abstracts | talks - embo · lyase belongs to the emerging superfamily of radical...

abstracts | talks

A genomic binding atlas for MBD proteins reveals methylation-dependent and -independent targeting principles

Tuncay Baubeca,*, Robert Ivanekb, Florian Lienertc, Dirk Schübelera

a Friedrich Miescher Instituteb Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerlandc Department of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA

Recent advances in sequencing have identified the genomic distribution of DNA methylation inunprecedented resolution while our knowledge about the binding of trans- acting factors still lacksbehind. We have established a genome-wide comparative mapping approach that enablesquantification of protein structure contribution to genomic localization of DNA binding factors. Thiswe utilized to generate a comprehensive binding atlas for the entire methyl-CpG binding domain(MBD) family of proteins, including disease-relevant mutants, domain deletions and tissue-specificisoforms, revealing modular binding preferences determined by structural composition.By comparing wild type with mutant MBD proteins we demonstrate that the recognition ofmethyl-CpGs by functional MBD domains is necessary and sufficient for targeting MBD proteins tomethylated regions in vivo. Quantitative assessment and modeling of DNA methylation-dependenttargeting reveals that binding occurs as a linear function of local methylation density regardless ofcell type, sequence composition or histone modifications. By systematic analysis of domaincontribution to genomic binding, we further identified specific preferences that are determined bydomains required for interactions with DNA or protein complexes. Notably, these recruit a subset ofMBD proteins to tissue-specific regulatory regions in a DNA methylation-independent manner.In summary, this quantitative framework exposes a complex binding behaviour determined bystructural composition and revises current models of DNA methylation readout through MBDproteins.

EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Spore Photoproduct Lyase, a radically different DNA repairenzyme for bacterial spore extreme UV-resistance

Alhosna Benjdiaa,*, Korbinian Heilb, Thomas Barendsa, Thomas Carellb, IlmeSchlichtinga

a Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg,Germanyb Department of Chemistry, Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians University,Butenandtstrasse 5-13, 81377 Munich, Germany

Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to aunique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase). Unlike DNA photolyases whichuse light energy and a fully reduced flavin cofactor to repair UV-induced DNA damages, SP lyaseemploys an alternative and light-independent direct reversal mechanism to repair photolesions. SPlyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes andinvolves a reduced [4Fe-4S] cluster and SAM to initiate the repair reaction. We solved the firstcrystal structure of this enigmatic enzyme in complex with its [4Fe-4S] cluster and its SAM cofactor,in the absence and presence of the DNA lesion, the spore photoproduct (1). The high resolutionstructures provide fundamental insights into the DNA binding mode and the enzyme active site. TheDNA lesion recognition involves a β-hairpin structure and its binding is mostly driven byinteractions with the bases. In the active site, we show that SAM and a conserved cysteine residueare perfectly positioned for hydrogen atom abstraction from the 5'-ring of the lesion and donation tothe 3'-ring of the spore photoproduct, respectively. Based on our structural and biochemicalcharacterizations, we substantiate the role of this cysteine as a hydrogen atom donor. In addition, weidentify another critical residue, tyrosine 98, which is likely involved in the regeneration of theenzyme cofactor for the next repair cycle. Our structure reveals how SP lyase, in a unique fashion,combines specific features of radical SAM and DNA repair enzymes to enable a complexradical-based repair reaction to take place (2).

(1) Benjdia* et al., Nucleic Acids Res. 2012 ;40(18):9308-18.(2) Benjdia*, Curr Opin Struct Biol. 2012 ;22(6):[email protected]


Chemical approaches to Biology and Medicine

Gonçalo Bernardesa,*, Dario Nerib

a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United KingdomInstitute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon,Portugal.b Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

This abstract features reaction engineering for chemical site-selective protein modification, theconstruction of homogeneous, vascular targeting antibody-drug conjugates (ADCs) for cancertherapy and the development of novel carbon monoxide (CO) releasing molecules (CORMs) for thedelivery of therapeutic CO in vivo.

Reaction engineering at cysteine: From disulfides to thioethers, a range of novel reactions andmethods that enable controlled, specific modification of proteins at cysteine were developed.[1,2]

Traceless, homogeneous ADCs for cancer therapy: Using a vascular targeting approach, it wasdemonstrated that a chemically defined, non-internalizing antibody-drug conjugates (ADCs) may beused to mediate strong anti-tumor activity in vivo.[3,4]

Delivery of therapeutic CO in vivo: CO-releasing molecules (CO-RMs) have the potential toconstitute safe treatments if CO release in vivo can be controlled in a spatial and temporal manner.[5]First, we studied the interaction of known CORM-3 with proteins. This results in the loss of achloride ion, glycinate, and one CO ligand.[6] These findings drove the development of a novelCORM with improved solubility and stability that distributes preferentially to the liver and fullyprotects mice against severe forms of malaria.[7]

[1] G. J. L. Bernardes et al., J. Am. Chem. Soc. 2008, 130, 5052-5053.[2] J. M. Chalker, G. J. L. Bernardes, B. G. Davis, Acc. Chem. Res. 2011, 44, 730-741.[3] G. J. L. Bernardes et al. Angew. Chem. Int. Ed. 2012, 51, 941-944.[4] M. Steiner, G. J. L. Bernardes* et al., Chem. Sci. 2013, 4, 297-302.[5] C. C. Romão, W. A. Blättler, J. D. Seixas, G. J. L. Bernardes*, Chem. Soc. Rev. 2012, 41,3571-3583.[6] T. Santos-Silva, A., G. J. L. Bernardes* et al., J. Am. Chem. Soc. 2011, 133, 1192-1195.[7] A. C. Pena, G. J. L. Bernardes* et al., Antimicrob. Agents Chemother. 2012, 56, 1281-1290.


Non-invasive high speed imaging of fast physiological processesin awake and freely moving mice

Oliver Brunsa,*, Thomas Bischofa, Daniel Harrisa, Moungi Bawendia

MIT - Department of Chemistry

Non-invasive imaging of physiological processes like heart rate, breathing motion, blood perfusionand dynamic behavior of the gastro intestinal tract is of great interest forpreclinical research relatedto cardiovascular disease, hypertension or behavioral sciences.

So far the imaging techniques available to study this processes in high resolution all require animalsto be fixed in one position. Therefore anesthesia is used if live animals are studied. This has greatlimitations in terms of experiment time. More importantly, the anesthesia massively disturbs thephysiological processes desired to study.

SWIR or NIR-II imaging (between 900 nm and 1700 nm) had been shown to be the ideal modalityfor non-invasive imaging. Due to the suboptimal emission properties of the materials used previously(e.g. single walled carbon nanotubes (SWCNTs)) fast imaging rates of 25 to 50 frames per secondcould not be archived.

Here we use InAs/CdZnSe core-shell quantum dots, which emit in the SWIR region with a quantumyield of 30% after functionalization in physiologic solution. This emission intensity is two orders ofmagnitude higher than that of SWCNTs used previously for non-invasive SWIR imaging. With thisquantum dots we have enough emission signal to move away from anaesthetized mice to studyphysiological processes.

Now we can image physiological parameters like heart rate, breathing rate and perfusion as well asenergy metabolism in conscious and freely behaving mice.


