Linking DNA repair and cell cycle progression through serine ADP-ribosylation of histones.

Although serine ADP-ribosylation (Ser-ADPr) by Poly(ADP-ribose)-polymerases is a cornerstone of the DNA damage response, how this regulates DNA repair and genome stability is unknown. Here, we exploit the ability to manipulate histone genes in Dictyostelium to identify that ADPr of the histone variant H3b at S10 and S28 maintains genome stability by integrating double strand break (DSB) repair with mitotic entry. Given the critical requirement for mitotic H3S10/28 phosphorylation, we develop separation of function mutations that maintain S10 phosphorylation whilst disrupting ADPr. Mechanistically, this reveals a requirement for H3bS10/28 ADPr in non-homologous end-joining by recruiting Ku to DSBs. Moreover, this also identifies H3bS10/S28 ADPr is critical to prevent premature mitotic entry with unresolved DNA damage, thus maintaining genome stability. Together, these data demonstrate how serine ADPr of histones coordinates DNA repair with cell cycle progression to maintain genome stability.

Moving the Research Forward: The Best of British Biology Using the Tractable Model System Dictyostelium discoideum.

The social amoeba Dictyostelium discoideum provides an excellent model for research across a broad range of disciplines within biology. The organism diverged from the plant, yeast, fungi and animal kingdoms around 1 billion years ago but retains common aspects found in these kingdoms. Dictyostelium has a low level of genetic complexity and provides a range of molecular, cellular, biochemical and developmental biology experimental techniques, enabling multidisciplinary studies to be carried out in a wide range of areas, leading to research breakthroughs. Numerous laboratories within the United Kingdom employ Dictyostelium as their core research model. This review introduces Dictyostelium and then highlights research from several leading British research laboratories, covering their distinct areas of research, the benefits of using the model, and the breakthroughs that have arisen due to the use of Dictyostelium as a tractable model system.

Possible Involvement of the Nutrient and Energy Sensors mTORC1 and AMPK in Cell Fate Diversification in a Non-Metazoan Organism.

mTORC1 and AMPK are mutually antagonistic sensors of nutrient and energy status that have been implicated in many human diseases including cancer, Alzheimer's disease, obesity and type 2 diabetes. Starved cells of the social amoeba Dictyostelium discoideum aggregate and eventually form fruiting bodies consisting of stalk cells and spores. We focus on how this bifurcation of cell fate is achieved. During growth mTORC1 is highly active and AMPK relatively inactive. Upon starvation, AMPK is activated and mTORC1 inhibited; cell division is arrested and autophagy induced. After aggregation, a minority of the cells (prestalk cells) continue to express much the same set of developmental genes as during aggregation, but the majority (prespore cells) switch to the prespore program. We describe evidence suggesting that overexpressing AMPK increases the proportion of prestalk cells, as does inhibiting mTORC1. Furthermore, stimulating the acidification of intracellular acidic compartments likewise increases the proportion of prestalk cells, while inhibiting acidification favors the spore pathway. We conclude that the choice between the prestalk and the prespore pathways of cell differentiation may depend on the relative strength of the activities of AMPK and mTORC1, and that these may be controlled by the acidity of intracellular acidic compartments/lysosomes (pHv), cells with low pHv compartments having high AMPK activity/low mTORC1 activity, and those with high pHv compartments having high mTORC1/low AMPK activity. Increased insight into the regulation and downstream consequences of this switch should increase our understanding of its potential role in human diseases, and indicate possible therapeutic interventions.

Dictyostelium discoideum as a Model to Assess Genome Stability Through DNA Repair.

Preserving genome integrity through repair of DNA damage is critical for human health and defects in these pathways lead to a variety of pathologies, most notably cancer. The social amoeba Dictyostelium discoideum is remarkably resistant to DNA damaging agents and genome analysis reveals it contains orthologs of several DNA repair pathway components otherwise limited to vertebrates. These include the Fanconi Anemia DNA inter-strand crosslink and DNA strand break repair pathways. Loss of function of these not only results in malignancy, but also neurodegeneration, immune-deficiencies and congenital abnormalities. Additionally, D. discoideum displays remarkable conservations of DNA repair factors that are targets in cancer and other therapies, including poly(ADP-ribose) polymerases that are targeted to treat breast and ovarian cancers. This, taken together with the genetic tractability of D. discoideum, make it an attractive model to assess the mechanistic basis of DNA repair to provide novel insights into how these pathways can be targeted to treat a variety of pathologies. Here we describe progress in understanding the mechanisms of DNA repair in D. discoideum, and how these impact on genome stability with implications for understanding development of malignancy.

