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  • ItemOpen AccessPublished version Peer-reviewed
    Combinational Treatment of Trichostatin A and Vitamin C Improves the Efficiency of Cloning Mice by Somatic Cell Nuclear Transfer.
    (MyJove Corporation, 2018-04-26) Azuma, Rika; Miyamoto, Kei; Oikawa, Mami; Yamada, Masayasu; Anzai, Masayuki
    Somatic cell nuclear transfer (SCNT) provides a unique opportunity to directly produce a cloned animal from a donor cell, and it requires the use of skillful techniques. Additionally, the efficiencies of cloning have remained low since the successful production of cloned animals, especially mice. There have been many attempts to improve the cloning efficiency, and trichostatin A (TSA), a histone deacetylase inhibitor, has been widely used to enhance the efficiency of cloning. Here, we report a dramatically improved cloning method in mice. This somatic cell nuclear transfer method involves usage of Hemagglutinating virus of Japan Envelope (HVJ-E), which enables easy manipulation. Moreover, the treatment using two small molecules, TSA and vitamin C (VC), with deionized bovine serum albumin (dBSA), is highly effective for embryonic development. This approach requires neither additional injection nor genetic manipulation, and thus presents a simple, suitable method for practical use. This method could become a technically feasible approach for researchers to produce genetically modified animals from cultured cells. Furthermore, it might be a useful way for the rescue of endangered animals via cloning.
  • ItemOpen AccessPublished version Peer-reviewed
    Expression of functional inhibitory neurotransmitter transporters GlyT1, GAT-1, and GAT-3 by astrocytes of inferior colliculus and hippocampus.
    (Springer Science and Business Media LLC, 2018-01-25) Ghirardini, Elsa; Wadle, Simon L; Augustin, Vanessa; Becker, Jasmin; Brill, Sina; Hammerich, Julia; Seifert, Gerald; Stephan, Jonathan; Stephan, Jonathan [0000-0003-4869-8092]
    Neuronal inhibition is mediated by glycine and/or GABA. Inferior colliculus (IC) neurons receive glycinergic and GABAergic inputs, whereas inhibition in hippocampus (HC) predominantly relies on GABA. Astrocytes heterogeneously express neurotransmitter transporters and are expected to adapt to the local requirements regarding neurotransmitter homeostasis. Here we analyzed the expression of inhibitory neurotransmitter transporters in IC and HC astrocytes using whole-cell patch-clamp and single-cell reverse transcription-PCR. We show that most astrocytes in both regions expressed functional glycine transporters (GlyTs). Activation of these transporters resulted in an inward current (IGly) that was sensitive to the competitive GlyT1 agonist sarcosine. Astrocytes exhibited transcripts for GlyT1 but not for GlyT2. Glycine did not alter the membrane resistance (RM) arguing for the absence of functional glycine receptors (GlyRs). Thus, IGly was mainly mediated by GlyT1. Similarly, we found expression of functional GABA transporters (GATs) in all IC astrocytes and about half of the HC astrocytes. These transporters mediated an inward current (IGABA) that was sensitive to the competitive GAT-1 and GAT-3 antagonists NO711 and SNAP5114, respectively. Accordingly, transcripts for GAT-1 and GAT-3 were found but not for GAT-2 and BGT-1. Only in hippocampal astrocytes, GABA transiently reduced RM demonstrating the presence of GABAA receptors (GABAARs). However, IGABA was mainly not contaminated by GABAAR-mediated currents as RM changes vanished shortly after GABA application. In both regions, IGABA was stronger than IGly. Furthermore, in HC the IGABA/IGly ratio was larger compared to IC. Taken together, our results demonstrate that astrocytes are heterogeneous across and within distinct brain areas. Furthermore, we could show that the capacity for glycine and GABA uptake varies between both brain regions.
  • ItemOpen AccessPublished version Peer-reviewed
    Extracellular Monomeric and Aggregated Tau Efficiently Enter Human Neurons through Overlapping but Distinct Pathways.
