9317701 Blackman Our experiments will explore two modes of gene regulation involving promoter-enhancer interactions. In the first, we will look at the intragenic regulation of the Drosophila decapentaplegic (dpp) gene, as we try to explain how its complex network of imaginal disk- specific enhancers search out their correct promoters. These experiments will test our view that this occurs through a mechanism of promoter competition. Our second line of experimentation will investigate the nature of the regulatory autonomy of adjacent genes. Even though the dpp enhancers can activate their own promoters up to 60 kb away, they are indifferent to the promoter of the adjacent tnd (transcript near dpp) gene which is located just a few kb away. Two models, one involving chromatin domains and the other promoter specificity, are commonly invoked to describe how genes can remain autonomous and we are in an excellent position to decide which of these, if either, is at work in the dpp/tnd system. Through our proposed experiments: 1) we will examine the promoter preferences of different disk-specific enhancers and determine whether these choices are established through a mechanism of promoter competition. Much of this work will involve transfection assays in tissue culture cells but these results will also be confirmed by in vivo and analysis. 2) we will characterize the regulatory sequences required for tnd transcription. We will use reporter gene constructs and interspecies sequence comparisons to identify the functionally important elements comprising the promoter and enhancer regions of tnd. 3) we will establish the mechanism by which tnd and dpp remain autonomous. We will use "promoter swap" experiments to decide whether the regulatory independence is mediated via chromatin domains or promoter specificity. For these experiments, we will use in vivo site- directed mutagenesis to completely replace the tnd promoter with another. 4) we will determine precisely w hich chromosomal sequences are responsible for this regulatory isolation. We will use molecular, molecular genetic, and biochemical techniques to identify the elements involved. %%% How genes are regulated in eukaryotic systems is not yet clearly understood. Sequences known as enhancers interact with specific promoter sequences to induce the expression of the gene controlled by the promoter but since the enhancer can be at a distance and in either orientation, how the specific sequences interact is not yet understood. Because of the novel features of this system, coupled with the powerful molecular genetic techniques available for Drosophila, the results of this work should provide one of the clearest understandings of how enhancers discriminate between alternative promoters. ***
