Genomic instability is one of the most important factors driving cancer progression and resistance to therapy. Oncogenes, by inducing DNA replication stress, are a critical factor underlying genomic instability in cancer, but the mechanisms by which oncogenes cause DNA replication stress have remained largely elusive. A major impediment has been mapping DNA replication initiation sites in the human genome, which is a prerequisite for understanding how oncogenes affect DNA replication. In this study, using a sensitive assay to monitor nascent DNA synthesis in early S phase, we identified thousands of replication initiation sites in cells before and after induction of the oncogenes CCNE1 or MYC. Remarkably, both oncogenes induced the firing of many novel, overlapping DNA replication origins that mapped within highly transcribed genes. These ectopic origins were normally suppressed by transcription during G1, but precocious entry into S phase due to oncogene activation allowed origin firing within genic regions that had not yet been transcribed. Forks from oncogene-induced origins were prone to collapse, as a result of conflicts between replication and transcription, and were associated with DNA double-strand break formation and chromosomal rearrangement breakpoints both in our experimental system and in a large cohort of human cancers. Thus, firing of ectopic origins caused by premature S phase entry represents a mechanism of oncogene-induced DNA replication stress that is relevant for genomic instability in human cancer.

Agenda:

 *   An introduction to the PacBio SMRT Sequencing technology and

its applications for Human Biomedical Research (45 min)

 by Déborah Moine and David Stucki, PacBio Field Application Scientists

  *   PacBio implementation at the NGS platform (20 min) 

by Sonia Lameiras, NGS platform

  *   Data analysis of full length RNA sequencing using IsoSeq (20 min) 

by Marc Deloger, Bioinformatics platform

Pacific Biosciences developed a sequencing system belonging to the 3rd generation of sequencers. This technology provides new fields of application that complement conventional Illumina short reads sequencing. Single Molecule, Real-Time (SMRT) Sequencing delivers long continuous reads (>20 kb), high consensus accuracy (up to 99.9999% QV50), uniform coverage (even across high GC content regions), along with simultaneous epigenetics characterisation. Among the main applications, whole genome or targeted sequencing approaches can be very efficient performed to better identify structural variants, copy number variations and SNVs with allelic resolution and phasing. In addition, RNA Sequencing, using isoform sequence (Iso-Seq®) analysis, provides full-length transcripts to understand alternative spliced gene isoforms— eliminating the need for transcriptome reconstruction using isoform-inference algorithms. It also delivers information about poly-adenylation sites for transcripts up to 10 kb in length across the full complement of isoforms within targeted genes or the entire transcriptome. The Sequel sequencing system is already running at the NGS platform of the Institut Curie and pilot studies are ongoing on main applications with respect to specific interests of the Institute.

Affiche du séminaire

Natural Killer (NK) cells play a key role in tumor control, and recognition by the activating receptor NKG2D of its ligands is particularly important for this activity, since although absent on healthy cells, NKG2D ligands (NKG2D-L) are expressed by stressed cells in different pathological situations. NKG2D-L can also be shed to the extracellular milieu in exosomes or as soluble proteins, leading to reduced cytotoxic activity. Therefore, the role of NKG2D and its ligands in anti-tumor control depends on many factors. In this seminar, different aspects of NKG2D-L biology will be addressed: at a molecular level, the specific biochemical characteristics and the GPI anchoring of the NKG2D-L, MICA, affecting its release in exosomes; at a cellular level, exploring the communication mediated by the transfer of intact NKG2D-L from the target cell to the NK cell and how this acquisition affect NK function; and finally, in a model of metastatic melanoma cells, the effects of chemotherapeutic agents on NKG2D-L expression, reducing NK cell recognition, and how it can be reversed with drug combinations. These data together, increase our understanding of NKG2D-L biology and show the complexity of the NKG2D system.