    (Elsevier BV, 2018-03-27) Evans, Lewis D; Wassmer, Thomas; Fraser, Graham; Smith, James; Perkinton, Michael; Billinton, Andrew; Livesey, Frederick J; Smith, James [0000-0001-9131-5849]; Livesey, Frederick [0000-0001-6128-3372]
    In Alzheimer's disease, neurofibrillary tangle pathology appears to spread along neuronal connections, proposed to be mediated by the release and uptake of abnormal, disease-specific forms of microtubule-binding protein tau MAPT. It is currently unclear whether transfer of tau between neurons is a toxic gain-of-function process in dementia or reflects a constitutive biological process. We report two entry mechanisms for monomeric tau to human neurons: a rapid dynamin-dependent phase typical of endocytosis and a second, slower actin-dependent phase of macropinocytosis. Aggregated tau entry is independent of actin polymerization and largely dynamin dependent, consistent with endocytosis and distinct from macropinocytosis, the major route for aggregated tau entry reported for non-neuronal cells. Anti-tau antibodies abrogate monomeric tau entry into neurons, but less efficiently in the case of aggregated tau, where internalized tau carries antibody with it into neurons. These data suggest that tau entry to human neurons is a physiological process and not a disease-specific phenomenon.
  • ItemOpen AccessPublished version Peer-reviewed
    Mixed analytical-stochastic simulation method for the recovery of a Brownian gradient source from probability fluxes to small windows.
    (Elsevier BV, 2018-02-15) Dobramysl, U; Holcman, D; Dobramysl, Ulrich [0000-0001-9363-654X]
    Is it possible to recover the position of a source from the steady-state fluxes of Brownian particles to small absorbing windows located on the boundary of a domain? To address this question, we develop a numerical procedure to avoid tracking Brownian trajectories in the entire infinite space. Instead, we generate particles near the absorbing windows, computed from the analytical expression of the exit probability. When the Brownian particles are generated by a steady-state gradient at a single point, we compute asymptotically the fluxes to small absorbing holes distributed on the boundary of half-space and on a disk in two dimensions, which agree with stochastic simulations. We also derive an expression for the splitting probability between small windows using the matched asymptotic method. Finally, when there are more than two small absorbing windows, we show how to reconstruct the position of the source from the diffusion fluxes. The present approach provides a computational first principle for the mechanism of sensing a gradient of diffusing particles, a ubiquitous problem in cell biology.
  • ItemOpen AccessAccepted version Peer-reviewed
    Stochastic population dynamics in spatially extended predator-prey systems
    (IOP Publishing, 2018) Dobramysl, U; Mobilia, M; Pleimling, M; Tauber, UC; Dobramysl, U [0000-0001-9363-654X]; Mobilia, M [0000-0002-1424-567X]; Pleimling, M [0000-0003-3191-3390]; Tauber, UC [0000-0001-7854-2254]
    Spatially extended population dynamics models that incorporate intrinsic noise serve as case studies for the role of fluctuations and correlations in biological systems. Including spatial structure and stochastic noise in predator-prey competition invalidates the deterministic Lotka-Volterra picture of neutral population cycles. Stochastic models yield long-lived erratic population oscillations stemming from a resonant amplification mechanism. In spatially extended predator-prey systems, one observes noise-stabilized activity and persistent correlations. Fluctuation-induced renormalizations of the oscillation parameters can be analyzed perturbatively. The critical dynamics and the non-equilibrium relaxation kinetics at the predator extinction threshold are characterized by the directed percolation universality class. Spatial or environmental variability results in more localized patches which enhances both species densities. Affixing variable rates to individual particles and allowing for trait inheritance subject to mutations induces fast evolutionary dynamics for the rate distributions. Stochastic spatial variants of cyclic competition with rock-paper-scissors interactions illustrate connections between population dynamics and evolutionary game theory, and demonstrate how space can help maintain diversity. In two dimensions, three-species cyclic competition models of the May-Leonard type are characterized by the emergence of spiral patterns whose properties are elucidated by a mapping onto a complex Ginzburg-Landau equation. Extensions to general food networks can be classified on the mean-field level, which provides both a fundamental understanding of ensuing cooperativity and emergence of alliances. Novel space-time patterns emerge as a result of the formation of competing alliances, such as coarsening domains that each incorporate rock-paper-scissors competition games.
