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We report the discovery, via a unique high-throughput screening strategy, of a novel bioactive anticancer compound: Thiol Alkylating Compound Inducing Massive Apoptosis (TACIMA)-218. We demonstrate that this molecule engenders apoptotic cell death in genetically diverse murine and human cancer cell lines, irrespective of their p53 status, while sparing normal cells. TACIMA-218 causes oxidative stress in the absence of protective antioxidants normally induced by Nuclear factor erythroid 2–related factor 2 activation. As such, TACIMA-218 represses RNA translation and triggers cell signaling cascade alterations in AKT, p38, and JNK pathways. In addition, TACIMA-218 manifests thiol-alkylating properties resulting in the disruption of redox homeostasis along with key metabolic pathways. When administered to immunocompetent animals as a monotherapy, TACIMA-218 has no apparent toxicity and induces complete regression of pre-established lymphoma and melanoma tumors. In sum, TACIMA-218 is a potent oxidative stress inducer capable of selective cancer cell targeting.

Supplementary data.

Supplementary Figure S3 shows expression of BRCA1, BRCA2, and Rad51 in ovarian cancer cell lines.

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines.Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561–73. ©2018 AACR.

Supplementary Table S1 documents clinical and genetic characteristics of ovarian cancer cell lines.

Cockayne syndrome group B (CSB) protein has been implicated in the repair of a variety of DNA lesions that induce replication stress. However, little is known about its role at stalled replication forks. Here, we report that CSB is recruited to stalled forks in a manner dependent upon its T1031 phosphorylation by CDK. While dispensable for MRE11 association with stalled forks in wild-type cells, CSB is required for further accumulation of MRE11 at stalled forks in BRCA1/2-deficient cells. CSB promotes MRE11-mediated fork degradation in BRCA1/2-deficient cells. CSB possesses an intrinsic ATP-dependent fork reversal activity in vitro, which is activated upon removal of its N-terminal region that is known to autoinhibit CSB’s ATPase domain. CSB functions similarly to fork reversal factors SMARCAL1, ZRANB3 and HLTF to regulate slowdown in fork progression upon exposure to replication stress, indicative of a role of CSB in fork reversal in vivo. Furthermore, CSB not only acts epistatically with MRE11 to facilitate fork restart but also promotes RAD52-mediated break-induced replication repair of double-strand breaks arising from cleavage of stalled forks by MUS81 in BRCA1/2-deficient cells. Loss of CSB exacerbates chemosensitivity in BRCA1/2-deficient cells, underscoring an important role of CSB in the treatment of cancer lacking functional BRCA1/2.

Eukaryotic cells have evolved highly orchestrated protein catabolic machineries responsible for the timely and selective disposal of proteins and organelles, thereby ensuring amino acid recycling. However, how protein degradation is coordinated with amino acid supply and protein synthesis has remained largely elusive. Here we show that the mammalian proteasome undergoes liquid-liquid phase separation in the nucleus upon amino acid deprivation. We termed these proteasome condensates SIPAN (Starvation-Induced Proteasome Assemblies in the Nucleus) and show that these are a common response of mammalian cells to amino acid deprivation. SIPAN undergo fusion events, rapidly exchange proteasome particles with the surrounding milieu and quickly dissolve following amino acid replenishment. We further show that: (i) SIPAN contain K48-conjugated ubiquitin, (ii) proteasome inhibition accelerates SIPAN formation, (iii) deubiquitinase inhibition prevents SIPAN resolution and (iv) RAD23B proteasome shuttling factor is required for SIPAN formation. Finally, SIPAN formation is associated with decreased cell survival and p53-mediated apoptosis, which might contribute to tissue fitness in diverse pathophysiological conditions., Upon starvation, cells coordinate protein disposal to recycle amino acids, although the role of the proteasome has been unclear. Here, the authors show that in the mammalian nucleus, proteasomes form condensates that dissolve following nutrient replenishment.

We report the discovery, via a unique high-throughput screening strategy, of a novel bioactive anticancer compound: Thiol Alkylating Compound Inducing Massive Apoptosis (TACIMA)-218. We demonstrate that this molecule engenders apoptotic cell death in genetically diverse murine and human cancer cell lines, irrespective of their p53 status, while sparing normal cells. TACIMA-218 causes oxidative stress in the absence of protective antioxidants normally induced by Nuclear factor erythroid 2–related factor 2 activation. As such, TACIMA-218 represses RNA translation and triggers cell signaling cascade alterations in AKT, p38, and JNK pathways. In addition, TACIMA-218 manifests thiol-alkylating properties resulting in the disruption of redox homeostasis along with key metabolic pathways. When administered to immunocompetent animals as a monotherapy, TACIMA-218 has no apparent toxicity and induces complete regression of pre-established lymphoma and melanoma tumors. In sum, TACIMA-218 is a potent oxidative stress inducer capable of selective cancer cell targeting.

