| RNA Polymerase (Pol) III transcribes small noncoding RNAs (e.g., tRNAs) important for translational capacity. Maf1 is a repressor of Pol III conserved from yeast to human, required for repression of Pol III in response to multiple environmental stresses, such as nutrient deprivation. Interestingly, Maf1 is a phosphoprotein, being phosphorylated in good growth conditions and dephosphorylated in poor growing conditions. The phosphatase acting on Maf1 has not been determined. I investigated the identity of the phosphatase in yeast Saccharomyces cerevisiae using a genetic Maf1-Pol III fusion construct in combination with molecular and biochemical assays. I queried members of Protein Phosphatase 2A and 4 complexes (PP2A and PP4, respectively) for their role in dephosphorylation of Maf1 and determined that PP4---containing Pph3 and Psy2, together with accessory factors Rrd1 and Tip41---is the major Maf1 phosphatase, acting in response to multiple stresses. Maf1 interacts with Pph3 in vivo, and biochemical purification of TAP-tagged Pph3-bound complex shows activity on purified, endogenously phosphorylated Maf1 in vitro, suggesting that PP4 is a direct phosphatase of Maf1.;In human cells, regulation of Pol III also involves chromatin regulation, not apparent in studies of Pol III in yeast. The full repertoire of Pol III-transcribed genes in human cells has not been defined. I determined the full Pol III transcriptome in human (HeLa) cells by chromatin immunoprecipitation (ChIP), coupled with microarray (ChIP-array) or high throughput sequencing (ChIP-seq), for Pol III subunit Rpc32, and Pol III transcription factors Brf1, Brf2 and TFIIIC63. I also determined the Pol III transcriptome in other cell types to address the possibility of cell type-specific activation of Pol III genes. Although many active Pol III genes were shared between all cell types assayed, a large number of genes were differentially enriched with Pol III. I compared enrichment of Pol III with chromatin modifications, transcription factors, and Pol II ChIP-seq profiles and found a significant correlation for active Pol III genes with active chromatin modifications and occupancy of transcription factors and Pol II. These results suggest that active chromatin gates Pol III accessibility to the human genome. |
