Project description:In Saccharomyces cerevisiae, specific genes physically relocate from the nucleoplasm to the nuclear periphery concomitant with transcriptional activation, where they associate with the nuclear pore complex (NPC). We took a genomics approach in order to gain insight into the universality and physiological relevance of the interaction between active genes and the NPC. Using synthetic genetic array (SGA) approach, we identified interactions between components of the SAGA histone acetyltransferase complex and the Mlp and Nup60 subunits of the NPC. Cells lacking these SAGA and NPC components display growth defects under optimal growth conditions, in which cells are grown on rich medium containing the preferred sugar glucose as a carbon source and incubated at their optimal temperature. That growth defects were observed under these non-stress conditions suggests that these interactions are indicative of defects in normal cell physiology. These results are consistent with a model where physical interactions between the NPC and SAGA are important for transcription of constitutively expressed genes. To test this hypothesis, we used microarray analysis to assess changes global transcript levels in the absence of Nup60, Ada2, or Nup60 and Ada2. Microarray analysis reveals that the growth defect in these double mutants is correlated with a synthetic reduction in steady-state transcript levels for numerous genes that are strongly expressed in wildtype cells. These results suggest that SAGA and Nup60 cooperate in transcriptional regulation, and are consistent with a model for global regulation of SAGA-dependent transcription at the NPC. Wildype, single deletion, and double deletion cells were inoculated to equal starting ODs in 50 mL YEPD media and grown at 30C with shaking. Cells were collected at mid-log phase, washed with chilled water, and stored at -80C for later processing. RNA was isolated using TRIzol reagent according to the manufacturer's protocol, with modification for yeast cells.
Project description:TFIID and SAGA are the only two known yeast complexes that modify chromatin and deliver TBP to promoters. Previous genome wide expression studies indicated that TFIID and SAGA positively regulate most but not all yeast genes. Using a relatively low noise microarray approach, we have re-examined the genome-wide dependence on TFIID and SAGA. We find that TFIID and SAGA contribute to the expression of virtually the entire genome, with TFIID being preferred at ~90% of the genes, and SAGA being preferred at ~10%. SAGA-dominated genes were found to overlap substantially with a previously described set of highly active genes that are attenuated in part by the TBP regulator NC2, and an auto-inhibitory function of TFIID. These SAGA-dominated genes also encompass most of the previously reported “TAF-independent” genes. These results build upon and refine the generally held view that activators recruit either TFIID or SAGA to promoters which then bind and acetylate nucleosomes locally, thereby enhancing TBP delivery to the TATA box. Promoter-specific differences in the ability to alleviate auto-inhibitory activities associated with TFIID and SAGA might contribute to the preferential use one complex versus the other. Keywords = Chromatin Immunoprecipitation Keywords = genome-wide binding Keywords: other