The Geminin and Idas coiled coils form a preferentiallyheterodimeric structure which affects Geminin function in DNAreplication licensing

Christophe Caillata,*, Eleftheria-Dafni Pefanib, Peter J. Gillespiec, Zoi Lygeroub,Anastassis Perrakisd, et al.a Unit for Virus Host-Cell Interactions (UVHCI), 38042 Grenoble, Franceb Laboratory of Biology, School of Medicine, University of Patras, 26505 Rio, Patras, Greecec Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, UnitedKingdomd Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands

In metazoans Geminin is an important regulator of proliferation and differentiation. Gemininprevents genome over-replication by inhibiting the DNA replication licensing factor Cdt1, but themechanisms coordinating cell proliferation to differentiation processes remain unclear. Two proteinscontaining a coiled-coil domain similar to Geminin : Idas (or Multicillin) and GEMC1 were recentlyidentified. Idas and GEMC1 being also involved in cell proliferation and/or cell differentiation weinvestigated their possible cooperation with Geminin. We show that Geminin preferentially formsstable coiled-coil mediated heterodimers with its homologue, Idas. In contrast to Idas:Gemininheterodimers, the Idas homodimers are thermodynamically unstable and Idas is unlikely to exist as astable species on its own under physiological conditions. The crystal structure of a complex of thehomology regions of Idas and Geminin forms a tight head-to-head heterodimeric coiled-coil thatexplains well the properties of the complex. This Idas:Geminin heterodimer binds Cdt1 less stronglythan Geminin:Geminin, still with high affinity, and with notably different thermodynamic properties.Consistently, in Xenopus egg extracts, Idas:Geminin is less active in licensing inhibition compared toa Geminin:Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limitedover-replication, which is counter-acted by Geminin co-expression. The properties of theIdas:Geminin interaction suggest it as a possible mechanism to modulate Geminin activity in cells.


Rapid reactivation of X-linked genes upon reprogramming ofhuman somatic cells by cell fusion

Irene Cantonea,*, Hakan Bagcia, Matthias Merkenschlagera, Neil Brockdorffb,Amanda G Fishera

a MRC Clinical Sciences Centre, Imperial College of London, London (UK)b Department of Biochemistry, University of Oxford, Oxford (UK)

X chromosome inactivation is a dosage compensation mechanism by which female somatic cellsensure gene expression from only one of the two X chromosomes in mammals. Repression of geneson the inactive X chromosome (Xi) is a major example of stable silencing that is maintained forlifetime. Reactivation of the Xi is associated with pluripotency and has been widely used as a modelto study the erasure of epigenetic memory in mouse cells. In human, the status of the two Xchromosomes in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) showconsiderable epigenetic variation depending on culture conditions, thus preventing the analysis of themolecular mechanisms that lead to X reactivation in pluripotency and reprogramming.Here, we used an inter-species cell fusion system whereby diploid female human fibroblasts (hF) arereprogrammed via fusion with mouse ESCs and showed that chromatin structural changes andbiallelic expression of X-linked genes on the Xi are detected in the human nucleus of heterokaryonsearly during reprogramming.Delocalization of XIST and loss of H3K27me3 enrichment from the Xi are the first events that occurtwo days after fusion, concomitantly with de novo expression of pluripotency genes. SequentialRNA/DNA-FISH and SNP-expression analysis show that these changes on the Xi are followed bypromoter DNA demethylation and reactivation of at least some X-linked genes. Biallelic expressionof X-linked genes occurs in a subset of cells in which XIST has been delocalized, thus suggestingthat the reorganization of XIST/H3K27me3 chromatin domain is not sufficient, although necessary,for gene reactivation. Our findings suggest a step-wise reactivation process that may entail onlyspecific heterochromatic regions of the Xi. Future studies will therefore investigate the reactivationof genes along the Xi by analyzing the expression of polymorphisms, during reprogramming ofclonal fibroblast populations, via allele-specific RNA sequencing approaches.


Real time dynamics of RNA Polymerase II clustering in livehuman cells

Ibrahim Cissea,*, Ignacio Izeddinb, Sebastien Causseb, Lydia Boudareneb, AdrienSenecalb, et al.a Howard Hughes Medical Institute, Chevy Chase, MD, USEcole Normale Superieure, 46 Rue d'Ulm, 75005 Paris, Franceb Ecole Normale Superieure, 46 Rue d'Ulm, 75005 Paris, France

Transcription has been reported to be spatially compartmentalized in transcription factorieshypothesized as clusters of RNA Polymerase II (Pol II). However, little is known of when these fociassemble, and how stable they are. We report a single-cell quantitative method to characterize proteinclustering both spatially and temporally, with single-molecule sensitivity in live eukaryotic cells. Weobtained super-resolution images consistent with clustered Pol II in the nucleus. Yet, Pol II clustersformed very transiently, with an average lifetime of 5.1 (± 0.4) s, refuting their notion as staticallyassembled nuclear substructures. Stimuli affecting the cells transcriptional state yielded orders ofmagnitude changes in the distribution and dynamics of Pol II clusters, implying that clustering isregulated and plays a role in the cells ability to effect rapid response to external signals. Ourobservations suggest transient crowding of enzymes may aid in rate-limiting steps of multi-component macromolecular assembly in vivo.


Cell type-specific Gene Regulatory Network of the PolycombRepressive Complex 2 (PRC2) in Arabidopsis

Miguel De Lucasa,*, li pua, François Roudierb, Siobhan bradya

a Department of Plant Biology and Genome Center, University of California - Davis, Davis, USA.b Institut de Biologie de l'Ecole Normale Supérieure, Centre National de la Recherche Scientifique (CNRS), Paris, France

Chromatin is a dynamic structure that responds to numerous stimuli to regulate DNA accessibility.The covalent modification of histones is the principal process used by the cell to modulatenucleosome mobility and turnover. The Polycomb Repressive Complex 2 (PRC2) catalyzes thetrimethylation of Histone 3 at lysine 27 (H3K27me3), which can act as a repressive chromatin mark.The plethora of key developmental processes regulated by PRC2 proteins has raised interest from awide range of fields.

PRC2 composition is conserved from humans to plants, but their spatiotemporal regulation andfunction has not been comprehensively characterized. Arabidopsis thaliana has 12 homologs ofDrosophila PRC2 subunits. The three PRC2 complexes described in Arabidopsis control theexpression of key genes involved in developmental transitions. It is also known that PRC2complexes are required to maintain organ identity and to keep cells appropriately differentiated.However, their role in these processes remains unknown.

Our goal is to describe the role of PRC2 in controlling cell proliferation and differentiation in theArabidopsis root.

We demonstrate that PRC2 subunits are differentially expressed among the different root cell types,being highly present in the dividing/pluripotent cells of the meristem and the cambium. To betterunderstand PRC2 spatiotemporal regulation and function, we have identified a transcriptionalregulatory network that controls PRC2 expression in the root vasculature. We have also generatedvascular-specific profiling of the H3K27me3 and H3K4me3 marks and correlated it with vascular-specific gene expression. This multi-layered transcriptional regulatory network (TF-PRC2genes-H3k27me3) will allow us to understand how epigenetic modifications are dynamically regulated inspace and time to control cell type and tissue specification, differentiation and function.


