Mechanisms of Gene Silencing in Plants: functional genomics and forward and reverse genetics to study paramutation, transposable elements, transgene silencing, and chromatin control of gene expression
My research program investigates the regulation of gene expression. The anthocyanin biosynthetic pathway in maize provides an exceptionally tractable system for genetic, biochemical and molecular approaches. A major emphasis in our research is to investigate how the regulatory genes of this pathway are controlled. These regulatory genes, which encode transcription factors that activate the anthocyanin biosynthetic genes, have multiple alleles that produce distinct developmental and tissue-specific patterns of anthocyanin pigments. A major emphasis is to use this system to investigate mechanisms of gene silencing, which has a fundamental role in development and has recently become a major problem with genetic engineering approaches to crop improvement. We use both forward and reverse genetic approaches to study paramutation, the regulation of transposable elements and transgene silencing. Paramutation is a mitotically and meiotically heritable change in gene expression that is induced by allele interactions. We have demonstrated that the heritable change is accompanied by a ten- to twenty-fold reduction in transcription. We used a combination of classical genetics, genomics, and molecular methods to identify and characterize the minimal sequences required for paramutation, which are 7 non-coding tandem repeats located within 95-102 kbp upstream of the transcription initiation site. These non-coding repeats are transcribed and are both necessary and sufficient to mediate paramutation. We have also identified multiple mutations in other genes required for the establishment and maintenance of paramutation. We have shown that these mutants also activate previously silent transposable elements and transgenes, indicating that the wild type proteins are required for multiple gene-silencing processes. The cloning of two of these genes, has revealed that paramutation involves RNA-mediated chromatin silencing mechanisms. As heritable changes in chromatin structure are clearly involved in the establishment and maintenance of distinct transcription states we are also pursuing a functional genomics approach to understand chromatin-level control of gene expression in maize.