  • ItemOpen AccessPublished version Peer-reviewed
    Fank1 and Jazf1 promote multiciliated cell differentiation in the mouse airway epithelium
    (Company of Biologists, 2018-04-16) Johnson, Jo-Anne; Watson, Julie Karen; Nikolic, Marko Z; Rawlins, EL; Nikolic, Marko [0000-0001-6304-6848]; Rawlins, Emma [0000-0001-7426-3792]
    The airways are lined by secretory and multiciliated cells which function together to remove particles and debris from the respiratory tract. The transcriptome of multiciliated cells has been extensively studied, but the function of many of the genes identified is unknown. We have established an assay to test the ability of over-expressed transcripts to promote multiciliated cell differentiation in mouse embryonic tracheal explants. Overexpression data indicated that Fank1 (Fibronectin type 3 and ankyrin repeat domains 1) and Jazf1 (JAZF zinc finger 1) promoted multiciliated cell differentiation alone, and cooperatively with the canonical multiciliated cell transcription factor Foxj1. Moreover, knock-down of Fank1 or Jazf1 in adult mouse airway epithelial cultures demonstrated that these factors are both required for ciliated cell differentiation in vitro. This analysis identifies Fank1 and Jazf1 as novel regulators of multiciliated cell differentiation. Moreover, we show that they are likely to function downstream of IL6 signalling and upstream of Foxj1 activity in the process of ciliated cell differentiation. In addition, our in vitro explant assay provides a convenient method for preliminary investigation of over-expression phenotypes in the developing mouse airways.
  • ItemOpen AccessPublished version Peer-reviewed
    Cyclin B1 is essential for mitosis in mouse embryos, and its nuclear export sets the time for mitosis.
    (Rockefeller University Press, 2018-01-02) Strauss, Bernhard; Harrison, Andrew; Coelho, Paula Almeida; Yata, Keiko; Zernicka-Goetz, Magdalena; Pines, Jonathon; Harrison, Andrew [0000-0002-3778-8415]; Coelho, Paula Almeida [0000-0003-0614-7575]; Yata, Keiko [0000-0001-7648-4898]; Pines, Jonathon [0000-0002-5227-6004]
    There is remarkable redundancy between the Cyclin-Cdk complexes that comprise the cell cycle machinery. None of the mammalian A-, D-, or E-type cyclins are required in development until implantation, and only Cdk1 is essential for early cell divisions. Cyclin B1 is essential for development, but whether it is required for cell division is contentious. Here, we used a novel imaging approach to analyze Cyclin B1-null embryos from fertilization onward. We show that Cyclin B1-/- embryos arrest in G2 phase after just two divisions. This is the earliest arrest of any Cyclin known and places Cyclin B1 with cdk1 as the essential regulators of the cell cycle. We reintroduced mutant proteins into this genetically null background to determine why Cyclin B1 is constantly exported from the nucleus. We found that Cyclin B1 must be exported from the nucleus for the cell to prevent premature entry to mitosis, and retaining Cyclin B1-Cdk1 at the plasma membrane precludes entry to mitosis.
  • ItemOpen AccessPublished version Peer-reviewed
    Broad Chromatin Domains: An Important Facet of Genome Regulation.
