Re-evaluating enhancer modularity and boundaries with quantitative approaches in Drosophila

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Transcriptional enhancers control in which cells, at what time, and how much a given gene is transcribed from a core promoter. The precision of this process is for instanceb necessary for normal embryonic development of all multicellular organisms. Classically, enhancers have been revealed and defined in reporter assays, where the regulatory activity of a DNA segment is tested in cell culture, or in transgenic organisms. This approach has painted a textbook picture where enhancers are short (100-1000 bp), discrete stretches of DNA sequence, working as independent modules. Using a different and quantitative approach to map enhancers, we have recently found that enhancers of the pigmentation gene yellow, driving activity in the wings of flies (Drosophila) appear much broader (~4000 bp) and entangled than the literature predicts. Preliminary evidence indicates that the results extend to another enhancer of yellow driving in the fly body. This situation undermines the notion of enhancer modularity, a key concept to understand Developmental and Evolutionary Biology. With this proposal, we would like to first thoroughly describe the degree to which regulatory information is intermingled among these different enhancers. We then want to understand through which mechanisms enhancers achieve their functional modularity. We suspect that regulatory information may be selectively available in different tissues by means of regulated accessibility of the DNA. We will specifically test this hypothesis by investingating the determinants of enhancer accessibility, in cis and in trans, for the body and wing enhancers. We will combine reporter assays in transgenic Drosophila, computational and quantitative image analysis, and genomic methods to achieve these goals. We expect our results to seed the redefinition of transcriptional enhancers, but also to reveal how regulatory information from different, overlapping enhancers is mobilized in a selective manner.