A multiplicity of coactivators is required by Gcn4p at individual promoters in vivo.
Transcriptional activators interact with multisubunit coactivators that modify chromatin structure or recruit the general transcriptional machinery to their target genes. Budding yeast cells respond to amino acid starvation by inducing an activator of amino acid biosynthetic genes, Gcn4p. We conducted a comprehensive analysis of viable mutants affecting known coactivator subunits from the Saccharomyces Genome Deletion Project for defects in activation by Gcn4p in vivo. The results confirm previous findings that Gcn4p requires SAGA, SWI/SNF, and SRB mediator (SRB/MED) and identify key nonessential subunits of these complexes required for activation. Among the numerous histone acetyltransferases examined, only that present in SAGA, Gcn5p, was required by Gcn4p. We also uncovered a dependence on CCR4-NOT, RSC, and the Paf1 complex. In vitro binding experiments suggest that the Gcn4p activation domain interacts specifically with CCR4-NOT and RSC in addition to SAGA, SWI/SNF, and SRB/MED. Chromatin immunoprecipitation experiments show that Mbf1p, SAGA, SWI/SNF, SRB/MED, RSC, CCR4-NOT, and the Paf1 complex all are recruited by Gcn4p to one of its target genes (ARG1) in vivo. We observed considerable differences in coactivator requirements among several Gcn4p-dependent promoters; thus, only a subset of the array of coactivators that can be recruited by Gcn4p is required at a given target gene in vivo.
Swanson
Mark J
MJ
Laboratory of Gene Regulation and Development, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA.
Qiu
Hongfang
H
Sumibcay
Laarni
L
Krueger
Anna
A
Kim
Soon-ja
SJ
Natarajan
Krishnamurthy
K
Yoon
Sungpil
S
Hinnebusch
Alan G
AG
D001483:Base Sequence; D001665:Binding Sites; D002843:Chromatin / Q000235:genetics / Q000378:metabolism; D004271:DNA, Fungal / Q000235:genetics; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017353:Gene Deletion; D005800:Genes, Fungal; D046911:Macromolecular Substances; D056892:Mediator Complex; D009154:Mutation; D009687:Nuclear Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D010957:Plasmids / Q000235:genetics; D011401:Promoter Regions, Genetic; D011494:Protein Kinases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017434:Protein Structure, Tertiary; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012260:Ribonucleases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000235:genetics* / Q000378:metabolism*; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015534:Trans-Activators / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D014157:Transcription Factors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D002843:Chromatin; D004271:DNA, Fungal; D004268:DNA-Binding Proteins; C058337:GAL11 protein, S cerevisiae; D046911:Macromolecular Substances; D056892:Mediator Complex; D009687:Nuclear Proteins; C097698:PAF1 protein, S cerevisiae; D021122:Protein Subunits; C103337:RSC complex, S cerevisiae; D011994:Recombinant Proteins; D029701:Saccharomyces cerevisiae Proteins; D015534:Trans-Activators; D014157:Transcription Factors; D011494:Protein Kinases; C078657:CCR4 protein, S cerevisiae; D012260:Ribonucleases