Autophagy promotes cell survival by maintaining NAD levels.

Autophagy is an essential catabolic process that promotes the clearance of surplus or damaged intracellular components. Loss of autophagy in age-related human pathologies contributes to tissue degeneration through a poorly understood mechanism. Here, we identify an evolutionarily conserved role of autophagy from yeast to humans in the preservation of nicotinamide adenine dinucleotide (NAD) levels, which are critical for cell survival. In respiring mouse fibroblasts with autophagy deficiency, loss of mitochondrial quality control was found to trigger hyperactivation of stress responses mediated by NADases of PARP and Sirtuin families. Uncontrolled depletion of the NAD(H) pool by these enzymes ultimately contributed to mitochondrial membrane depolarization and cell death. Pharmacological and genetic interventions targeting several key elements of this cascade improved the survival of autophagy-deficient yeast, mouse fibroblasts, and human neurons. Our study provides a mechanistic link between autophagy and NAD metabolism and identifies targets for interventions in human diseases associated with autophagic, lysosomal, and mitochondrial dysfunction.

Kel1 is a phosphorylation-regulated noise suppressor of the pheromone signaling pathway.

Mechanisms have evolved that allow cells to detect signals and generate an appropriate response. The accuracy of these responses relies on the ability of cells to discriminate between signal and noise. How cells filter noise in signaling pathways is not well understood. Here, we analyze noise suppression in the yeast pheromone signaling pathway and show that the poorly characterized protein Kel1 serves as a major noise suppressor and prevents cell death. At the molecular level, Kel1 prevents spontaneous activation of the pheromone response by inhibiting membrane recruitment of Ste5 and Far1. Only a hypophosphorylated form of Kel1 suppresses signaling, reduces noise, and prevents pheromone-associated cell death, and our data indicate that the MAPK Fus3 contributes to Kel1 phosphorylation. Taken together, Kel1 serves as a phospho-regulated suppressor of the pheromone pathway to reduce noise, inhibit spontaneous activation of the pathway, regulate mating efficiency, and prevent pheromone-associated cell death.

Resolution of R-loops by INO80 promotes DNA replication and maintains cancer cell proliferation and viability.

Collisions between the DNA replication machinery and co-transcriptional R-loops can impede DNA synthesis and are a major source of genomic instability in cancer cells. How cancer cells deal with R-loops to proliferate is poorly understood. Here we show that the ATP-dependent chromatin remodelling INO80 complex promotes resolution of R-loops to prevent replication-associated DNA damage in cancer cells. Depletion of INO80 in prostate cancer PC3 cells leads to increased R-loops. Overexpression of the RNA:DNA endonuclease RNAse H1 rescues the DNA synthesis defects and suppresses DNA damage caused by INO80 depletion. R-loops co-localize with and promote recruitment of INO80 to chromatin. Artificial tethering of INO80 to a LacO locus enabled turnover of R-loops in cis. Finally, counteracting R-loops by INO80 promotes proliferation and averts DNA damage-induced death in cancer cells. Our work suggests that INO80-dependent resolution of R-loops promotes DNA replication in the presence of transcription, thus enabling unlimited proliferation in cancers.

The INO80 remodeller in transcription, replication and repair.

The accessibility of eukaryotic genomes to the action of enzymes involved in transcription, replication and repair is maintained despite the organization of DNA into nucleosomes. This access is often regulated by the action of ATP-dependent nucleosome remodellers. The INO80 class of nucleosome remodellers has unique structural features and it is implicated in a diverse array of functions, including transcriptional regulation, DNA replication and DNA repair. Underlying these diverse functions is the catalytic activity of the main ATPase subunit, which in the context of a multisubunit complex can shift nucleosomes and carry out histone dimer exchange. In vitro studies showed that INO80 promotes replication fork progression on a chromatin template, while in vivo it was shown to facilitate replication fork restart after stalling and to help evict RNA polymerase II at transcribed genes following the collision of a replication fork with transcription. More recent work in yeast implicates INO80 in the general eviction and degradation of nucleosomes following high doses of oxidative DNA damage. Beyond these replication and repair functions, INO80 was shown to repress inappropriate transcription at promoters in the opposite direction to the coding sequence. Here we discuss the ways in which INO80's diverse functions help maintain genome integrity.This article is part of the themed issue 'Chromatin modifiers and remodellers in DNA repair and signalling'.

