distinct features of CLK/CYC function. Microarray studies have identified numerous rhythmically expressed transcripts, some of which are potential direct CLK targets. Here we demonstrate a circadian function for one such target, a bHLH-Orange repressor, CG17100/CLOCKWORK ORANGE (CWO). cwo is rhythmically expressed, and levels are reduced in Clk mutants, suggesting that cwo is CLK activated in vivo. cwo mutants display reduced-amplitude molecular and behavioral rhythms with lengthened periods. Molecular analysis suggests that CWO acts, in part, by repressing CLK target genes. We propose that CWO acts as a transcriptional and behavioral rhythm amplifier.
| S-EPMC1963421 | biostudies-literature
Project description: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.
| S-EPMC1899476 | biostudies-literature
Project description:CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (RNA-Seq)
| PRJNA693024 | ENA
Project description:CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (ChIP-Seq)
| PRJNA693023 | ENA
Project description:The circadian clock generates rhythms of approximately 24 h through periodic expression of the clock genes. In insects, the major clock genes period (per) and timeless (tim) are rhythmically expressed upon their transactivation by CLOCK/CYCLE, with peak levels in the early night. In Drosophila, clockwork orange (cwo) is known to inhibit the transcription of per and tim during the daytime to enhance the amplitude of the rhythm, but its function in other insects is largely unknown. In this study, we investigated the role of cwo in the clock mechanism of the cricket Gryllus bimaculatus. The results of quantitative RT-PCR showed that under a light/dark (LD) cycle, cwo is rhythmically expressed in the optic lobe (lamina-medulla complex) and peaks during the night. When cwo was knocked down via RNA interference (RNAi), some crickets lost their locomotor rhythm, while others maintained a rhythm but exhibited a longer free-running period under constant darkness (DD). In cwoRNAi crickets, all clock genes except for cryptochrome 2 (cry2) showed arrhythmic expression under DD; under LD, some of the clock genes showed higher mRNA levels, and tim showed rhythmic expression with a delayed phase. Based on these results, we propose that cwo plays an important role in the cricket circadian clock.
| S-EPMC7659126 | biostudies-literature
Project description:Many organisms use circadian clocks to keep temporal order and anticipate daily environmental changes. In Drosophila, the master clock gene Clock promotes the transcription of several key target genes. Two of these gene products, PER and TIM, repress CLK-CYC-mediated transcription. To recognize additional direct CLK target genes, we designed a genome-wide approach and identified clockwork orange (cwo) as a new core clock component. cwo encodes a transcriptional repressor that synergizes with PER and inhibits CLK-mediated activation. Consistent with this function, the mRNA profiles of CLK direct target genes in cwo mutant flies manifest high trough values and low amplitude oscillations. Because behavioral rhythmicity fails to persist in constant darkness (DD) with little or no effect on average mRNA levels in flies lacking cwo, transcriptional oscillation amplitude appears to be linked to rhythmicity. Moreover, the mutant flies are long period, consistent with the late repression indicated by the RNA profiles. These findings suggest that CWO acts preferentially in the late night to help terminate CLK-CYC-mediated transcription of direct target genes including cwo itself. The presence of mammalian homologs with circadian expression features (Dec1 and Dec2) suggests that a similar feedback mechanism exists in mammalian clocks.
| S-EPMC1899475 | biostudies-literature