CLOCKWORK ORANGE promotes CLOCK-CYCLE activation via the Drosophila ortholog of CLOCK INTERACING PROTEIN, CIRCADIAN (ChIP-Seq)
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE165042 | GEO | 2021/07/23
REPOSITORIES: GEO
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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:Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. Prime examples are the circadian clock TFs CLOCK (CLK) and CYCLE (CYC or BMAL1): while they control a core transcriptional circuit throughout animal bodies, downstream clock target genes and circadian physiology are tissue-specific. Here, we use ChIP-seq to determine the regulatory targets of Drosophila CLK and CYC, which we epitope-tagged by homologous recombination. Both TFs have distinct binding sites in heads versus bodies, suggesting that they directly control tissue-specific downstream target genes. Analysis of these context-specific binding sites revealed distinct sequence motifs for putative clock partner factors, including a motif for the GATA factor SERPENT (SRP). SRP indeed synergistically enhances CLK/CYC-mediated activity of a cis-regulatory region bound by CLK/CYC specifically in bodies. These results reveal how universal clock circuits can generate tissue-specific outputs and demonstrate an approach to dissect regulatory interactions more generally. We sequenced ChIP and input samples, as well as M-bM-^@M-^\mockM-bM-^@M-^] samples for which we performed ChIP with the V5 antibody from wildtype w- flies (not carrying the V5 tag) for two independent biological replicates each, summing to 24 libraries in total.
2013-06-01 | E-GEOD-40467 | biostudies-arrayexpress
Project description: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
2007-04-24 | GSE7569 | GEO
Project description:CLOCK (CLK) is a master transcriptional regulator of the circadian clock in Drosophila. To identify CLK direct target genes and address circadian transcriptional regulation in Drosophila, we performed chromatin immunoprecipitation-tiling array assays (ChIP-chip) with a number of circadian proteins. CLK binding cycles on at least 800 sites with maximal binding in the early night. The CLK partner protein CYCLE (CYC) is on most of these sites. The CLK/CYC heterodimer is joined 4-6 hrs later by the transcriptional repressor PER, indicating that the majority of CLK targets are regulated similarly to core circadian genes (Menet et al. 2010). About 30% of target genes also show cycling Pol II binding. Many of these generate cycling RNAs despite not being documented in prior RNA cycling studies. This is due in part to different RNA isoforms and to fly head tissue heterogeneity. CLK has specific targets in different tissues, implying that important CLK partner proteins and/or mechanisms contribute to gene-specific and tissue-specific regulation.
2011-11-15 | GSE32613 | GEO
Project description:Background: Broadly expressed transcriptions factors (TFs) control tissue-specific programs of gene expression through interactions with local TF networks. A prime example is the circadian clock: although the conserved TFs CLOCK (CLK) and CYCLE (CYC) control a transcriptional circuit throughout animal bodies, rhythms in behavior and physiology are generated tissue specifically. Yet, how CLK and CYC determine tissue-specific clock programs has remained unclear. Results: Here, we use a functional genomics approach to determine the cis-regulatory requirements for clock specificity. We first determine CLK and CYC genome-wide binding targets in heads and bodies by ChIP-seq and show that they have distinct DNA targets in the two tissue contexts. Computational dissection of CLK/CYC context-specific binding sites reveals sequence motifs for putative partner factors, which are predictive for individual binding sites. Among them, we show that the opa and GATA motifs, differentially enriched in head and body binding sites respectively, can be bound by OPA and SERPENT (SRP) and act synergistically with CLK/CYC in the Drosophila feedback loop, suggesting that they help to determine their direct targets and therefore orchestrate tissue-specific clock outputs. In addition, using in vivo transgenic assays, we validate that GATA motifs are required for proper tissue-specific gene expression in the adult fat body, midgut, and Malpighian tubules, revealing a cis-regulatory signature for enhancers of the peripheral circadian clock. Conclusions: Our results reveal how universal clock circuits can regulate tissue-specific rhythms and, more generally, provide insights into the mechanism by which universal TFs can be modulated to drive tissue-specific programs of gene expression.