Séminaire proposé par U932

The natural history of cancer involves interactions between the tumor and the immune defense mechanism of the host. The crucial role of the patient’s own immune system on clinical outcome is demonstrated by the immunological characterization of the tumor microenvironment. The analysis of type, location and density of tumor infiltrating immune cell components has identified immune cell types that can be either beneficial or harmful to patients showing also that immune cells can infiltrate the core (center) of the tumor and/or remain located in peritumoral areas and that lymphocytic infiltration of tumor or peritumoral tissue can be a favorable prognostic factor in a range of tumor types. In order to better understand the tumor microenvironment and its architecture, recent advances in the field of tissue imaging resulted in the development of multispectral imaging and analysis of up to six immunofluorescence markers within intact tissue sections. This novel technique combines imaging with spectroscopy allowing quantitative assessment of cellular phenotype and activity in a way similar to flow cytometry, while simultaneously providing tissue context and information about cell-to-cell usually difficult or impossible to obtain by other methods. We developed different 7-plex immunofluorescence multispectral panels and applied this novel technology to multiple tumor types in order to identify and quantify the densities of different immune cell subtypes. Furthermore, we have developed an analytical pipeline for first- and second-order spatial analysis of multispectral imaging data, which enables a high-definition characterization of the tumor microenvironment architecture including the spatial distribution of the different immune cell subtypes.

The genome is propagated through cell division cycles by the faithful duplication and segregation of chromosomes into two new daughter cells during mitotic divisions. In addition, so-called "epigenetic" chromosome structures that maintain functional chromosomes and that "memorize" the transcriptional state of a cell lineage are also maintained through mitotic and sometimes even meiotic divisions. Although the mechanism of inheritance of DNA sequences has been worked out decades ago, how the more fluid epigenetic information of gene activities and chromosome structure is maintained in time is not understood. We are interested in resolving this. My lab aims at three major questions 1) What is the basis of chromatin-based memory at the human centromere? The histone H3 variant CENP-A is critical for the faithful inheritance and propagation of centromeres and is dependent on the stable transmission and self-templating capacity of CENP-A chromatin. 2) Can chromatin contribute to the maintenance of active gene expression? We have developed methods to directly determine the rate of histone H3.1 and H3.3 retention at specific loci in human somatic cells and aim to determine their contribution to transcriptional memory. Finally, 3) we use heritable gene silencing in yeast to determine whether and how epigenetic states can impact on the evolution of a species. 

For more information: www.jansenlab.org

***

See biosketch attached.

***
 

***

See biosketch attached.

***
 

Multiple cytosolic innate sensors form large oligomers following activation, yet this assembly needs to be tightly regulated to avoid accumulation of misfolded aggregates. We observed that the eIF2α kinase of the highly conserved integrated stress response (ISR) pathway, heme-regulated inhibitor (HRI), controlled the oligomerization of Nod1 and Nod2, and was essential for innate signaling by these bacterial sensors. In vivo, HRI- deficient mice displayed blunted responses to intraperitoneal injection of Nod ligands and were susceptible to intragastric infection with Citrobacter rodentium. We identified HSPB8 as an HRI-induced chaperone that mediated the effects of HRI by interacting with Nod1 and Nod2, thereby preventing aggregation of Nod signalosomes. Furthermore, HRI licensed activation of multiple innate immune signaling pathways that require oligomerization, such as MAVS, TIFA and NLRP3, but was dispensable for oligomerization-independent sensors, including STING and Toll-like receptors (TLRs). More generally, HRI was essential to limit proteotoxic stress induced by proteasome inhibition, and to prevent á-synuclein aggregation, showing that HRI acts as a general sensor of cytosolic protein misfolding. Thus, HRI, eIF2α and HSPB8 define a novel cytosolic unfolded protein response (cUPR) functionally homologous to the PERK/eIF2α/HSPA5 axis of the endoplasmic reticulum UPR. We propose that the cUPR is a critical protein surveillance system that controls cytosolic protein oligomerization and innate immune signaling, which may represent a druggable target for inflammatory diseases and pathologies displaying cytotoxic protein aggregation.

