DNA replication checkpoint control of Wee1 stability by vertebrate Hsl7

G2/M checkpoints prevent mitotic entry upon DNA damage or replication inhibition by targeting the Cdc2 regulators Cdc25 and Wee1. Although Wee1 protein stability is regulated by DNA-responsive checkpoints, the vertebrate pathways controlling Wee1 degradation have not been elucidated. In budding yeast, stability of the Wee1 homologue, Swe1, is controlled by a regulatory module consisting of the proteins Hsl1 and Hsl7 (histone synthetic lethal 1 and 7), which are targeted by the morphogenesis checkpoint to prevent Swe1 degradation when budding is inhibited. We report here the identification of Xenopus Hsl7 as a positive regulator of mitosis that is controlled, instead, by an entirely distinct checkpoint, the DNA replication checkpoint. Although inhibiting Hsl7 delayed mitosis, Hsl7 overexpression overrode the replication checkpoint, accelerating Wee1 destruction. Replication checkpoint activation disrupted Hsl7-Wee1 interactions, but binding was restored by active polo-like kinase. These data establish Hsl7 as a component of the replication checkpoint and reveal that similar cell cycle control modules can be co-opted for use by distinct checkpoints in different organisms.     

The Morphogenesis Checkpoint in Saccharomyces cerevisiae: Cell Cycle Control of Swe1p Degradation by Hsl1p and Hsl7p

In Saccharomyces cerevisiae, the Wee1 family kinase Swe1p is normally stable during G(1) and S phases but is unstable during G(2) and M phases due to ubiquitination and subsequent degradation. However, perturbations of the actin cytoskeleton lead to a stabilization and accumulation of Swe1p. This response constitutes part of a morphogenesis checkpoint that couples cell cycle progression to proper bud formation, but the basis for the regulation of Swe1p degradation by the morphogenesis checkpoint remains unknown. Previous studies have identified a protein kinase, Hsl1p, and a phylogenetically conserved protein of unknown function, Hsl7p, as putative negative regulators of Swe1p. We report here that Hsl1p and Hsl7p act in concert to target Swe1p for degradation. Both proteins are required for Swe1p degradation during the unperturbed cell cycle, and excess Hsl1p accelerates Swe1p degradation in the G(2)-M phase. Hsl1p accumulates periodically during the cell cycle and promotes the periodic phosphorylation of Hsl7p. Hsl7p can be detected in a complex with Swe1p in cell lysates, and the overexpression of Hsl7p or Hsl1p produces an effective override of the G(2) arrest imposed by the morphogenesis checkpoint. These findings suggest that Hsl1p and Hsl7p interact directly with Swe1p to promote its recognition by the ubiquitination complex, leading ultimately to its destruction.     

A conserved motif common to the histone acetyltransferase Esa1 and the histone deacetylase Rpd3

Post-translational modification of histones enables dynamic regulation of chromatin structure in eukaryotes. Histone acetyltransferase (HAT) and histone deacetylase (HDAC) modify the N-terminal tails of histones by adding or removing acetyl groups to specific lysine residues. A particular pair of HAT (Esa1) and HDAC (Rpd3) is proposed to modify the same lysine residue in vitro and in vivo. Thus, HAT and HDAC might have similar structural and functional motifs. Here we show that HAT (Esa1 family) and HDAC (Rpd3 family) have similar amino acid stretches in the primary structures through evolution. We refer to this region as the "ER (Esa1-Rpd3) motif." In the tertiary structure of Esa1, the ER motif is located near the active center. In Rpd3, for which the tertiary structure remains unclear, we demonstrate that the ER motif contains the same secondary structure as found in Esa1 by circular dichroism analysis. We did alanine-scanning mutagenesis and found that the ER motif regions of Esa1 or Rpd3 are required for HAT activity of Esa1 or HDAC activity of Rpd3, respectively. Our discovery of the ER motif present in the pair of enzymes (HAT and HDAC) indicates that HAT and HDAC have common structural bases, although they catalyze the reaction with opposite functions.     

Genetic analysis implicates the Set3/Hos2 histone deacetylase in the deposition and remodeling of nucleosomes containing H2A.Z

