We have identified a novel cis-regulatory element, the Promoter Targeting Sequence (PTS) from the Drosophiht Bithorax Complex. The PTS has an anti-insulator activity, allowing an otherwise blocked enhancer to activate its promoter over an intervening insulator; it also has a promoter-targeting activity, selectively targeting only one out of wo possible promoters. The PTS is located in the Abdominal-B(Abd-B) gene where the regulatory region is organized into segmental-specific domains by insulator DNAs such as Fab-7 and Fab-8. Mutations in the PTS result in the loss of Abd-B expression and homeotic transformations in the abdomen. We propose that PTS mediates enhancer-promoter interactions by overcoming the enhancer-blocking activity of the Fab insulators, thus converting the Fab insulators into local domain boundary elements. Our working hypothesis is that PTS functions by forming a stable association between DNA around the enhancer and the DNA near the promoter through a mechanism that is insensitive to insulator block, epigenetically stable, and independent of activators that interact with the enhancer. Given the striking similarity in Hox clusters between Drosophila and vertebrate, we have reason to believe that PTS represent a new class of cis-regulatory elements that regulate long-range enhancer-promoter interactions in the Hox gene clusters in both invertebrate and vertebrate animals. We propose to dissect the function of the PTS by testing several hypothesis related to our """"""""stable association model"""""""".
In Specific Aim la we will test the prediction that formation of a heritable stable enhancer promoter interaction is independent of enhancer identity and enhancer-binding proteins. In lb we will test whether or not the PTS inactivates an insulator, and whether or not the insulator contributes to the promoter-targeting activity of the PTS. Finally, in Aim lc, we will determine how the relative position of the PTS to an enhancer, promoter and insulator affects its anti-insulator and promoter-targeting activities, and how the PTS affects enhancer-promoter specificity in Abd-B. In the second specific aim, we will determine if any of the known Hox gene regulators mediate or antagonize PTS activities. We will also investigate the role of chromatin modification in PTS function. And lastly, we will conduct genetic screens to isolate genes or proteins that function through the PTS element. We will mainly focus on a comprehensive F1 FIp-FRT screen to identify both dominant and recessive mutations. Mutations that modify PTS activity will be mapped to identify the modifier gene encoded proteins. Identification of the proteins mediating PTS activities is essential for determining the molecular mechanisms of these activities and will guide future studies aimed at understanding how enhancer-promoter interactions are regulated in complex developmental genes.