The intestinal immune system has the challenging role of tolerating foreign nutrients and the luminal microbiome, while excluding or eliminating pathogens; failure in these processes leads to a range of intestinal diseases such as food allergies, Celiac Disease, Inflammatory Bowel Diseases and gastrointestinal infections. However, how the immune system decides to mount a tolerogenic or inflammatory response remains incompletely understood. We observed that a wide range of immune responses could be achieved by topological segregation of immune reactions into different gut draining lymph nodes, according to compartmentalized lymphatic drainage from functionally distinct gut segments. This represents a novel axis of diet and gut-microbiome to host communication, and explains how tolerogenic and immunogenic decisions in mLNs can occur simultaneously, thus shedding light on how adaptive immune homeostasis is achieved in the gut- and how it may fail if compartments are mixed or perturbed.

     Interplay between DNA repair of the oxidatively modified base 8-oxo-7,8-dihydroguanine (OG) and transcriptional activation has been observed in the VEGF, TNF-α, SIRT1, and BCL2 genes.  We synthesized OG into the VEGF potential G-quadruplex sequence (PQS) in the coding strand of a luciferase promoter to identify that base excision repair (BER) unmasked the G-quadruplex (G4) fold for gene activation.(1)  OG was site-specifically synthesized into a luciferase reporter plasmid to follow the time-dependent expression in mammalian cells when OG in the VEGF PQS context was located in the coding vs. template strand of the luciferase promoter.  Removal of OG from the coding strand by OG-glycosylase 1 (OGG1)-mediated BER upregulated transcription.  When OG was in the template strand in the VEGF PQS context, transcription was downregulated by a BER-independent process.(2)  By following the change in transcription up to 72 h, we could follow luciferase expression back to the background level that identified repair on the template strand was more efficient than on the coding strand of the promoter.  Reporters were also synthesized with an OG:A base pair that requires repair on both strands in order to return the sequence to a G:C base pair.  By monitoring the up/down luciferase expression, we followed the repair of an OG:A base pair occurring on both strands in mammalian cells.  Depending on the strand in which OG resides, this modification is an up/down regulator of transcription by coupling DNA repair with transcriptional activation.  The recent results further support a growing hypothesis that OG is an epigenetic-like DNA modification.(3-5)  Given that 50% of gene promoters in the DNA repair pathway may be controlled by G-quadruplexes, the concept emerges that these G-rich sequences act as sensors of oxidative stress providing redox-regulatory activity for gene expression. (6)

Affiche du séminaire

    Thierry Voet, co-founder and group leader of the Sanger Institute-EBI Single-Cell Genomics Centre, Hinxton, UK  and Associate Professor of the KU Leuven, Belgium, will be visiting the Institut Curie on Feb 2 and giving a seminar entitled: “Single-cell (multi-omics) sequencing to study cellular heterogeneity in health and disease.”

He has developed very impressive methodology to combine single cell DNA sequencing at high coverage with single cell RNAseq (Mcaulay…Voet, Nature Protocols, 2016). Using this technology, he addresses a wide range of important questions from early development to cancer and disease.

Affiche du séminaire

The goal of this seminar is to give an overview on my past work, as well as to introduce the questions that my group will aim at addressing.

I will showcase few single-cell technologies that my colleagues and I have developed at Stanford University, which apply to multiplexed imaging, mass cytometry and sequencing.

I will then briefly touch upon two biological questions that I have being trying to address. One of these two will also be the main focus of our laboratory: why is the immune system not capable of eradicating cancer cells? Our angle on such popular question (these days) focuses onto the gene regulatory mechanisms underlying T-lymphocytes exhaustion.  

This seminar is intended to serve as primer for discussion later-on the same day, with anyone interested in meeting with us.

Gerlind Wallon is the deputy director of the European Molecular Biology Organization (EMBO). A German native, she graduated with a PhD in Biochemistry from Brandeis University, Waltham, Massachusetts, USA, followed by a post-doctoral period at EMBL in Heidelberg.

At EMBO she is responsible for the EMBO Young Investigator Programme, the EMBO Installation Grants, the EMBO Courses and Workshops and the Women in Science activities. Gerlind Wallon has developed the EMBO Laboratory Management Courses for leadership development and authored studies on the selection processes and the effects of gender on application success at EMBO and a report on the use of quota in academia.