Histone variants and histone modification complexes act to regulate the functions of chromatin. In Saccharomyces cerevisiae the histone variant H2A.Z is encoded by HTZ1. Htz1 is dispensable for viability in budding yeast, but htz1Δ is synthetic sick or lethal with the null alleles of about 200 nonessential genes. One of the strongest of these interactions is with the deletion of SET3, which encodes a subunit of the Set3/Hos2 histone deacetylase complex. Little is known about the functions of Set3, and interpreting these genetic interactions remains a highly challenging task. Here we report the results of a forward genetic screen to identify bypass suppressors of the synthetic slow-growth phenotype of htz1Δ set3Δ. Among the identified loss-of-function suppressors are genes encoding subunits of the HDA1 deacetylase complex, the SWR1 complex, the H2B deubiquitination module of SAGA, the proteasome, Set1, and Sir3. This constellation of suppressor genes is uncommon among the global set of htz1Δ synthetic interactions. BDF1, AHC1, RMR1, and CYC8 were identified as high-copy suppressors. We also identified interactions with SLX5 and SLX8, encoding the sumoylation-targeted ubiquitin ligase complex. In the context of htz1Δ set3Δ, suppressors in the SWR1 and the H2B deubiquitination complexes show strong functional similarity, as do suppressors in the silencing genes and the proteasome. Surprisingly, while both htz1Δ set3Δ and swr1Δ set3Δ have severe slow-growth phenotypes, the htz1Δ swr1Δ set3Δ triple mutant grows relatively well. We propose that Set3 has previously unrecognized functions in the dynamic deposition and remodeling of nucleosomes containing H2A.Z.     

A monitor for bud emergence in the yeast morphogenesis checkpoint

Cell cycle transitions are subject to regulation by both external signals and internal checkpoints that monitor satisfactory progression of key cell cycle events. In budding yeast, the morphogenesis checkpoint arrests the cell cycle in response to perturbations that affect the actin cytoskeleton and bud formation. Herein, we identify a step in this checkpoint pathway that seems to be directly responsive to bud emergence. Activation of the kinase Hsl1p is dependent upon its recruitment to a cortical domain organized by the septins, a family of conserved filament-forming proteins. Under conditions that delayed or blocked bud emergence, Hsl1p recruitment to the septin cortex still took place, but hyperphosphorylation of Hsl1p and recruitment of the Hsl1p-binding protein Hsl7p to the septin cortex only occurred after bud emergence. At this time, the septin cortex spread to form a collar between mother and bud, and Hsl1p and Hsl7p were restricted to the bud side of the septin collar. We discuss models for translating cellular geometry (in this case, the emergence of a bud) into biochemical signals regulating cell proliferation.     

A glycolytic burst drives glucose induction of global histone acetylation by picNuA4 and SAGA

Little is known about what enzyme complexes or mechanisms control global lysine acetylation in the amino-terminal tails of the histones. Here, we show that glucose induces overall acetylation of H3 K9, 18, 27 and H4 K5, 8, 12 in quiescent yeast cells mainly by stimulating two KATs, Gcn5 and Esa1. Genetic and pharmacological perturbation of carbon metabolism, combined with ¹H-NMR metabolic profiling, revealed that glucose induction of KAT activity directly depends on increased glucose catabolism. Glucose-inducible Esa1 and Gcn5 activities predominantly reside in the picNuA4 and SAGA complexes, respectively, and act on chromatin by an untargeted mechanism. We conclude that direct metabolic regulation of globally acting KATs can be a potent driving force for reconfiguration of overall histone acetylation in response to a physiological cue.     

Functional and physical interactions between yeast 14-3-3 proteins, acetyltransferases, and deacetylases in response to DNA replication perturbations

The highly conserved 14-3-3 proteins participate in many biological processes in different eukaryotes. The BMH1 and BMH2 genes encode the two functionally redundant Saccharomyces cerevisiae 14-3-3 isoforms. In this work we provide evidence that defective 14-3-3 functions not only impair the ability of yeast cells to sustain DNA replication in the presence of sublethal concentrations of methyl methanesulfonate (MMS) or hydroxyurea (HU) but also cause S-phase checkpoint hyperactivation. Inactivation of the catalytic subunit of the histone acetyltransferase NuA4 or of its interactor Yng2, besides leading to S-phase defects and persistent checkpoint activation in the presence of genotoxic agents, is lethal for bmh mutants. Conversely, the lack of the histone deacetylase subunit Rpd3 or Sin3 partially suppresses the hypersensitivity to HU of bmh mutants and restores their ability to complete DNA replication in the presence of MMS or HU. These data strongly suggest that reduced acetyltransferase functionality might account for the S-phase defects of bmh mutants in the presence of genotoxic agents. Consistent with a role of 14-3-3 proteins in acetyltransferase and deacetylase regulation, we find that acetylation of H3 and H4 histone tails is reduced in temperature-sensitive bmh mutants shifted to the restrictive temperature. Moreover, Bmh proteins physically interact, directly or indirectly, with the Esa1 acetyltransferase throughout the cell cycle and with the Rpd3 deacetylase specifically during unperturbed S phase and after HU treatment. Taken together, our results highlight a novel role for 14-3-3 proteins in the regulation of histone acetyltransferase and deacetylase functions in the response to replicative stress.     

