A Multiplicity of Coactivators Is Required by Gcn4p at Individual Promoters In Vivo

Abstract

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.Eukaryotic activator proteins stimulate transcription by binding to their target genes and carrying out two general functions: (i) altering the locations or structures of nucleosomes and (ii) recruiting TATA-binding protein (TBP), other general transcription factors (GTFs), and RNA polymerase II (RNA PolII) to the promoter. Most activators carry out these functions indirectly by recruiting multisubunit complexes, collectively called coactivators (39,70,90). One class of coactivators uses ATP hydrolysis to displace nucleosomes and thereby expose or obscure protein binding sites in the promoter (91,124). Each of the nucleosome remodeling complexes of Saccharomyces cerevisiae, known as SWI/SNF, RSC, ISW1, and ISW2, contains a different subunit harboring the ATPase activity of the complex (reviewed in references 70 and 91). Although each has been implicated in transcriptional activation in vivo (5,38,51,85,123), only the nonessential SWI/SNF complex has been shown to interact physically with activators (93, 139) and be recruited to a promoter for nucleosome remodeling and transcriptional activation in vitro (45,96,139). Recruitment of the SWI/SNF complex by yeast activators has also been demonstrated in living yeast cells by chromatin immunoprecipitation (ChIP) assays (24, 126).Another class of coactivators alters chromatin structure by acetylation of lysines in the amino-terminal tails of histones. This modification destabilizes higher-order chromatin structure (116) and also may stimulate binding of other coactivator proteins containing a bromodomain (9,91,120,135). The SAGA complex is the best-characterized yeast coactivato...

Recruitment of the ArgR/Mcm1p repressor is stimulated by the activator Gcn4p: A self-checking activation mechanism

“…They were produced by the Saccharomyces Genome Deletion Project from BY4741 (MATa his3⌬1 leu2⌬0 met15⌬0 ura3⌬0) and purchased from Research Genetics͞ Invitrogen (Carlsbad, CA), or they were derived from such strains. All deletion alleles were confirmed by PCR amplification (13). We also confirmed that the arg80⌬ and arg81⌬ strains are defective for arginine-repression of an ARG1-lacZ fusion on plasmid YCp87-ARG1-lacZ (data not shown), and that the arg11⌬ strain is an arginine auxotroph.…”

“…We also confirmed that the arg80⌬ and arg81⌬ strains are defective for arginine-repression of an ARG1-lacZ fusion on plasmid YCp87-ARG1-lacZ (data not shown), and that the arg11⌬ strain is an arginine auxotroph. Insertion of the coding sequences for 13 myc epitopes at the C termini of ARG80, ARG81, ARG82, and MCM1 was conducted as described (13), and the myc-tagged alleles were verified by PCR amplification and Western analysis of whole-cell extracts (WCEs), using anti-myc antibodies (data not shown). The MCM1-myc strain grew indistinguishably from the parental untagged strain.…”

“…ChIP assays were conducted as described (14) by using the same primers utilized to amplify the ARG1 upstream activation sequence (UAS) (13), SNZ1 UAS (14), or ARG4 UAS (15). Western analysis of WCEs from cells treated with trichloroacetic acid was conducted as described (14).…”

Genetic Interactions Between Mediator and the Late G1-Specific Transcription Factor Swi6 in Saccharomyces cerevisiae

“…Gcn4 is required for the full induction of .500 genes (Natarajan et al 2001). Four native Gcn4 target genes have been analyzed for their inducibility in a panel of 80 deletion mutants, each of which lacked one of the known coactivators or corepressors of transcription (Swanson et al 2003). Each of the Gcn4 targets displayed a significantly different pattern of dependency, indicating that a different subset of coregulators are recruited by Gcn4 to these promoters.…”

Comparative Genomics of Transcriptional Control in the Human Malaria Parasite Plasmodium falciparum

“…1). The CCR4-NOT coactivator possesses cytoplasmic mRNA deadenylase activity (Tucker et al 2001), and sequences homologous to the core components of this complex, including homologs of CCR4-which serves as the catalytic subunit (Swanson et al 2003), are present in the parasite (Fig. 2).…”