Affiche du séminaire

After living cells are exposed to ionizing radiation, a variety of chemical modifications of DNA are induced either directly by ionization of DNA or indirectly through interactions with water-derived radicals. The DNA lesions include single strand breaks (SSB), base lesions, sugar damage, and apurinic/apyrimidinic sites (AP sites). It has been postulated that a significant amount of clustered DNA damage could be generated by ionizing radiation especially by that of high LET. Clustered DNA damage is defined as two or more lesions within one to two helical turns of DNA induced by a single radiation track. A double strand break (DSB) of DNA is a type of clustered DNA damage, in which single strand breaks are formed on opposite strands in close proximity. Formation and repair of DSBs have been studied in great detail over the years as they have been linked to important biological endpoints, such as cell death, loss of genetic material, and chromosome aberration. We are investigating non-DSB type of clustered DNA damage, as it has received less attention and its significance remained largely unknown. Our studies on the yield of non-DSB clustered damage induced by ion-particle irradiation as well as the biological consequences of non-DSB clustered DNA damage will be presented.

Affiche du séminaire

Recent papers: - Gardner B. M., Castanzo D. T., Chowdhury S., Stjepanovic G., Stefely M. S., Hurley J. H., Lander G. C., Martin A. The peroxisomal AAA-ATPase Pex1/Pex6 unfolds substrates by processive threading Nat. Commun. 9 135 (2018) - Schoneberg J., Lee I. H., Iwasa J. H., Hurley J. H. Reverse-topology membrane scission by the ESCRT proteins Nat. Rev. Mol. Cell Biol. 18 5-17 (2017) - Young L. N., Cho K., Lawrence R., Zoncu R., Hurley J. H. Dynamics and architecture of the NRBF2-containing phosphatidylinositol 3-kinase complex I of autophagy Proc Natl Acad Sci U S A 113 8224-8229 (2016) - Schulze-Gahmen U., Echeverria I., Stjepanovic G., Bai Y., Lu H., Schneidman-Duhovny D., Doudna J. A., Zhou Q., Sali A., Hurley J. H. Insights into HIV-1 proviral transcription from integrative structure and dynamics of the Tat:AFF4:P-TEFb:TAR complex Elife 5 (2016) - Carlson L. A., Bai Y., Keane S. C., Doudna J. A., Hurley J. H. Reconstitution of selective HIV-1 RNA packaging in vitro by membrane-bound Gag assemblies Elife 5 (2016) - Lee I. H., Kai H., Carlson L. A., Groves J. T., Hurley J. H. Negative membrane curvature catalyzes nucleation of endosomal sorting complex required for transport (ESCRT)-III assembly Proc Natl Acad Sci U S A 112 15892-15897 (2015)  

*******************************************************************************

*******************************************************************************

Batf3-dependent CD8α+ dendritic cells (cDC1) capture and present antigens derived from tumors and virally infected cells, a process called cross-presentation. We established a functional CRISPR screen for novel regulators of cross-presentation in primary cDC1 generated by FLT3L cultures of Cas9 transgenic Kit-high bone marrow progenitors and a candidate based selection of target genes. We identified a BEACH-domain containing that was essential for cross-presentation of cell-associated antigens by cDC1, not required for MHC class II presentation or cross-presentation by monocyte-derived DCs.  Mice deficient in this gene develop normal populations of cDC1 that produce IL-12 and can protect against Toxoplasma gondii infection, but fail to prime virus-specific CD8+ T cells in vivo or induce tumor rejection.  Current findings on this gene will be discussed.

From its beginnings as a mere metaphase finder, over the last 3 decades Metafer has grown into a versatile slide scanner for various applications. It mainly targets (clinical) cytogenetics, toxicology, radiation biodosimetry, pathology and (currently under development) microbiology, but the wide array of image processing operations and measurement features that Metafer offers allow it to adapt to all kinds of scientific tasks based around the analysis of cells on slides in brightfield and fluorescence.

