Abstract
There are many examples within gene complexes of transcriptional enhancers interacting with only a subset of target promoters. A number of molecular mechanisms including promoter competition, insulators and chromatin looping are thought to play a role in regulating these interactions. At the Drosophila bithorax complex (BX-C), the IAB5 enhancer specifically drives gene expression only from the Abdominal-B (Abd-B) promoter, even though the enhancer and promoter are 55 kb apart and are separated by at least three insulators. In previous studies, we discovered that a 255 bp cis-regulatory module, the promoter tethering element (PTE), located 5′ of the Abd-B transcriptional start site is able to tether IAB5 to the Abd-B promoter in transgenic embryo assays. In this study we examine the functional role of the PTE at the endogenous BX-C using transposon-mediated mutagenesis. Disruption of the PTE by P element insertion results in a loss of enhancer-directed Abd-B expression during embryonic development and a homeotic transformation of abdominal segments. A partial deletion of the PTE and neighboring upstream genomic sequences by imprecise excision of the P element also results in a similar loss of Abd-B expression in embryos. These results demonstrate that the PTE is an essential component of the regulatory network at the BX-C and is required in vivo to mediate specific long-range enhancer-promoter interactions.
Highlights
To ensure a high fidelity of gene expression patterns in embryos a very strict functional requirement exists for the interaction of cisregulatory modules (CRMs) in the genome of animals during development [1,2,3,4]
Knowing that expression from the endogenous Abd-B m promoter was perturbed in Abd-BT2N mutants, we examined whether the enhancers from the bithorax complex (BX-C) were re-directed to the intact ectopic Abd-B m promoter, which drives lacZ, on the P element insertion
Germ-band elongation stage embryos is consistent with a loss of IAB-enhancer directed expression from the Abd-B m promoter [29]. This suggests that deletion of the Abd-B promoter tethering sequence (PTE) and the neighboring 1.1 kb 59 sequence in the Abd-BDPTE-UP line leads to a disruption of the long-range interactions between the Abd-B m promoter and enhancers from the iab-5, iab-6 and iab-7 regions in PS10, 11 and 12, respectively
Summary
To ensure a high fidelity of gene expression patterns in embryos a very strict functional requirement exists for the interaction of cisregulatory modules (CRMs) in the genome of animals during development [1,2,3,4]. At the Drosophila bithorax complex (BX-C), an extensive network of CRMs located in over 300 kb of infraabdominal (iab) non-genic sequence is responsible for directing embryonic expression of just three homeotic genes; Ultrabithorax (Ubx), abdominal-A (abd-A) and Abdominal-B (Abd-B) (Fig. 1) [13,14]. These three homeotic genes are critical for establishing cellular identities in the presumptive thoracic and abdominal segments during development [2,15]. Deletion of either of the characterized PTSs from the endogenous BX-C did not result in a significant phenotype [8,18,19], suggesting that there might be other sequences capable of mediating the longrange enhancer-promoter specificity at the BX-C
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