Methylation-directed acetylation of histone H3 regulates developmental sensitivity to histone deacetylase inhibition.

Hydroxamate-based lysine deacetylase inhibitors (KDACis) are approved for clinical use against certain cancers. However, intrinsic and acquired resistance presents a major problem. Treatment of cells with hydroxamates such as trichostatin A (TSA) leads to rapid preferential acetylation of histone H3 already trimethylated on lysine 4 (H3K4me3), although the importance of this H3K4me3-directed acetylation in the biological consequences of KDACi treatment is not known. We address this utilizing Dictyostelium discoideum strains lacking H3K4me3 due to disruption of the gene encoding the Set1 methyltransferase or mutations in endogenous H3 genes. Loss of H3K4me3 confers resistance to TSA-induced developmental inhibition and delays accumulation of H3K9Ac and H3K14Ac. H3K4me3-directed H3Ac is mediated by Sgf29, a subunit of the SAGA acetyltransferase complex that interacts with H3K4me3 via a tandem tudor domain (TTD). We identify an Sgf29 orthologue in Dictyostelium with a TTD that specifically recognizes the H3K4me3 modification. Disruption of the gene encoding Sgf29 delays accumulation of H3K9Ac and abrogates H3K4me3-directed H3Ac. Either loss or overexpression of Sgf29 confers developmental resistance to TSA. Our results demonstrate that rapid acetylation of H3K4me3 histones regulates developmental sensitivity to TSA. Levels of H3K4me3 or Sgf29 will provide useful biomarkers for sensitivity to this class of chemotherapeutic drug.

Microbe Profile: Dictyostelium discoideum: model system for development, chemotaxis and biomedical research.

The social amoeba Dictyostelium discoideum is a versatile organism that is unusual in alternating between single-celled and multi-celled forms. It possesses highly-developed systems for cell motility and chemotaxis, phagocytosis, and developmental pattern formation. As a soil amoeba growing on microorganisms, it is exposed to many potential pathogens; it thus provides fruitful ways of investigating host-pathogen interactions and is emerging as an influential model for biomedical research.

Human Platelet Protein Ubiquitylation and Changes following GPVI Activation.

Platelet activators stimulate post-translational modification of signalling proteins to change their activity or their molecular interactions leading to signal propagation. One covalent modification is attachment of the small protein ubiquitin to lysine residues in target proteins. Modification by ubiquitin can either target proteins for degradation by the proteasome or act as a scaffold for other proteins. Pharmacological inhibition of deubiquitylases or the proteasome inhibition of platelet activation by collagen, demonstrating a role for ubiquitylation, but relatively few substrates for ubiquitin have been identified and the molecular basis of inhibition is not established. Here, we report the ubiquitome of human platelets and changes in ubiquitylated proteins following stimulation by collagen-related peptide (CRP-XL). Using platelets from six individuals over three independent experiments, we identified 1,634 ubiquitylated peptides derived from 691 proteins, revealing extensive ubiquitylation in resting platelets. Note that 925 of these peptides show an increase of more than twofold following stimulation with CRP-XL. Multiple sites of ubiquitylation were identified on several proteins including Syk, filamin and integrin heterodimer sub-units. This work reveals extensive protein ubiquitylation during activation of human platelets and opens the possibility of novel therapeutic interventions targeting the ubiquitin machinery.

Dictyostelium as a Model to Assess Site-Specific ADP-Ribosylation Events.