Identification of biological variation between single-cells usingRNA-Seq

Idan Efronia,*, Ip Pui-Lenga, Nawy Tala, Mello Alisona, Birnbaum Kennetha

New York University, Center for Genomics and Systems Biology, 12 Waverly Place, 10003, USA

Global profiling of single cells has the potential to reveal coordinated transcriptional regulation at thecellular level that is otherwise obscured by sampling of heterogeneous cell populations. However,single-cell experiments are characterized by high levels of technical noise, and traditional methodsfor identification of differentially expressed genes are inapplicable to single-cell assays, as they relyon biological replicates. We developed an approach to identify biological variation that exceedstechnical noise in single-cell experiments. Technical noise distribution is determined empiricallyfrom pooled-and-split cell samples, and the resulting probability function is used to deriveconfidence measures for biological cell-to-cell variation. We applied this method to cells from twotissues of the Arabidopsis root: the highly homogenous Quiescent Center (QC), which supports theroot stem cell niche, and from the meristematic stele, which includes vascular cells at early stages ofdifferentiation. Cell-type specific markers, including transcription factors with known functions,were among the most highly expressed genes, and the identity of single cells could be robustlydetermined. Using our statistical approach, we have detected hundreds of genes that varysignificantly among cells of the same cell type. Notably, we detected multiple coherent gene modulesthat varied together, such as ribosomal proteins and auxin biosynthesis genes in the QC, andvalidated this coregulation with single root data. Thus, our method allows the detection of biologicalvariation among single-cell profiles and shows promise in the characterization of novel cellularstates.


Thymic epithelial cell expansion through matricellular proteinCYR61 boosts progenitor homing and T-cell output

Yalin Emrea,*, Magali Irlaa, Isabelle Dunand-Sauthiera, Romain Balleta, ChristianVesinb, et al.a University of Geneva, Pathology and Immunology, Switzerlandb University of Geneva, Physiology and Metabolism, Switzerland

Thymic epithelial cells (TEC) are phenotypically and functionally heterogeneous stromal cells thatgenerate microenvironments required for the formation of T-cells within the thymus. TEC control themigration, proliferation and selection of differentiating thymocytes, and defects in TEC impair thenormal development of T-cells leading to immunodeficiency or autoimmunity. Here we show thattreatment with CYR61 (cysteine-rich protein 61), a matricellular protein implicated in cellproliferation and migration, improves thymus function and T-cell development. We identify TEC asthe major source of CYR61 and show that binding of CYR61 to LFA1, ICAM1 and integrin α6supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobeswith recombinant CYR61 expands the stromal compartment by inducing thymocyte-independentproliferation of TEC via activation of the Akt signaling pathway. In addition, engraftment of CYR61overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output viaexpansion of TEC. This increases the space for the recruitment of circulating hematopoieticprogenitors and the development of T-cells. Our discovery paves the way for therapeuticinterventions designed to restore thymus stroma and T-cell generation.


Role of par-1 serine/threonin kinase in synaptic transmission

Clara Essmanna,*, Emma Hileya, Stephen Nurrisha

MRC LMCB University College London

During my EMBO long-term fellow ship I worked with Stephen Nurrish at the MRC Laboratory forMolecular and Cellular Biology in London to study regulation of synaptic function using C. elegans.I have been able to demonstrate a novel function of the polarity gene PAR-1 in control of activity atthe neuromuscular junction. Splicing produces many different PAR-1 isoforms. I identified amutation (nz90) as being defective in the expression of a new spliceform, PAR-1L. PAR-1 belongs tothe family of par-proteins (“partitioning defective” par-1-6) that are highly conserved key players toestablish cell polarity during the first cell division. Moreover, par-proteins and their orthologues havebeen shown to mediate and maintain cell polarity in polarized cells such as neurons, where they arethought to be involved in the development of Alzheimers disease. However, until now, a role forPAR-1 in C. elegans neuronal function has not been demonstrated. Contraction of C. elegans musclesis caused by release of acetylcholine (ACh) by cholinergic motorneurons, relaxation is caused byrelease of GABA from GABA-ergic motorneurons. par-1(nz90) has a defect in Ach-release asdemonstrated by both resistance to acetylcholinesterase inhibitors, and by electrophysiologicalrecordings of the muscle. However, rescue experiments demonstrate that PAR-1 acts in theGABA-neurons. Rescue does not require PAR-1 kinase activity suggesting PAR-1 acts as a scaffoldin neuronal signalling, again a novel property for the PAR-1 protein. Experiments with synapticmarkers suggests a small but significant increase in pre-synaptic release sites in the GABA-neuronsof par-1(nz90) mutants. Given the effect on Ach-release by par-1(nz90) we speculate that theGABA-neurons are forming extra synapses onto the cholinergic motorneurons and cause theirinhibition. In support of our model par-1(nz90) inhibition of Ach-release requires GABA-synthesis.Currently we are identifying the GABA-receptors by which the par-1(nz90) mutation acts to inhibitACh release.


A two-step mechanism for epigenetic specification of centromereidentity and function

Daniele Fachinettia,*, Diego Folcoa, Arshad Desaia, Lars Jansenb, Don Clevelanda

a Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California atSan Diego, La Jolla, CA 92093, USAb Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, P-2780-156, Oeiras, Portugal

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotesby an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containingthe centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark thatacts through a two-step mechanism to identify, maintain and propagate centromere functionindefinitely. Initially, centromere position is replicated and maintained by chromatin assembled withthe centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleationof kinetochore assembly onto CATD-containing chromatin is shown to require either CENP-A’samino- or carboxy-terminal tails for recruitment of inner kinetochore proteins, including stabilizingCENP-B binding to human centromeres or direct recruitment of CENP-C


Identification of new pathways inducing axonal degeneration

Konstantin Feinberga,*, Alessandro Dattib, David Kaplana, Freda Millera

a The Hospital for Sick Children, Molecular Biology Department, Toronto, Canadab Samuel Lunenfeld Research Institute - Mount Sinai Hospital, Toronto, Canada

The cognitive and motor dysfunction that occurs in neurodegenerative disorders and neurotrauma isdue to the loss of appropriate functional neural connections as a consequence of the neuronal deathand/or degeneration of axons. My objectives are to identify the molecular mechanisms that underlieaxonal degeneration and to discover an effective way to prevent it. To address this, I asked whetherkinase inhibitors could be identified that prevent axon degeneration in cultured primary sympatheticor sensory neurons withdrawn from survival factors. Using a library of 480 kinase inhibitors,including over 110 in clinical trials or use, I identified a number of drugs that dramatically preventedaxon degeneration, including inhibitors of the activities of growth factor receptors such as EGFR,VEGFR and c-MET, intracellular kinases including GSK3β and c-Abl, and kinases that regulate thecell cycle and DNA replication, such as Chk and Cdk. Three of the most potent drugs used for cancerindications and that are known to cross the blood brain barrier, Ponatinib and Bosutinib (Abl/Srcinhibitors) and Foretinib (VEGFR/c-MET inhibitor) were chosen for further analysis. These drugsdelayed or prevented axonal death for up to seven days when applied only to axons incompartmented chambers. Foretinib also delayed neuronal degeneration caused by chemotherapeutictreatment, and delayed Wallerian degeneration, where axons are physically detached from their cellbodies. We are now identifying using phosphoproteomics the targets of these drugs in axons, anddetermining whether they prevent axon degeneration in rodent models of Parkinson’s Disease anddiabetic neuropathy, and the degeneration of septal cholinergic neurons that are preferentiallyvulnerable in Alzheimer’s disease (fimbria-fornix lesions). We believe that this work will result inthe discovery of novel treatments for neurodegeneration caused by disease or trauma.