Nucleotide excision repair (NER) eliminates highly-genotoxic solar UV-induced DNA photoproducts that otherwise stimulate malignant melanoma development. Here, a genome-wide loss-of-function screen, coupling CRISPR/Cas9 technology with a flow cytometry-based DNA repair assay, was used to identify novel genes required for efficient NER in primary human fibroblasts. Interestingly, the screen revealed multiple genes encoding proteins, with no previously known involvement in UV damage repair, that significantly modulate NER uniquely during S phase of the cell cycle. Among these, we further characterized Dyrk1A, a dual specificity kinase that phosphorylates the proto-oncoprotein cyclin D1 on threonine 286 (T286), thereby stimulating its timely cytoplasmic relocalization and proteasomal degradation required for proper regulation of the G1-S phase transition and control of cellular proliferation. We demonstrate that in UV-irradiated HeLa cells, depletion of Dyrk1A leading to overexpression of cyclin D1 causes inhibition of NER uniquely during S phase and reduced cell survival. Consistently, expression/nuclear accumulation of nonphosphorylatable cyclin D1 (T286A) in melanoma cells strongly interferes with S phase NER and enhances cytotoxicity post-UV. Moreover, the negative impact of cyclin D1 (T286A) overexpression on repair is independent of cyclin-dependent kinase activity but requires cyclin D1-dependent co-stabilization of p21. Our data indicate that inhibition of NER during S phase might represent a previously unappreciated non-canonical mechanism by which oncogenic cyclin D1 fosters melanomagenesis.

InSaccharomyces cerevisiae, newly synthesized histone H3 are acetylated on lysine 56 (H3 K56ac) by the Rtt109 acetyltransferase prior to their deposition on nascent DNA behind replication forks. Two deacetylases of the sirtuin family, Hst3 and Hst4, remove H3 K56ac from chromatin following S phase.hst3Δhst4Δ cells present constitutive H3 K56ac, which sensitizes cells to replicative stress via mechanisms that remain unclear. We performed a screen to identify genes that influence cell fitness upon nicotinamide (NAM)-induced inhibition of sirtuins. The screen revealed thatDBF4heterozygosity causes NAM sensitivity.DBF4andCDC7encode subunits of the Dbf4-dependent kinase, which activates origins of DNA replication. We show that i) cells harboring thedbf4-1orcdc7-4hypomorphic alleles are sensitive to NAM, ii) Rif1, an inhibitor of Cdc7-dependent activation of origins, causes DNA damage and replication defects in NAM-treated cells andhst3Δhst4Δ mutants, and iii)cdc7-4 hst3Δhst4Δ cells display synthetic temperature sensitivity associated with delayed initiation of DNA replication. Such replication defects are not due to activation of the intra-S phase checkpoint but require Rtt109-dependent H3 K56ac. Overall, these results suggest that persistent H3 K56ac sensitizes cells to replicative stress in part by negatively influencing replication origin activity.

Helix-destabilizing DNA lesions induced by environmental mutagens such as UV light cause genomic instability by strongly blocking the progression of DNA replication forks (RF). At blocked RF, single-stranded DNA (ssDNA) accumulates and is rapidly bound by Replication Protein A (RPA) complexes. Such stretches of RPA-ssDNA constitute platforms for recruitment/activation of critical factors that promote DNA synthesis restart. However, during periods of severe replicative stress, RPA availability may become limiting due to inordinate sequestration of this multifunctional complex on ssDNA, thereby negatively impacting multiple vital RPA-dependent processes. Here, we performed a genome-wide screen to identify factors which restrict the accumulation of RPA-ssDNA during UV-induced replicative stress. While this approach revealed some expected “hits” acting in pathways such as nucleotide excision repair, translesion DNA synthesis, and the intra-S phase checkpoint, it also identifed SCAI, whose role in the replicative stress response was previously unappreciated. Upon UV exposure, SCAI knock-down caused elevated accumulation of RPA-ssDNA during S phase, accompanied by reduced cell survival and compromised RF progression. These effects were independent of the previously reported role of SCAI in 53BP1-dependent DNA double-strand break repair. We also found that SCAI colocalized with stalled RF, and that its depletion promoted nascent DNA degradation. Finally, we (i) provide evidence that EXO1 is the major nuclease underlying ssDNA formation and consequent DNA replication defects in SCAI knockout cells and, consistent with this, (ii) demonstrate that SCAI inhibits EXO1 activity on a ssDNA gap in vitro. Taken together, our data establish SCAI as a novel regulator of the replicative stress response in human cells.

