Abstract
Steroid hormones drive the transcription of developmental genes by activating distinct sets of enhancers across tissues and developmental contexts, but the mechanisms that target specific enhancers remain incompletely understood, even in model organisms. It has been proposed that selective binding of nuclear receptors occurs at regulatory sites primed by other classes of DNA-binding transcription factors. However, direct studies of cooperation between nuclear receptors and different transcription factors remain limited. A previous study suggested that the GATA family factor SRP/dGATAb primes regulatory sites in S2 Schneider cells for activation by 20-hydroxyecdysone (20E), the principal steroid hormone in Drosophila development. Yet the genome-wide impact of SRP/dGATAb depletion on transcriptional responses to 20E has not been examined. We investigated the role of SRP/dGATAb in the response of S2 Schneider cells to 20E using SRP/dGATAb depletion via RNA interference. Combined RNA-Seq and ChIP-Seq analyses identified primary targets of SRP/dGATAb that (i) are transcriptionally induced by 20E and (ii) contain binding sites for both EcR and SRP/dGATAb. SRP/dGATAb depletion altered the transcription of different 20E-induced genes in opposite ways. For one subset of 20E-activated genes whose expression decreased upon depletion, SRP/dGATAb regulated active regulatory sites marked by H3K27Ac and enriched with SRP/dGATAb motifs. In these loci, SRP/dGATAb depletion reduced EcR binding at co-bound sites, demonstrating a priming role for this GATA family protein. In contrast, for a second subset of 20E-activated but SRP/dGATAb-suppressed genes (i.e., genes whose expression increased upon SRP/dGATAb depletion), SRP/dGATAb and EcR co-bound sites exhibited undisturbed EcR binding. Notably, the overall level of H3K27 acetylation at these loci increased upon SRP/dGATAb depletion. Our data indicate that SRP/dGATAb positively regulates 20E-inducible transcription in S2 Schneider cells for some genes, functioning as a priming factor that facilitates EcR recruitment and chromatin acetylation. In contrast, for another subset of 20E-inducible genes, SRP/dGATAb exerts a negative regulatory effect, restraining activity of the regulatory sites.