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation.

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.

DNA repair choice defines a common pathway for recruitment of chromatin regulators.

DNA double-strand break repair is essential for maintenance of genome stability. Recent work has implicated a host of chromatin regulators in the DNA-damage response, and although several functional roles have been defined, the mechanisms that control their recruitment to DNA lesions remain unclear. Here we find that efficient double-strand break recruitment of the INO80, SWR-C, NuA4, SWI/SNF and RSC enzymes is inhibited by the non-homologous end-joining machinery, and that their recruitment is controlled by early steps of homologous recombination. Strikingly, we find no significant role for H2A.X phosphorylation in the recruitment of chromatin regulators, but rather their recruitment coincides with reduced levels of H2A.X phosphorylation. Our work indicates that cell cycle position has a key role in DNA repair pathway choice and that recruitment of chromatin regulators is tightly coupled to homologous recombination.

Chromatin and the genome integrity network.

The maintenance of genome integrity is essential for organism survival and for the inheritance of traits to offspring. Genomic instability is caused by DNA damage, aberrant DNA replication or uncoordinated cell division, which can lead to chromosomal aberrations and gene mutations. Recently, chromatin regulators that shape the epigenetic landscape have emerged as potential gatekeepers and signalling coordinators for the maintenance of genome integrity. Here, we review chromatin functions during the two major pathways that control genome integrity: namely, repair of DNA damage and DNA replication. We also discuss recent evidence that suggests a novel role for chromatin-remodelling factors in chromosome segregation and in the prevention of aneuploidy.

Global regulation of H2A.Z localization by the INO80 chromatin-remodeling enzyme is essential for genome integrity.

INO80 is an evolutionarily conserved, ATP-dependent chromatin-remodeling enzyme that plays roles in transcription, DNA repair, and replication. Here, we show that yeast INO80 facilitates these diverse processes at least in part by controlling genome-wide distribution of the histone variant H2A.Z. In the absence of INO80, H2A.Z nucleosomes are mislocalized, and H2A.Z levels at promoters show reduced responsiveness to transcriptional changes, suggesting that INO80 controls H2A.Z dynamics. Additionally, we demonstrate that INO80 has a histone-exchange activity in which the enzyme can replace nucleosomal H2A.Z/H2B with free H2A/H2B dimers. Genetic interactions between ino80 and htz1 support a model in which INO80 catalyzes the removal of unacetylated H2A.Z from chromatin as a mechanism to promote genome stability.

The Ino80 chromatin-remodeling enzyme regulates replisome function and stability.

Previous studies have demonstrated essential roles for ATP-dependent chromatin-remodeling and chromatin-modifying enzymes in gene transcription and DNA repair, but few studies have addressed how the replication machinery deals with chromatin. Here we show that the Ino80 remodeling enzyme is recruited to replication origins as cells enter S phase. Inducible degradation of Ino80 shows that it is required continuously for efficient progression of forks, especially when cells are confronted with low levels of replication stress. Furthermore, we show that stalling of replication forks in an ino80 mutant is a lethal event, and that much of the replication machinery dissociates from the stalled fork. Our data indicate that the chromatin-remodeling activity of Ino80 regulates efficient progression of replication forks and that Ino80 has a crucial role in stabilizing a stalled replisome to ensure proper restart of DNA replication.

Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptation in response to DNA damage.

Ino80 and Swr1 are ATP-dependent chromatin remodeling enzymes that have been implicated in DNA repair. Here we show that Ino80 is required for cell cycle checkpoint adaptation in response to a persistent DNA double-strand break (DSB). The failure of cells lacking Ino80 to escape checkpoint arrest correlates with an inability to maintain high levels of histone H2AX phosphorylation and an increased incorporation of the Htz1p histone variant into chromatin surrounding the DSB. Inactivation of Swr1 eliminates this DNA damage-induced Htz1p incorporation and restores H2AX phosphorylation and checkpoint adaptation. We propose that Ino80 and Swr1 function antagonistically at chromatin surrounding a DSB, and that they regulate the incorporation of different histone H2A variants that can either promote or block cell cycle checkpoint adaptation.

