Chromatin structure is believed to be important for a number of cellular processes, including transcription. However, the role of nucleosomes in transcription is not well understood. We have identified the yeast histone locus HTB1-HTB1, encoding histones H2A and H2B, as a suppressor of solo 8 insertion mutations that inhibit adjacent gene expression. The HTA1-HTB1 locus causes suppression either when present on a high-copy-number plasmid or when mutant. These changes in HTA1-HTB1 alter transcription of the genes adjacent to the 8 insertions. On the basis of this result, we have examined the effects of increased and decreased histone gene dosage for all four yeast histone loci. From the types of histone gene dosage changes that cause suppression of insertion mutations, we conclude that altered stoichiometry of histone dimer sets can alter transcription in yeast. Many studies have demonstrated the presence of altered chromatin structure in transcriptionally active regions of the genome (for reviews, see Mathis et al. 1980;Pederson et al. 1986). However, other work has shown that the level of transcription does not affect chromatin structure at certain loci (Nasmyth 1982; for review, see Mathis et al. 1980). Thus, the cause-and-effect relationship between chromatin structure and transcription remains unclear.The major subunit of chromatin is the nucleosome, which consists of DNA wrapped around a histone octamer that is composed of two H2A-H2B dimers and an H3-H4 tetramer (for review, see Pederson et al. 1986). Since histones are the protein components of nucleosomes, it is reasonable to hypothesize that alteration of histone levels would affect chromatin structure. A genetic approach to study the effect of chromatin structure on transcription and other cellular processes would be to alter histone levels by mutation or by gene amplification and then to screen for resulting mutant phenotypes.Changes in histone levels have been reported to cause various phenotypes. Meeks-Wagner and Hartwell (1986) showed that a high copy number of the genes encoding H2A and H2B or of the genes encoding H3 and H4 causes increased chromosome loss during mitosis in Saccharomyces cerevisiae. Yeast cells depleted for H2B arrest in 3Present address: Program in Molecular Biology and Virology, Memorial

The Saccharomyces cerevisiae Sln1 protein is a 'twocomponent' regulator involved in osmotolerance. Twocomponent regulators are a family of signal-transduction molecules with histidine kinase activity common in prokaryotes and recently identified in eukaryotes. Phosphorylation of Sln1p inhibits the HOG1 MAP kinase osmosensing pathway via a phosphorelay mechanism including Ypd1p and the response regulator, Ssk1p. SLN1 also activates an MCM1-dependent reporter gene, P-lacZ, but this function is independent of Ssk1p. We present genetic and biochemical evidence that Skn7p is the response regulator for this alternative Sln1p signaling pathway. Thus, the yeast Sln1 phosphorelay is actually more complex than appreciated previously; the Sln1 kinase and Ypd1 phosphorelay intermediate regulate the activity of two distinct response regulators, Ssk1p and Skn7p. The established role of Skn7p in oxidative stress is independent of the conserved receiver domain aspartate, D427. In contrast, we show that Sln1p activation of Skn7p requires phosphorylation of D427. The expression of TRX2, previously shown to exhibit Skn7p-dependent oxidative-stress activation, is also regulated by the SLN1 phosphorelay functions of Skn7p. The identification of genes responsive to both classes of Skn7p function suggests a central role for Skn7p and the SLN1-SKN7 pathway in integrating and coordinating cellular response to various types of environmental stress.

To identify genes required for the synthesis of glycosyl phosphatidylinositol (GPI) membrane anchors in yeast, we devised a way to isolate GPI anchoring mutants in which colonies are screened for defects in [3H]-inositol incorporation into protein. The gpi1 mutant, identified in this way, is temperature sensitive for growth and defective in vitro in the synthesis of GlcNAc-phosphatidylinositol, the first intermediate in GPI biosynthesis (Leidich, S. D., Drapp, D. A., and Orlean, P. (1994) J. Biol. Chem. 269, 10193-10196). We report the isolation of two more conditionally lethal mutants, gpi2 and gpi3, which, like gpi1, have a temperature-sensitive defect in the incorporation of [3H]inositol into protein and which lack in vitro GlcNAc-phosphatidylinositol synthetic activity. Haploid Saccharomyces cerevisiae strains containing any pairwise combination of the gpi1, gpi2, and gpi3 mutations are inviable. The GPI2 gene, cloned by complementation of the gpi2 mutant's temperature sensitivity, encodes a hydrophobic 269-amino acid protein that resembles no other gene product known to participate in GPI assembly. Gene disruption experiments show that GPI2 is required for vegetative growth. Overexpression of the GPI2 gene causes partial suppression of the gpi1 mutant's temperature sensitivity, a result that suggests that the Gpi1 and Gpi2 proteins interact with one another in vivo. The gpi3 mutant is defective in the SPT14 gene, which encodes a yeast protein similar to the product of the mammalian PIG-A gene, which complements a GlcNAc-phosphatidylinositol synthesis-defective human cell line. In yeast, at least three gene products are required for the first step in GPI synthesis, as is the case in mammalian cells, and utilization of several different proteins at this stage is therefore likely to be a general characteristic of the GPI synthetic pathway.

