TFIID and Spt-Ada-Gcn5-Acetyltransferase Functions Probed by Genome-wide Synthetic Genetic Array Analysis Using a Saccharomyces cerevisiae taf9-ts Allele

Abstract

TAF9 is a TATA-binding protein associated factor (TAF) conserved from yeast to humans and shared by two transcription coactivator complexes, TFIID and SAGA. The essentiality of the TAFs has made it difficult to ascertain their roles in TFIID and SAGA function. Here we performed a genomic synthetic genetic array analysis using a temperature-sensitive allele of TAF9 as a query. Results from this experiment showed that TAF9 interacts genetically with: (1) genes for multiple transcription factor complexes predominantly involving Mediator, chromatin modification/remodeling complexes, and regulators of transcription elongation; (2) virtually all nonessential genes encoding subunits of the SWR-C chromatin-remodeling complex and both TAF9 and SWR-C required for expressing the essential housekeeping gene RPS5; and (3) key genes for cell cycle control at the G 1 /S transition, as well as genes involved in cell polarity, cell integrity, and protein synthesis, suggesting a link between TAF9 function and cell growth control. We also showed that disruption of SAGA by deletion of SPT20 alters histone-DNA contacts and phosphorylated forms of RNA polymerase II at coding sequences. Our results raise the possibility of an unappreciated role for TAF9 in transcription elongation, perhaps in the context of SAGA, and provide further support for TAF9 involvement in cell cycle progression and growth control.

ELM1 Is Required for Multidrug Resistance in Saccharomyces cerevisiae

“…The genotypes of yeast strains used in this study are listed in Table 1 (Wolfger et al 1997;Gao et al 2004) and described previously for deletion strains derived from BY4741 (Milgrom et al 2005). The null alleles introduced into pdr1-3 were carried out by PCRmediated allele transfer from deletion alleles of nonessential genes available from the collection of synthetic genetic arrays (Tong et al 2001) or by PCR-based gene deletion using modification cassettes as previously described (Longtine et al 1998).…”

“…was induced by treatment of cells for 45 min in YPD medium containing 0.2 mg/ml (0.71 mm) cycloheximide (CYH) as described (Gao et al 2004;Milgrom et al 2005).…”

“…Ethylmethane sulfonate mutagenesis and identification of elm1-300 mutation: The ethylmethane sulfonate (EMS) mutagenesis protocol was performed as described (Lawrence 1991;Milgrom et al 2005), using a concentration of EMS (15 ml/ml of cell culture, final 15 mm) that gave 50% cell survival. Log-phase pdr1-3 cells ($5 3 10 7 cells/ml of WCS261, Table 1) were treated with EMS, and 8 3 10 4 cells were screened for mutants that failed to grow on 1.0 mg/ml CYH by replica plating.…”

“…Two major transcriptional activators, Pdr1 and Pdr3, control the level of many drug transporters in S. cerevisiae (Gao et al 2004;Milgrom et al 2005 and references therein). These homologous proteins belong to the Gal4 superfamily with Cys 6 -Zn(II) DNAbinding domains (Poch 1997;Kolaczkowski et al 1998;Bauer et al 1999).…”

“…These alternations reflect changes in the interactions between Pdr1 and PDRE and are associated with PDR5 transcriptional activation (Gao et al 2004). Moreover, we showed that proper interactions between histones and PDR5 coding sequences specifically require the transcription factor Spt-Ada-Gcn5-acetyltransferase (SAGA) (Milgrom et al 2005). However, factors other than Pdr1 and SAGA that are required for proper transcriptional regulation of PDR5 remain to be identified.…”