Spt3 and Mot1 cooperate in nucleosome remodeling independently of TBP recruitment.

We have investigated the requirements for nucleosome remodeling upon transcriptional induction of the GAL1 promoter. We found that remodeling was dependent on two SAGA complex components, Gcn5 and Spt3. The involvement of the latter was surprising as its function has been suggested to be directly involved in TATA-binding protein (TBP) recruitment. We demonstrated that this novel function was in fact independent of TBP recruitment and this was further validated using a Gal4-driven synthetic promoter. Most importantly, we showed that the involvement of Spt3 in chromatin remodeling was independent of transcription, as it was also observed for a nonpromoter nucleosome located next to an activator-binding site. In an effort to explore how the Spt3 function was elicited, we found that Mot1, an ATPase of the Snf2 family that genetically interacts with Spt3, was also required for nucleosome remodeling independently of TBP recruitment. Interestingly enough, Spt3 and Mot1 were recruited on the GAL1 promoter as well as on the nonpromoter site in an interdependent manner. These findings show that the two proteins cooperate in nucleosomal transactions.

The Snf1 kinase controls glucose repression in yeast by modulating interactions between the Mig1 repressor and the Cyc8-Tup1 co-repressor.

Among lower eukaryotes, glucose repression is a conserved, widely spread mechanism regulating carbon catabolism. The yeast Snf1 kinase, the Mig1 DNA-binding repressor and the Mig1-interacting co-repressor complex Cyc8(Ssn6)-Tup1 are central components of this pathway. Previous experiments suggested that cytoplasmic translocation of Mig1, upon its phosphorylation by Snf1 in the nucleus, is the key regulatory step for releasing glucose repression. In this report we re-evaluate this model. We establish the coordinated repressive action of Mig1 and Cyc8-Tup1 on GAL1 transcription, but we find that Cyc8-Tup1 is not tethered by Mig1 to the promoter DNA. We demonstrate that both negative regulators occupy GAL1 continuously under either repression or activation conditions, although the majority of the Mig1 is redistributed to the cytoplasm upon activation. We show that Snf1-dependent phosphorylation of Mig1 abolishes interaction with Cyc8-Tup1, and we propose that regulation of this interaction, not the Mig1 cytoplasmic localization, is the molecular switch that controls transcriptional repression/de-repression.

Post-TATA binding protein recruitment clearance of Gcn5-dependent histone acetylation within promoter nucleosomes.

Transcriptional activation of eukaryotic genes often requires the function of histone acetyltransferases (HATs), which is expected to result in the hyperacetylation of histones within promoter nucleosomes. In this study we show that, in Saccharomyces cerevisiae, the steady-state levels of Gcn5-dependent histone acetylation within a number of transcriptionally active promoters are inversely related to the rate of transcription. High acetylation levels were measured only when transcription was attenuated either by TATA element mutations or in a strain carrying a temperature-sensitive protein component of RNA polymerase II. In addition, we show that in one case the low levels of histone acetylation depend on the function of the Rpd3 histone deacetylase. These results point to the existence of an unexpected interplay of two opposing histone-modifying activities which operate on promoter nucleosomes following the initiation of RNA synthesis. Such interplay could ensure rapid turnover of chromatin acetylation states in continuously reprogrammed transcriptional systems.

Cti6, a PHD domain protein, bridges the Cyc8-Tup1 corepressor and the SAGA coactivator to overcome repression at GAL1.

The yeast Cyc8 and Tup1 proteins form a corepressor complex that, when tethered to DNA, turns off transcription. Release of the Cyc8-Tup1 corepressor from a promoter has been considered as a prerequisite for subsequent transcriptional activation. Contrasting this, we demonstrate that Cyc8-Tup1 is continuously associated with target promoters under both repressive and inducing conditions. At the GAL1 promoter, Cyc8-Tup1 facilitates recruitment of SAGA (Spt-Ada-Gcn5-acetyltranferase) via Cti6, a PHD domain protein that physically links the Cyc8-Tup1 and SAGA complexes. Lack of functional corepressor renders GAL1 transcription largely independent of specific SAGA subunits. Thus, corepressor's release is not the mechanism of derepression; instead, it is the coactivator complex that alleviates Cyc8-Tup1-mediated repression under induction conditions.