The long term goal of the proposed research is to better understand molecular mechanisms in the highly regulated production of ribosomes, which more than any other cellular event is directly correlated with the growth status of the cell. In a rapidly growing yeast cell (Saccharomyces cerevisiae), which is the model system used herein, 60 percent of total transcription is devoted to the synthesis of ribosomal RNA by RNA polymerase I and ribosomes are made at a rate of 2000 per minute. Thus, it is necessary to coordinately and tightly regulate these genes, which use a significant fraction of the cell's resources. Although a great deal is known about the molecules that participate in Pol I transcription, there are still large holes in our understanding. Much of this is due to the unusual properties of the Pol I system, in which a multi-gene family is transcribed by a polymerase that is solely devoted to that purpose, such that regulation is possible either by adjusting the number of active genes or by adjusting the activity level of those genes. Although it is difficult to distinguish between these two levels of regulation using genetic, molecular and biochemical techniques, the information is accessible to an electron microscopic (EM) approach. Thus, the immediate goal of the proposed research is to add to the current repertoire of available tools for the study of yeast rRNA transcription by applying the Miller chromatin spreading technique for the direct visualization of active rDNA genes and inactive nucleolar chromatin. The approach is straightforward and combines the power of yeast genetics with the power of a picture. The EM approach will be used to (Aim 1) characterize normal patterns of up- and down-regulation of Pol I transcription, such as across a typical growth curve and as nutritional status is improved, (Aim 2) determine the role of Pol I transcription factors and the Pol I enhancer to normal patterns of transcription by visualizing transcription as they are being depleted or when mutated, and (Aim 3) determine the contribution of chromatin structure and rDNA silencing to Pol I transcription by localizing histone modifications in rDNA chromatin and by visualizing rRNA transcription in the absence of histone deacetylases known to have a nucleolar role. The approach holds promise to answer fundamental questions regarding gene regulation in this important multi-gene family that have eluded biochemical and genetic approaches but can easily and unambiguously be addressed by direct visualization. Given the direct positive correlation between rRNA synthesis, nucleolar size and growth rate of a cell, with all three being up-regulated in cancer cells, it seems likely that up-regulation of rRNA synthesis is a contributing factor to tumorigenesis. Furthermore, the recently uncovered role of the nucleolus in aging may be elucidated further by the proposed experiments.
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