RESUMO
In Saccharomyces cerevisiae, transcription of several drug transporter genes, including the major transporter gene PDR5, has been shown to peak during mitosis. The significance of this observation, however, remains unclear. PDR1 encodes the primary transcription activator of multiple drug transporter genes in S. cerevisiae, including PDR5. Here, we show that in synchronized PDR1 and pdr1-3 (multidrug resistant) strains, cellular efflux of a known substrate of ATP-binding-cassette transporters, doxorubicin (a fluorescent anticancer drug), is highest during mitosis when PDR5 transcription peaks. A genetic screen performed to identify regulators of multidrug resistance revealed that a truncation mutation in ELM1 (elm1-300) suppressed the multidrug resistance of pdr1-3. ELM1 encodes a serine/threonine protein kinase required for proper regulation of multiple cellular kinases, including those involved in mitosis, cytokinesis, and cellular morphogenesis. elm1-300 as well as elm1Delta mutations in a pdr1-3 strain also caused elongated bud morphology (indicating a G2/M delay) and reduction of PDR5 transcription under induced and noninduced conditions. Interestingly, mutations in several genes functionally related to ELM1, including cla4Delta, gin4Delta, and cdc28-C127Y, also caused drastic reductions in drug resistance and PDR5 transcription. Collectively, these data show that ELM1, and genes encoding related serine/threonine protein kinases, are required for regulation of multidrug resistance involving, at least in part, control of PDR5 transcription.
Assuntos
Transportadores de Cassetes de Ligação de ATP/genética , Sequência de Aminoácidos , Farmacorresistência Fúngica Múltipla/genética , Proteínas Quinases/genética , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Deleção de Sequência , Transportadores de Cassetes de Ligação de ATP/biossíntese , Divisão Celular/genética , Fase G2/genética , Regulação Fúngica da Expressão Gênica/genética , Proteínas Quinases/metabolismo , Saccharomyces cerevisiae/enzimologia , Proteínas de Saccharomyces cerevisiae/biossíntese , Proteínas de Saccharomyces cerevisiae/metabolismo , Especificidade da Espécie , Transcrição GênicaRESUMO
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 G1/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.
Assuntos
Alelos , Genoma Fúngico , Histona Acetiltransferases/fisiologia , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/genética , Fatores Associados à Proteína de Ligação a TATA/genética , Fator de Transcrição TFIID/fisiologia , Transportadores de Cassetes de Ligação de ATP/metabolismo , Ciclo Celular/genética , Cromatina/metabolismo , Proteínas de Ligação a DNA/fisiologia , Análise em Microsséries , Proteínas Ribossômicas/biossíntese , Proteínas Ribossômicas/genética , Saccharomyces cerevisiae/crescimento & desenvolvimento , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores Associados à Proteína de Ligação a TATA/metabolismo , Fator de Transcrição TFIID/genética , Fator de Transcrição TFIID/metabolismo , Fatores de Transcrição/fisiologia , Transcrição Gênica/fisiologiaRESUMO
Drug resistance as a result of overexpression of drug transporter genes presents a major obstacle in the treatment of cancers and infections. The molecular mechanisms underlying transcriptional up-regulation of drug transporter genes remains elusive. Employing Saccharomyces cerevisiae as a model, we analyzed here transcriptional regulation of the drug transporter gene PDR5 in a drug-resistant pdr1-3 strain. This mutant bears a gain-of-function mutation in PDR1, which encodes a transcriptional activator for PDR5. Similar to the well studied model gene GAL1, we provide evidence showing that PDR5 belongs to a group of genes whose transcription requires the Spt-Ada-Gcn5 acetyltransferase (SAGA) complex. We also show that the drugindependent PDR5 transcription is associated with enhanced promoter occupancy of coactivator complexes, including SAGA, Mediator, chromatin remodeling SWI/SNF complex, and TATA-binding protein. Analyzed by chromatin immunoprecipitations, loss of contacts between histones and DNA occurs at both promoter and coding sequences of PDR5. Consistently, micrococcal nuclease susceptibility analysis revealed altered chromatin structure at the promoter and coding sequences of PDR5. Our data provide molecular description of the changes associated with constitutive PDR5 transcription, and reveal the molecular mechanism underlying drug-independent transcriptional up-regulation of PDR5.
