Analysis of rDNA Chromatin in Aging Yeast Cells

University Of Virginia Charlottesville, Charlottesville VA

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Abstract

The long term goal of this project is to understand how changes in rDNA chromatin and its capacity for rRNA transcription contribute to the aging process in yeast cells. The main hypothesis to be tested is that aging in yeast is in part regulated by the status of the rDNA chromatin structure, and that RNA polymerase I (Pol I) activity plays a role in this process. Since all eukaryotic organisms, including humans, express rRNA from a conserved rDNA tandem array and utilize RNA polymerase I, the information gained from the yeast system is predicted to be applicable to the study of the human aging problem. In the first specific aim of this pilot proposal, the actual changes in Pol I transcription and post-translational histone modification status that occur in middle-aged and old yeast cells will be determined. Age-sorted cell populations will be isolated and subjected to Northern blot and psoralen crosslinking analyses to measure the number of rDNA repeats that are transcribed by Pol I and the rate of transcription. Electron microscopy of Miller chromatin spreads will determine the amount of Pol I activity on each active rDNA repeat. Histone modifications at the rDNA and control chromosomal regions will be examined using chromatin immunoprecipitations. In the second specific aim, the changes in replicative life-span caused by mutations in relevant histone residues that undergo an age-related modification will be determined. In addition, the role of RNA polymerase I in regulating replicative life-span will be directly tested throught the use of Pol I mutants that are known to influence rDNA silencing. This approach is expected to yield important basic information about the link between aging and rDNA chromatin, and lead to an expanded RO1 application.