IntroductionReplication of the nuclear genome is an essential step for cell division. Pathogenic variants in genes coding for highly conserved components of the DNA replication machinery cause Meier-Gorlin syndrome (MGORS).ObjectiveIdentification of novel genes associated with MGORS.MethodsExome sequencing was performed to investigate the genotype of an individual presenting with prenatal and postnatal growth restriction, a craniofacial gestalt of MGORS and coronal craniosynostosis. The analysis of the candidate variants employed bioinformatic tools, in silico structural protein analysis and modelling in budding yeast.ResultsA novel homozygous missense variant NM_016095.2:c.341G>T, p.(Arg114Leu), in GINS2 was identified. Both non-consanguineous healthy parents carried this variant. Bioinformatic analysis supports its classification as pathogenic. Functional analyses using yeast showed that this variant increases sensitivity to nicotinamide, a compound that interferes with DNA replication processes. The phylogenetically highly conserved residue p.Arg114 localises at the docking site of CDC45 and MCM5 at GINS2. Moreover, the missense change possibly disrupts the effective interaction between the GINS complex and CDC45, which is necessary for the CMG helicase complex (Cdc45/MCM2–7/GINS) to accurately operate. Interestingly, our patient’s phenotype is strikingly similar to the phenotype of patients with CDC45-related MGORS, particularly those with craniosynostosis, mild short stature and patellar hypoplasia.ConclusionGINS2 is a new disease-associated gene, expanding the genetic aetiology of MGORS.

DNA fiber fluorography is widely employed to study the kinetics of DNA replication, but the usefulness of this approach has been limited by the lack of freely-available automated analysis tools. Quantification of DNA fibers usually relies on manual examination of immunofluorescence microscopy images, which is laborious and prone to inter- and intra-operator variability. To address this, we developed an unbiased, fully automated algorithm that quantifies length and color of DNA fibers from fluorescence microscopy images. Our fiber quantification method, termed FiberQ, is an open-source image processing tool based on edge detection and a novel segment splicing approach. Here, we describe the algorithm in detail, validate our results experimentally, and benchmark the analysis against manual assessments. Our implementation is offered free of charge to the scientific community under the General Public License.

Replication of the nuclear genome is an essential step for cell division. Pathogenic variants in genes coding for highly conserved components of the DNA replication machinery cause Meier-Gorlin syndrome (MGORS).Identification of novel genes associated with MGORS.Exome sequencing was performed to investigate the genotype of an individual presenting with prenatal and postnatal growth restriction, a craniofacial gestalt of MGORS and coronal craniosynostosis. The analysis of the candidate variants employed bioinformatic tools,A novel homozygous missense variant NM_016095.2:c.341GT, p.(Arg114Leu), in

USP16 (also known as UBP-M) has emerged as a histone H2AK119 deubiquitylase (DUB) implicated in the regulation of chromatin-associated processes and cell cycle progression. Despite this, available evidence suggests that this DUB is also present in the cytoplasm. How the nucleo-cytoplasmic transport of USP16, and hence its function, is regulated has remained elusive. Here, we show that USP16 is predominantly cytoplasmic in all cell cycle phases. We identified the nuclear export signal (NES) responsible for maintaining USP16 in the cytoplasm. We found that USP16 is only transiently retained in the nucleus following mitosis and then rapidly exported from this compartment. We also defined a non-canonical nuclear localization signal (NLS) sequence that plays a minimal role in directing USP16 into the nucleus. We further established that this DUB does not accumulate in the nucleus following DNA damage. Instead, only enforced nuclear localization of USP16 abolishes DNA double-strand break (DSB) repair, possibly due to unrestrained DUB activity. Thus, in contrast to the prevailing view, our data indicate that USP16 is actively excluded from the nucleus and that this DUB might indirectly regulate DSB repair.This article has an associated First Person interview with the first author of the paper.

