Starvation for amino acids induces Gcn4p, a transcriptional activator of amino acid biosynthetic genes in Saccharomyces cerevisiae. In an effort to identify all genes regulated by Gcn4p during amino acid starvation, we performed cDNA microarray analysis. Data from 21 pairs of hybridization experiments using two different strains derived from S288c revealed that more than 1,000 genes were induced, and a similar number were repressed, by a factor of 2 or more in response to histidine starvation imposed by 3-aminotriazole (3AT). Profiling of a gcn4⌬ strain and a constitutively induced mutant showed that Gcn4p is required for the full induction by 3AT of at least 539 genes, termed Gcn4p targets. Genes in every amino acid biosynthetic pathway except cysteine and genes encoding amino acid precursors, vitamin biosynthetic enzymes, peroxisomal components, mitochondrial carrier proteins, and autophagy proteins were all identified as Gcn4p targets. Unexpectedly, genes involved in amino acid biosynthesis represent only a quarter of the Gcn4p target genes. Gcn4p also activates genes involved in glycogen homeostasis, and mutant analysis showed that Gcn4p suppresses glycogen levels in amino acid-starved cells. Numerous genes encoding protein kinases and transcription factors were identified as targets, suggesting that Gcn4p is a master regulator of gene expression. Interestingly, expression profiles for 3AT and the alkylating agent methyl methanesulfonate (MMS) overlapped extensively, and MMS induced GCN4 translation. Thus, the broad transcriptional response evoked by Gcn4p is produced by diverse stress conditions. Finally, profiling of a gcn4⌬ mutant uncovered an alternative induction pathway operating at many Gcn4p target genes in histidine-starved cells.In response to environmental perturbations, Saccharomyces cerevisiae cells elicit rapid transcriptional reprogramming involving both activation and repression of gene expression. Transcriptional activator proteins function by binding to specific promoter elements, called upstream activating sequences (UASs) in yeast cells, and recruiting the transcriptional machinery. Thus, transcriptional stimulation requires the expression and function of an activator and the appropriate UAS element in the promoters of its target genes. A plethora of mechanisms are known to regulate the activity or expression of transcriptional activators in response to specific signals. For example, in cells grown on glucose, Gal80p inhibits the ability of Gal4p to activate transcription of genes encoding galactosemetabolizing enzymes, whereas Gal3p alleviates this inhibition on galactose medium (83). The transcriptional activators Pho4p, Swi5p, and Yap1p are regulated by the coupling of their nuclear localization to the levels of inorganic phosphate, cell cycle and mother-daughter status, or oxidative stress, respectively (reviewed in reference 52). Starvation for amino acids, purines, and glucose limitation induces the synthesis of Gcn4p, a bZIP transcriptional activator of amino acid biosynthetic gene...

Set2-mediated methylation of histone H3 at Lys 36 (H3K36me) is a co-transcriptional event that is necessary for the activation of the Rpd3S histone deacetylase complex, thereby maintaining the coding region of genes in a hypoacetylated state. In the absence of Set2, H3K36 or Rpd3S acetylated histones accumulate on open reading frames (ORFs), leading to transcription initiation from cryptic promoters within ORFs. Although the co-transcriptional deacetylation pathway is well characterized, the factors responsible for acetylation are as yet unknown. Here we show that, in yeast, co-transcriptional acetylation is achieved in part by histone exchange over ORFs. In addition to its function of targeting and activating the Rpd3S complex, H3K36 methylation suppresses the interaction of H3 with histone chaperones, histone exchange over coding regions and the incorporation of new acetylated histones. Thus, Set2 functions both to suppress the incorporation of acetylated histones and to signal for the deacetylation of these histones in transcribed genes. By suppressing spurious cryptic transcripts from initiating within ORFs, this pathway is essential to maintain the accuracy of transcription by RNA polymerase II.

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Iron homeostasis is highly regulated in organisms across evolutionary time scale as iron is essential for various cellular processes. In a computational screen, we identified the Yap/bZIP domain family in Candida clade genomes. Cap2/Hap43 is essential for C. albicans growth under iron-deprivation conditions and for virulence in mouse. Cap2 has an amino-terminal bipartite domain comprising a fungal-specific Hap4-like domain and a bZIP domain. Our mutational analyses showed that both the bZIP and Hap4-like domains perform critical and independent functions for growth under iron-deprivation conditions. Transcriptome analysis conducted under iron-deprivation conditions identified about 16% of the C. albicans ORFs that were differentially regulated in a Cap2-dependent manner. Microarray data also suggested that Cap2 is required to mobilize iron through multiple mechanisms; chiefly by activation of genes in three iron uptake pathways and repression of iron utilizing and iron storage genes. The expression of HAP2, HAP32, and HAP5, core components of the HAP regulatory complex was induced in a Cap2-dependent manner indicating a feed-forward loop. In a feed-back loop, Cap2 repressed the expression of Sfu1, a negative regulator of iron uptake genes. Cap2 was coimmunoprecipitated with Hap5 from cell extracts prepared from iron-deprivation conditions indicating an in vivo association. ChIP assays demonstrated Hap32-dependent recruitment of Hap5 to the promoters of FRP1 (Cap2-induced) and ACO1 (Cap2-repressed). Together our data indicates that the Cap2-HAP complex functions both as a positive and a negative regulator to maintain iron homeostasis in C. albicans.

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...