Gene Silencing Mechanisms in Drosophila

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Genes transformed back into the organism of origin often cause silencing of dispersed homologous copies, including the endogenous gene. Two types of silencing occur. One is transcriptional and involves changes in chromatin structure as evidenced by the accumulation of Polycomb Group proteins on the silenced transgene insertion sites. The other involves post-transcriptional RNA turnover in a similar manner to RNA interference. Emerging evidence has indicated potential connections among RNA interference, post-transcriptional silencing and transcriptional transgene silencing. In this project, a genetic and molecular approach will be undertaken to determine any relationship among these types of silencing. Transgenes of the Alcohol dehydrogenase (Adh) and white (w) eye color genes in Drosophila will be used as a model system. First, a series of constructs will be transformed that do or do not exhibit gene expression to determine whether transcriptional silencing requires the expression of the silencing gene to occur. If silencing can only occur when the homologous sequences are expressed, an RNA mediated process would be implicated in causing the chromatin changes that are associated with the cases of transcriptional silencing. Secondly, the interrelationship between RNAi-like mechanisms and transcriptional silencing will be examined. A construct predicted to induce post-transcriptional silencing of Adh will be tested for its ability to produce either type of silencing. Next, mutations that block post-transcriptional silencing will be tested for their influence on pairing sensitive silencing. Further, a transgene predicted to generate double stranded RNA homologous to Adh promoter regions will be produced and tested for its ability to induce transcriptional silencing. Studies of gene silencing are likely to reveal new information about gene expression and nuclear dynamics in multicellular eukaryotes. One role of these silencing mechanisms is as a protection against viruses and transposons, although many of the genes that have been identified in the process are also known to affect developmental mechanisms. Understanding the basic processes of gene silencing has the potential to lead to virus protection and to have applications in gene therapy.