Dissecting Promoter Function in Vivo

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Many fundamental cellular processes are controlled at the level of transcription. The extensive knowledge that has been acquired over the past years on the in vivo properties of the trans-acting regulators is contrasted by how little is still known about the in vivo organization, evolution and function of cis-acting regulatory elements. Previous studies on the control of maize flavonoid biosynthesis have provided unique tools to investigate promoter structure in its normal in vivo environment. As a model to understand promoter function in vivo, the regulatory region of a structural gene of the pathway (A1) that has been extensively dissected in vitro, and that is a target of at least two well-defined regulatory systems will be studied. The organization of cis-acting regulatory elements in the A1 promoter will be further-investigated by using in vivo footprinting strategies to identify the sequences recognized by the regulators in normal chromatin environments. Unique genetic resources will be used to study in vivo the result of deletions of cis-acting sequences on A1 transcription. The in vivo effect of point mutations and small insertions in regulatory elements that have been refractive to in vitro analysis will be analyzed. In two very well genetically-characterized cases, the regulation of A1 is transferred to transposable elements. Moreover, epigenetic modifications of the transposons directly affect A1 transcription. Our unique understanding of the regulation of A1 makes it ideal to interpret transposable element gene control systems providing important insights into the molecular bases of epigenetic phenomena. Whether transposons can promote A1 transcription in a regulator-independent fashion, and whether elements of the transposon entirely replace the normal cis-acting regulatory elements of the gene will be examined. Finally, the epigenetic effect that transposons often have on gene expression, by studying the in vivo methylation status of the A1 gene promoter when its expression is controlled by transposable element insertions will be investigatedTaken together, these studies should provide one of the most comprehensive in vivo studies done on the regulatory region of a plant gene. Because of the uniqueness of the genetic and molecular tools available, the findings derived from these studies could be fundamental to the general field of regulation of gene expression.