Transcription from non-protein-coding DNA (ncDNA), including intergenic DNA, is widespread among eukaryotes. However, the consequences of this transcription are only beginning to be investigated. The long-term objective for this project is to understand how transcription of ncDNA regulates gene expression. This grant focuses on our recent discovery of a previously uncharacterized mechanism of gene regulation: repression by intergenic transcription. In Saccharomyces cerevisiae, we showed that expression of an intergenic transcript, SRG1, represses the transcription of the adjacent SER3 gene. However, we found that it is the act of transcription, not the SRG1 ncRNA product, which mediates repression by a transcription interference mechanism. In this proposal, we plan to extend these studies by using a combination of genetic, molecular, genomic and biochemical approaches. First, we will identify and characterize factors involved in both the activation and repression of SER3. The results of these studies will provide important insights into the mechanism of repression by intergenic transcription and establish tools to characterize the function of new cases of intergenic transcription. Second, we will begin to dissect the molecular details of this repression mechanism. Using both gene expression analysis and chromatin immunoprecipitation methods, we will determine the relationship between the rate of intergenic transcription and repression of the adjacent gene and test the hypothesis that intergenic transcription modifies chromatin structure to repress the adjacent gene. Third, we will identify and characterize new cases of repression by intergenic transcription. This will allow us to extend our analysis to more genes in order to understand the general features of this repression mechanism. We will focus our analysis on a subset of intergenic DNAs that represent strong candidates for repression by intergenic transcription based on results from our genome- wide RNA Pol II ChIP studies and more recent yeast whole-genome expression studies. It is becoming increasingly clear that transcription of ncDNA plays an important role in regulating normal cell growth and development in humans and thus is likely to be involved in the prevention of human diseases, including cancer. Given that factors and mechanisms of transcription regulation are largely conserved among eukaryotes, our analysis of repression by intergenic transcription in yeast may provide new insight into the roles of transcription of ncDNA in humans.