    (Wiley, 2017-12) Carelli, Francesco N; Sharma, Garima; Ahringer, Julie; Carelli, Francesco N [0000-0003-4280-4923]; Sharma, Garima [0000-0001-8698-3682]; Ahringer, Julie [0000-0002-7074-4051]
    Chromatin composition differs across the genome, with distinct compositions characterizing regions associated with different properties and functions. Whereas many histone modifications show local enrichment over genes or regulatory elements, marking can also span large genomic intervals defining broad chromatin domains. Here we highlight structural and functional features of chromatin domains marked by histone modifications, with a particular emphasis on the potential roles of H3K27 methylation domains in the organization and regulation of genome activity in metazoans.
  • ItemOpen AccessAccepted version Peer-reviewed
    Bioinformatics challenges and perspectives when studying the effect of epigenetic modifications on alternative splicing
    (Royal Society of London) Koziol, MJ; Pacini, C; Koziol, Magdalena [0000-0002-4123-4343]; Pacini, Clare [0000-0001-7791-0940]
    It is widely known that epigenetic modifications are important in regulating transcription, but several have also been reported in alternative splicing. The regulation of pre-mRNA splicing is important to explain proteomic diversity and the misregulation of splicing has been implicated in many diseases. Here, we give a brief overview of the role of epigenetics in alternative splicing and disease. We then discuss the bioinformatics methods that can be used to model interactions between epigenetic marks and regulators of splicing. These models can be used to identify alternative splicing and epigenetic changes across different phenotypes.
  • ItemOpen AccessAccepted version Peer-reviewed
    Developmental mechanisms and adult stem cells for therapeutic lung regeneration.
    (Elsevier BV, 2018-01-15) Lee, Joo-Hyeon; Rawlins, Emma L; Lee, Joo [0000-0002-7364-6422]; Rawlins, Emma [0000-0001-7426-3792]
    Chronic degenerative lung diseases are essentially untreatable pathological conditions. By contrast, the healthy lung has numerous mechanisms that allow for rapid repair and restoration of function following minor acute injuries. We discuss the normal endogenous processes of lung development, homeostatic maintenance and repair and consider the research strategies required for the development of methods for human therapeutic lung regeneration.
  • ItemOpen AccessPublished version Peer-reviewed
    An Alternative STAT Signaling Pathway Acts in Viral Immunity in Caenorhabditis elegans.
    (American Society for Microbiology, 2017-09-05) Tanguy, Mélanie; Véron, Louise; Stempor, Przemyslaw; Ahringer, Julie; Sarkies, Peter; Miska, Eric A; Tanguy, Melanie [0000-0003-4862-0981]; Stempor, Przemyslaw [0000-0002-9464-7475]; Ahringer, Julie [0000-0002-7074-4051]; Miska, Eric [0000-0002-4450-576X]
    Across metazoans, innate immunity is vital in defending organisms against viral infection. In mammals, antiviral innate immunity is orchestrated by interferon signaling, activating the STAT transcription factors downstream of the JAK kinases to induce expression of antiviral effector genes. In the nematode Caenorhabditis elegans, which lacks the interferon system, the major antiviral response so far described is RNA interference (RNAi), but whether additional gene expression responses are employed is not known. Here we show that, despite the absence of both interferon and JAK, the C. elegans STAT homolog STA-1 orchestrates antiviral immunity. Intriguingly, mutants lacking STA-1 are less permissive to antiviral infection. Using gene expression analysis and chromatin immunoprecipitation, we show that, in contrast to the mammalian pathway, STA-1 acts mostly as a transcriptional repressor. Thus, STA-1 might act to suppress a constitutive antiviral response in the absence of infection. Additionally, using a reverse genetic screen, we identify the kinase SID-3 as a new component of the response to infection, which, along with STA-1, participates in the transcriptional regulatory network of the immune response. Our work uncovers novel physiological roles for two factors in viral infection: a SID protein acting independently of RNAi and a STAT protein acting in C. elegans antiviral immunity. Together, these results illustrate the complex evolutionary trajectory displayed by innate immune signaling pathways across metazoan organisms.IMPORTANCE Since innate immunity was discovered, a diversity of pathways has arisen as powerful first-line defense mechanisms to fight viral infection. RNA interference, reported mostly in invertebrates and plants, as well as the mammalian interferon response and JAK/STAT pathway are key in RNA virus innate immunity. We studied infection by the Orsay virus in Caenorhabditis elegans, where RNAi is known to be a potent antiviral defense. We show that, in addition to its RNAi pathway, C. elegans utilizes an alternative STAT pathway to control the levels of viral infection. We identify the transcription factor STA-1 and the kinase SID-3 as two components of this response. Our study defines C. elegans as a new example of the diversity of antiviral strategies.
