Wei-Chun Au

Altered Dosage and Mislocalization of Histone H3 and Cse4p Lead to Chromosome Loss in Saccharomyces cerevisiae

Cse4p is an essential histone H3 variant in Saccharomyces cerevisiae that defines centromere identity and is required for proper segregation of chromosomes. In this study, we investigated phenotypic consequences of Cse4p mislocalization and increased dosage of histone H3 and Cse4p, and established a direct link between histone stoichiometry, mislocalization of Cse4p, and chromosome segregation. Overexpression of the stable Cse4p mutant, cse4K16R, resulted in its mislocalization, increased association with chromatin, and a high rate of chromosome loss, all of which were suppressed by constitutive expression of histone H3 (Δ16H3). We determined that Δ16H3 did not lead to increased chromosome loss; however, increasing the dosage of histone H3 (GALH3) resulted in significant chromosome loss due to reduced levels of centromere (CEN)-associated Cse4p and synthetic dosage lethality (SDL) in kinetochore mutants. These phenotypes were suppressed by GALCSE4. We conclude that the chromosome missegregation of GALcse4K16R and GALH3 strains is due to mislocalization and a functionally compromised kinetochore, respectively. Suppression of these phenotypes by histone Δ16H3 and GALCSE4 supports the conclusion that proper stoichiometry affects the localization of histone H3 and Cse4p and is thus essential for accurate chromosome segregation.

A Role for Histone H4K16 Hypoacetylation in Saccharomyces cerevisiae Kinetochore Function

Hypoacetylated H4 is present at regional centromeres; however, its role in kinetochore function is poorly understood. We characterized H4 acetylation at point centromeres in Saccharomyces cerevisiae and determined the consequences of altered H4 acetylation on chromosome segregation. We observed low levels of tetra-acetylated and K16 acetylated histone H4 (H4K16Ac) at centromeres. Low levels of H4K16Ac were also observed at noncentromeric regions associated with Cse4p. Inhibition of histone deacetylases (HDAC) using nicotinamide (NAM) caused lethality in cse4 and hhf1-20 kinetochore mutants and increased centromeric H4K16Ac. Overexpression of Sas2-mediated H4K16 acetylation activity in wild-type cells led to increased rates of chromosome loss and synthetic dosage lethality in kinetochore mutants. Consistent with increased H4K16 acetylation as a cause of the phenotypes, deletion of the H4K16 deacetylase SIR2 or a sir2-H364Y catalytic mutant resulted in higher rates of chromosome loss compared to wild-type cells. Moreover, H4K16Q acetylmimic mutants displayed increased rates of chromosome loss compared to H4K16R nonacetylatable mutants and wild-type cells. Our work shows that hypoacetylated centromeric H4 is conserved across eukaryotic centromeres and hypoacetylation of H4K16 at centromeres plays an important role in accurate chromosome segregation.

Loss of SOD1 and LYS7 sensitizes Saccharomyces cerevisiae to hydroxyurea and DNA damage agents and downregulates MEC1 pathway effectors.

ABSTRACT Aerobic metabolism produces reactive oxygen species, including superoxide anions, which cause DNA damage unless removed by scavengers such as superoxide dismutases. We show that loss of the Cu,Zn-dependent superoxide dismutase, SOD1, or its copper chaperone, LYS7, confers oxygen-dependent sensitivity to replication arrest and DNA damage in Saccharomyces cerevisiae. We also find that sod1Δ strains, and to a lesser extent lys7Δ strains, when arrested with hydroxyurea (HU) show reduced induction of the MEC1 pathway effector Rnr3p and of Hug1p. The HU sensitivity of sod1Δ and lys7Δ strains is suppressed by overexpression of TKL1, a transketolase that generates NADPH, which balances redox in the cell and is required for ribonucleotide reductase activity. Our results suggest that the MEC1 pathway in sod1Δ mutant strains is sensitive to the altered cellular redox state due to increased superoxide anions and establish a new relationship between SOD1, LYS7, and the MEC1-mediated checkpoint response to replication arrest and DNA damage in S. cerevisiae.

