Functional Analysis of Repressor Binding Sites in the iab-2 Regulatory Region of the abdominal-A Homeotic Gene

Abstract

Spatial boundaries of homeotic gene expression are initiated and maintained by two sets of transcriptional repressors: the gap gene products and the Polycomb group proteins. Previously, the Hunchback (HB) protein has been implicated in setting the anterior expression limit of the UBX homeotic protein in parasegment 6. Here we investigate DNA elements and trans-acting repressors that control spatial expression of the Abdominal-A (ABD-A) homeotic protein. Analysis of a 1.7-kb enhancer element [iab-2(1.7)] from the iab-2 regulatory region shows that in contrast to Ubx enhancer elements, both HB and Krüppel (KR) are required to set the ABD-A anterior boundary in parasegment 7. DNase I footprinting and site-directed mutagenesis show that HB 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 that KR repression occurs through a local quenching mechanism. We also show that the gap repressor Giant (GT) initially establishes a posterior expression limit at PS9, which shifts posteriorly after the blastoderm stage. Finally, we show that this iab-2 enhancer contains multiple binding sites for the Polycomb group protein Pleiohomeotic (PHO). These iab-2 PHO sites are required in vivo for chromosome pairing-dependent repression of a mini-white reporter. However, the PHO sites are not sufficient to maintain repression of a homeotic reporter gene anterior to PS7. Full maintenance at late embryonic stages requires additional sequences adjacent to the iab-2(1.7) enhancer.

Extraction of Functional Binding Sites from Unique Regulatory Regions: The Drosophila Early Developmental Enhancers

“…total), Caudal (15), Ftz (25), Hunchback (43), Knirps (47), Krüppel (21), and Tramtrak (7). We also narrowed down the number of regulatory regions to 10, each containing at least two of the seven types of sites: engrailed intron (enint; Kassis et al 1989;Florence et al 1997), even-skipped stripe 2 (eve2; Stanojevic et al 1991;Small et al 1992;Arnosti et al 1996), even-skipped stripe 3+7 (eve3+7; Small et al 1996), fushi-tarazu proximal enhancer (ftzprox; Han et al 1993Han et al , 1998Yu et al 1999), hairy stripe 6 enhancer (hairy6; Langeland et al 1994), hairy stripe 7 enhancer (hairy7; La Rosee et al 1997Rosee et al , 1999, abdominal-A enhancer (iab2; Shimell et al 2000), Krüppel region 730 (kr730; Hoch et al 1991), spalt early enhancer (sal; Kuhnlein et al 1997;Barrio et al 1999;de Celis et al 1999), and tailless enhancer (tll; Hoch et al 1992;Liaw et al 1995).…”

Architectural and Functional Diversity of Polycomb Group Response Elements in Drosophila

“…PREs from a number of genes have been closely examined, including four PREs from the engrailed (en) and invected (inv) genes (Americo et al 2002;Cunningham et al 2010); bxd, iab2, and iab7 (also known as Fab-7) PREs from the bithorax complex (BX-C; Hagstrom et al 1997;Shimell et al 2000;Busturia et al 2001;Mishra et al 2001;Dejardin et al 2005); and an evenskipped (eve) PRE (Fujioka et al 2008). PREs are made up of binding sites for many different proteins including Pho/Phol (Brown et al 1998(Brown et al , 2003Fritsch et al 1999), Spps (Sp1 factor for pairing-sensitive silencing) ; GAGA factor (GAF); and Dsp1.…”

Genome-wide Mapping of in Vivo Targets of the Drosophila Transcription Factor Krüppel

“…The majority of the newly isolated Krü ppel target sites lack Krü ppel binding site clusters as revealed in cis-acting elements of the Krü ppel-dependent segmentation genes (4,7,21,25,55). However, the isolated and subsequently tested set of DNA fragments is enriched in Krü ppel-associated chromatin, as has been found with the eve stripe 2 element, which contains clustered Krü ppel target sites (21).…”

“…Studies concerning the biological function of Krü ppel were focused on its role in segmentation and on target genes that are controlled by Krü ppel during early body pattern formation (7,(17)(18)(19)(20)(21)(22)(23)(24)(25)(26). In addition, a few Krü ppel-regulated genes have been identified on the basis of altered gene expression patterns in Kr mutant embryos, by in vitro studies showing that Krü ppel binds to the respective cis-acting control elements (14,27,28), and by genetic modifier screens involving the dominant Kr mutation Irregular facets (If) (29,30).…”