RNA-Seq of Drosophila tim promoter/enhancer mutants around the clock
Ontology highlight
ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE259245 | GEO | 2024/02/28
REPOSITORIES: GEO
Similar Datasets
Project description:To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E box region and an upstream E box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. To examine the effect of promoter E-box deletions on Clk binding, we performed Clk-ChIP around the clock in wild-type and 126 mutant flies.
2024-02-28 | GSE259247 | GEO
Project description:To address the contribution of transcriptional regulation to Drosophila clock gene expression and to behavior, we generated a series of CRISPR-mediated deletions within two regions of the circadian gene timeless (tim), an intronic E box region and an upstream E box region that are both recognized by the key transcription factor Clock (Clk) and its heterodimeric partner Cycle. The upstream deletions but not an intronic deletion dramatically impact tim expression in fly heads; the biggest upstream deletion reduces peak RNA levels and tim RNA cycling amplitude to about 15% of normal, and there are similar effects on tim protein (TIM). The cycling amplitude of other clock genes is also strongly reduced, in these cases due to increases in trough levels. These data underscore the important contribution of the upstream E box enhancer region to tim expression and of TIM to clock gene transcriptional repression in fly heads. Surprisingly, tim expression in clock neurons is only modestly affected by the biggest upstream deletion and is similarly affected by a deletion of the intronic E box region. This distinction between clock neurons and glia is paralleled by a dramatically enhanced accessibility of the intronic enhancer region within clock neurons. This distinctive feature of tim chromatin was revealed by ATAC-seq (Assay for Transposase-Accessible Chromatin with sequencing) assays of purified neurons and glia as well as of fly heads. The enhanced cell type-specific accessibility of the intronic enhancer region explains the resilience of clock neuron tim expression and circadian behavior to deletion of the otherwise more prominent upstream tim E box region.
2024-02-28 | GSE259243 | GEO
Project description:In order to identify genes affected in our model of PANK defficiency we profiled fly heads of female flies control or expressing fumble RNAi by the use of the tim-gal4 driver Female fly heads of the indicated strains were collected at 2 different circadian timepoints, RNA extracted, labeled and hybridized to Affymetrix oligonucleotide microarrays.
2015-01-12 | E-GEOD-42037 | biostudies-arrayexpress
Project description:The timing of behavior under natural light-dark conditions is a function of circadian clocks and photic input pathways. Yet a mechanistic understanding of how these pathways collaborate in animals is lacking. Here we demonstrate in Drosophila that the Phosphatase of Regenerating Liver-1 (PRL-1) sets period length and behavioral phase gated by photic signals. PRL-1 knockdown in PDF clock neurons dramatically lengthens circadian period. PRL-1 mutants exhibit allele-specific interactions with the light- and clock-regulated gene timeless (tim). Moreover, we show that PRL-1 promotes TIM accumulation and dephosphorylation. Interestingly, the PRL-1 mutant period lengthening is suppressed in constant light and PRL-1 mutants display a delayed phase under short, but not long, photoperiod conditions. Thus, our studies reveal that PRL-1 dependent dephosphorylation of TIM is a core mechanism of the clock that sets period length and phase in darkness, enabling the behavioral adjustment to changing day-night cycles.
2020-11-11 | PXD020986 | Pride
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:Circadian rhythms are daily oscillations in metabolism and physiology and are generated by the circadian clock. In fruit fly Drosophila, the circadian clock is generated by a transcription-translation feedback loop in which the positive arm components Clock and Cycle activate the expression of the Period and Timeless genes of negative arm, as well as other circadian clock-regulated genes. After being retained in the cytoplasm, the Period and Timeless proteins then migrate to the nucleus to inhibit the Clock/Cycle transactivity by protein-protein interactions (PPIs). The endogenous circadian clock is synchronized with the geological (solar) clock via photoreceptors. Drosophila Cryptochrome protein functions as a circadian photoreceptor. In the early morning, exposure of Cryptochrome to light causes a conformational change in it which results in the formation of new PPIs. Light-activated Cryptochrome interacts with the core clock protein Timeless and the E3 ubiquitin ligase-substrate adaptor protein Jetlag, which results in the ubiquitylation of Timeless by Jetlag-E3 ligase complex and then degradation of Timeless within minutes by proteasome system. Rapid degradation of Timeless and then its partner protein Period, because of its instability in the absence of Timeless, relieves the inhibition on the Clock/Cycle transcription factors suddenly. Therefore, Clock/Cycle-driven expression of circadian clock-regulated genes are induced again, which is the restart of the circadian oscillation or the resetting of the clock. Following Timeless degradation, Cryptochrome is also degraded so the photoreceptor mechanism does not start a new resetting signal until all the required factors are re-synthesized in a circadian manner. Light-dependent degradation of Drosophila Cryptochrome can be observed in Drosophila S2 cell line in culture. In this project, we aimed at finding the interactome of Cryptochrome protein in Drosophila S2 cell line under light and in the dark using proximity labeling method. Because of the fast kinetics of Cryptochrome degradation, we chose the enzymes that can saturate in less than one hour. TurboID (TID) and APEX2 enzymes label proteins with biotin in the proximity even though they work with different mechanisms. They were fused to Cryptochrome protein, and proximity labeling was performed in the dark or under light. We have identified novel light-dependent or -independent interactors of Drosophila Cryptochrome and confirmed some of them using classical coimmunoprecipitation technique.
2024-02-05 | PXD038052 | Pride
Project description:Many different functions are regulated by circadian rhythms, including those orchestrated by discrete clock neurons within animal brains. To comprehensively characterize and assign cell identity to the 75 pairs of Drosophila circadian neurons, we optimized a single cell RNA sequencing method and assayed clock neuron gene expression at different times of day. The data identify at least 17 clock neuron categories with striking spatial regulation of gene expression. Transcription factor regulation is prominent and likely contributes to the robust circadian oscillation of many transcripts, including those that encode cell-surface proteins previously shown to be important for cell recognition and synapse formation during development. The many other clock-regulated genes also constitute an important resource for future mechanistic and functional studies between clock neurons and/or for temporal signaling to circuits elsewhere in the fly brain.
2021-01-12 | GSE157504 | 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:In order to identify genes affected in our model of PANK defficiency we profiled fly heads of female flies control or expressing fumble RNAi by the use of the tim-gal4 driver
2015-01-12 | GSE42037 | GEO
Project description:The circadian clock is comprised of proteins that form negative feedback loops, which regulate the timing of global gene expression in a coordinated 24 hour cycle. As a result, the plant circadian clock is responsible for regulating numerous physiological processes central to growth and survival. To date, most plant circadian clock studies have relied on diurnal transcriptome changes to elucidate molecular connections between the circadian clock and observable phenotypes in wild-type plants. Here, we have combined high-throughput RNA-sequencing and mass spectrometry to comparatively characterize the lhycca1, prr7prr9, gi and toc1 circadian clock mutant rosette transcriptome and proteome at the end-of-day and end-of-night.
2018-01-02 | PXD004763 | Pride