A broad spectrum of spontaneous and genotoxin-induced DNA lesions impede replication fork progression. The DNA damage response that acts to promote completion of DNA replication is associated with dynamic changes in chromatin structure that include two distinct processes which operate genome-wide during S-phase. The first, often referred to as histone recycling or parental histone segregation, is characterized by the transfer of parental histones located ahead of replication forks onto nascent DNA. The second, known as de novo chromatin assembly, consists of the deposition of new histone molecules onto nascent DNA. Because these two processes occur at all replication forks, their potential to influence a multitude of DNA repair and DNA damage tolerance mechanisms is considerable. The purpose of this review is to provide a description of parental histone segregation and de novo chromatin assembly, and to illustrate how these processes influence cellular responses to DNA replication roadblocks.

Resveratrol is a natural product associated with wide-ranging effects in animal and cellular models, including lifespan extension. To identify the genetic target of resveratrol in human cells, we conducted genome-wide CRISPR-Cas9 screens to pinpoint genes that confer sensitivity or resistance to resveratrol. An extensive network of DNA damage response and replicative stress genes exhibited genetic interactions with resveratrol and its analog pterostilbene. These genetic profiles showed similarity to the response to hydroxyurea, an inhibitor of ribonucleotide reductase that causes replicative stress. Resveratrol, pterostilbene, and hydroxyurea caused similar depletion of nucleotide pools, inhibition of replication fork progression, and induction of replicative stress. The ability of resveratrol to inhibit cell proliferation and S phase transit was independent of the histone deacetylase sirtuin 1, which has been implicated in lifespan extension by resveratrol. These results establish that a primary impact of resveratrol on human cell proliferation is the induction of low-level replicative stress.

The Saccharomyces cerevisiae genome encodes five sirtuins (Sir2 and Hst1–4), which constitute a conserved family of NAD-dependent histone deacetylases. Cells lacking any individual sirtuin display mild growth and gene silencing defects. However, hst3Δ hst4Δ double mutants are exquisitely sensitive to genotoxins, and hst3Δ hst4Δ sir2Δ mutants are inviable. Our published data also indicate that pharmacological inhibition of sirtuins prevents growth of several fungal pathogens, although the biological basis is unclear. Here, we present genome-wide fitness assays conducted with nicotinamide (NAM), a pan-sirtuin inhibitor. Our data indicate that NAM treatment causes yeast to solicit specific DNA damage response pathways for survival, and that NAM-induced growth defects are mainly attributable to inhibition of Hst3 and Hst4 and consequent elevation of histone H3 lysine 56 acetylation (H3K56ac). Our results further reveal that in the presence of constitutive H3K56ac, the Slx4 scaffolding protein and PP4 phosphatase complex play essential roles in preventing hyperactivation of the DNA damage-response kinase Rad53 in response to spontaneous DNA damage caused by reactive oxygen species. Overall, our data support the concept that chromosome-wide histone deacetylation by sirtuins is critical to mitigate growth defects caused by endogenous genotoxins.

Intrinsic and acquired resistance to cisplatin remains a primary hurdle to treatment of high-grade serous ovarian cancer (HGSOC). Cisplatin selectively kills tumor cells by inducing DNA crosslinks that block replicative DNA polymerases. Single-stranded DNA (ssDNA) generated at resulting stalled replication forks (RF) is bound and protected by heterotrimeric replication protein A (RPA), which then serves as a platform for recruitment and activation of replication stress response factors. Cells deficient in this response are characterized by extensive ssDNA formation and excessive RPA recruitment that exhausts the available pool of RPA, which (i) inhibits RPA-dependent processes such as nucleotide excision repair (NER) and (ii) causes catastrophic failure of blocked RF. Here, we investigated the influence of RPA availability on chemosensitivity using a panel of human HGSOC cell lines. Our data revealed a striking correlation among these cell lines between cisplatin sensitivity and the inability to efficiently repair DNA via NER, specifically during S phase. Such defects in NER were attributable to RPA exhaustion arising from aberrant activation of DNA replication origins during replication stress. Reduced RPA availability promoted Mre11-dependent degradation of nascent DNA at stalled RF in cell lines exhibiting elevated sensitivity to cisplatin. Strikingly, defective S-phase NER, RF instability, and cisplatin sensitivity could all be rescued by ectopic overexpression of RPA. Taken together, our findings indicate that RPA exhaustion represents a major determinant of cisplatin sensitivity in HGSOC cell lines. Significance: The influence of replication protein A exhaustion on cisplatin sensitivity harbors important implications toward improving therapy of various cancers that initially respond to platinum-based agents but later relapse due to intrinsic or acquired drug resistance. Cancer Res; 78(19); 5561–73. ©2018 AACR.