Tollip modulates EGFR signaling

Malgorzata Garstkaa,*, Ilana Berlina, Hans Janssena, Magdalena Szczygiela, JacquesNeefjesa, et al.NKI-AVL, Amsterdam, The Netherlands

Tight control of Epidermal Growth Factor Receptor (EGFR) signaling is required for proper cellgrowth and development. To terminate EGFR signaling, receptor is ubiquitylated and delivered toendosomes where upon action of ESCRT proteins EGFR is incorporated into intralumenal vesicles ofthe Multivesicular Body.

Tollip depletion results in increased EGFR surface levels and delays EGFR degradation. Tollipdepletion also causes significant morphological changes of the endocytic pathway - abnormallyenlarged MVB compartment characterized by a decreased amount of intralumenal vesicles comparedto the control.

Tollip localizes to cytosol and throughout the endocytic pathway – from the plasma membrane tolysosomes. Tollip binds to Tom1L1, and two of ESCRT components - Eps15 and Hrs, andparticipates in the delivery of ubiquitylated EGFR from the plasma membrane to the endosomes.Membrane binding, ubiquitin binding and dimerization of Tollip are required for its function.

Tollip controls the vesiculation at the Multivesicular Body and is involved in ESCRT-dependentEGFR delivery to lysosomes.


SwissSidechain: a molecular and structural database ofnon-natural sidechains

David Gfellera,*, Olivier Michielina, Vincent Zoetea

Molecular Modeling, Swiss Institute of Bioinformatics, Lausanne, Switzerland

Amino acids form the building blocks of all proteins. Naturally occurring amino acids are restrictedto a few tens of sidechains. Yet, the potential chemical diversity of amino acid sidechains is nearlyinfinite. Exploiting this diversity using non-natural sidechains has recently found widespreadapplications. With the SwissSidechain database (http://www.swisssidechain.ch), we offer a centraland curated platform for non-natural sidechains. SwissSidechain provides biophysical, structural andmolecular data for hundreds of commercially available non-natural amino acid sidechains. Wefurther provide plugins to seamlessly insert non-natural sidechains into peptides and proteins usingmolecular visualization software, as well as topologies and parameters compatible with molecularmechanics software. Recent experimental results on a phage display optimized ligand of urokinaseplasminogen activator show how expanding the building blocks of peptides and proteins withnon-natural sidechains is powerful to develop potent inhibitors of protein interactions.


p27Kip1 is a microtubule-associated protein that promotesmicrotubule polymerisation during neuron migration

Juliette Godina,*, Sophie Laguessea, Noémie Thomasa, Lina Malinouskayaa, PierreCloseb, et al.a GIGA-NeurosciencesInterdisciplinary Cluster for Applied Genoproteomics (GIGA-R), University of Liège, C.H.U. Sart Tilman, Liège 4000,Belgiumb GIGA-Signal TransductionInterdisciplinary Cluster for Applied Genoproteomics (GIGA-R), University of Liège, C.H.U. Sart Tilman, Liège 4000,Belgium

The migration of cortical interneurons is characterized by extensive morphological changes thatresult from successive cycles of nucleokinesis and neurite branching. Their molecular bases remainelusive and the present work describes how p27Kip1 controls cell cycle unrelated signaling pathwaysto regulate these morphological remodelings. Live imaging reveals that interneurons lackingp27Kip1 show delayed tangential migration resulting from defects in both nucleokinesis anddynamic branching of the leading process. At the molecular level, we described p27Kip1 as a novelmicrotubule-associated protein that promotes polymerisation of microtubules in extending neurites,thereby contributing to tangential migration. Furthermore, we show that p27Kip1 controlsactomyosin contractions that drive both forward translocation of the nucleus as well as growth conesplitting. Thus, p27Kip1 cell-autonomously controls nucleokinesis and neurite branching byregulating both actin and microtubule cytoskeletons.


Mitochondrial protein import caught in the act: a study byelectron cryo-tomography

Vicki Golda,*, Raffaele Ievab, Martin van der Laanb, Nikolaus Pfannerb, WernerKühlbrandta

a Max-Planck-Institute of Biophysics, Frankfurt, Germanyb Institute for Biochemistry and Molecular Biology, Freiburg, Germany

Mitochondria import a plethora of proteins into four distinct compartments, via complex andversatile networks of dynamic targeting pathways. A major gateway into mitochondria is via theTOM and TIM23 translocases, which form macromolecular complexes spanning the outer and innermembranes respectively. Due to an intensive biochemical effort, the fundamentals of proteintransport by TOM/TIM23 are understood, yet there is inadequate structural information. Our goal isto directly visualise the protein import machinery in action within whole mitochondria, by thetechnique of electron cryo-tomography. This is achieved by stalling a pre-protein substrate throughboth channels concurrently, creating the bona fide import supercomplex.

A novel substrate has been designed, which by the virtue of a tightly folded domain (DHFR), has theability to arrest whilst crossing both complexes and membranes simultaneously. It contains a specificC-terminal electron dense tag to assist localisation on the mitochondrial surface when viewed in themicroscope. Overexpression and purification of the substrate has been successfully optimised in E.coli. Biochemically, this preprotein has been shown to be functional by in vitro import into energisedS. cerevisae mitochondria and subsequent detection of the TOM/TIM23 supercomplex bynative-PAGE.

Tomograms have been collected which show the number and distribution of pre-protein import siteson the mitochondrial surface. Early data pinpoints specific sites of protein translocation, which wenow aim to investigate in more detail. The protein complexes found at these sites will be analysedwith the ultimate goal of carrying out subtomogram averaging. This information will be used todetermine the three dimensional organisation of translocation sites in situ.


Structure determination of membrane proteins in a nativeenvironment

Franz Hagna,*, Manuel Etzkorna, Thomas Raschlea, Gerhard Wagnera

Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, MA 02115, USA

Structural studies of membrane proteins are still hampered by difficulties in finding appropriatemembrane mimicking media that maintain protein structure and function. Here, I will describe anovel membrane system, called phospholipid nanodiscs, that consists of a patch of lipids and twocopies of a lipid-binding protein that wrap around these lipids and form disc-shaped particles. Usingthis system, membrane proteins can be investigated in a real detergent-free and native-like lipidenvironment. I will show our recent success in performing structure determination of membraneproteins in this system with NMR spectroscopy. Furthermore, I will show unpublished data onstructural studies of mitochondrial membrane proteins that play a key role in diseases like heatfailure and cancer. Finally, I will present data on our studies of G-proteins that bind to thepharmaceutically important class of G-protein-coupled receptors and will eventually outline ourplans for future work.