The amoeba Dictyostelium discoideum is a single-cell organism that can undergo a simple developmental program, making it an excellent model to study the molecular mechanisms of cell motility, signal transduction, and cell-type differentiation. A variety of human genes that are absent or show limited conservation in other invertebrate models have been identified in this organism. This includes ADP-ribosyltransferases, also known as poly-ADP-ribose polymerases (PARPs), a family of proteins that catalyze the addition of single or poly-ADP-ribose moieties onto target proteins. The genetic tractability of Dictyostelium and its relatively simple genome structure makes it possible to disrupt PARP gene combinations, in addition to specific ADP-ribosylation sites at endogenous loci. Together, this makes Dictyostelium an attractive model to assess how ADP-ribosylation regulates a variety of cellular processes including DNA repair, transcription, and cell-type specification. Here we describe a range of techniques to study ADP-ribosylation in Dictyostelium, including analysis of ADP-ribosylation events in vitro and in vivo, in addition to approaches to assess the functional roles of this modification in vivo.

Site-specific ADP-ribosylation of histone H2B in response to DNA double strand breaks.

ADP-ribosyltransferases (ARTs) modify proteins with single units or polymers of ADP-ribose to regulate DNA repair. However, the substrates for these enzymes are ill-defined. For example, although histones are modified by ARTs, the sites on these proteins ADP-ribosylated following DNA damage and the ARTs that catalyse these events are unknown. This, in part, is due to the lack of a eukaryotic model that contains ARTs, in addition to histone genes that can be manipulated to assess ADP-ribosylation events in vivo. Here we exploit the model Dictyostelium to identify site-specific histone ADP-ribosylation events in vivo and define the ARTs that mediate these modifications. Dictyostelium histones are modified in response to DNA double strand breaks (DSBs) in vivo by the ARTs Adprt1a and Adprt2. Adprt1a is a mono-ART that modifies H2BE18 in vitro, although disruption of this site allows ADP-ribosylation at H2BE19. Although redundancy between H2BE18 and H2BE19 ADP-ribosylation is also apparent following DSBs in vivo, by generating a strain with mutations at E18/E19 in the h2b locus we demonstrate these are the principal sites modified by Adprt1a/Adprt2. This identifies DNA damage induced histone mono-ADP-ribosylation sites by specific ARTs in vivo, providing a unique platform to assess how histone ADP-ribosylation regulates DNA repair.

Emerging models for DNA repair: Dictyostelium discoideum as a model for nonhomologous end-joining.

DNA double strand breaks (DSBs) are a particularly cytotoxic variety of DNA lesion that can be repaired by homologous recombination (HR) or nonhomologous end-joining (NHEJ). HR utilises sequences homologous to the damage DNA template to facilitate repair. In contrast, NHEJ does not require homologous sequences for repair but instead functions by directly re-joining DNA ends. These pathways are critical to resolve DSBs generated intentionally during processes such as meiotic and site-specific recombination. However, they are also utilised to resolve potentially pathological DSBs generated by mutagens and errors during DNA replication. The importance of DSB repair is underscored by the findings that defects in these pathways results in chromosome instability that contributes to a variety of disease states including malignancy. The general principles of NHEJ are conserved in eukaryotes. As such, relatively simple model organisms have been instrumental in identifying components of these pathways and providing a mechanistic understanding of repair that has subsequently been applied to vertebrates. However, certain components of the NHEJ pathway are absent or show limited conservation in the most commonly used invertebrate models exploited to study DNA repair. Recently, however, it has become apparent that vertebrate DNA repair pathway components, including those involved in NHEJ, are unusually conserved in the amoeba Dictyostelium discoideum. Traditionally, this genetically tractable organism has been exploited to study the molecular basis of cell type specification, cell motility and chemotaxis. Here we discuss the use of this organism as an additional model to study DNA repair, with specific reference to NHEJ.

Nonhomologous end-joining promotes resistance to DNA damage in the absence of an ADP-ribosyltransferase that signals DNA single strand breaks.