The deubiquitinase (DUB) and tumor suppressor BAP1 catalyzes ubiquitin removal from histone H2A Lys-119 and coordinates cell proliferation, but how BAP1 partners modulate its function remains poorly understood. Here, we report that BAP1 forms two mutually exclusive complexes with the transcriptional regulators ASXL1 and ASXL2, which are necessary for maintaining proper protein levels of this DUB. Conversely, BAP1 is essential for maintaining ASXL2, but not ASXL1, protein stability. Notably, cancer-associated loss of BAP1 expression results in ASXL2 destabilization and hence loss of its function. ASXL1 and ASXL2 use their ASXM domains to interact with the C-terminal domain (CTD) of BAP1, and these interactions are required for ubiquitin binding and H2A deubiquitination. The deubiquitination-promoting effect of ASXM requires intramolecular interactions between catalytic and non-catalytic domains of BAP1, which generate a composite ubiquitin-binding interface (CUBI). Notably, the CUBI engages multiple interactions with ubiquitin involving (i) the ubiquitin carboxyl hydrolase catalytic domain of BAP1, which interacts with the hydrophobic patch of ubiquitin, and (ii) the CTD domain, which interacts with a charged patch of ubiquitin. Significantly, we identified cancer-associated mutations of BAP1 that disrupt the CUBI and notably an in-frame deletion in the CTD that inhibits its interaction with ASXL1/2 and DUB activity and deregulates cell proliferation. Moreover, we demonstrated that BAP1 interaction with ASXL2 regulates cell senescence and that ASXL2 cancer-associated mutations disrupt BAP1 DUB activity. Thus, inactivation of the BAP1/ASXL2 axis might contribute to cancer development.

O-GlcNAcylation is a posttranslational modification catalyzed by the O-Linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) and reversed by O-GlcNAcase (OGA). Numerous transcriptional regulators, including chromatin modifying enzymes, transcription factors, and co-factors, are targeted by O-GlcNAcylation, indicating that this modification is central for chromatin-associated processes. Recently, OGT-mediated O-GlcNAcylation was reported to be a novel histone modification, suggesting a potential role in directly coordinating chromatin structure and function. In contrast, using multiple biochemical approaches, we report here that histone O-GlcNAcylation is undetectable in mammalian cells. Conversely, O-GlcNAcylation of the transcription regulators Host Cell Factor-1 (HCF-1) and Ten-Eleven Translocation protein 2 (TET2) could be readily observed. Our study raises questions on the occurrence and abundance of O-GlcNAcylation as a histone modification in mammalian cells and reveals technical complications regarding the detection of genuine protein O-GlcNAcylation. Therefore, the identification of the specific contexts in which histone O-GlcNAcylation might occur is still to be established.

International audience; Retromer is a multimeric protein complex that mediates endosome-to-trans-Golgi network (TGN) and endosome-to-plasma membrane trafficking of integral membrane proteins. Dysfunction of this complex has been linked to Alzheimer's disease and Parkinson's disease. The recruitment of retromer to endosomes is regulated by Rab7 (also known as RAB7A) to coordinate endosome-to-TGN trafficking of cargo receptor complexes. Rab7 is also required for the degradation of internalized integral membrane proteins, such as the epidermal growth factor receptor (EGFR). We found that Rab7 is palmitoylated and that this modification is not required for membrane anchoring. Palmitoylated Rab7 colocalizes efficiently with and has a higher propensity to interact with retromer than nonpalmitoylatable Rab7. Rescue of Rab7 knockout cells by expressing wild-type Rab7 restores efficient endosome-to-TGN trafficking, while rescue with nonpalmitoylatable Rab7 does not. Interestingly, Rab7 palmitoylation does not appear to be required for the degradation of EGFR or for its interaction with its effector, Rab-interacting lysosomal protein (RILP). Overall, our results indicate that Rab7 palmitoylation is required for the spatiotemporal recruitment of retromer and efficient endosome-to-TGN trafficking of the lysosomal sorting receptors.

Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.

Antibodies that recognize specific histone modifications are invaluable tools to study chromatin structure and function. There are numerous commercially available antibodies that recognize a remarkable diversity of histone modifications. Unfortunately, many of them fail to work in certain applications or lack the high degree of specificity required of these reagents. The production of affinity-purified polyclonal antibodies against histone modifications demands a little effort but, in return, provides extremely valuable tools that overcome many of the concerns and limitations of commercial antibodies. We present a series of protocols and guidelines for the production and use of large amounts of polyclonal antibodies that recognize modifications of canonical histones. Our protocols can be applied to obtain antibodies that occur in histone variants and proteins other than histones. In addition, some of our protocols are compatible with the production of monoclonal or recombinant antibodies.

