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

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

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

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

Interplay between Ino80 and Swr1 chromatin remodeling enzymes regulates cell cycle checkpoint adaptationin 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

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