The abdominal-B promoter tethering element mediates promoter-enhancer specificity at the Drosophila bithorax complex

Abstract

At the Drosophila bithorax complex many distinct classes of cis-regulatory modules work collectively during development to control gene expression. Abdominal-B (Abd-B) is one of three homeotic genes in the BX-C and is expressed in specific presumptive abdominal segments in the embryo. The transcription of Abd-B is tightly controlled by an array of cis-regulatory modules that direct its expression over extended genomic distances. These regulatory modules include promoters, insulators, silencers, enhancers, promoter targeting sequences and the recently identified promoter tethering element (PTE). To activate gene expression at the endogenous complex, enhancers located >50 kb away must bypass intervening insulators to interact with the Abd-B promoter. The molecular mechanisms that allow enhancers to bypass insulators are not currently well understood. In this short article, we report on a novel mechanism for insulator bypass involving the PTE. In addition, we use bioinformatic analysis across twelve Drosophila genomes to identify putative cis-regulatory sequences that may be capable of facilitating specific promoter-enhancer interactions at the bithorax complex and propose a model for their molecular function during development.

Keywords: Abdominal-B; Drosophila; bithorax; cis regulation; enhancer; promoter.

Figure 1

Promoter tethering element contains anti-insulator activity. On the B^ΔPTE-Fab8-A-5 construct (top diagram), the Fab-8 insulator was placed between two homeotic promoters, Abd-B and abd-A. In this arrangement, the IAB5 enhancer is directed to the abd-A promoter. The abd-A-lacZ reporter gene exhibits a three stripe IAB5 expression pattern in presumptive abdominal segments 5, 7 and 9 that is readily detected in blastoderm-stage embryos (B), while no CAT expression is detected (A). When the 255bp promoter tethering element is inserted upstream of the Abd-B promoter (B-Fab- 8-A5 construct, bottom diagram) the IAB5 enhancer bypasses the proximal abd-A promoter and is now redirected to the Abd-B promoter, as strong CAT expression is detected (C), while no lacZ expression is detected (D).

Figure 2

Conservation of a clustered hexamer sequence in Abd-B regulatory regions. The 3′ regulatory region that controls Abd-B expression is shown with the genomic locations of the enhancers (blue boxes), insulators (black ellipses) and PTE (yellow box). The PTE contains a highly conserved 24-mer (49112–49135) that is a tandem array of four TGGT(T/C)(C/T) motifs, that was identified using cis-Decoder software (http://evoprinter.ninds.nih.gov/cisdecoder/index.htm). In the PTE sequence (top box) the nucleotides in lowercase are not conserved, while the nucleotides in bold caps are conserved across all twelve Drosophila species. The colored nucleotides represent sequences conserved in all species except; D. willistoni (orange), D. pseudoobscura (blue), D. mojavensis (green), D. grimshawi (red). Fly Enhancer software (http://genomeenhancer.org/fly) was used to search for clusters of the hexamer motif within 150 kb of the Abd-B transcription start site. This program allowed for a stringent search for the occurrence of at least two of the (TGGTCT) or (TGGTTC) motifs in a 50 bp window. In addition to the cluster in the PTE, there are four other locations in the Abd-B 3′ regulatory region that met this stringent criterion. One highly conserved cluster is located 5 kb 3′ of the IAB5 enhancer (109929–109940) and contains two tandem conserved hexamer sequences. A second highly conserved cluster is located between the IAB7a and IAB7b enhancers (75042–75067). Two other clusters (gray arrows) are found in the Abd-B 3′ regulatory regions, one in the iab-6 region (86869–86901) and one in the iab-7 region close to the IAB7b enhancer (66397–66417). All genomic co-ordinates refer to Genbank sequence DM31961.

Figure 3

Promoter tethering element regulates long-range promoter-enhancer interactions. Model for the molecular mechanism for the function of the Abd-B PTE involves the homomeric interaction of protein factors (orange ellipses) bound to conserved clusters of hexamer motif sequences (red line) within the PTE (yellow box) and close to the IAB5 enhancer (blue box). The interactions of these regulatory proteins facilitate a chromatin loop configuration that brings the IAB5 enhancer to the Abd-B promoter. The interactions shown here represent the situation in cells expressing Abd-B in the presumptive fifth abdominal segment in the embryo, where transcription is activated by the IAB5 enhancer. In cells from different segments the enhancer(s) specific to that segment will be tethered to the promoter; eighth segment (IAB8), seventh segment (IAB7a and IAB7b) and sixth segment (IAB6).