Replacement of Fab-7 by the gypsy or scs insulator disrupts long-distance regulatory interactions in the Abd-B gene of the bithorax complex.

Chromatin domain boundaries, like scs or gypsy insulators in Drosophila, have been identified in transgene assays through their enhancer-blocking activity. Boundary elements in the bithorax complex (BX-C), such as Fab-7 and Fab-8, have been identified genetically and been shown to have insulator activity in transgene assays. However, it is not clear whether boundary elements identified in transgene assays will function appropriately in chromosomal contexts such as BX-C. Using gene conversion, we have substituted the scs or gypsy insulators for Fab-7. We find that both scs and gypsy are very potent insulators in the ectoderm, but surprisingly, the insulating activity of gypsy (but not scs) is lost in the CNS. Our results reveal that the Fab-7 boundary must have special properties that scs and gypsy lack, which allow it to function appropriately in BX-C regulation.

The iab-7 polycomb response element maps to a nucleosome-free region of chromatin and requires both GAGA and pleiohomeotic for silencing activity.

In the work reported here we have undertaken a functional dissection of a Polycomb response element (PRE) from the iab-7 cis-regulatory domain of the Drosophila melanogaster bithorax complex (BX-C). Previous studies mapped the iab-7 PRE to an 860-bp fragment located just distal to the Fab-7 boundary. Located within this fragment is an approximately 230-bp chromatin-specific nuclease-hypersensitive region called HS3. We have shown that HS3 is capable of functioning as a Polycomb-dependent silencer in vivo, inducing pairing-dependent silencing of a mini-white reporter. The HS3 sequence contains consensus binding sites for the GAGA factor, a protein implicated in the formation of nucleosome-free regions of chromatin, and Pleiohomeotic (Pho), a Polycomb group protein that is related to the mammalian transcription factor YY1. We show that GAGA and Pho interact with these sequences in vitro and that the consensus binding sites for the two proteins are critical for the silencing activity of the iab-7 PRE in vivo.

The Fab-8 boundary defines the distal limit of the bithorax complex iab-7 domain and insulates iab-7 from initiation elements and a PRE in the adjacent iab-8 domain.

The Drosophila bithorax complex Abdominal-B (Abd-B) gene specifies parasegmental identity at the posterior end of the fly. The specific pattern of Abd-B expression in each parasegment (PS) determines its identity and, in PS10-13, Abd-B expression is controlled by four parasegment-specific cis-regulatory domains, iab-5 to iab-8, respectively. In order to properly determine parasegmental identity, these four cis-regulatory domains must function autonomously during both the initiation and maintenance phases of BX-C regulation. The studies reported here demonstrate that the (centromere) distal end of iab-7 domain is delimited by the Fab-8 boundary. Initiators that specify PS12 identity are located on the proximal iab-7 side of Fab-8, while initiators that specify PS13 identity are located on the distal side of Fab-8, in iab-8. We use transgene assays to demonstrate that Fab-8 has enhancer blocking activity and that it can insulate reporter constructs from the regulatory action of the iab-7 and iab-8 initiators. We also show that the Fab-8 boundary defines the realm of action of a nearby iab-8 Polycomb Response Element, preventing this element from ectopically silencing the adjacent domain. Finally, we demonstrate that the insulating activity of the Fab-8 boundary in BX-C is absolutely essential for the proper specification of parasegmental identity by the iab-7 and iab-8 cis-regulatory domains. Fab-8 together with the previously identified Fab-7 boundary delimit the first genetically defined higher order domain in a multicellular eukaryote.

Transvection in the Drosophila Abd-B domain: extensive upstream sequences are involved in anchoring distant cis-regulatory regions to the promoter.

The Abd-B gene, one of the three homeotic genes in the Drosophila bithorax complex (BX-C), is required for the proper identity of the fifth through the eighth abdominal segments (corresponding to parasegments 10-14) of the fruitfly. The morphological difference between these four segments is due to the differential expression of Abd-B, which is achieved by the action of the parasegment-specific cis-regulatory regions infra-abdominal-5 (iab-5), -6, -7 and -8. The dominant gain-of-function mutation Frontabdominal-7 (Fab-7) removes a boundary separating two of these cis-regulatory regions, iab-6 and iab-7. As a consequence of the Fab-7 deletion, the parasegment 12- (PS12-) specific iab-7 is ectopically activated in PS11. This results in the transformation of the sixth abdominal segment (A6) into the seventh (A7) in Fab-7 flies. Here we report that point mutations of the Abd-B gene in trans suppress the Fab-7 phenotype in a pairing-dependent manner and thus represent a type of transvection. We show that the observed suppression is the result of trans-regulation of the defective Abd-B gene by the ectopically activated iab-7. Unlike previously demonstrated cases of trans-regulation in the Abd-B locus, trans-suppression of Fab-7 is sensitive to heterozygosity for chromosomal rearrangements that disturb homologous pairing at the nearby Ubx locus. However, in contrast to Ubx, the transvection we observed in the Abd-B locus is insensitive to the allelic status of zeste. Analysis of different deletion alleles of Abd-B that enhance trans-regulation suggests that an extensive upstream region, different from the sequences required for transcription initiation, mediates interactions between the iab cis-regulatory regions and the proximal Abd-B promoter. Moreover, we find that the amount of DNA deleted in the upstream region is roughly proportional to the strength of trans-interaction, suggesting that this region consists of numerous discrete elements that cooperate in tethering the iab regulatory domains to Abd-B. Possible implications of the tethering complex for the regulation of Abd-B are discussed. In addition, we present evidence that the tenacity of trans-interactions in the Abd-B gene may vary, depending upon the tissue and stage of development.