  • ItemOpen AccessAccepted version Peer-reviewed
    Nuclear transplantation, the conservation of the genome, and prospects for cell replacement.
    (Wiley-Blackwell, 2017-01) Gurdon, JB; Gurdon, John [0000-0002-5621-3799]
    Initial nuclear transplantation experiments in Xenopus eggs provided the first evidence for the conservation of the genome after cellular differentiation. This Discovery-in-Context Review recounts the early experiments that led to successful nuclear transfer in amphibians and the establishment of totipotency of a differentiated cell and shows how these discoveries paved the way for similar cloning experiments in other organisms.
  • ItemOpen AccessAccepted version Peer-reviewed
    Muscle Stem Cells Exhibit Distinct Clonal Dynamics in Response to Tissue Repair and Homeostatic Aging.
    (Elsevier BV, 2018-01-04) Tierney, Matthew T; Stec, Michael J; Rulands, Steffen; Simons, Benjamin D; Sacco, Alessandra; Rulands, Steffen [0000-0001-6398-1553]; Simons, Benjamin [0000-0002-3875-7071]
    The clonal complexity of adult stem cell pools is progressively lost during homeostatic turnover in several tissues, suggesting a decrease in the number of stem cells with distinct clonal origins. The functional impact of reduced complexity on stem cell pools, and how different tissue microenvironments may contribute to such a reduction, are poorly understood. Here, we performed clonal multicolor lineage tracing of skeletal muscle stem cells (MuSCs) to address these questions. We found that MuSC clonal complexity is maintained during aging despite heterogenous reductions in proliferative capacity, allowing aged muscle to mount a clonally diverse, albeit diminished, response to injury. In contrast, repeated bouts of tissue repair cause a progressive reduction in MuSC clonal complexity indicative of neutral drift. Consistently, biostatistical modeling suggests that MuSCs undergo symmetric expansions with stochastic fate acquisition during tissue repair. These findings establish distinct principles that underlie stem cell dynamics during homeostatic aging and muscle regeneration.
  • ItemOpen AccessAccepted version Peer-reviewed
    Live imaging of neurogenesis in the adult mouse hippocampus.
    (American Association for the Advancement of Science (AAAS), 2018-02-09) Pilz, Gregor-Alexander; Bottes, Sara; Betizeau, Marion; Jörg, David J; Carta, Stefano; April, Simon; Simons, Benjamin D; Helmchen, Fritjof; Jessberger, Sebastian; Pilz, Gregor-Alexander [0000-0002-6161-4976]; Bottes, Sara [0000-0003-1801-9247]; Betizeau, Marion [0000-0002-0851-292X]; Jörg, David J [0000-0001-5960-0260]; Carta, Stefano [0000-0001-7691-7550]; April, Simon [0000-0002-8941-7990]; Simons, Benjamin D [0000-0002-3875-7071]; Helmchen, Fritjof [0000-0002-8867-9569]; Jessberger, Sebastian [0000-0002-0056-8275]
    Neural stem and progenitor cells (NSPCs) generate neurons throughout life in the mammalian hippocampus. We used chronic in vivo imaging and followed genetically labeled individual NSPCs and their progeny in the mouse hippocampus for up to 2 months. We show that NSPCs targeted by the endogenous Achaete-scute homolog 1 (Ascl1) promoter undergo limited rounds of symmetric and asymmetric divisions, eliciting a burst of neurogenic activity, after which they are lost. Further, our data reveal unexpected asymmetric divisions of nonradial glia-like NSPCs. Cell fates of Ascl1-labeled lineages suggest a developmental-like program involving a sequential transition from a proliferative to a neurogenic phase. By providing a comprehensive description of lineage relationships, from dividing NSPCs to newborn neurons integrating into the hippocampal circuitry, our data offer insight into how NSPCs support life-long hippocampal neurogenesis.
