The bithorax complex iab-7 Polycomb response element has a novel role in the functioning of the Fab-7 chromatin boundary

Abstract

Expression of the three bithorax complex homeotic genes is orchestrated by nine parasegment-specific regulatory domains. Autonomy of each domain is conferred by boundary elements (insulators). Here, we have used an in situ replacement 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-71 deleted four nuclease hypersensitive sites, HS*, HS1, HS2, and HS3, located between the iab-6 and iab-7 regulatory domains. Transgenic and P-element excision experiments mapped the boundary to HS*+HS1+HS2, while HS3 was shown to be the iab-7 Polycomb response element (PRE). Recent replacement experiments showed that HS1 is both necessary and sufficient for boundary activity when HS3 is also present 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 binding in 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 association in vivo, depend 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+HS3mPho combination is lost when the flies are heterozygous for a mutation in the GAF gene. Taken together, these results reveal a novel function for the iab-7 PREs in chromosome architecture.

Complete reconstitution of bypass and blocking functions in a minimal artificialFab-7insulator fromDrosophila bithoraxcomplex

“…Binding Sites for the Architectural Proteins Cooperate with dHS1 in Organization of the Functional Boundary. We previously found that the distal 240 bp of the HS1 region (dHS1) is critical for the boundary activity of Fab-7 and contains the LBC recognition sequences (38,44,46). However, dHS1 alone has only some residual boundary function (Fig.…”

“…Since dHS1 is bound by LBC in nuclear extracts, an important question is whether LBC is responsible (at least in part) for the bypass activity of this sequence in vivo. The minimal Fab-7 recognition sequence binding this very large complex was ∼65 bp long (38,46). Although the GAGAG motifs in the small 65-bp probes are critical for LBC binding, they are less important when larger dHS1 sequences are used for EMSA probes, because neighboring sequences may contain additional binding sites for proteins that stabilize LBC recruitment.…”

“…Rather, the likely role of LBC is to provide a scaffold for the association of other, as-yet unknown factors. The reason for this is that LBC binds to sites elsewhere in the genome that have quite different functions (43,46). We would expect that the bypass activity of dHS1 might require not only an LBC association, but also other DNA-binding proteins.…”

“…Elba and Insensitive bind to sites in HS*, HS1, and HS2 (38,(42)(43)(44), while Pita binds to two sites in HS2 (14,27,45). HS1 and HS3 contain recognition sequences for LBC (38,46). LBC is a very large (>1,000 kDa) complex and is unusual in that the minimal recognition sequence in electrophoretic mobility shift assays (EMSAs) for probes from Fab-7 is ∼65 bp long.…”

The insulator functions of theDrosophilapolydactyl C2H2 zinc finger protein CTCF: Necessity versus sufficiency

“…In addition, as in vertebrates, dCTCF is one of dozens of polydactyl C2H2 zinc finger proteins encoded in the fly genome (10,11). However, unlike vertebrates, several of these polydactyl C2H2 zinc finger protein family members [Su(Hw), Pita, Zipic, Zw5, CLAMP, and Opbp] have been shown to play roles in chromosomal architecture in flies, and it is likely that other members of this large protein family will have similar functions (12)(13)(14)(15)(16)(17). Moreover, like dCTCF, two of these zinc finger proteins, Su(Hw) and Pita, are components of boundaries in BX-C and are important in the proper regulation of the BX-C homeotic genes (18)(19)(20).…”