Proximity-dependent biotin labelling reveals CP190 as an EcR/Usp molecular partner

Abstract

proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila. Two labelling enzymes (BioID2 and APEX2) were fused to EcR or Usp to biotin label the surrounding proteins. the ecR/Usp heterodimer was found to collaborate with nuclear pore subunits, chromatin remodelers, and architectural proteins. Many proteins identified through proximity-dependent labelling with ecR/Usp were described previously as functional components of an ecdysone response, corroborating the potency of this labelling method. A link to ecdysone response was confirmed for some newly discovered regulators by immunoprecipitation of prepupal nuclear extract with anti-ecR antibodies and functional experiments in Drosophila S2 cells. 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. CP190 was found to co-immunoprecipitate with the EcR subunit of EcR/Usp in a 20E-independent manner. ChIP-Seq experiments revealed only partial overlapping between CP190 and EcR bound sites in the Drosophila genome and complete absence of CP190 binding at 20E-dependent enhancers. Analysis of Hi-C data demonstrated an existence of remote interactions between 20E-dependent enhancers and CP190 sites which suggests formation of a protein complex between EcR/Usp and CP190 through the space. Our results support the previous concept that CP190 has a role in stabilization of specific chromatin loops for proper activation of transcription of genes regulated by 20E hormone.www.nature.com/scientificreports www.nature.com/scientificreports/ rate (e.g., promoter-bound transcriptional complexes). Recently, an additional directed mutagenesis procedure of BirA has allowed for the development of the fast TurboID enzyme 7 .An alternative to the BioID2 technology is a combination of APEX2, a plant ascorbate peroxidase, and a biotin-phenol substrate. In the presence of hydrogen peroxide, the APEX2 enzyme can catalyse the substrate conversion (biotin-phenol) into active and short-lived radicals which can modify the surrounding proteins 3 . Although the method is less common than the BioID technique, APEX2 labelling requires significantly less time to biotinylate surrounding proteins, making it more suited for researching unstable complexes 8,9 . For example, it was recently integrated with the CRISPR/Cas9 system to investigate DNA-protein interactions, as an alternative to ChIP-Seq 10 .The main research interest of our group is to accurately describe the molecular mechanisms of transcriptional activation in eukaryotes. The ultimate goal is to understand the general mechanism for most genes, including the cooperative function of hundreds of transcriptional factors. As an experimental approach we use Drosophila melanogaster developmental genes (e.g., 20-hydroxyecdysone (20E)-dependent), which are a well-regulated physiological system that is convenient to study 11,12 . The main molecular sensor of an ecdysone system is the EcR/Us...

The negative elongation factor NELF promotes induced transcriptional response of Drosophila ecdysone-dependent genes

“…1 A). We obtained specific antibodies against 3 out of 4 subunits of Drosophila NELF: NELF-A, NELF-B and NELF-E. Antibodies against NELF-A and NELF-E were previously briefly characterized 2 , 3 . In this report we provide their additional description together with information on properties of newly obtained antibodies against NELF-B (Fig.…”

“…GRO-Seq data were described previously and deposited as GSM3274627 and GSM3274628 29 . ChIP-Seqs of FLAG-EcR from S2 Schneider cells were deposited and described previously as GSE139316 3 . RNA-Seqs from 1 h DMSO- or 20E-treated S2 Schneider cells were described previously but were not deposited in GEO (thus now we provide these data in GSE156847 1 .…”

“…Pol II S2P and Pol II S5P antibodies were purchased from Sigma-Aldrich (Ab5095 and Ab5131). For the ChIP-Seq analysis of CBP/Neijre, Rpb3, Spt5, NELF-A and NELF-E we used previously described antibodies 2 , 3 . Antibodies against NELF-B (epitope corresponding to 150–594 amino acid residues) were generated in this study.…”

“…For this purpose, we chose a set of developmental genes that can be rapidly induced by the addition of 20-hydroxyecdysone (20E) hormone in Drosophila cell culture 1 . In our previous works we have described in detail the primary stages of 20E-dependent induction and found new molecular partners of the EcR/Usp ecdysone sensor 2 , 3 . The present study aims to characterize the molecular complex responsible for the organization of the Pol II pause at 20E-dependent genes.…”

“…In other words, we showed that 20E-dependent genes are regulated via Pol II pausing. In a recent work we revealed an interaction between the EcR/Usp sensor and the NELF-A subunit of the Negative elongation factor (NELF) and hypothesized that the latter may be responsible for Pol II pausing at 20E-dependent promoters 3 .…”