Gga2 Mediates Sequential Ubiquitin-independent and Ubiquitin-dependent Steps in the Trafficking of ARN1 from the trans-Golgi Network to the Vacuole

In Saccharomyces cerevisiae, ARN1 encodes a transporter for the uptake of ferrichrome, an important nutritional source of iron. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network (TGN) to the vacuolar lumen via the vacuolar protein-sorting pathway. Arn1p is mis-sorted to the plasma membrane in cells lacking Gga2p, a monomeric clathrin-adaptor protein involved in vesicular transport from the TGN. Although Ggas have been characterized as ubiquitin receptors, we show here that ubiquitin binding by Gga2 was not required for the TGN-to-endosome trafficking of Arn1, but it was required for subsequent sorting of Arn1 into the multivesicular body. In a ubiquitin-binding mutant of Gga2, Arn1p accumulated on the vacuolar membrane in a ubiquitinated form. The yeast epsins Ent3p and Ent4p were also involved in TGN-to-vacuole sorting of Arn1p. Amino-terminal sequences of Arn1p were required for vacuolar protein sorting, as mutation of ubiquitinatable lysine residues resulted in accumulation on the vacuolar membrane, and mutation of either a THN or YGL sequence resulted in mis-sorting to the plasma membrane. These studies suggest that Gga2 is involved in sorting at both the TGN and multivesicular body and that the first step can occur without ubiquitin binding.

Phosphorylation of centromeric histone H3 variant regulates chromosome segregation in Saccharomyces cerevisiae

Cse4 is posttranslationally modified in Saccharomyces cerevisiae. Ipl1 contributes to Cse4 phosphorylation in vivo and in vitro. Phosphorylation of Cse4 at centromeres is enhanced in response to nocodazole or reduced cohesion. The results suggest that phosphorylation of Cse4 ensures faithful chromosome segregation.

Insights into assembly and regulation of centromeric chromatin in Saccharomyces cerevisiae

At the core of chromosome segregation is the centromere, which nucleates the assembly of a macromolecular kinetochore (centromere DNA and associated proteins) complex responsible for mediating spindle attachment. Recent advances in centromere research have led to identification of many kinetochore components, such as the centromeric-specific histone H3 variant, CenH3, and its interacting partner, Scm3. Both are essential for chromosome segregation and are evolutionarily conserved from yeast to humans. CenH3 is proposed to be the epigenetic mark that specifies centromeric identity. Molecular mechanisms that regulate the assembly of kinetochores at specific chromosomal sites to mediate chromosome segregation are not fully understood. In this review, we summarize the current literature and discuss results from our laboratory, which show that restricting the localization of budding yeast CenH3, Cse4, to centromeres and balanced stoichiometry between Scm3 and Cse4, contribute to faithful chromosome transmission. We highlight our findings that, similar to other eukaryotic centromeres, budding yeast centromeric histone H4 is hypoacetylated, and we discuss how altered histone acetylation affects chromosome segregation. This article is part of a Special Issue entitled: Chromatin in time and space.

Phosphatidylserine Is Involved in the Ferrichrome-induced Plasma Membrane Trafficking of Arn1 in Saccharomyces cerevisiae

Arn1 is an integral membrane protein that mediates the uptake of ferrichrome, an important nutritional source of iron, in Saccharomyces cerevisiae. In the absence of ferrichrome, Arn1p is sorted directly from the trans-Golgi network to the vacuolar lumen for degradation. In the presence of low levels of ferrichrome, the siderophore binds to a receptor domain on Arn1, triggering the redistribution of Arn1 to the plasma membrane. When extracellular ferrichrome levels are high, Arn1 cycles between the plasma membrane and intracellular vesicles. To further understand the mechanisms of trafficking of Arn1p, we screened 4580 viable yeast deletion mutants for mislocalization of Arn1-GFP using synthetic genetic array technology. We identified over 100 genes required for trans-Golgi network-to-vacuole trafficking of Arn1-GFP and only two genes, SER1 and SER2, required for the ferrichrome-induced plasma membrane trafficking of Arn1-GFP. SER1 and SER2 encode two enzymes of the major serine biosynthetic pathway, and the Arn1 trafficking defect in the ser1Δ strain was corrected with supplemental serine or glycine. Plasma membrane trafficking of Hxt3, a structurally related glucose transporter, was unaffected by SER1 deletion. Serine is required for the synthesis of multiple cellular components, including purines, sphingolipids, and phospholipids, but of these only phosphatidylserine corrected the Arn1 trafficking defects of the ser1Δ strain. Strains with defects in phospholipid synthesis also exhibited alterations in Arn1p trafficking, indicating that the intracellular trafficking of some transporters is dependent on the phospholipid composition of the cellular membranes.