Structural basis of the translational regulation of msl2 mRNA bySXL and UNR during dosage compensation in Drosophila

Janosch Henniga,*, Cristina Milittib, Gregorz Popowiczc, Iren Wanga, MiriamSonntaga, et al.a Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany &Center for Integrated Protein Science Munich (CiPSM) at Biomolecular NMR, Department Chemie, Technische UniversitätMünchen, 85747 Garching, Germanyb Centre for Genomic Regulation (CRG), Gene Regulation, Stem Cells and Cancer Programme, 08003 Barcelona, Spainc Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany

The protein Upstream of N-Ras (UNR) is a key regulator of gene expression at the translational levelin both humans and Drosophila. In Drosophila, the role of UNR in dosage compensation is wellcharacterized. UNR and the female-specific protein Sex-lethal (SXL) bind cooperatively to the 3’UTR of msl2 mRNA, which encodes the rate-limiting subunit of the dosage compensation complex.This interaction represses the translation of msl2 mRNA thereby blocking dosage compensation infemale flies. To unravel the molecular mechanisms of these interactions we studied the structuralbasis for the assembly of the SXL-UNR-msl2 ribonucleoprotein complex.

UNR consists of five cold-shock domains, where only the first of these domains (CSD1) is requiredfor the interaction with msl2 mRNA and SXL. On the other hand, only the RNA binding domains ofSXL, contained in a fragment called dRBD4, are required for the interaction.

Here, we present the crystal structure of the complex of dRBD4-CSD1 bound to its interaction regionin msl2 mRNA, validated by NMR spectroscopy and small angle neutron/X-ray scattering. Thestructure reveals new insights into how the cooperative and novel interaction between an RNArecognition motif (Sxl), a cold shock domain, and mRNA triggers translational repression in thedevelopment of fruit flies. Based on this structure, a detailed mutational analysis provided furtherinsights which will be presented.


Uncoupling of the endocannabinoid signalling complex in amouse model of fragile X syndrome

KM Junga, M Sepersb, Chris Henstridgec,*, O Lassalled, D Neuhoferd, et al.a Department of Anatomy and Neurobiology, University of California, Irvine, California 92697, USA.b INSERM U862, Circuit and Dendritic Mechanisms Underlying Cortical Plasticity Group, Neurocentre Magendie, 146Rue Léo-Saignat, F 33077 Bordeaux Cedex, France.c Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary.d INSERM U901, Marseille 13009, France.

Fragile X syndrome (FRAX), the most common inherited form of mental disability, is caused by lossof fragile X mental retardation protein (FMRP), an RNA-binding protein that controls glutamatemGlu5 receptor-dependent signaling. Enhanced long-term depression (LTD) is widely considered asthe major synaptic pathophysiological consequence of exacerbated mGlu5 signaling. However,unexpectedly, we found that another mGlu5- and also endocannabinoid-dependent form of LTD iscompletely absent in a mouse model of FRAX. We found that spatial and functional uncoupling ofthe endocannabinoid signalosome, a perisynaptic macromolecular complex present in dendriticspines that links mGlu5 receptors to the 2-arachidonoylglycerol (2-AG)-producing enzymediacylglycerol lipase-α, accounts for this impaired synaptic plasticity at excitatory synapses inseveral forebrain areas. To assess anatomical uncoupling, we performed a detailed electronmicroscopy study, which revealed that the perisynaptic accumulation of mGlu5 remained intact inboth groups. In contrast, DGL-α distribution was concentrated perisynaptically at glutamatergicsynapses in wild type, but not in FMRP-KO mice. Instead, the levels of DGL-α within the spine headcytoplasm were significantly higher in FMRP-KO animals and intriguingly, increased DGL-αlabeling was found in the spine neck in FMRP-KO mice. This spatial alteration in theendocannabinoid signalosome may explain reduced versus enhanced endocannabinoid signaling atglutamatergic or GABAergic synapses reported earlier, respectively, and may lead to a shift in thebalance of excitatory/inhibitory synaptic plasticity in FMRP-knockout animals. To test whetherpharmacological enhancement of 2-AG signaling normalizes this perturbed plasticity in FMRP-KOmice, we blocked either MGL or ABHD6, two 2-AG degrading enzymes, which restored LTD atglutamatergic synapses and reversed behavioural abnormalities in the FMRP-KO. Thus, theregulation of 2-AG metabolism may provide a new approach to alleviate some phenotypicconsequences of FRAX.


Toward a structural understanding of kinetochore architecture

Jenny Kellera,*, Arsen Petrovica, Anika Altenfelda, Andrea Musacchioa

Max Planck Institute of Molecular Physiology, Otto-Hahn-str.11, 44227 Dortmund, Germany

High fidelity of chromosome segregation during mitosis is essential for maintaining the integrity ofthe genome. If the process is perturbed, cells can gain or lose chromosomes and become aneuploid.In some cases, surviving cells may proliferate without regulation leading to chromosome instability(CIN) and tumor formation (Cleveland et al., 2003; Diaz-Rodriguez et al., 2008). Deciphering themolecular mechanisms of accurate chromosome segregation is therefore important to understandtumorigenesis. Kinetochores are multi-protein complexes that play a central role in the correctseparation of chromosomes during mitosis (reviewed in Santaguida and Musacchio, 2009). Aconserved complex known as the KMN (for Knl1 complex, Mis12 complex, Ndc80 complex)network is essential for kinetochore/microtubule attachment and for the feedback mechanisms(Spindle Assembly Checkpoint, SAC) that help to maintain ploidy in mitosis. At present,high-resolution structural information on the KMN network is only available for the NDC80complex (whose structure was determined in the Musacchio laboratory), whereas the remaining 6subunits have not been characterized. In the work presented, we show the structural organization ofthe KMN network and its role in the recruitment of the Rod-Zw10-Zwilch (RZZ) complex, which isessential for the SAC.


Host cell entry of Respiratory Syncytial Virus involvesmacropinocytosis followed by proteolytic activation of the Fprotein

Magdalena Krzyzaniaka,*, Ari Heleniusa

ETH Zurich, Institute of Biochemistry, Schafmattsrasse 18, Zurich 8093, Switzerland

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae thatcauses severe respiratory tract infections. Reports in the literature have indicated that to infect cellsthe incoming viruses either fuse their envelope directly with the plasma membrane or exploitclathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa,A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to followvirus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. Avariety of perturbants were employed to characterize the cellular processes involved.We found that immediately after binding to cells RSV activated a signaling cascade involving theEGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actinrearrangements; the cells rounded up, plasma membrane blebs were formed, and there was asignificant increase in fluid uptake. If these effects were inhibited using compounds targetingNa+/H+ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV wasrapidly and efficiently internalized by an actin-dependent process that had all hallmarks ofmacropinocytosis. Rather than fusing with the plasma membrane, the viruses thus enteredRab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average50 min after internalization. Rab5 was required for infection.To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses,we analyzed the fusion protein, F, and could show that, although already cleaved by a furin familyprotease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavageby a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious.