  • ItemOpen AccessPublished version Peer-reviewed
    Developmental excitatory-to-inhibitory GABA-polarity switch is disrupted in 22q11.2 deletion syndrome: a potential target for clinical therapeutics.
    (Springer Science and Business Media LLC, 2017-11-16) Amin, Hayder; Marinaro, Federica; De Pietri Tonelli, Davide; Berdondini, Luca; Amin, Hayder [0000-0002-6515-2991]; Marinaro, Federica [0000-0001-6609-1134]; De Pietri Tonelli, Davide [0000-0001-9537-8900]; Berdondini, Luca [0000-0001-9521-2812]
    Individuals with 22q11.2 microdeletion syndrome (22q11.2 DS) show cognitive and behavioral dysfunctions, developmental delays in childhood and risk of developing schizophrenia and autism. Despite extensive previous studies in adult animal models, a possible embryonic root of this syndrome has not been determined. Here, in neurons from a 22q11.2 DS mouse model (Lgdel +/-), we found embryonic-premature alterations in the neuronal chloride cotransporters indicated by dysregulated NKCC1 and KCC2 protein expression levels. We demonstrate with large-scale spiking activity recordings a concurrent deregulation of the spontaneous network activity and homeostatic network plasticity. Additionally, Lgdel +/- networks at early development show abnormal neuritogenesis and void of synchronized spontaneous activity. Furthermore, parallel experiments on Dgcr8 +/- mouse cultures reveal a significant, yet not exclusive contribution of the dgcr8 gene to our phenotypes of Lgdel +/- networks. Finally, we show that application of bumetanide, an inhibitor of NKCC1, significantly decreases the hyper-excitable action of GABAA receptor signaling and restores network homeostatic plasticity in Lgdel +/- networks. Overall, by exploiting an on-a-chip 22q11.2 DS model, our results suggest a delayed GABA-switch in Lgdel +/- neurons, which may contribute to a delayed embryonic development. Prospectively, acting on the GABA-polarity switch offers a potential target for 22q11.2 DS therapeutic intervention.
  • ItemOpen AccessAccepted version Peer-reviewed
    Promoter-bound METTL3 maintains myeloid leukaemia by m6A-dependent translation control.