2013-06-01 | GSE40467 | GEO
Project description:The transcription factor Mef2 regulates activity-dependent neuronal plasticity and morphology in mammals, and clock neurons are reported to experience activity-dependent circadian remodeling in Drosophila. We show here that Mef2 is required for this daily fasciculation-defasciculation cycle. Moreover, the master circadian transcription complex CLK/CYC directly regulates Mef2 transcription. ChIP-Chip analysis identified numerous Mef2 target genes implicated in neuronal plasticity, including the cell-adhesion gene Fas2. Genetic epistasis experiments support this transcriptional regulatory hierarchy, CLK/CYC->Mef2-> Fas2, indicate that it influences the circadian fasciculation cycle within pacemaker neurons and suggest that this cycle also contributes to circadian behavior. Mef2 therefore transmits clock information to machinery involved in neuronal remodeling, which contributes to locomotor activity rhythms. Mef2 ChIP-chip samples collected at 6 timepoints, input and IP samples
2013-05-03 | E-GEOD-46576 | biostudies-arrayexpress
Project description:The transcription factor Mef2 regulates activity-dependent neuronal plasticity and morphology in mammals, and clock neurons are reported to experience activity-dependent circadian remodeling in Drosophila. We show here that Mef2 is required for this daily fasciculation-defasciculation cycle. Moreover, the master circadian transcription complex CLK/CYC directly regulates Mef2 transcription. ChIP-Chip analysis identified numerous Mef2 target genes implicated in neuronal plasticity, including the cell-adhesion gene Fas2. Genetic epistasis experiments support this transcriptional regulatory hierarchy, CLK/CYC->Mef2-> Fas2, indicate that it influences the circadian fasciculation cycle within pacemaker neurons and suggest that this cycle also contributes to circadian behavior. Mef2 therefore transmits clock information to machinery involved in neuronal remodeling, which contributes to locomotor activity rhythms.
2013-05-03 | GSE46576 | GEO
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:Feeding on high sugar diet reprograms metabolism, partially mediated through a gene regulatory network controlled by intracellular sugar sensor Mondo/ChREBP-Mlx. Sugar-induced gene expression changes include activation of genes encoding enzymes of glycolysis, pentose phosphate pathways, and de novo lipogenesis, collectively driving the flux of sugar-derived carbon into triacylglycerides. These sugar-induced metabolic changes are likely to trigger compensatory responses to avoid depletion of necessary metabolites, but they remain poorly characterized. Through a global temporal clustering analysis of sugar-induced gene expression in Drosophila we identify new gene expression programs controlled by sugar feeding. These include rapid and transient downregulation of ribosome biogenesis genes, known targets of transcription factor Myc. Through in silico motif predictions and experimental validation, we identify transcriptional repressor Clockwork orange (CWO) as a mediator of this response. CWO expression is directly activated by Mondo-Mlx and it counteracts Myc through repression of its expression as well as through binding to overlapping regulatory regions of ribosome biogenesis genes. Loss of CWO function leads to depletion of UDP-GlcNAc pools and impaired viability on high sugar diet. Analysis of CWO orthologs, BHLHE40 and BHLHE41, in mouse hepatocytes revealed a conserved role for BHLHE41 in repressing ribosome biogenesis genes. Collectively, our data uncover a gene regulatory circuit balancing the activities of lipid and protein biosynthesis to maintain homeostasis during high sugar feeding.
2023-06-01 | GSE207198 | GEO
Project description:Feeding on high sugar diet reprograms metabolism, partially mediated through a gene regulatory network controlled by intracellular sugar sensor Mondo/ChREBP-Mlx. Sugar-induced gene expression changes include activation of genes encoding enzymes of glycolysis, pentose phosphate pathways, and de novo lipogenesis, collectively driving the flux of sugar-derived carbon into triacylglycerides. These sugar-induced metabolic changes are likely to trigger compensatory responses to avoid depletion of necessary metabolites, but they remain poorly characterized. Through a global temporal clustering analysis of sugar-induced gene expression in Drosophila we identify new gene expression programs controlled by sugar feeding. These include rapid and transient downregulation of ribosome biogenesis genes, known targets of transcription factor Myc. Through in silico motif predictions and experimental validation, we identify transcriptional repressor Clockwork orange (CWO) as a mediator of this response. CWO expression is directly activated by Mondo-Mlx and it counteracts Myc through repression of its expression as well as through binding to overlapping regulatory regions of ribosome biogenesis genes. Loss of CWO function leads to depletion of UDP-GlcNAc pools and impaired viability on high sugar diet. Analysis of CWO orthologs, BHLHE40 and BHLHE41, in mouse hepatocytes revealed a conserved role for BHLHE41 in repressing ribosome biogenesis genes. Collectively, our data uncover a gene regulatory circuit balancing the activities of lipid and protein biosynthesis to maintain homeostasis during high sugar feeding.
2023-06-01 | GSE207197 | GEO