Strong heterogeneity in mutation rate causes misleadinghallmarks of natural selection on indel mutations

Erika Kvikstada,*, Laurent Duretb

a The Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdomb LBBE, UCBL- Universite Lyon 1, Lyon, France

Elucidating the mechanisms of mutation accumulation and fixation is critical to understand thenature of genetic variation and its contribution to genome evolution. Of particular interest is therelative impact of selection vs. non-adaptive forces operating on insertions and deletions (indels).Here, we take advantage of recent population-scaled sequencing efforts, together with whole-genomesequence alignments, that provide unprecedented datasets of indel polymorphism and divergence forthe analysis of indel fixation dynamics. Briefly, we investigated the derived allele frequency (DAF)spectra for short (1-50 base pairs) indel variation identified in the 1000 Genomes Pilot 1 data. Inconjunction with comparisons of indel diversity vs. divergence in a modified McDonald-Kreitmanapproach, our analyses revealed an apparently higher fixation probability for insertions thandeletions. This fixation bias scales with the local mutation rate and is particularly pronounced atindel hotspot loci. In non-repetitive (NR) regions, the DAF and McDonald-Kreitman tests areincongruent with the action of widespread selection on indels. Furthermore, we identified anunprecedented number of loci in NR contexts with evidence for multiple indel events in the primatephylogeny. According to a model of uniform indel rate evolution, such loci are 60-fold more frequentthan expected, thus providing evidence of as yet unidentified “cryptic” indel mutation hotspots. Wepropose that indel homoplasy, at known and cryptic hotspots, produces systematic errors indetermination of derived vs. ancestral alleles that in turn lead to misidentification of insertions vs.deletions via parsimony. Given the strong heterogeneity in indel mutation rate across the genome,classic selection tests should be interpreted with caution due to the sensitivity of parsimony methodsto indel homoplasy. These results are predicted to have great impact on additional studies seeking toinfer evolutionary forces operating on indels observed in closely related species, since suchmutations are traditionally presumed to be homoplasy-free.


Angiogenesis controls neurogenesis in the developing cerebralcortex

Christian Langea,*, Ruben Boona, Christine Wub, Calvin Kuob, Peter Carmelieta

a Laboratory for Neurogenesis and the Neurovascular Link, Vesalius Research Center, VIB and KU Leuven, Leuven,Belgiumb Division of Hematology, School of Medicine, Stanford University, San Francisco, USA

Neural stem cells (NSCs) regulate their expansion and differentiation according to instructive signalsfrom their cellular microenvironment, the stem cell niche. Blood vessels are thought to be an integralpart of the stem cell niches in the of the developing and the adult brain. However, the functionalsignificance of blood vessels for NSC maintenance and the underlying molecular mechanisms arepoorly defined.Here, we show that angiogenesis is coupled with the switch from NSC expansion to neurogenesisduring development of the mouse cerebral cortex. Moreover, specific inhibition of brain angiogenesisreduces the switch to neurogenesis in the cortex, demonstrating for the first time a causal role ofblood vessels in this process.Contrary to previous models, we find that reduction of blood vessels in the NSC niches augmentsproliferation and expansion specifically of NSCs, while reducing their differentiation. Finally, ourresults suggest oxygen delivery as a major mechanism of blood vessel influence on the switch toneurogenesis since mimicking hypoxia signaling in the NSCs, phenocopies the effect of angiogenicinhibition. Taken together, these findings provide novel, unprecedented insights how blood vesselsregulate NSC proliferation and differentiation in their in vivo niche.


Bone-derived Osteocalcin regulates muscle mass and function

Kathrin Julia Lauea,*, Olga Sumaraa, Marc McKeeb, Gerard Karsentya

a Columbia University, New York, USb McGill University, Montreal, Quebec, Canada

Sarcopenia, the decline of muscle mass and function, is a main characteristic of organismal aging anda major public health problem. Remarkably, the onset of muscle decline during aging is concurrentwith a loss of bone mass suggesting that both tissues could share one or several regulatorymechanisms. However, the signals that coordinate the functions of the musculo-skeletal system arepoorly understood.Osteocalcin, a peptide hormone secreted by osteoblasts, regulates physiological processes known todecline with age such as glucose homeostasis and male fertility. During exercise, the circulatinglevels of active osteocalcin increase. We therefore tested whether osteocalcin and its putativereceptor, Gpcr6a, could regulate muscle mass and function.Indeed, upon physical challenge on a treadmill, the distance and time Osteocalcin-/- mice can run aresignificantly lower compared to their wild type littermates. Accordingly, these mice exhibitdecreased oxidative capacity and defects in the uptake of nutrients in early adulthood. At later stages,osteocalcin-deficient mice show a decrease in muscle mass and myofiber diameter as well as areduction in structural protein levels. In addition, osteocalcin is required for muscle regenerationafter chemical-induced injury.Our findings identify osteocalcin as one of the long sought after molecules linking bone and muscletissue suggesting that a decrease in osteocalcin level during aging could contribute to thedevelopment of sarcopenia.


Structure of Biological Supramolecular Assemblies Solved bySolid-State NMR

Antoine Loqueta,*, Nikolas Sgourakisb, Karin Gillera, Jean Philippe Demersa,Rashmi Guptac, et al.a Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germanyb University of Washington, Seattle, WA 98195, USAc Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany

The assembly of multiple protein subunits into large supramolecular complexes plays a key role inmany cellular processes, notably in bacterial virulence, neurodegenerative disease-relatedmechanisms, and as a part of nanomachines. 3D structures of these supramolecular assemblies arestill rare as they remain often recalcitrant for atomic-resolution techniques. First, their elongatedshape, complexity, and lack of long-range order are severe obstacles to grow single crystalsamenable to X-ray crystallography. Second, their inherent insolubility restricts the use of solutionNMR.

Here, I will show that state-of-the-art Solid-State NMR methodology (1-3) is able to reveal thesupramolecular interfaces and ultimately the complete atomic structure of a protein assembly. Ourapproach is demonstrated on the Salmonella typhimurium Type III Secretion System Needle, abacterial filament that mediates the injection of pathogen effector proteins to the cytosol of host cellsduring bacterial infection. Using an integrated approach combining Solid-State NMR, STEM, andcomputational modeling, we present an atomic resolution model of the Needle (4) in its nativefilamentous state.

(1) Loquet et al., J. Am. Chem. Soc. 2010(2) Loquet et al., J. Am. Chem. Soc. 2011(3) Loquet et al., Acc. Chem. Res. 2013(4) Loquet et al., Nature 2012


The yeast Mph1 helicase alters telomere structure by disruptingthe protective fold-back structure

Sarah Luke-Glasera,*, Brian Lukea

Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69112Heidelberg, Germany

The yeast helicase, Mph1 is similar in sequence to human FANCM and displays many overlapping invitro biochemical properties with human RTEL1. Recently, RTEL1 has been implicated in theresolution of t-loop structures at telomeres. Through chromatin immunoprecipitation experiments wefound that Mph1 localizes to yeast telomeres. Furthermore, over-expression of Mph1 results in theloss of a fold-back structure at natural telomere 6R. Consistent with the fold-back structure providinga protective role, we observed toxicity in CST mutants and rapid senescence in telomerase mutantsupon Mph1 overexpression due to exacerbated nuclease-mediated resection within the telomericrepeats. Importantly, when we overexpressed an allele of Mph1 (Mph1 delta 60) that is unable tointeract with Smc5, we did not detect telomere dysfunction phenotypes. The SMC5/6 proteincomplex forms a cohesion-like ring structure that has also been shown to localize to telomeres.smc5/6 temperature sensitive mutants display rapid cellular senescence in the absence of telomeraseand this is rescued upon the further deletion Mph1. Furthermore, smc5/6 mutants, like Mph1over-expression, show a defect in telomeric fold-back structure. Together these data suggest that theSMC5/6 complex may be responsible for the tight regulation of the Mph1 helicase at telomeres inorder to resolve the telomere fold-back structure at the appropriate time and place.