    (Springer Science and Business Media LLC, 2017-12-07) Barbieri, Isaia; Tzelepis, Konstantinos; Pandolfini, Luca; Shi, Junwei; Millán-Zambrano, Gonzalo; Robson, Samuel C; Aspris, Demetrios; Migliori, Valentina; Bannister, Andrew J; Han, Namshik; De Braekeleer, Etienne; Ponstingl, Hannes; Hendrick, Alan; Vakoc, Christopher R; Vassiliou, George S; Kouzarides, Tony; Barbieri, Isaia [0000-0003-3035-8970]; Tzelepis, Konstantinos [0000-0002-4865-7648]; Pandolfini, Luca [0000-0003-1444-8167]; Bannister, Andrew [0000-0002-6312-4436]; Han, Namshik [0000-0002-7741-6384]; Hendrick, Alan [0000-0002-8604-0462]; Vassiliou, George [0000-0003-4337-8022]; Kouzarides, Tony [0000-0002-8918-4162]
    N6-methyladenosine (m6A) is an abundant internal RNA modification in both coding and non-coding RNAs that is catalysed by the METTL3-METTL14 methyltransferase complex. However, the specific role of these enzymes in cancer is still largely unknown. Here we define a pathway that is specific for METTL3 and is implicated in the maintenance of a leukaemic state. We identify METTL3 as an essential gene for growth of acute myeloid leukaemia cells in two distinct genetic screens. Downregulation of METTL3 results in cell cycle arrest, differentiation of leukaemic cells and failure to establish leukaemia in immunodeficient mice. We show that METTL3, independently of METTL14, associates with chromatin and localizes to the transcriptional start sites of active genes. The vast majority of these genes have the CAATT-box binding protein CEBPZ present at the transcriptional start site, and this is required for recruitment of METTL3 to chromatin. Promoter-bound METTL3 induces m6A modification within the coding region of the associated mRNA transcript, and enhances its translation by relieving ribosome stalling. We show that genes regulated by METTL3 in this way are necessary for acute myeloid leukaemia. Together, these data define METTL3 as a regulator of a chromatin-based pathway that is necessary for maintenance of the leukaemic state and identify this enzyme as a potential therapeutic target for acute myeloid leukaemia.
  • ItemOpen AccessPublished version Peer-reviewed
    FAN1 interaction with ubiquitylated PCNA alleviates replication stress and preserves genomic integrity independently of BRCA2
    (Springer Nature, 2017-10-20) Porro, Antonio; Berti, Matteo; Pizzolato, Julia; Bologna, Serena; Kaden, Svenja; Saxer, Anja; Ma, Yue; Nagasawa, Kazuo; Sartori, Alessandro A; Jiricny, Josef; Bologna, Serena [0000-0003-1555-0817]
    Interstrand cross-link (ICL) hypersensitivity is a characteristic trait of Fanconi anemia (FA). Although FANCD2-associated nuclease 1 (FAN1) contributes to ICL repair, FAN1 mutations predispose to karyomegalic interstitial nephritis (KIN) and cancer rather than to FA. Thus, the biological role of FAN1 remains unclear. Because fork stalling in FAN1-deficient cells causes chromosomal instability, we reasoned that the key function of FAN1 might lie in the processing of halted replication forks. Here, we show that FAN1 contains a previously-uncharacterized PCNA interacting peptide (PIP) motif that, together with its ubiquitin-binding zinc finger (UBZ) domain, helps recruit FAN1 to ubiquitylated PCNA accumulated at stalled forks. This prevents replication fork collapse and controls their progression. Furthermore, we show that FAN1 preserves replication fork integrity by a mechanism that is distinct from BRCA2-dependent homologous recombination. Thus, targeting FAN1 activities and its interaction with ubiquitylated PCNA may offer therapeutic opportunities for treatment of BRCA-deficient tumors.
  • ItemOpen AccessAccepted version Peer-reviewed
    Human primary liver cancer–derived organoid cultures for disease modeling and drug screening
    (Nature Publishing Group, 2017-12) Broutier, LJR; Gianmarco Mastrogiovanni; Monique M.A. Verstegen; Hayley E. Francies; Lena Morrill Gavarró; Charles R Bradshaw; George E Allen; Robert Arnes-Benito; Olga Sidorova; Marcia P. Gaspersz; Nikitas Georgakopoulos; Bon-Kyoung Koo; Sabine Dietmann; Susan E. Davies; Raaj K. Praseedom; Ruby Lieshout; Jan N. M. 10 IJzermans; Stephen J Wigmore; Kourosh Saeb-Parsy; Mathew J. Garnett; Luc J.W. van der Laan; Meritxell Huch
    Human liver cancer research currently lacks in vitro models that can faithfully recapitulate the pathophysiology of the original tumor. We recently described a novel, near-physiological organoid culture system, wherein primary human healthy liver cells form long-term expanding organoids that retain liver tissue function and genetic stability. Here we extend this culture system to the propagation of primary liver cancer (PLC) organoids from three of the most common PLC subtypes: hepatocellular carcinoma (HCC), cholangiocarcinoma (CC) and combined HCC/CC (CHC) tumors. PLC-derived organoid cultures preserve the histological architecture, gene expression and genomic landscape of the original tumor, allowing for discrimination between different tumor tissues and subtypes, even after long-term expansion in culture in the same medium conditions. Xenograft studies demonstrate that the tumorogenic potential, histological features and metastatic properties of PLC-derived organoids are preserved in vivo. PLC-derived organoids are amenable for biomarker identification and drug-screening testing and led to the identification of the ERK inhibitor SCH772984 as a potential therapeutic agent for primary liver cancer. We thus demonstrate the wide-ranging biomedical utilities of PLC-derived organoid models in furthering the understanding of liver cancer biology and in developing personalized-medicine approaches for the disease.
