Drosophila Ecdysone Receptor Study
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ABSTRACT:
ORGANISM(S): Drosophila melanogaster
PROVIDER: GSE9156 | GEO | 2008/05/01
SECONDARY ACCESSION(S): PRJNA102707
REPOSITORIES: GEO
Similar Datasets
Project description:We used the DamID method to systematically identify the binding sites of Ecdysone Receptor and its heterodimeric partner USP across the whole genome in Drosophila Kc cells. We find that the EcR sites are a subset of the USP sites and that only a proportion are ecdysone regulated from an accompanying ecdysone profiling study. The role of EcR/USP in the ecdysone network appears to be coordinated by the recruitment of many transcription factors as well as signaling molecules. Keywords: DamID, chromatin profiling, DNA microarray In this study we mapped the genomic binding sites of steroid hormone nuclear receptor EcR and USP in Kc167 cells using the DamID method. DamID involves the low level expression of a fusion protein consisting of DNA adenine methyltransferase (Dam) and a chromatin protein of interest. This fusion protein is targeted to the native binding sites of the chromatin protein, where Dam methylates adenines in the surrounding DNA. The methylated DNA fragments were isolated and amplified by selective PCR, labeled with a fluorescent dye and hybridized to whole genome tiling microarrays. In this study,experiments were done with samples obtained from independent experiments and include dye swaps.
2008-05-01 | E-GEOD-9156 | biostudies-arrayexpress
Project description:Methoxyfenozide (RH-2485) represents the group of non-steroidal ecdysteroid agonists with a dibenzoylhydrazine structure, that are used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ~1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis is also predicted to gain insights into the mechanism of resistance and the crosstalk between ecdysteroid signaling and cell proliferation-linked processes.
2010-11-08 | GSE17748 | GEO
Project description:Ecdysone signaling has long been studied as a paradigm for how hormones can synchronize development events in tissues throughout the animal. At the genetic level, ecdysone acts through its receptor, EcR, which has been classically thought to act at a relatively small number of canonical, ecdysone-responsive genes which then go on to activate the effectors of cellular processes. However, in spite of the decades of work studying EcR, the understanding of how EcR promotes changes in gene expression genome-wide in vivo remains incomplete. To examine EcR’s role in promoting changes in gene expression, we use a tissue-specific GAL4 driver to knockdown EcR throughout wing development and perform RNAseq on both WT wings and EcR-RNAi wings at -6h after puparium formation (APF) and +6hAPF. We find that EcR is required to promote thousands of gene expression changes genome-wide. To determine whether EcR is directly effecting these changes, we perform CUT&RUN in wings to identify EcR binding sites genome-wide. We find that EcR directly binds thousands of sites genome-wide, including many genes important for wing development. Collectively, these data suggest that EcR does not only act the top of a signaling cascade, but, instead, directly effects the response to ecdysone at multiple levels.
2019-04-24 | GSE124254 | GEO
Project description:In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its receptor, a heterodimer of the EcR and Usp proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates metamorphosis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. We find that EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.
2022-09-12 | GSE202809 | GEO
Project description:In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its receptor, a heterodimer of the EcR and Usp proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates metamorphosis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. We find that EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.
2022-09-12 | GSE202808 | GEO
Project description:In Drosophila, the ecdysone steroid hormone is essential for coordinating developmental timing across physically separated tissues. Ecdysone directly impacts genome function through its receptor, a heterodimer of the EcR and Usp proteins. Ligand binding to EcR triggers a transcriptional cascade, including activation of a set of primary response transcription factors. The hierarchical organization of this pathway has left the direct role of EcR in mediating ecdysone responses obscured. Here, we investigate the role of EcR in controlling tissue-specific ecdysone responses, focusing on two tissues that diverge in their response to rising ecdysone titers: the larval salivary gland, which undergoes programmed destruction, and the wing imaginal disc, which initiates metamorphosis. We find that EcR functions bimodally, with both gene repressive and activating functions, even at the same developmental stage. We find that EcR DNA binding profiles are highly tissue-specific, and transgenic reporter analyses demonstrate that EcR plays a direct role in controlling enhancer activity. Despite a strong correlation between tissue-specific EcR binding and tissue-specific open chromatin, we find that EcR does not control chromatin accessibility at genomic targets. We conclude that EcR contributes extensively to tissue-specific ecdysone responses. However, control over access to its binding sites is subordinated to other transcription factors.
2022-09-12 | GSE202807 | GEO
Project description:The subject of the current study is the finding of possible molecular partners of Drosophila EcR receptor. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. All fused proteins were expressed using the Act5C promoter in Drosophila S2 cells. To ensure functionality of the generated proteins, we verified their ability to bind EcR and Usp sites in the Drosophila genome with the ChIP-Seq. Our results demonstrate that EcR and Usp fusions can be recruited to genomic sites endogenous for the EcR/Usp proteins. Conversely, unfused BioID2 and APEX2 enzymes do not bind to EcR/Usp sites. A more in-depth study was conducted to clarify the association of EcR/Usp with one of the detected proteins, CP190, a well-described cofactor of Drosophila insulators. ChIP-Seq experiments revealed an enrichment of CP190 binding on transcription start and end sites of 20E-dependent genes but not at EcR/Usp-bound enhancers.