Polo kinase Cdc5 associates with centromeres to facilitate the removal of centromeric cohesin during mitosis

Cdc5 associates with centromeric chromatin during mitosis. Cdc5 plays a critical role in the differential removal of cohesin from centromeric chromatin compared to chromosome arms.

Yeast hEST1A/B (SMG5/6)- Like proteins contribute to environment-sensing adaptive gene expression responses

During its natural life cycle, budding yeast (Saccharomyces cerevisiae) has to adapt to drastically changing environments, but how environmental-sensing pathways are linked to adaptive gene expression changes remains incompletely understood. Here, we describe two closely related yeast hEST1A-B (SMG5-6)–like proteins termed Esl1 and Esl2 that contain a 14-3-3–like domain and a putative PilT N-terminus ribonuclease domain. We found that, unlike their metazoan orthologs, Esl1 and Esl2 were not involved in nonsense-mediated mRNA decay or telomere maintenance pathways. However, in genome-wide expression array analyses, absence of Esl1 and Esl2 led to more than two-fold deregulation of ∼50 transcripts, most of which were expressed inversely to the appropriate metabolic response to environmental nutrient supply; for instance, normally glucose-repressed genes were derepressed in esl1Δ esl2Δ double mutants during growth in a high-glucose environment. Likewise, in a genome-wide synthetic gene array screen, esl1Δ esl2Δ double mutants were synthetic sick with null mutations for Rim8 and Dfg16, which form the environmental-sensing complex of the Rim101 pH response gene expression pathway. Overall, these results suggest that Esl1 and Esl2 contribute to the regulation of adaptive gene expression responses of environmental sensing pathways.

N-terminal Sumoylation of Centromeric Histone H3 Variant Cse4 Regulates Its Proteolysis To Prevent Mislocalization to Non-centromeric Chromatin

Stringent regulation of cellular levels of evolutionarily conserved centromeric histone H3 variant (CENP-A in humans, CID in flies, Cse4 in yeast) prevents its mislocalization to non-centromeric chromatin. Overexpression and mislocalization of CENP-A has been observed in cancers and leads to aneuploidy in yeast, flies, and human cells. Ubiquitin-mediated proteolysis of Cse4 by E3 ligases such as Psh1 and Sumo-Targeted Ubiquitin Ligase (STUbL) Slx5 prevent mislocalization of Cse4. Previously, we identified Siz1 and Siz2 as the major E3 ligases for sumoylation of Cse4. In this study, we have identified lysine 65 (K65) in Cse4 as a site that regulates sumoylation and ubiquitin-mediated proteolysis of Cse4 by Slx5. Strains expressing cse4 K65R exhibit reduced levels of sumoylated and ubiquitinated Cse4 in vivo. Furthermore, co-immunoprecipitation experiments reveal reduced interaction of cse4 K65R with Slx5, leading to increased stability and mislocalization of cse4 K65R under normal physiological conditions. Based on the increased stability of cse4 K65R in psh1∆ strains but not in slx5∆ strains, we conclude that Slx5 targets sumoylated Cse4 K65 for ubiquitination-mediated proteolysis independent of Psh1. In summary, we have identified and characterized the physiological role of Cse4 K65 in sumoylation, ubiquitin-mediated proteolysis, and localization of Cse4 for genome stability.