ADP-ribosylation of proteins at DNA lesions by ADP-ribosyltransferases (ARTs) is an early response to DNA damage. The best defined role of ADP-ribosylation in the DNA damage response is in repair of single strand breaks (SSBs). Recently, we initiated a study of how ADP-ribosylation regulates DNA repair in Dictyostelium and found that two ARTs (Adprt1b and Adprt2) are required for tolerance of cells to SSBs, and a third ART (Adprt1a) promotes nonhomologous end-joining (NHEJ). Here we report that disruption of adprt2 results in accumulation of DNA damage throughout the cell cycle following exposure to agents that induce base damage and DNA SSBs. Although ADP-ribosylation is evident in adprt2(-) cells exposed to methylmethanesulfonate (MMS), disruption of adprt1a and adprt2 in combination abolishes this response and further sensitises cells to this agent, indicating that in the absence of Adprt2, Adprt1a signals MMS-induced DNA lesions to promote resistance of cells to DNA damage. As a consequence of defective signalling of SSBs by Adprt2, Adprt1a is required to assemble NHEJ factors in chromatin, and disruption of the NHEJ pathway in combination with adprt2 increases sensitivity of cells to MMS. Taken together, these data indicate overlapping functions of different ARTs in signalling DNA damage, and illustrate a critical requirement for NHEJ in maintaining cell viability in the absence of an effective SSB response.

Investigation of DNA repair pathway activity.

DNA is constantly being damaged from endogenous and exogenous sources and efficient repair of different types of DNA lesions is essential for the survival of the organism. Dictyostelium is highly resistant to DNA damage and its genome sequence has revealed the presence of multiple repair pathways conserved with vertebrates but lost in other genetically tractable invertebrate models. As such, Dictyostelium is a powerful model organism to study selected human DNA repair pathways and may provide insights into the molecular basis of how cells become resistant to DNA damage. Here we describe a range of assays used to study DNA repair in Dictyostelium. Genes required for repair of DNA damage can be identified and analyzed by comparing the ability of control or mutant cells to survive exposure to genotoxic agents that induce different types of DNA lesion. We also describe assays that assess the presence of markers for DNA repair within chromatin either in the form of posttranslational modification of proteins at sites of damage or the recruitment of repair factors to DNA lesions. Finally, we also describe more direct assays to assess repair of DNA double-strand breaks by either homologous recombination or non-homologous end joining.

Dynamic acetylation of lysine-4-trimethylated histone H3 and H3 variant biology in a simple multicellular eukaryote.

Dynamic acetylation of all lysine-4-trimethylated histone H3 is a complex phenomenon involved in Immediate-early gene induction in metazoan eukaryotes. Higher eukaryotes express repeated copies of three closely related H3 variants, inaccessible to genetic analysis. We demonstrate conservation of these phenomena in Dictyostelium which has three single-copy H3 variant genes. Although dynamic acetylation is targeted to two H3 variants which are K4-trimethylated, K9-acetylation is preferentially targeted to one. In cells lacking Set1 methyltransferase and any detectable K4-trimethylation, dynamic acetylation is lost demonstrating a direct link between the two. Gene replacement to express mutated H3 variants reveals a novel interaction between K4-trimethylation on different variants. Cells expressing only one variant show defects in growth, and in induction of a UV-inducible gene, demonstrating the functional importance of variant expression. These studies confirm that dynamic acetylation targeted to H3K4me3 arose early in evolution and reveal a very high level of specificity of histone variant utilization in a simple multicellular eukaryote.

The role of ADP-ribosylation in regulating DNA double-strand break repair.

ADP-ribosylation is the post translational modification of proteins catalysed by ADP-ribosyltransferases (ARTs). ADP-ribosylation has been implicated in a wide variety of cellular processes including cell growth and differentiation, apoptosis and transcriptional regulation. Perhaps the best characterised role, however, is in DNA repair and genome stability where ADP-ribosylation promotes resolution of DNA single strand breaks. Although ADP-ribosylation also occurs at DNA double strand breaks (DSBs), which ARTs catalyse this reaction and the molecular basis of how this modification regulates their repair remains a matter of debate. Here we review recent advances in our understanding of how ADP-ribosylation regulates DSB repair. Specifically, we highlight studies using the genetic model organism Dictyostelium, in addition to vertebrate cells that identify a third ART that accelerates DSB repair by non-homologous end-joining through promoting the interaction of repair factors with DNA lesions. The implications of these data with regards to how ADP-ribosylation regulates DNA repair and genome stability are discussed.

PARP regulates nonhomologous end joining through retention of Ku at double-strand breaks.