The mechanism of action of valproate (VPA), a widely prescribed short chain fatty acid with anticonvulsant and anticancer properties, remains poorly understood. Here, the yeast Saccharomyces cerevisiae was used as model to investigate the biological consequences of VPA exposure. We found that low pH strongly potentiates VPA-induced growth inhibition. Transcriptional profiling revealed that under these conditions, VPA modulates the expression of genes involved in diverse cellular processes including protein folding, cell wall organisation, sexual reproduction, and cell cycle progression. We further investigated the impact of VPA on selected processes and found that this drug: i) activates markers of the unfolded protein stress response such as Hac1 mRNA splicing; ii) modulates the cell wall integrity pathway by inhibiting the activation of the Slt2 MAP kinase, and synergizes with cell wall stressors such as micafungin and calcofluor white in preventing yeast growth; iii) prevents activation of the Kss1 and Fus3 MAP kinases of the mating pheromone pathway, which in turn abolishes cellular responses to alpha factor; and iv) blocks cell cycle progression and DNA replication. Overall, our data identify heretofore unknown biological responses to VPA in budding yeast, and highlight the broad spectrum of cellular pathways influenced by this chemical in eukaryotes.

SummaryYeast Rtt109 promotes nucleosome assembly and genome stability by acetylating K9, K27, and K56 of histone H3 through interaction with either of two distinct histone chaperones, Vps75 or Asf1. We report the crystal structure of an Rtt109-AcCoA/Vps75 complex revealing an elongated Vps75 homodimer bound to two globular Rtt109 molecules to form a symmetrical holoenzyme with a ∼12 Å diameter central hole. Vps75 and Rtt109 residues that mediate complex formation in the crystals are also important for Rtt109-Vps75 interaction and H3K9/K27 acetylation both in vitro and in yeast cells. The same Rtt109 residues do not participate in Asf1-mediated Rtt109 acetylation in vitro or H3K56 acetylation in yeast cells, demonstrating that Asf1 and Vps75 dictate Rtt109 substrate specificity through distinct mechanisms. These studies also suggest that Vps75 binding stimulates Rtt109 catalytic activity by appropriately presenting the H3-H4 substrate within the central cavity of the holoenzyme to promote H3K9/K27 acetylation of new histones before deposition.

(2009). Histone acetylation floods the human cell cycle and DNA damage response. Cell Cycle: Vol. 8, No. 12, pp. 1818-1822.

Nucleotide excision repair (NER) is a highly conserved pathway that removes helix-distorting DNA lesions induced by a plethora of mutagens, including UV light. Our laboratory previously demonstrated that human cells deficient in either ATM and Rad3-related (ATR) kinase or translesion DNA polymerase η (i.e. key proteins that promote the completion of DNA replication in response to UV-induced replicative stress) are characterized by profound inhibition of NER exclusively during S phase. Toward elucidating the mechanistic basis of this phenomenon, we developed a novel assay to quantify NER kinetics as a function of cell cycle in the model organism Saccharomyces cerevisiae. Using this assay, we demonstrate that in yeast, deficiency of the ATR homologue Mec1 or of any among several other proteins involved in the cellular response to replicative stress significantly abrogates NER uniquely during S phase. Moreover, initiation of DNA replication is required for manifestation of this defect, and S phase NER proficiency is correlated with the capacity of individual mutants to respond to replicative stress. Importantly, we demonstrate that partial depletion of Rfa1 recapitulates defective S phase-specific NER in wild type yeast; moreover, ectopic RPA1-3 overexpression rescues such deficiency in either ATR- or polymerase η-deficient human cells. Our results strongly suggest that reduction of NER capacity during periods of enhanced replicative stress, ostensibly caused by inordinate sequestration of RPA at stalled DNA replication forks, represents a conserved feature of the multifaceted eukaryotic DNA damage response.

