Unknown,Transcriptomics,Genomics,Proteomics

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Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide

ABSTRACT: 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 In the resistant cells, the ecdysteroid receptor (EcR/USP) complex was no longer active in the presence of methoxyfenozide.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.

ORGANISM(S): Drosophila melanogaster

SUBMITTER: Guy Smagghe

PROVIDER: E-GEOD-17748 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide.

Mosallanejad Hadi H Badisco Liesbeth L Swevers Luc L Soin Thomas T Knapen Dries D Vanden Broeck Jozef J Smagghe Guy G

Journal of insect physiology 20100909 12

Methoxyfenozide (RH-2485) is a non-steroidal ecdysteroid agonist with a dibenzoylhydrazine structure, representing a group 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 ...[more]

PMID: 20816975

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Project description:Dosage compensation refers to the equalization of most X-linked gene products between males and females. In Drosophila, it is mediated by the MSL complex that preferentially associates with numerous sites on the X chromosome in somatic cells of males, and is responsible for an enhancement of the transcriptional rate of a substantial number of X-linked genes. Here we show that topoisomerase II (Topo II) is an integral part of the mechanistic basis of dosage compensation and we highlight a novel function for this enzyme. A widely accepted model of transcription postulates that the moving bubble generated by an RNA polymerase elongating complex induces positive DNA supercoiling in the region ahead of the complex and negative supercoiling in its wake. These transitory changes in supercoiling are resolved by the action of topoisomerases. We have investigated the role of Topo II in dosage compensation by RNAi-mediated depletion, and we have used chromatin immunoprecipitation to determine its genomic distribution and relative abundance on X-linked genes. Topo II is enriched on dosage compensated genes and this enrichment is independent from the approximate two-fold enhancement in transcription of these genes. We have demonstrated an RNA-dependent association of Topo II with MLE, the ATPase-helicase subunit of the MSL complex. Our results indicate a role for Topo II that is additional to and different from its function in restoring normal DNA superhelicity during the transcription process. We suggest that the enhanced level of Topo II alters the DNA supercoiling of compensated gene units to facilitate transcription and could provide a basis for the recent report that the MSL complex enhances transcription by increasing the rate of elongation of RNA polymerase II. Investigation of dosage compensated gene expression levels in S2 and S2 Topo II RNAi knockdown cells. mRNA was isolated from S2 cells and S2 cells with RNAi knockdown of Topo II. The mRNA was then converted to cDNA and hybridized to a NimbleGen D. melanogaster gene expression array. 2 replicates each.

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Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide

Publications

Ecdysone signaling and transcript signature in Drosophila cells resistant against methoxyfenozide.

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