Poly adenosine diphosphate (ADP)-ribosylation (PARylation) by poly ADP-ribose (PAR) polymerases (PARPs) is an early response to DNA double-strand breaks (DSBs). In this paper, we exploit Dictyostelium discoideum to uncover a novel role for PARylation in regulating nonhomologous end joining (NHEJ). PARylation occurred at single-strand breaks, and two PARPs, Adprt1b and Adprt2, were required for resistance to this kind of DNA damage. In contrast, although Adprt1b was dispensable for PARylation at DSBs, Adprt1a and, to a lesser extent, Adprt2 were required for this event. Disruption of adprt2 had a subtle impact on the ability of cells to perform NHEJ. However, disruption of adprt1a decreased the ability of cells to perform end joining with a concomitant increase in homologous recombination. PAR-dependent regulation of NHEJ was achieved through promoting recruitment and/or retention of Ku at DSBs. Furthermore, a PAR interaction motif in Ku70 was required for this regulation and efficient NHEJ. These data illustrate that PARylation at DSBs promotes NHEJ through recruitment or retention of repair factors at sites of DNA damage.

DNA double-strand break repair pathway choice in Dictyostelium.

DNA double-strand breaks (DSBs) can be repaired by homologous recombination (HR) or non-homologous end joining (NHEJ). The mechanisms that govern whether a DSB is repaired by NHEJ or HR remain unclear. Here, we characterise DSB repair in the amoeba Dictyostelium. HR is the principal pathway responsible for resistance to DSBs during vegetative cell growth, a stage of the life cycle when cells are predominantly in G2. However, we illustrate that restriction-enzyme-mediated integration of DNA into the Dictyostelium genome is possible during this stage of the life cycle and that this is mediated by an active NHEJ pathway. We illustrate that Dclre1, a protein with similarity to the vertebrate NHEJ factor Artemis, is required for NHEJ independently of DNA termini complexity. Although vegetative dclre1(-) cells are not radiosensitive, they exhibit delayed DSB repair, further supporting a role for NHEJ during this stage of the life cycle. By contrast, cells lacking the Ku80 component of the Ku heterodimer that binds DNA ends to facilitate NHEJ exhibit no such defect and deletion of ku80 suppresses the DSB repair defect of dclre1(-) cells through increasing HR efficiency. These data illustrate a functional NHEJ pathway in vegetative Dictyostelium and the importance of Ku in regulating DSB repair choice during this phase of the life cycle.

Submaximal inhibition of protein kinase C restores ADP-induced dense granule secretion in platelets in the presence of Ca2+.

Protein kinase C (PKC) is a family of serine/threonine kinases that play isoform-specific inhibitory and stimulatory roles in platelet activation. We show here that the pan-PKC inhibitor Ro31-8220 can be used to dissect these events following platelet activation by ADP. Submaximal concentrations of Ro31-8220 potentiated aggregation and dense granule secretion to ADP in plasma anticoagulated with citrate, in D-Phe-Pro-Arg-chloromethyl ketone-anticoagulated plasma, which has physiological levels of Ca(2+), and in washed platelets. Potentiation was retained on inhibition of cyclooxygenase and was associated with an increase in intracellular Ca(2+). Potentiation of aggregation and secretion was abolished by a maximally effective concentration of Ro31-8220, consistent with a critical role of PKC in secretion. ADP-induced secretion was potentiated in the presence of an inhibitor of PKCß but not in the presence of available inhibitors of other PKC isoforms in human and mouse platelets. ADP-induced secretion was also potentiated in mouse platelets deficient in PKCε but not PKC. These results demonstrate that partial blockade of PKC potentiates aggregation and dense granule secretion by ADP in association with increased Ca(2+). This provides a molecular explanation for the inability of ADP to induce secretion in plasma in the presence of physiological Ca(2+) concentrations, and it reveals a novel role for PKC in inhibiting platelet activation by ADP in vivo. These results also demonstrate isoform-specific inhibitory effects of PKC in platelets.

Targets downstream of Cdk8 in Dictyostelium development.