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56Ac) is present in newly synthesized histones deposited throughout the genome during DNA replication. The sirtuins Hst3 and Hst4 deacetylate H3K56 after S phase, and virtually all histone H3 molecules are K56 acetylated throughout the cell cycle in hst3∆ hst4∆ mutants. Failure to deacetylate H3K56 causes thermosensitivity, spontaneous DNA damage, and sensitivity to replicative stress via molecular mechanisms that remain unclear. Here we demonstrate that unlike wild-type cells, hst3∆ hst4∆ cells are unable to complete genome duplication and accumulate persistent foci containing the homologous recombination protein Rad52 after exposure to genotoxic drugs during S phase. In response to replicative stress, cells lacking Hst3 and Hst4 also displayed intense foci containing the Rfa1 subunit of the single-stranded DNA binding protein complex RPA, as well as persistent activation of DNA damage–induced kinases. To investigate the basis of these phenotypes, we identified histone point mutations that modulate the temperature and genotoxic drug sensitivity of hst3∆ hst4∆ cells. We found that reducing the levels of histone H4 lysine 16 acetylation or H3 lysine 79 methylation partially suppresses these sensitivities and reduces spontaneous and genotoxin-induced activation of the DNA damage-response kinase Rad53 in hst3∆ hst4∆ cells. Our data further suggest that elevated DNA damage–induced signaling significantly contributes to the phenotypes of hst3∆ hst4∆ cells. Overall, these results outline a novel interplay between H3K56Ac, H3K79 methylation, and H4K16 acetylation in the cellular response to DNA damage.

Ultraviolet (UV) light causes highly genotoxic DNA lesions that are removed by nucleotide excision repair (NER), and NER is critical for preventing UV-associated skin cancers. UV-induced DNA lesions block the progression of DNA polymerases, leading to replicative stress and genomic instability. Upon UV, the ataxia-telangectasia and rad3 related (ATR) kinase is rapidly activated, leading to cell-cycle arrest, inhibition of replication origin firing, and stabilization of blocked replication forks. Intriguingly, previous data indicated that reduced ATR activity causes profound NER defects specifically in S phase cells. Moreover many model cancer cell lines, including a majority of melanoma lines, present striking S phase-specific NER defects. We used the yeast S. cerevisiae as model to investigate NER activity during S phase. We optimized a novel flow cytometry-based assay to quantify NER as a function of cell cycle in this organism. Using this assay we demonstrate that, as for human cells, deletion of the yeast ATR homolog Mec1 causes NER defects uniquely in S, and that initiation of DNA replication is prerequisite for manifestation of this defect. S phase-specific NER was perturbed by mutations in various genes encoding key DNA damage response factors that regulate, e.g. cell cycle checkpoints, chromatin remodeling, and homologous recombination. Our data also reveal a strong correlation between S phase-specific NER defects and elevated RPA focus formation in mutants that are sensitive to replicative stress. Finally we demonstrate that modulation of RPA levels strongly influences NER activity specifically during S in human cancer cell lines. Overall, our data indicate that mutations in a multitude of DNA damage response pathways cause inordinate sequestration of RPA at stalled replication forks, thereby reducing the availability of this factor to perform its essential role in NER. This has important implications for our understanding of UV-induced skin cancer development in humans. Citation Format: Elliot Drobetsky, François Bélanger, Jean-Philippe Angers, Émile Fortier, Santiago Costantino, Hugo Wurtele. Exploring the interplay between nucleotide excision repair and DNA replicative stress [abstract]. In: Proceedings of the AACR Special Conference on DNA Repair: Tumor Development and Therapeutic Response; 2016 Nov 2-5; Montreal, QC, Canada. Philadelphia (PA): AACR; Mol Cancer Res 2017;15(4_Suppl):Abstract nr A32.

In Saccharomyces cerevisiae, histone H3 lysine 56 acetylation (H3K56ac) occurs in newly synthesized histones that are deposited throughout the genome during DNA replication. Defects in H3K56ac sensitize cells to genotoxic agents, suggesting that this modification plays an important role in the DNA damage response. However, the links between histone acetylation, the nascent chromatin structure, and the DNA damage response are poorly understood. Here we report that cells devoid of H3K56ac are sensitive to DNA damage sustained during transient exposure to methyl methanesulfonate (MMS) or camptothecin but are only mildly affected by hydroxyurea. We demonstrate that, after exposure to MMS, H3K56ac-deficient cells cannot complete DNA replication and eventually segregate chromosomes with intranuclear foci containing the recombination protein Rad52. In addition, we provide evidence that these phenotypes are not due to defects in base excision repair, defects in DNA damage tolerance, or a lack of Rad51 loading at sites of DNA damage. Our results argue that the acute sensitivity of H3K56ac-deficient cells to MMS and camptothecin stems from a failure to complete the repair of specific types of DNA lesions by recombination and/or from defects in the completion of DNA replication.

