Role of Mod(mdg4)-67.2 Protein in Interactions between Su(Hw)-Dependent Complexes and Their Recruitment to Chromatin.

Su(Hw) belongs to the class of proteins that organize chromosome architecture, determine promoter activity, and participate in formation of the boundaries/insulators between the regulatory domains. This protein contains a cluster of 12 zinc fingers of the C2H2 type, some of which are responsible for binding to the consensus site. The Su(Hw) protein forms complex with the Mod(mdg4)-67.2 and the CP190 proteins, where the last one binds to all known Drosophila insulators. To further study functioning of the Su(Hw)-dependent complexes, we used the previously described su(Hw)E8 mutation with inactive seventh zinc finger, which produces mutant protein that cannot bind to the consensus site. The present work shows that the Su(Hw)E8 protein continues to directly interact with the CP190 and Mod(mdg4)-67.2 proteins. Through interaction with Mod(mdg4)-67.2, the Su(Hw)E8 protein can be recruited into the Su(Hw)-dependent complexes formed on chromatin and enhance their insulator activity. Our results demonstrate that the Su(Hw) dependent complexes without bound DNA can be recruited to the Su(Hw) binding sites through the specific protein-protein interactions that are stabilized by Mod(mdg4)-67.2.

Insulator protein Su(Hw) recruits SAGA and Brahma complexes and constitutes part of Origin Recognition Complex-binding sites in the Drosophila genome.

Despite increasing data on the properties of replication origins, molecular mechanisms underlying origin recognition complex (ORC) positioning in the genome are still poorly understood. The Su(Hw) protein accounts for the activity of best-studied Drosophila insulators. Here, we show that Su(Hw) recruits the histone acetyltransferase complex SAGA and chromatin remodeler Brahma to Su(Hw)-dependent insulators, which gives rise to regions with low nucleosome density and creates conditions for ORC binding. Depletion in Su(Hw) leads to a dramatic drop in the levels of SAGA, Brahma and ORC subunits and a significant increase in nucleosome density on Su(Hw)-dependent insulators, whereas artificial Su(Hw) recruitment itself is sufficient for subsequent SAGA, Brahma and ORC binding. In contrast to the majority of replication origins that associate with promoters of active genes, Su(Hw)-binding sites constitute a small proportion (6%) of ORC-binding sites that are localized preferentially in transcriptionally inactive chromatin regions termed BLACK and BLUE chromatin. We suggest that the key determinants of ORC positioning in the genome are DNA-binding proteins that constitute different DNA regulatory elements, including insulators, promoters and enhancers. Su(Hw) is the first example of such a protein.

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

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.