Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action.
Project description:In yeast, ribosome production is controlled transcriptionally by tight coregulation of the 138 ribosomal protein genes (RPGs). RPG promoters display limited sequence homology, and the molecular basis for their coregulation remains largely unknown. Here we identify two prevalent RPG promoter types, both characterized by upstream binding of the general transcription factor (TF) Rap1 followed by the RPG-specific Fhl1/Ifh1 pair, with one type also binding the HMG-B protein Hmo1. We show that the regulatory properties of the two promoter types are remarkably similar, suggesting that they are determined to a large extent by Rap1 and the Fhl1/Ifh1 pair. Rapid depletion experiments allowed us to define a hierarchy of TF binding in which Rap1 acts as a pioneer factor required for binding of all other TFs. We also uncovered unexpected features underlying recruitment of Fhl1, whose forkhead DNA-binding domain is not required for binding at most promoters, and Hmo1, whose binding is supported by repeated motifs. Finally, we describe unusually micrococcal nuclease (MNase)-sensitive nucleosomes at all RPG promoters, located between the canonical +1 and M-bM-^@M-^S1 nucleosomes, which coincide with sites of Fhl1/Ifh1 and Hmo1 binding. We speculate that these M-bM-^@M-^XM-bM-^@M-^XfragileM-bM-^@M-^YM-bM-^@M-^Y nucleosomes play an important role in regulating RPG transcriptional output. Genome-wide examination of binding sites for transcription factors controlling ribosomal protein genes expression and nucleosome positions in budding yeast cells
Project description:Genome wide mapping of RNA polymearase III binding sites in Saccharomyces cerevisiae under normal growth and nutrient starved condition using ChIP-seq. Chromatin Immuno-precipitation (ChIP) was performed for FLAG tagged version of pol III subunit RPC128 after crosslinking the log-phase cells with formaldehyde. MOCK and IP DNA was sequenced and coverage of pol III was calculated at each base of the genome.
Project description:The target of rapamycin (TOR) plays a central role in eukaryotic cell growth control. With prevalent hyper-activation of the mTOR pathway in human cancers, novel strategies to enhance TOR pathway inhibition are highly desirable. We used a yeast-based high-throughput chemical genetic screen to identify small-molecule enhancers of rapamycin (SMERs) and used whole genome expression analysis to identify their mechanisms of action.