Candida albicans is a major fungal pathogen that causes serious systemic and mucosal infections in immunocompromised individuals. In yeast, histone H3 Lys56 acetylation (H3K56ac) is an abundant modification regulated by enzymes that have fungal-specific properties, making them appealing targets for antifungal therapy. Here we demonstrate that H3K56ac in C. albicans is regulated by the RTT109 and HST3 genes, which respectively encode the H3K56 acetyltransferase (Rtt109p) and deacetylase (Hst3p). We show that reduced levels of H3K56ac sensitize C. albicans to genotoxic and antifungal agents. Inhibition of Hst3p activity by conditional gene repression or nicotinamide treatment results in a loss of cell viability associated with abnormal filamentous growth, histone degradation and gross aberrations in DNA staining. We show that genetic or pharmacological alterations in H3K56ac levels reduce virulence in a mouse model of C. albicans infection. Our results demonstrate that modulation of H3K56ac is a unique strategy for treatment of C. albicans and, possibly, other fungal infections. -¬ 2010 Nature America, Inc. All rights reserved

A two-pronged approach using specialized peptide detection and clustering tools was developed to profile changes in histone post-translational modifications (PTMs). The extent and nature of modification was inferred by comparing the mass profiles of intact core histones from nano LC-MS experiments. Histones displaying changes in their intact mass profiles were fractionated, derivatized with propionic anhydride, and digested with trypsin prior to nano LC-MS analyses. Our methodology was validated by comparing the abundance of histone PTMs in wild type and mutant strains of Saccharomyces cerevisiae lacking the histone acetyltransferase Rtt109 and a nucleosome assembly factor known as Asf1. Both Rtt109 and Asf1 were previously found to be essential for acetylation of histone H3 lysine 56 (H3K56ac), a modification that plays an important role in the response to genotoxic agents that interfere with DNA replication. The generation of ion abundance distribution plots enabled a rapid and comprehensive profiling of histone peptides. Our analytical methodology and data mining approach showed that most common histone PTMs were unaffected in mutant yeast cells lacking Rtt109 and Asf1. However, a subpopulation representing 17% of all H3 histones in wild type cells showed an acetylated K56 residue that was significantly reduced in both mutant strains. Our generic strategy for histone PTM profiling can be applied to assess global changes in histone PTMs across sample sets and to establish structure-function relationships.

Rtt109, also known as KAT11, is a recently characterized fungal-specific histone acetyltransferase (HAT) that modifies histone H3 lysine 56 (H3K56) to promote genome stability. Rtt109 does not show sequence conservation with other known HATs and depends on association with either of two histone chaperones, Asf1 or Vps75, for HAT activity. Here we report the X-ray crystal structure of an Rtt109-acetyl coenzyme A complex and carry out structure-based mutagenesis, combined with in vitro biochemical studies of the Rtt109-Vps75 complex and studies of Rtt109 function in vivo. The Rtt109 structure reveals noteworthy homology to the metazoan p300/CBP HAT domain but exhibits functional divergence, including atypical catalytic properties and mode of cofactor regulation. The structure reveals a buried autoacetylated lysine residue that we show is also acetylated in the Rtt109 protein purified from yeast cells. Implications for understanding histone substrate and chaperone binding by Rtt109 are discussed.

Spatial proximity between genomic loci can play important roles in their function and regulation. We have developed an open-ended method based on Chromosome Conformation Capture technology allowing us to perform genome-wide scanning of the loci that form the spatial environment of a given locus at a given time. As a proof of principle we present the use of this methodology to investigate the dynamics of the spatial environment of the HoxB1 gene before and after the induction of its expression in mouse embryonic stem cells. Our results indicate that the HoxB1 locus' immediate spatial environment can be divided roughly into three parts: a first part is represented by a domain of immediate proximity on each side of the HoxB1 locus covering approximately 110 kb, a second part extends to a domain of 800 kb and a third part consists of distal intra-chromosomal and inter-chromosomal interactions. Consistent with FISH studies showing the decondensation and repositioning of HoxB1 outside of its chromosomal territory during its expression, the proportion of inter-chromosomal interactions between HoxB1 and the rest of the genome increases after its induction, while interactions with distal intra-chromosomal loci become less frequent. These results indicate that this technique can be used to determine the dynamics of loci interactions on a genome-wide scale.

The packaging of DNA into chromatin creates a number of significant barriers to the detection of DNA lesions and their timely and accurate repair. Eukaryotic cells have evolved a number of enzymes that modulate chromatin structure and facilitate DNA repair. Recent research illustrates how nucleosome remodelling enzymes cooperate with both DNA-damage-inducible and constitutive histone modifications to promote many facets of the cellular response to DNA damage.