The long term objective of this proposal is to elucidate the molecular mechanism of rDNA silencing, which is the SIR2- dependent suppression of recombination and RNA polymerase II (Pol II)-mediated transcription in the yeast ribosomal DNA (rDNA) locus. SIR2 is the founding member of an evolutionarily conserved gene family that has been implicated in transcriptional silencing, DNA repair, and meiosis checkpoint regulation in yeast. RDNA silencing was only recently identified and therefore provides a novel point of view to the investigation of chromatin effects on gene repression. Silencing of various tumor suppressor genes is well established as a step leading to cancer formation. Furthermore, regulation of ribosomal RNA transcription has also been implicated in cellular growth control. Therefore, understanding the basic mechanism of gene silencing in the rDNA will provide important insights to human disease processes and also the function of SIR2. Molecular genetic and biochemical approaches will be used to define the epigenetic factors controlling rDNA silencing and to classify it relative to the other known forms of silencing in yeast, telomere position effect (TPE) and repression of the silent mating type loci (HML and HMR). These approaches will also be used to determine the role of RNA polymerase I-mediated rRNA transcription in the silencing of Pol II-transcribed genes position within the rDNA. Finally, the role of NAD metabolism in regulating rDNA silencing will be molecularly dissected. The proposed work will also provide a framework for the future functional analysis of multiple rDNA silencing factors that have been identified.
