The bithorax complexiab-7Polycomb Response Element has a novel role in the functioning of theFab-7chromatin boundary
Authors: Kyrchanova Olga , Kurbidaeva Amina , Sabirov Marat , Postika Nikolay , Wolle Daniel , Aoki Tsutomu , Maksimenko Oksana , Mogila Vladic , Schedl Paul , Georgiev Pavel 
Published: May 2018

Expression of the threeBithoraxcomplex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used anin situreplacement strategy to reanalyze the sequences required for the functioning of one of the best-characterized fly boundaries,Fab-7. It was initially identified by a deletion,Fab-71, that transformed parasegment (PS) 11 into a duplicate copy of PS12.Fab-71deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located in between theiab-6andiab-7regulatory domains. Transgene andP-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be theiab-7Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also presented in the replacement construct. Surprisingly, while HS1+HS3 combination has full boundary activity, we discovered that HS1 alone has only minimal function. Moreover, when combined with HS3, only the distal half of HS1, dHS1, is needed. A ∼1,000 kD multiprotein complex containing the GAF protein, called the LBC, binds to the dHS1 sequence and we show that mutations in dHS1 that disrupt LBC binding in nuclear extracts eliminate boundary activity and GAF bindingin vivo. HS3 has binding sites for GAF and Pho proteins that are required for PRE silencing. In contrast, HS3 boundary activity only requires the GAF binding sites. LBC binding with HS3 in nuclear extracts, and GAF associationin vivodepend upon the HS3 GAF sites, but not the Pho sites. Consistent with a role for the LBC in HS3 boundary activity, the boundary function of the dHS1+HS3mPhocombination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for theiab-7PREs in chromosome architecture.

Author Summary

Polycomb group proteins (PcG) are important epigenetic regulators of developmental genes in all higher eukaryotes. InDrosophila,these proteins are bound to specific regulatory DNA elements called Polycomb group Response Elements (PREs). PcG support proper patterns of homeotic gene expression throughout development.DrosophilaPREs are made up of binding sites for a complex array of DNA binding proteins, including GAF and Pho. In the regulatory region of the bithorax complex (BX-C), the boundary/insulator elements organize the autonomous regulatory domains, and their active or repressed states are regulated by PREs. Here, we studied the domain organization of theFab-7boundary and the neighboring PRE, which separate theiab-6andiab-7domains involved in transcription of theAbd-Bgene. It was previously thought that PRE recruits PcG proteins that inhibit activation of theiab-7enhancers in the inappropriate domains. However, here we found that PRE contributes to boundary activity and in combination with a key 242 bpFab-7region (dHS1) can form a completely functional boundary. Late Boundary Complex (LBC) binds not only to dHS1 but also to PRE and is required for the boundary activity of both elements. At the same time, mutations of Pho binding sites strongly diminish recruiting of PcG but do not considerably affect boundary function, suggesting that these activities can be separated in PRE.