Investigation of phosphorylation mediated signaling in mitosisby targeted mass spectrometry approaches

Alessio Maiolicaa,*, Fabrizio Villab, Erwin Schoofc, De Metondo Mariaa, RuediAebersolda, et al.a ETH, Zurich, Switzerlandb IEO, Milano, Italyc Technical University of Denmark, Lyngby, Denmark

The mitotic cell cycle is a strictly ordered sequence of events that determine dramatic changes in thecells.A central regulatory system accurately coordinates, mainly by means of phosphorylation and proteindegradation the ordering of all the steps by initiating each event at the appropriate time.Advances in phosphoproteomics made possible the identification of thousands regulated phosphosites in mitosis, offering a view of the mitotic phosphoproteome regulation at a “systems level”.However very little is known about the contribution of every kinase to the complex regulatorylandscape of all mitotic events. Phosphorylation mediated signaling events are, virtually always,organized as cascades of reactions where each kinase or phosphatase can modulate the activation ofother signaling molecules by phosphorylation or de-phosphorylation events. Reproduciblequantification of every phosphorylation site is therefore required for a complete understanding ofbiological process such as mitosis.Here we report a novel strategy, base on kinase reaction screening, for the identification of kinases’substrates by targeted mass spectrometry methods.

The approach consists of four phases:

1)Kinase purificationFunctional enzymes are purified from stable cell lines inducibly expressing affinity-tagged kinasesand/or recombinantly expressed in ectopic system.

2)Peptide library constructionSubstrate peptides are opportunely selected form the literature and chemical synthetized in theirunphosphorylated form on a large scale.

3)Multiplex in vitro kinase assay on peptide librariesThe phosphopeptides, formed by the in vitro reaction with specific kinase, are identified by shotgunmass spectrometry.

4)Validation of candidate substratesThe peptides library is used in its phosphorylated and unphosphorylated forms to develop assays forselected reaction monitoring (SRM) and SWATH. The assays are then used for detection andvalidation of the phosphorylation sites in cellular systems.

The approach has been used for the identification of novel substrates of different mitotic kinasesnamely: AuroraB, Plk1, Cdk1 and Haspin.


Control of colonic epithelium damage by NAIP

Kendle Maslowskia,*, Jurg Tschoppa

University of Lausanne, Department of Biochemistry, Switzerland

Neuronal apoptosis inhibitory protein (NAIP, or BIRC1) is a member of the IAP (inhibitor ofapoptosis) and NLR (Nod-like receptor) families. Humans contain one functional copy of the NAIPgene, whereas, in mice, multiple homologues of Naip exist which varies depending on thebackground (the C57B/6 mouse strain posses five: Naip 1, 2, 5, 6 and 7). We have developedcomplete Naip knockout mice (Naip1-7–/–), which have enabled us to study the in vivo function ofthe Naip family. In humans and mice, NAIP is highly expressed in the colonic epithelium, whichsuggests a role of Naip within the colon. The AOM/DSS model of colitis-associated CRC was usedto induce colonic tumors in Naipfl/fl and Naip1-7–/– mice. Naip1-7–/– mice showed an increase intumor burden, despite having reduced colitis. In tumors from Naipfl/fl mice, expression of Naip wasdecreased, compared to the surrounding colonic tissue. This suggests that a reduction of Naip is afeature of tumor development. Whilst inflammation is most often linked directly to tumorprogression, certain protective factors during colitis have tumor-promoting properties, such as IL22,which we found was increased in Naip1-7–/– mice. AOM induces O6-methylguanine adducts,resulting in G to A nucleotide transitions; early after administration a wave of apoptosis induced byaccumulation of damaged DNA occurs. Failure to repair DNA or clear AOM-initiated colonicepithelium can lead to increased tumorigenesis. Therefore, we analyzed colonic extracts 8 hrs afterAOM injection and found that Naip1-7–/– had defects in DNA-damage response, which couldunderpin the increased tumorigenesis observed in these mice. In summary, we have generated novelNaip1-7–/– mice, which has enabled us to determine a critical role for Naip in regulating responsesto epithelial damage and induction of colitis and colorectal cancer.


Coordinating epithelial cell division with tissue organization andpolarity

Eurico Morais-de-Sáa,*, Claudio Sunkela

IBMC, Porto, Portugal

Epithelial cells form a monolayered tissue that controls exchanges between the differentcompartments of multicellular organisms. The architecture and function of this tissue relies on theasymmetric localization of cellular junctions and the formation of cortical domains polarized alongthe apico-basal axis. During proliferation, epithelial cells have to coordinate the re-organization oftheir polarity with the cell shape changes associated with mitosis. Here, we use clonal analysis andlive imaging of the Drosophila follicular epithelium to explore how the polarity machinery isremodelled during cell division. Furthermore, we show that cytokinesis is asymmetric along thedivision plane of follicle epithelial cells, positioning the midbody near the apical domain. In thiscontext, asymmetric cytokinesis is independent of the intrinsic asymmetry of actomyosin ringcomponents. Instead, it relies on apical adherens junctions formed between the mitotic andneighboring cells, which maintain the contact with the contractile ring during cytokinesis anddetermine midbody position in partially depolarized daughter cells. We further show that ectopicmidbody localization alters epithelial shape due to the mispositioning of the new apical interfaceformed between daughter cells. Thus, we propose that the AJ acts as a positional cue maintainedwithin a proliferating tissue, which transfers cell architecture to the new daughter cells via thepositioning of the cytokinetic machinery.


How SR proteins connect splicing to mRNA export in vivo

Michaela Müller-McNicolla,*, Michaela Steinera, Valentina Bottia, Holger Brandla,Karla Neugebauera

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany

The SR protein family comprises seven regulatory RNA-binding proteins that play essential roles inpre-mRNA splicing. Some SR protein family members connect splicing to subsequent steps in geneexpression, such as mRNA export and translation, but their endogenous mRNA targets are largelyunknown. To systematically investigate the role of individual SR proteins in mRNA export in vivo,we established murine P19 stem cell lines, expressing seven GFP-tagged SR proteins from bacterialartificial chromosomes (BACs) without overexpression. As a prerequisite for a function in mRNAexport, we first quantified the capacity of individual SR proteins to shuttle between the nucleus andthe cytoplasm. We observed that in contrast to HeLa cells, where SRSF2 and SRSF5 do not shuttle,in P19 cells all SR proteins shuttle, however with different shuttling capacities. Investigating thisfurther, we found that the shuttling capacities correlate with the phosphorylation status of individualSR proteins, their interaction with the mRNA export factor NXF1 and their presence in translatingpolyribosomes. To identify mRNAs that depend on individual SR proteins for their export to thecytoplasm, we performed knockdown and cell fractionation followed by microarrays or RNA-Seq.We identified export targets for all SR proteins and observed that the shuttling capacities correlatewell with the number and expression levels of exported mRNAs. Furthermore, the export targets arefunctionally related and point to an important function of SR proteins in the regulation of differentcell fates. To confirm that export targets bind directly to their cognate SR protein we performed UVcrosslinking and immunoprecipitation (iCLIP) followed by deep sequencing. Our study reveals thebasis for the differential contribution of individual SR proteins to mRNA export in P19 cells andpresents for the first time endogenous export targets for all SR proteins.


