Su(Hw) primes 66D and 7F Drosophila chorion genes loci for amplification through chromatin decondensation

Abstract

Suppressor of Hairy wing [Su(Hw)] is an insulator protein that participates in regulating chromatin architecture and gene repression in Drosophila. In previous studies we have shown that Su(Hw) is also required for pre-replication complex (pre-RC) recruitment on Su(Hw)-bound sites (SBSs) in Drosophila S2 cells and pupa. Here, we describe the effect of Su(Hw) on developmentally regulated amplification of 66D and 7F Drosophila amplicons in follicle cells (DAFCs), widely used as models in replication studies. We show Su(Hw) binding co-localizes with all known DAFCs in Drosophila ovaries, whereas disruption of Su(Hw) binding to 66D and 7F DAFCs causes a two-fold decrease in the amplification of these loci. The complete loss of Su(Hw) binding to chromatin impairs pre-RC recruitment to all amplification regulatory regions of 66D and 7F loci at early oogenesis (prior to DAFCs amplification). These changes coincide with a considerable Su(Hw)-dependent condensation of chromatin at 66D and 7F loci. Although we observed the Brm, ISWI, Mi-2, and CHD1 chromatin remodelers at SBSs genome wide, their remodeler activity does not appear to be responsible for chromatin decondensation at the 66D and 7F amplification regulatory regions. We have discovered that, in addition to the CBP/Nejire and Chameau histone acetyltransferases, the Gcn5 acetyltransferase binds to 66D and 7F DAFCs at SBSs and this binding is dependent on Su(Hw). We propose that the main function of Su(Hw) in developmental amplification of 66D and 7F DAFCs is to establish a chromatin structure that is permissive to pre-RC recruitment.

Su(Hw) interacts with Combgap to establish long-range chromatin contacts

“…Consistent with previous research on BEAF32 IBP indirect chromatin binding 7 , we show that indirect binding of Su(Hw) to chromatin can re ect the long-range chromatin interactions in which Su(Hw) participates. Previously, we identi ed a cluster of indirect Su(Hw) ChIP-Seq peaks in Drosophila ovaries that contain a GTGT-motif instead of the Su(Hw) DNA-binding motif 17 . Here, we found that these indirect Su(Hw) peaks are occupied by Combgap, a protein known to recruit PcG group proteins to chromatin 13,14 .…”

“…Our ChIP-Seq data from wild-type and Su(Hw) LOF Drosophila ovaries con rmed this observation. We showed that direct Su(Hw) ChIP-Seq peaks can be classi ed into two clusters: those enriched with FAIRE signal and active chromatin proteins (Su(Hw) direct FAIRE + peaks), and those lacking these features (Su(Hw) direct FAIRE-peaks) 17 . In situ Hi-C experiments demonstrated that both Su(Hw)-dependent LRIs and domain border insulation are only characteristic of Su(Hw) direct FAIRE + peaks (Fig.…”

“…In our previous study, we demonstrated that direct Su(Hw) ChIP-Seq peaks in Drosophila ovaries can be classi ed into two clusters based on enrichment with the FAIRE signal and active chromatin features, such as chromatin remodelers and Gcn5 histone acetyltransferase (hereon we refer to this cluster as Su(Hw) direct FAIRE + peaks) or the lack of such enrichment (Su(Hw) direct FAIRE-peaks) 17 . We analysed average LRIs for these two clusters separately.…”

“…The following previously described ChIP-Seq data for the S2 Drosophila cell line were used: H3K27me3 23 (GSE41440), Combgap 19 (GSE200213), Su(Hw) 17 (GSE30740), Rpb3, NELF-A 21 (GSE156847). For the ovaries of egg chamber stages 1-8 we used the following the ChIP-Seq and MNase-Seq data previously described: ChIP-Seq for Su(Hw), Brm, ISWI, Mi2, CHD1 17 (GSE168894) and MNase-Seq data for follicular cells from egg chambers stages 1-8 26 (NCBI-SRA BioProject SRP057811). We have not used any gures or text from the manuscripts previously published, only analysed the data deposited at the free public access databases.…”

“…For the co-immunoprecipitations and ChIP-Seq experiments we used previously described antibodies against Combgap 19 , Su(Hw) 17 and Su(Hw) LOF ovaries. The image was generated using pyGenomeTracks 59 .…”