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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.
Highlights
Proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila
EcR and Usp fused to APEX2 and BioID2 enzymes bind genomic sites of EcR and Usp
BioID2 and APEX2 enzymes were marked with a 3xFLAG epitope and fused to EcR and Usp through a flexible 25 nm GS linker[4]
Summary
Proximity-dependent biotin labelling revealed undescribed participants of the ecdysone response in Drosophila. 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. In vivo proximity-dependent labelling technology was developed based on the directed mutagenesis of Escherichia coli biotin-ligase BirA2 This modified enzyme provides a higher level of promiscuous biotinylation than the original (e.g., an endogenous BirA can biotinylate only the BAP target). BioID has successfully investigated protein compounds of certain chromatin loci[5,6] This technique has predominantly been used for the analysis of stable protein agglomerates (e.g., heterochromatin) because BioID labelling requires hours to be affective. This feature makes BioID a poor method for the characterization of protein complexes with a high turnover
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