Gene regulation and signal transduction in development; Structure and function of transcriptional enhancers; Enhancer evolution. DNA sequences called cis-regulatory elements, or enhancers, can be located upstream or downstream of a gene, or even within it. They may be very near the promoters they regulate or many kilobases away--even on the far side of other genes. Enhancers control when, where, and how strongly genes will be expressed. They contain binding sites for transcription factors, proteins that bind DNA and turn genes on or off. A few highly conserved cell signaling pathways (e.g. Hedgehog, Wnt, Notch, TGF-β/BMP, RTK/Ras/MAPK) control the vast majority of cell fate decisions during animal development. The primary effects of cell signaling are changes in gene expression, mediated by signal-regulated transcription factors. These factors bind to the enhancers of target genes, and turn them on or off in response to pathway signaling. Faulty signaling can result in diseases such as cancer, diabetes, autoimmune disorders, and neurodegenerative diseases, as well as developmental defects. Signal-regulated enhancers have been intensively studied for years, but basic questions about these regulatory sequences remain unanswered. The gaps in our knowledge are best illustrated by the fact that "synthetic" versions of well-characterized enhancers (i.e., combinations of the known transcription factor binding sites) nearly always fail to drive gene expression in vivo. Therefore, it seems that we don't yet know all of the component parts of the enhancer, or its basic structure. These experiments raise the possibility that unidentified DNA-binding proteins, which may be functionally distinct from transcription factors, are essential for gene activation in higher eukaryotes. How do these proteins interact with signal-regulated transcription factors to activate transcription? What are the structural rules that govern these interactions? How do cis-regulatory sequences change during evolution? Using the Drosophila model system, we are employing genetic, biochemical, evolutionary, transgenic, and bioinformatics approaches to the study of these problems. We are actively researching the following aspects of enhancer activity: In vivo structure-function studies of developmental enhancers; Enhancer-promoter "looping" in vivo; Mechanisms of transcriptional regulation by the Hedgehog, Wnt, Notch, and MAPK pathways; Sub-nuclear localization of enhancers during gene activation; Dynamics of enhancer evolution; Reverse-engineering synthetic enhancer elements.