The yeast "two-component" osmotic stress phosphorelay consists of the histidine kinase, Sln1p, the phosphorelay intermediate, Ypd1p and two response regulators, Ssk1p and Skn7p, whose activities are regulated by phosphorylation of a conserved aspartyl residue in the receiver domain. Dephospho-Ssk1p leads to activation of the hyper-osmotic response (HOG) pathway, whereas phospho-Skn7p presumably leads to activation of hypo-osmotic response genes. The multifunctional Skn7 protein is important in oxidative as well as osmotic stress; however, the Skn7p receiver domain aspartate that is the phosphoacceptor in the SLN1 pathway is dispensable for oxidative stress. Like many well-characterized bacterial response regulators, Skn7p is a transcription factor. In this report we investigate the role of Skn7p in osmotic response gene activation. Our studies reveal that the Skn7p HSF-like DNA binding domain interacts with a cis-acting element identified upstream of OCH1 that is distinct from the previously defined HSE-like Skn7p binding site. Our data support a model in which Skn7p receiver domain phosphorylation affects transcriptional activation rather than DNA binding to this class of DNA binding site.

To understand the function of SPT13, a gene encoding a trans-acting factor involved in regulation of Ty-mediated gene expression, we (13,17,50,60,66). Ty element insertion mutations often reduce adjacent-gene transcription, whereas solo 8 insertion mutations often alter adjacent-gene transcription qualitatively by providing an alternate and preferred site for transcription initiation (29,59,69). In both cases, the change in the pattern of transcription of the adjacent gene can lead to a mutant phenotype. For example, Ty and 8 insertion mutations at the HIS4 and LYS2 genes frequently cause respectively (50,59,60,67). By selection for unlinked suppressors of Ty and 8 insertion mutations, we have identified 15 different SPT (suppressor of Ty insertion mutation) genes, each of which is likely to encode a protein involved in the regulation of Ty-mediated transcription and possibly in the transcription of other yeast genes (19,67,68).The upstream activation site, TATA, and initiation site sequences for Ty transcription are located in the 8 sequences (14,40). However, control of Ty and adjacent-gene transcription is also mediated by sequences located within the Ty transcription unit (18, 51). At the nucleotide sequence level, the internal regulatory sequences in Ty elements show similarity to the simian virus 40 enhancer core sequence (18,51). Functional studies also suggest an enhancerlike activity, since these sequences, located downstream of the Ty promoter, contribute to activation of Ty transcription (12,18,24,40,51

SummaryThe Saccharomyces cerevisiae Yap1p and Skn7p transcription factors collaborate in the activation of oxidative stress response (OSR) genes. Although Yap1p and Skn7p oxidative stress response elements (YRE, OSRE) have been characterized and identified in some OSR genes, many OSR genes lack such elements. In this study, the complex, oxidative responsive, CCP1 promoter was used as a model to investigate the cis -acting elements responsible for activation by oxidative stress. In addition to consensus YRE and OSRE sequences, novel Yap1p and Skn7p binding sites were identified in the CCP1 promoter. These new sites were found to mediate Yap1p-and Skn7p-dependent activation of OSR genes including TSA1 and CTT1 previously thought to lack Yap1p and Skn7p binding sites. The novel YREs and OSREs were found to be enriched in the promoter regions of a set of 179 OSR genes. The widespread existence of novel Yap1p and Skn7p binding sites strongly suggest that direct binding of Yap1p and Skn7p is responsible for activation of many more OSR genes than previously believed.

Sln1p is a plasma membrane-localized two-component histidine kinase that functions as an osmotic stress sensor in Saccharomyces cerevisiae. Changes in osmotic pressure modulate Sln1p kinase activity, which, together with Ypd1p, a phosphorelay intermediate, changes the phosphorylation status of two response regulators, Ssk1p and Skn7p. Ssk1p controls the activity of the HOG1 mitogen-activated protein kinase pathway. Skn7p is a nuclearly localized transcription factor that regulates genes involved in cell wall integrity and other processes. Subcellular compartmentalization may therefore play an important role in eukaryotic two-component pathway regulation. We have studied the subcellular localization of SLN1 pathway components and find that Ypd1p is a dynamic protein with a role in shuttling the osmotic stress signal from Sln1p to Ssk1p in the cytosol and to Skn7p in the nucleus. The need to translocate the signal into different intracellular compartments contributes a spatial dimension to eukaryotic two-component pathways compared to the prototypical two-component pathways of prokaryotes.

The bifunctional Saccharomyces cerevisiae Skn7 transcription factor regulates osmotic stress response genes as well as oxidative stress response genes; however, the mechanisms involved in these two types of regulation differ. Skn7 osmotic stress activity depends on the phosphorylation of the receiver domain aspartate, D427, by the Sln1 histidine kinase. In contrast, D427 and the SLN1-SKN7 phosphorelay are dispensable for the oxidative stress response, although the receiver domain is required. The majority of oxidative stress response genes regulated by Skn7 also are regulated by the redox-responsive transcription factor Yap1. It is therefore possible that the nuclearly localized Skn7 does not itself respond to the oxidant but simply cooperates with Yap1 when it translocates to the nucleus. We report here that oxidative stress leads to a phosphatase-sensitive, slow-mobility Skn7 variant. This suggests that Skn7 undergoes a posttranslational modification by phosphorylation following exposure to oxidant. Oxidant-dependent Skn7 phosphorylation was eliminated in strains lacking the Yap1 transcription factor. This suggests that the phosphorylation of Skn7 is regulated by Yap1. Mutations in the receiver domain of Skn7 were identified that affect its oxidative stress function. These mutations were found to compromise the association of Yap1 and Skn7 at oxidative stress response gene promoters. A working model is proposed in which the association of Yap1 with Skn7 in the nucleus is a prerequisite for Skn7 phosphorylation and the activation of oxidative stress response genes.