Summary 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 mis-localized, 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 novel 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 novel mechanism to promote genome stability.

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.

Supplemental material is available at http://www.genesdev.org.

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.

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.

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.

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. studies showed that INO80 promotes replication fork progression on a chromatin template, while 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'.

DNA double-strand break (DSB) 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 DSB 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 (γH2AX) in the recruitment of chromatin regulators, but rather their recruitment coincides with reduced levels of γH2AX. Our work indicates that cell cycle position plays a key role in DNA repair pathway choice and that recruitment of chromatin regulators is tightly coupled to homologous recombination.