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Sci Rep. 2020 Mar 16;10(1):4793. doi: 10.1038/s41598-020-61514-0.

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

Author information

1
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
2
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
3
Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Moscow, 119334, Russia.
4
Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia. vorobyeva@genebiology.ru.

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.

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