Institute of Gene Biology, Russian Academy of Sciences, Moscow, 119334, Russia.
Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997, Russia.
Sci Rep. 2020 Mar 16;10(1):4793. doi: 10.1038/s41598-020-61514-0.
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.
邻近依赖性生物素标记揭示了果蝇蜕皮激素反应中未被描述的参与者。将两种标记酶（BioID2 和 APEX2）融合到 EcR 或 Usp 上，以生物素标记周围的蛋白质。发现 EcR/Usp 异二聚体与核孔亚基、染色质重塑因子和结构蛋白协同作用。通过 EcR/Usp 邻近依赖性标记鉴定的许多蛋白质以前被描述为蜕皮激素反应的功能成分，这证实了这种标记方法的有效性。通过用抗 EcR 抗体免疫沉淀预蛹核提取物并在 Drosophila S2 细胞中进行功能实验，对一些新发现的调节剂与蜕皮激素反应的联系进行了更深入的研究。为了澄清 EcR/Usp 与检测到的一种蛋白质 CP190 的关联，进行了更深入的研究，CP190 是果蝇绝缘子的一种很好描述的辅助因子。发现 CP190 以 20E 非依赖性方式与 EcR/Usp 的 EcR 亚基共免疫沉淀。ChIP-Seq 实验仅揭示了 CP190 和 EcR 在果蝇基因组中结合位点之间的部分重叠，并且在 20E 依赖性增强子处完全不存在 CP190 结合。Hi-C 数据分析表明，20E 依赖性增强子和 CP190 位点之间存在远程相互作用，这表明通过空间形成 EcR/Usp 和 CP190 之间的蛋白质复合物。我们的结果支持了以前的概念，即 CP190 在稳定特定染色质环方面发挥作用，以正确激活受 20E 激素调节的基因的转录。
