Rpd3-medicated Nucleosome Deposition onto the rDNA Genes of Saccharomyces cerevisiae

Johnson, Joseph McKinney, Department of Biochemistry and Molecular Genetics, University of Virginia
Smith, Jeff, Department of Biochemistry and Molecular Genetics, University of Virginia
Auble, David, Department of Biochemistry and Molecular Genetics, University of Virginia
Beyer, Ann, Department of Microbiology, University of Virginia
Grant, Patrick, Department of Biochemistry and Molecular Genetics, University of Virginia

Ribosome biosynthesis depends on RNA Polymerase I (Pol I) transcription of ribosomal RNA. Transcribed from the ribosomal DNA (rDNA) genes, the production of rRNAs is a key step of ribosome production. In Saccharomyces cerevisiae, rDNA genes are organized in a tandem array of ~150 to 200 repeats. About 500f these genes are transcribed by Pol I at any given time during exponential growth. Both the rate of transcription and the number of active genes are reduced as cells approach stationary phase. In strains lacking the histone deacetylase Rpd3, Pol I transcription is repressed in stationary phase, but the percentage of rDNA genes that remain in an 'open' chromatin structure, as measured by psoralen crosslinking, remains ~50%. We hypothesized that the 'open' chromatin structure was caused by nucleosome depletion on actively transcribed genes and that Rpd3 was required for proper nucleosome reassembly when the genes were repressed. Indeed, H2B and H4 occupancy on the rDNA genes, as measured by ChIP, greatly increased in an Rpd3-dependent manner when cells transitioned from log into stationary phase. The apparent removal and redeposition of H2B suggested a role for the chromatin remodeling FACT complex. Consistent with this model, the Spt16 and Pob3 subunits of FACT were shown by ChIP to physically associate throughout the rDNA gene in log phase and during the diauxic shift. Psoralen crosslinking assays revealed Spt16 was important for the "closing" of rDNA genes in post-log phase, similar to Rpd3. Importantly, spt16-197 mutants were defective in reassembling H2B onto the body of rDNA genes, suggesting that FACT may utilize deacetylated H2A/H2B dimers for nucleosome reassembly onto repressed rDNA genes. Both in vivo and in vitro transcriptional assays revealed the regulation of Pol I iii transcription was defective during the transition between growth phases in an spt16-197 mutant. All together, this work further defines processes required to establish a dynamic equilibrium between 'open' and 'closed' rDNA genes due to nucleosome deposition. This is the first evidence that suggests that there are coordinated, perhaps cooperative, activities by Rpd3, a histone deacetylase, and yFACT, a chromatin remodeler, as part of the Pol I transcription cycle.

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PHD (Doctor of Philosophy)
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