Functional analysis of repressor binding sites in the iab-2 regulatory region of the abdominal-A homeotic gene

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the P olycomb group proteins. Previously, the Hunchback (HB) protein has been imp licated in setting the anterior expression limit of the UBX homeotic protei n in parasegment 6. Here we investigate DNA elements and trans-acting repre ssors that control spatial expression of the Abdominal-A (ABD-A) homeotic p rotein. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 r egulatory region shows that in contrast to Ubx enhancer elements, both HE a nd Kruppel (KR) are required to set the ABD-A anterior boundary in parasegm ent 7. DNase I footprinting and site-directed mutagenesis show that HE and KR are direct regulators of this iab-2 enhancer. The single KR site can be moved to a new location 100 bp away and still maintain repressive activity, whereas relocation by 300 bp abolishes activity. These results suggest tha t KR repression occurs through a local quenching mechanism. We also show th at the gap repressor Giant (GT) initially establishes a posterior expressio n limit at PS9, which shifts posteriorly after the blastoderm stage. Finall y, we show that this iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (PHO). These iab-2 PHO sites are requ ired in vivo for chromosome pairing-dependent repression of a mini-white re porter. However, the PHO sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryon ic stages requires additional sequences adjacent to the iab-2(1.7) enhancer . (C) 2000 Academic Press.