The seminar will give insight into the various possibilities that the Metafer slide scanning system offers.

The emergence of migratory cell populations within tumours represents a critical early stage during cancer metastasis. We have previously reported one such population, marked by CD66, in cervical cancers. It is unclear what broad mechanisms regulate such migratory populations.

Here, we describe the role of a Suv39H1-low heterochromatin state as a driver of cervical cancer migratory populations. Cervical cancer cells sorted based on migratory ability in vitro show low Suv39H1, and Suv39H1 knockdown enhances cell migration. Histopathology shows the emergence of migratory Suv39H1low populations in advanced carcinoma progression. Meta-analysis of data from The Cancer Genome Atlas (TCGA) reveals that Suv39H1-low tumours show migration and CD66 expression signatures, and correlate with lower patient survival. Lastly, genome-wide profiling of migrated populations using RNA-Seq and H3K9me3 ChIP-Seq reveals Suv39H1-linked transcriptome alterations and a broad loss of H3K9me3, suggesting an increase in chromatin plasticity in migrated populations. The understanding of such chromatin based regulation in migratory populations may prove valuable in efforts to develop anti-metastatic strategies.

Parkinson’s disease (PD) is characterized by the progressive loss of dopaminergic neurons in the substantia nigra (SN), associated to the aggregation of α-synuclein (α-Syn) that forms intraneuronal inclusions. In addition, mounting evidence suggest that neuro-immune interactions, favoured by cerebrovascular alterations, are involved in the pathomechanisms of PD. In this work, we focus on a myeloid population that surrounds blood vessels: perivascular macrophages (PVMs). We show that both in PD patients and in a mouse model of degenerative synucleinopathy, there is an increased proliferative recruitment of PVMs within the SN. We also found that specific ablation of PVMs aggravates the degenerative phenotype in synucleinopathic mice. PVMs-mediated neuroprotection is not likely linked to regulatory mechanisms of microglial cell response. Interestingly, the absence of PVMs is associated with higher vascular expression of VCAM-1 and enhanced infiltration of CD4+ and CD8+ T-cells, which have a toxic influence on dopaminergic neurons survival, as T-cell deficient mice are protected from α-Syn mediated toxicity. We also observe that PVMs deficiency leads to an increased spreading of α-Syn pathology following striatal injections of α-Syn fibrils. We hypothesize that PVMs might be essential for the clearance of toxic α-Syn species due to their phagocytic activity, but also to their importance in regulating interstitial and cerebrospinal fluid (CSF) circulation, as shown by analysing CSF movement via the infusion of fluorescent tracers in PVMs-deficient conditions. Overall, our data highlight the importance of PVMs in regulating the toxicity and propagation of synucleinopathy and provide a rationale for therapeutic strategies aimed at boosting PVMs functions in PD.

************************************************************

      Abstract to come      

The dynamics of genetic diversity in large clonally-evolving cell populations are poorly understood, despite having implications for the detection and treatment of cancers. I will describe work where we combine barcode lineage tracking, sequencing of adaptive clones, and mathematical modelling of mutational dynamics to understand how genetic diversity changes in an experimental system. We find that, despite differences in beneficial mutational mechanisms and fitness effects between environments, early adaptive genetic diversity increases predictably, driven by the expansion of many single-mutant lineages.

However, a crash in diversity follows, caused by highly-fit double-mutants fed from exponentially growing single-mutants, a process closely related to the classic Luria-Delbruck experiment. The diversity crash is likely to be a general feature of clonal evolution, however its timing and magnitude is stochastic. I will go onto describe more recent work applying some of these insights to quantitatively understand the clonal evolution that drives clonal hematopoiesis and their implications for predicting subsequent hematologic cancers.