BACKGROUND: Cdk8 is a component of the mediator complex which facilitates transcription by RNA polymerase II and has been shown to play an important role in development of Dictyostelium discoideum. This eukaryote feeds as single cells but starvation triggers the formation of a multicellular organism in response to extracellular pulses of cAMP and the eventual generation of spores. Strains in which the gene encoding Cdk8 have been disrupted fail to form multicellular aggregates unless supplied with exogenous pulses of cAMP and later in development, cdk8- cells show a defect in spore production. RESULTS: Microarray analysis revealed that the cdk8- strain previously described (cdk8-HL) contained genome duplications. Regeneration of the strain in a background lacking detectable gene duplication generated strains (cdk8-2) with identical defects in growth and early development, but a milder defect in spore generation, suggesting that the severity of this defect depends on the genetic background. The failure of cdk8- cells to aggregate unless rescued by exogenous pulses of cAMP is consistent with a failure to express the catalytic subunit of protein kinase A. However, overexpression of the gene encoding this protein was not sufficient to rescue the defect, suggesting that this is not the only important target for Cdk8 at this stage of development. Proteomic analysis revealed two potential targets for Cdk8 regulation, one regulated post-transcriptionally (4-hydroxyphenylpyruvate dioxygenase (HPD)) and one transcriptionally (short chain dehydrogenase/reductase (SDR1)). CONCLUSIONS: This analysis has confirmed the importance of Cdk8 at multiple stages of Dictyostelium development, although the severity of the defect in spore production depends on the genetic background. Potential targets of Cdk8-mediated gene regulation have been identified in Dictyostelium which will allow the mechanism of Cdk8 action and its role in development to be determined.

Control of cyclin C levels during development of Dictyostelium.

BACKGROUND: Cdk8 and its partner cyclin C form part of the mediator complex which links the basal transcription machinery to regulatory proteins. The pair are required for correct regulation of a subset of genes and have been implicated in control of development in a number of organisms including the social amoeba Dictyostelium discoideum. When feeding, Dictyostelium amoebae are unicellular but upon starvation they aggregate to form a multicellular structure which develops into a fruiting body containing spores. Cells in which the gene encoding Cdk8 has been deleted fail to enter aggregates due to a failure of early gene expression. PRINCIPAL FINDINGS: We have monitored the expression levels of cyclin C protein during development and find levels decrease after the multicellular mound is formed. This decrease is triggered by extracellular cAMP that, in turn, is working in part through an increase in intracellular cAMP. The loss of cyclin C is coincident with a reduction in the association of Cdk8 with a high molecular weight complex in the nucleus. Overexpression of cyclin C and Cdk8 lead to an increased rate of early development, consistent with the levels being rate limiting. CONCLUSIONS: Overall these results show that both cyclin C and Cdk8 are regulated during development in response to extracellular signals and the levels of these proteins are important in controlling the timing of developmental processes. These findings have important implications for the role of these proteins in controlling development, suggesting that they are targets for developmental signals to regulate gene expression.

Differential roles of the PKC novel isoforms, PKCdelta and PKCepsilon, in mouse and human platelets.

BACKGROUND: Increasing evidence suggests that individual isoforms of protein kinase C (PKC) play distinct roles in regulating platelet activation. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we focus on the role of two novel PKC isoforms, PKCdelta and PKCepsilon, in both mouse and human platelets. PKCdelta is robustly expressed in human platelets and undergoes transient tyrosine phosphorylation upon stimulation by thrombin or the collagen receptor, GPVI, which becomes sustained in the presence of the pan-PKC inhibitor, Ro 31-8220. In mouse platelets, however, PKCdelta undergoes sustained tyrosine phosphorylation upon activation. In contrast the related isoform, PKCepsilon, is expressed at high levels in mouse but not human platelets. There is a marked inhibition in aggregation and dense granule secretion to low concentrations of GPVI agonists in mouse platelets lacking PKCepsilon in contrast to a minor inhibition in response to G protein-coupled receptor agonists. This reduction is mediated by inhibition of tyrosine phosphorylation of the FcRgamma-chain and downstream proteins, an effect also observed in wild-type mouse platelets in the presence of a PKC inhibitor. CONCLUSIONS: These results demonstrate a reciprocal relationship in levels of the novel PKC isoforms delta and epsilon in human and mouse platelets and a selective role for PKCepsilon in signalling through GPVI.