Septin-dependent assembly of a cell cycle-regulatory module in Saccharomyces cerevisiae

Saccharomyces cerevisiae septin mutants have pleiotropic defects, which include the formation of abnormally elongated buds. This bud morphology results at least in part from a cell cycle delay imposed by the Cdc28p-inhibitory kinase Swe1p. Mutations in three other genes (GIN4, encoding a kinase related to the Schizosaccharomyces pombe mitotic inducer Nim1p; CLA4, encoding a p21-activated kinase; and NAP1, encoding a Clb2p-interacting protein) also produce perturbations of septin organization associated with an Swe1p-dependent cell cycle delay. The effects of gin4, cla4, and nap1 mutations are additive, indicating that these proteins promote normal septin organization through pathways that are at least partially independent. In contrast, mutations affecting the other two Nim1p-related kinases in S. cerevisiae, Hsl1p and Kcc4p, produce no detectable effect on septin organization. However, deletion of HSL1, but not of KCC4, did produce a cell cycle delay under some conditions; this delay appears to reflect a direct role of Hsl1p in the regulation of Swe1p. As shown previously, Swe1p plays a central role in the morphogenesis checkpoint that delays the cell cycle in response to defects in bud formation. Swe1p is localized to the nucleus and to the daughter side of the mother bud neck prior to its degradation in G(2)/M phase. Both the neck localization of Swe1p and its degradation require Hsl1p and its binding partner Hsl7p, both of which colocalize with Swe1p at the daughter side of the neck. This localization is lost in mutants with perturbed septin organization, suggesting that the release of Hsl1p and Hsl7p from the neck may reduce their ability to inactivate Swe1p and thus contribute to the G(2) delay observed in such mutants. In contrast, treatments that perturb actin organization have little effect on Hsl1p and Hsl7p localization, suggesting that such treatments must stabilize Swe1p by another mechanism. The apparent dependence of Swe1p degradation on localization of the Hsl1p-Hsl7p-Swe1p module to a site that exists only in budded cells may constitute a mechanism for deactivating the morphogenesis checkpoint when it is no longer needed (i.e., after a bud has formed).     

Feedback control of Swe1p degradation in the yeast morphogenesis checkpoint

Saccharomyces cerevisiae cells exposed to a variety of physiological stresses transiently delay bud emergence or bud growth. To maintain coordination between bud formation and the cell cycle in such circumstances, the morphogenesis checkpoint delays nuclear division via the mitosis-inhibitory Wee1-family kinase, Swe1p. Swe1p is degraded during G2 in unstressed cells but is stabilized and accumulates following stress. Degradation of Swe1p is preceded by its recruitment to the septin scaffold at the mother-bud neck, mediated by the Swe1p-binding protein Hsl7p. Following osmotic shock or actin depolymerization, Swe1p is stabilized, and previous studies suggested that this was because Hsl7p was no longer recruited to the septin scaffold following stress. However, we now show that Hsl7p is in fact recruited to the septin scaffold in stressed cells. Using a cyclin-dependent kinase (CDK) mutant that is immune to checkpoint-mediated inhibition, we show that Swe1p stabilization following stress is an indirect effect of CDK inhibition. These findings demonstrate the physiological importance of a positive-feedback loop in which Swe1p activity inhibits the CDK, which then ceases to target Swe1p for degradation. They also highlight the difficulty in disentangling direct checkpoint pathways from the effects of positive-feedback loops active at the G2/M transition.     

Molecular Dissection of the Checkpoint Kinase Hsl1p

Cell shape can influence cell behavior. In Saccharomyces cerevisiae, bud emergence can influence cell cycle progression via the morphogenesis checkpoint. This surveillance pathway ensures that mitosis always follows bud formation by linking degradation of the mitosis-inhibitory kinase Swe1p (Wee1) to successful bud emergence. A crucial component of this pathway is the checkpoint kinase Hsl1p, which is activated upon bud emergence and promotes Swe1p degradation. We have dissected the large nonkinase domain of Hsl1p by using evolutionary conservation as a guide, identifying regions important for Hsl1p localization, function, and regulation. An autoinhibitory motif restrains Hsl1p activity when it is not properly localized to the mother-bud neck. Hsl1p lacking this motif is active as a kinase regardless of the assembly state of cytoskeletal septin filaments. However, the active but delocalized Hsl1p cannot promote Swe1p down-regulation, indicating that localization is required for Hsl1p function as well as Hsl1p activation. We also show that the septin-mediated Hsl1p regulation via the novel motif operates in parallel to a previously identified Hsl1p activation pathway involving phosphorylation of the Hsl1p kinase domain. We suggest that Hsl1p responds to alterations in septin organization, which themselves occur in response to the local geometry of the cell cortex.