Martina Kutmon , Kristina Hanspers, Jonathan Mélius, Ryan Miller, Nuno Nunes, Anders Riutta,
Denise Slenter, Egon L. Willighagen, Chris T. Evelo, Alexander R. Pico

WikiPathways ( www.wikipathways.org ) is a community curated pathway databases that enables
researchers to capture rich, intuitive models of pathways. In this talk, I will highlight the latest
developments, newest features and ongoing projects of WikiPathways and the associated tools
pathvisio.js, PathVisio and the WikiPathways app for Cytoscape. The database and the associated
tools are developed as open source projects with a lot of community engagement.
Tools: The interactive JavaScript-based pathway viewer, pathvisio.js, is integrated in the
WikiPathways website and enables users to zoom in and click on pathway elements to show links
to other databases. The standalone pathway editor, analysis and visualization tool, PathVisio
provides easy-to-use drawing and annotation tools to capture identities, relationships, comments
and literature references for each pathway element and interaction. The WikiPathways app for
Cytoscape can be used to import biological pathways in Cytoscape for data visualization and
network analysis.
Data: The WikiPathways database is improved by continuous data curation and updates through
an expanding community: more than 630 individual contributors and more than 2,500 edits on
nearly 900 pathways in 2017. We have now reach a total number of pathways of 2,652 for 27
different species. Recently, we have decided to adopt the Creative Commons CC0 waiver for our
content on WikiPathways. Our data is available for download from our website, through our REST
webservice or in RDF format from our SPARQL endpoint. We are also in the progress of importing
our content into WikiData ( www.wikidata.org ).

Reactome (https://reactome.org) [1] is a free, open-source, open-data, curated and

peer-reviewed knowledge base of biomolecular pathways, currently covering 2,106
Pathways; 10,712 protein coding genes; 11,302 reactions; 27,452 literature references
and 1,800 small molecules. Pathways are arranged in a hierarchical structure, allowing
the user to navigate from high level concepts like immune system to detailed pathway
diagrams showing biomolecular events like membrane transport or phosphorylation.
The Reactome curation strategy focuses on the annotation of “normal” pathways in
human. However, we increasingly annotate disease-specific pathway modifications,
grouped in three major classes: loss of function (typically metabolic disease
phenotypes), gain of function (typically cancer phenotypes), and host-pathogen
interactions.
Here, we present new developments in the multi-scale Reactome visualization system
that facilitate navigation through the pathway hierarchy and enable efficient reuse of
Reactome visualizations for users' own research presentations and publications.
For the higher levels of the hierarchy, Reactome now provides scalable, interactive
textbook-style diagrams in SVG format, which are also freely downloadable and
editable (Fig 1). Repeated diagram elements like 'mitochondrion' or 'receptor' are freely
available as a library of graphic elements at https://reactome.org/icon-lib. Detailed
lower-level diagrams are now downloadable in editable PPTX format as sets of
interconnected objects, as well as in standard png format.
[1] Sidiropoulos K, Viteri G, Sevilla C, Jupe S, Webber M, Orlic-Milacic M, Jassal B,
May B, Shamovsky V, Duenas C, Rothfels K, Matthews L, Song H, Stein L, Haw R,
D'Eustachio P, Ping P, Hermjakob H, Fabregat A. Reactome enhanced pathway
visualization. Bioinformatics. 2017 Nov 1;33(21):3461-3467.
 

Epigenetic information is for example stored in the form of modified bases in the genome. The positions and the kind of the base modifications is one factor that determines the identity of the corresponding cell. Setting and erasing of epigenetic imprints is one factor that controls the development process from a zygote over an omnipotent stem cells to finally an adult specialized cell. The underlying biochemical mechanism are largely unknown. I am going to discuss results related to the function and distribution of the new epigenetic bases 5-hydroxymethylcytosine (hmC), 5-formylcytosine (fC), 5-carboxycytosine (caC), and 5-hydroxymethyluracil.[1] These nucleobases seem to control epigenetic programming of cells. Synthetic routes to these new bases and isotope derivatives will be discussed that enable the preparation of oligonucleotides and allow high end mass spectrometry studies to decipher the biological functions of the new bases.[2] In particular, results from quantitative mass spectrometry, new covalent-capture proteomics mass spectrometry and isotope tracing techniques will be reported.[3] Finally I am dicussing potential präbiotic origins of modified bases[4].