2020-03-17 | GSE139316 | GEO
Project description:Developmental abnormalities observed in Cornelia de Lange Syndrome (CdLS) have been genetically linked to mutations in the cohesin machinery. These findings raise the possibility that cohesin, in addition to its canonical function of mediating sister chromatid cohesion, might also be involved in regulating gene expression. We report that cleavage of cohesin’s kleisin subunit in post-mitotic Drosophila salivary glands induces major changes (both up and down) in the transcript levels of many genes. Kinetic analyses of changes in transcript levels upon cohesin cleavage reveal that a subset of genes responds to cohesin cleavage within a few hours. In addition, cohesin binds to most of these loci, suggesting that cohesin is directly regulating their expression. Amongst these genes are several that are regulated by the steroid hormone Ecdysone. Transcripts at EcR and Eip74EF, which encode an Ecdysone Receptor and an Ecdysone-regulated transcription factor, respectively, decline ten-fold within four hours of cohesin cleavage. Cytological visualization of transcription at selected Ecdysone-responsive genes reveals that puffing at Eip74EF ceases within an hour or two of cohesin cleavage, long before any decline in EcR associated with this locus. We conclude that cohesin regulates expression of a distinct set of genes, including those mediating the Ecdysone response DamID experiments for Rad21 were performed on gDNA from drosophila salivary glands. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. The experiment was done in duplicate in the reverse dye orientation.
2011-03-22 | E-GEOD-21874 | biostudies-arrayexpress
Project description:Steroid hormones regulate tissue development and physiology by modulating the transcription of a broad spectrum of genes. In insects, the principal steroid hormones, ecdysteroids, trigger the expression of thousands of genes through a cascade of transcription factors (TFs) to coordinate developmental transitions such as larval molting and metamorphosis. However, whether ecdysteroid signaling can bypass transcriptional hierarchies to exert its function in individual developmental processes is unclear. Here, we report that a single non-TF effector gene mediates the transcriptional output of ecdysteroid signaling in Drosophila myoblast fusion, a critical step in muscle development and differentiation. Specifically, we show that the 20-hydroxyecdysone (commonly referred to as “ecdysone”) secreted from an extraembryonic tissue, amnioserosa, acts on embryonic muscle cells to directly activate the expression of antisocial (ants), which encodes an essential scaffold protein enriched at the fusogenic synapse. Not only is ants transcription directly regulated by the heterodimeric ecdysone receptor complex composed of Ecdysone receptor (EcR) and Ultraspiracle (USP) via ecdysone response elements, but more strikingly, expression of ants alone is sufficient to rescue the myoblast fusion defect in ecdysone signaling-deficient mutants. We further show that EcR/USP and a muscle-specific transcription factor Twist synergistically activate ants expression in vitro and in vivo. Taken together, our study provides the first example of a steroid hormone directly activating the expression of a single key non-TF effector gene to regulate a developmental process via inter-organ signaling and provides a new paradigm for understanding steroid hormone signaling in other developmental and physiological processes.
2024-03-20 | GSE236183 | GEO
Project description:Methoxyfenozide (RH-2485) represents the group of non-steroidal ecdysteroid agonists with a dibenzoylhydrazine structure, that are used as novel biorational insecticides in the control of insect pests. Here we report on the selection of Drosophila melanogaster S2 cells for resistance to inhibition of cell proliferation by methoxyfenozide by ~1000-fold over 4 months. Cells were exposed to gradually increasing concentrations of methoxyfenozide and selected out based on the ecdysteroid-sensitive response for cell proliferation. In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide. But when resistant cells were relaxed from pressure in methoxyfenozide-free medium, induction of the reporter construct was observed. In parallel, EcR/USP functionality was also restored when resistant cells were rescued by a Drosophila EcR plasmid. However, it was striking that in the resistant cells the ecdysteroid-sensitive response for cell proliferation was not restored upon methoxyfenozide withdrawal, indicating permanent changes in the physiology of the cells during selection. To investigate changes in gene expression caused by inactivation of the EcR/USP complex in resistant cells, Drosophila oligo 14kv1 microarrays were used and probed with cDNAs from resistant cells in the presence and absence of ecdysone agonist on one hand and from unselected sensitive cells on the other hand. A selection of 324 differentially expressed genes was assigned covering diverse functions as transport, enzyme activity, cytoskeleton organization, cell cycle machinery, transcription/translation and ecdysteroid signaling. Besides the identification of (primary and secondary) target genes of the EcR/USP signaling pathway, this analysis is also predicted to gain insights into the mechanism of resistance and the crosstalk between ecdysteroid signaling and cell proliferation-linked processes. In this microarray experiment three biological conditions were under study: (i) untreated S2 cells (S2), (ii) a subpopulation of these untreated cells, which were grown under continuous pressure of methoxyfenozide and which were shown to become resistant to this compound (S2res) and (iii) a subpopulation of the resistant cells, which were again cultured without methoxyfenozide added to the medium (S2res-woMF). For each condition, two subpopulations (which were treated as biological replicates, although they are not exactly independent), were isolated and used for the RNA extractions. The overall experimental design was an n+2 A-optimal design. When each hybridisation is respresented by an arrow (Cy5 ? Cy3), the design can be respresented as follows: First loop = S2.1 ? S2res.1 ? S2.2 ? S2res.2 ? S2.1; Second loop = S2.1 ? S2res-woMF.1 ? S2.2 ? S2res-woMF.2 ? S2.1 (numbers following the condition name correspond to the biological replicate numbers). The two loops are thus connected by S2.1 and S2.2. Two analyses were performed: S2res compared to S2 and S2res-woMF compared to S2.
2010-11-08 | E-GEOD-17748 | biostudies-arrayexpress