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Role of SRP/dGATAb in the response of Drosophila S2 Schneider cells to 20-hydroxyecdysone

ABSTRACT: Background: The tissue-specific response to steroid hormones remains unclear even in model organisms. The mechanism is proposed to be based on the selective binding of nuclear hormone receptors to regulatory sites that are primed by tissue-specific factors. But studies directly describing the cooperative work of nuclear receptors with tissue-specific transcription factors remain sparse. Earlier, GATA family factor SRP/dGATAb was proposed to prime regulatory sites in S2 Schneider cells for their activation by 20-hydroxyecdysone (20E), the main steroid hormone of the Drosophila development. Highly specific expression of SRP/dGATAb in Drosophila tissues makes it a suitable candidate for a factor enabling tissue-specific response to 20E. Results: The role of SRP/dGATAb in the response of S2 Schneider cells to 20E was determined using SRP/dGATAb depletion via RNA interference. Combination RNA-Seq with ChIP-Seq analysis helped identify the primary targets of SRP/dGATAb whose transcription is induced in response to 20E treatment and whose loci contain EcR and SRP/dGATAb binding sites. SRP/dGATAb depletion was found to alter transcription of different 20E-induced genes in opposite ways. For 20E-activated genes whose transcription was decreased upon depletion, SRP/dGATAb was found to control active regulatory sites marked by the H3K27Ac and enriched with SRP/dGATAb motifs. In this group SRP/dGATAb depletion reduced EcR binding level at sites co-bound with EcR and SRP/dGATAb, demonstrating a priming role for GATA family protein. In contrast, for 20E-activated SRP/dGATAb-suppressed genes whose transcription was increased upon SRP/dGATAb depletion, SRP/dGATAb and EcR co-bound sites showed unchanged H3K27Ac levels and EcR binding. The overall level of H3K27 acetylation at the loci of the second group of genes showed increase upon SRP/dGATAb depletion. Conclusions: Obtained data showed a positive role of SRP/dGATAb in 20E-inducible transcription in S2 Schneider cells for some genes. SRP/dGATAb worked as a priming transcriptional factor for 20E-inducible regulatory sites controlling EcR recruitment and chromatin acetylation. But for another group of 20E-inducible genes, SRP/dGATAb had negative impact on transcription. Its recruitment through the protein-protein interactions restrained activity of the regulatory sites.

ORGANISM(S): Drosophila melanogaster

PROVIDER: GSE304853 | GEO | 2025/12/08

REPOSITORIES: GEO

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Project description:Juvenile hormone (JH) and 20-hydroxy-ecdysone (20E) are highly versatile hormones, coordinating development, growth, and reproduction in insects. Pulses of 20E provide key signals for initiating developmental and physiological transitions, while JH promotes or inhibits these signals in a stage-specific manner. Previous evidence suggests that JH and 20E might modulate innate immunity, but whether and how these hormones interact to regulate the immune response remains unclear. Here we show that JH and 20E have antagonistic effects on the expression of antimicrobial peptides (AMPs) in Drosophila melanogaster. In S2* cells challenged with bacterial peptidoglycans, 20E induces promoter activity and expression of AMPs in a dose-dependent manner, while JH III and its synthetic analogs (JHa) methoprene and pyriproxyfen abolish this 20E-dependent response. Using microarrays and GFP reporter gene assays in adult flies, we confirm that JH is a hormonal immuno-suppressor in vivo. When silencing both partners of the ecdysone receptor (EcR ) / ultraspiracle (USP) heterodimer with RNAi in S2* cells, 20E fails to activate Diptericin (Dpt) expression, suggesting that 20E regulates expression of this gene through EcR / USP signaling. In contrast, silencing methoprene-tolerant (MET), a candidate JH receptor, does not impair the immuno-suppressive action of JH III and JHa, indicating that in this context MET does not function as a JH receptor. Our results suggest that the balance of 20E and JH is a major determinant of immune homeostasis in insects. Experiment Overall Design: To examine the transcriptional response of y, w flies to treatment with exogenous JH, we performed a microarray experiment on females treated with JH or solvent (control). Flies were grown on regular yeast diet, switched to no-yeast food within one hour of eclosion, and yeast-starved for 5 days posteclosion to lower their endogenous JH titer and to synchronize their physiology (cf. Tu and Tatar, 2003; Gershman et al., 2007). Subsequently, we anesthetized flies on ice and topically treated them with 0.1 ïl of 187 mM JH III in acetone or with 0.1ï ïl 100% acetone (control) using a 1 ïl Hamilton syringe with a repeating dispenser. 12 hours after hormone administration, samples were snap-frozen in liquid nitrogen and stored at -80Â°C. RNA was isolated from samples (2 JH samples, 2 control samples, each with 30 females) by lysis, as described in Gershman et al. (2007). cDNA products were hybridized at the Brown University Genomics Core Facility to Affymetrix GeneChip Drosophila_1 Genome Arrays (2 replicate chips per treatment). The dataset consisted of 14,009 probe sets, with 6,142 probe sets annotated. Expression data were analyzed for significant over- or underrepresentation of gene ontology (GO) terms with the web application FatiGO (Al-Shahrour et al., 2004), using a two-fold change criterion. To test whether JH treatment significantly suppresses expression of AMPs we used t-tests implemented in JMP IN 5.1.

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Role of SRP/dGATAb in the response of Drosophila S2 Schneider cells to 20-hydroxyecdysone

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