  • ItemOpen AccessPublished version Peer-reviewed
    A Unifying Theory of Branching Morphogenesis
    (Elsevier, 2017-09-21) Simons, BD; Hannezo, E; Simons, Benjamin [0000-0002-3875-7071]
    The morphogenesis of branched organs remains a subject of abiding interest. Although much is known about the underlying signaling pathways, it remains unclear how macroscopic features of branched organs, including their size, network topology and spatial patterning, are encoded. Here we show that, in mouse mammary gland, kidney and human prostate, these features can be explained quantitatively within a single unifying framework of branching and annihilating random walks. Based on quantitative analyses of large-scale organ reconstructions and proliferation kinetics measurements, we propose that morphogenesis follows from the proliferative activity of equipotent tips that stochastically branch and randomly explore their environment, but compete neutrally for space, becoming proliferatively inactive when in proximity with neighboring ducts. These results show that complex branched epithelial structures in mammalian tissues develop as a self-organized process, reliant upon a strikingly simple, but generic, rule, without recourse to a rigid and deterministic sequence of genetically programmed events.
  • ItemOpen AccessAccepted version Peer-reviewed
    PGBD5 promotes site-specific oncogenic mutations in human tumors
    (Nature Publishing Group, 2017-07) Henssen, AG; Koche, R; Zhuang, J; Jiang, E; Reed, C; Eisenberg, A; Still, E; MacArthur, IC; Rodríguez-Fos, E; Gonzalez, S; Puiggròs, M; Blackford, AN; Mason, CE; de Stanchina, E; Gönen, M; Emde, A-K; Shah, M; Arora, K; Reeves, C; Socci, ND; Perlman, E; Antonescu, CR; Roberts, CWM; Steen, H; Mullen, E; Jackson, SP; Torrents, D; Weng, Z; Armstrong, SA; Kentsis, A; Jackson, Stephen [0000-0001-9317-7937]
    Genomic rearrangements are a hallmark of human cancers. Here, we identify the piggyBac transposable element derived 5 (PGBD5) gene as encoding an active DNA transposase expressed in the majority of childhood solid tumors, including lethal rhabdoid tumors. Using assembly-based whole-genome DNA sequencing, we found previously undefined genomic rearrangements in human rhabdoid tumors. These rearrangements involved PGBD5-specific signal (PSS) sequences at their breakpoints and recurrently inactivated tumor-suppressor genes. PGBD5 was physically associated with genomic PSS sequences that were also sufficient to mediate PGBD5-induced DNA rearrangements in rhabdoid tumor cells. Ectopic expression of PGBD5 in primary immortalized human cells was sufficient to promote cell transformation in vivo. This activity required specific catalytic residues in the PGBD5 transposase domain as well as end-joining DNA repair and induced structural rearrangements with PSS breakpoints. These results define PGBD5 as an oncogenic mutator and provide a plausible mechanism for site-specific DNA rearrangements in childhood and adult solid tumors.