Identifcation of novel molecular targets in colon cancer

Paloma Ordóñez-Morána,*, Joerg Huelskena

Swiss Institute for Cancer Research, École Polytechnique Fédérale de Lausanne, Switzerland

The Wnt pathway appears to be one of the most central mechanisms in colon cancer initiation basedon its frequent constitutive activation (~90%). We and others have reported that the main effector ofthis pathway β-catenin binds to different transcription factors enhancing the expression of alternativesets of target genes. Forkhead box O3 (FOXO3a), downstream effector of PI3K/AKT pathway, isone of those for which β-catenin acts as a transcriptional coactivator. In cancer, FOXO transcriptionfactors have been considered to be tumour suppressors because they can induce cell-cycle arrest andapoptosis.

Recently, we have described the function of these transcriptional effectors in colon cancerprogression. Unexpectedly, their simultaneous nuclear accumulation promotes cell scattering andmetastasis by regulating a defined set of target genes. Furthermore, β-catenin confers resistance toFOXO3a-mediated apoptosis induced by PI3K/AKT inhibitors. So, FOXO3a does not behave as atumor suppressor but rather as a metastasis inducer activated by PI3K/AKT inhibitors when acting inconcert with β-catenin. In this context, we have evaluated SPRY2 regulation by both proteins, andour results reveal SPRY2 as a new direct β-catenin/FOXO3a target gene that is expressed inWnt-activated cells in normal and tumoral tissues and correlates with shorter overall survival ofcolon cancer patients. Thus, we propose that it would be important to evaluate β-catenin status beforedeciding on treatment and that SPRY2 should therefore be considered as a prognosis marker.

Furthermore, intestinal stem cells have been described as the cells-of-origin of cancer. At present, weinvestigate undescribed β-catenin targets (possible tumour suppressors or oncogenes) that modulatethe intestinal stem cell compartment. For these targets we define their effects on stem cellself-renewal versus differentiation, long-term maintenance and lineage fate. We therefore areidentifying regulatory functions controlling cancer stem cell maintenance which could be relevant fortumourigenesis.


Asymmetric inheritance of primary ciliary membrane in dividingneural progenitors

Judith Paridaena,*, Michaela Wilsch-Brauningera, Wieland Huttnera

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

The primary cilium is a microtubule-based protrusion of the cell that is involved in mediatingextracellular signals, such as Sonic Hedgehog (Shh). The primary cilium is nucleated by thecentrosome containing the eldest centriole (mother centriole). Prior to mitosis, the primary cilium isdisassembled in order for the centrosomes to serve as the poles of the mitotic spindle. Recentfindings show that asymmetric inheritance of the centrosome containing the mother centriole isrelated to cell fate decisions during mammalian neurogenesis. However, it is unknown whethercentrosome-associated structures such as the primary cilium are involved in this asymmetricalregulation of cell fate. Therefore, we investigated the fate of the primary cilium upon cell division ofdeveloping neural progenitors and the possible involvement of the primary cilium in subsequent cellfate decisions in the developing mouse neocortex.We found that in mitotic neural progenitors and cultured cell lines, a membrane structure containingthe ciliary small GTPase Arl13b is associated with the centrosome that contains the mother centriole.By surface biotinylation of the apical membrane in explanted mouse telencephalic hemispheres, weshow that this structure contains ciliary membrane and is derived from the primary cilium that waspresent at the cell surface prior to mitosis. Using live imaging, we find that upon completion ofmitosis, this centrosome-associated ciliary membrane is asymmetrically inherited by one of thedaughter cells. Furthermore, we show that inheritance of the ciliary membrane causes earlierreestablishment of the primary cilium after division. Interestingly, the association of the ciliarymembrane with one centrosome in mitotic neural progenitors decreases as neurogenesis proceeds.We hypothesize that the observed asymmetry in primary cilium reassembly after mitosis mightdifferentially expose daughter cells to extracellular signals, such as Shh. Therefore, we speculate thatinheritance of the ciliary membrane affects cell fate decisions during mammalian neurogenesis.


Gabaergic projection neurons improve naive olfactorydiscrimination of close odors in drosophila

Moshe Parnasa,*, Wolf Huetterotha, Gero Miesenböcka

University of Oxford, UK

Most neurons involved in perceptual judgments are at least two synapses removed from sensoryreceptors. Psychophysical models that link perception to the physical qualities of external stimuli arethus black boxes. Opening these black boxes is challenging and requires comprehensive estimates ofactivity in many neurons carrying perceptually relevant signals. Because sensory representations aredistributed over large numbers of neurons, such estimates have generally remained elusive. Here, wetake advantage of the well-characterized olfactory system of fruit flies to relate knowledge of theneuronal population representations of odors to behavioral measures of odor discrimination. Fliesdetect odors using ~50 types of olfactory receptor neurons (ORNs). ORN axons segregateanatomically by receptor type and transmit signals via separate synaptic relays, to discrete classes ofexcitatory projection neurons (ePNs). Previously, ORN responses to odors and a transformationestimating PN spike rates from measured ORN spike rates were presented. ePNs project to themushroom body, and the lateral horn (LH). The LH, thought to be responsible of naïve behavior, alsoreceives input from a functionally uncharacterized group of GABAergic inhibitory PNs (iPNs). Thefact that iPNs target exclusively the LH hints at a possible function of these inhibitory neurons innaïve behavior. We formulate and test a simple model of innate odor discrimination that takes as itsinput the estimated PN signals projected onto the LH and generates as its output a prediction ofwhether two odors can be distinguished. We show that the main determinant of discriminability is thedistance between the PN activity patterns evoked by two odors. Experimental manipulations of thisdistance have graded predictable perceptual consequences. We further show that, inhibition by iPNsmakes closely related odors easier to distinguish, in all likelihood by imposing a high-pass filter onePN output that stretches the distances between partially overlapping odor representations.


Translational coordination of protein folding at the ribosome

Sebastian Pechmanna,*, Judith Frydmana

Department of Biology and BioX, Stanford University, USA

Newly synthesized proteins must be folded properly to become functional and avoid aggregation.Complex cellular mechanisms, including regulation of local translation rates and recruitment ofprotein folding and translocation components, influence co-translational folding events at theribosome. The choice of codons in the mRNA sequences can influence local translation kineticsduring protein synthesis. However, whether codon preference is generally linked to co-translationalregulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiencyscale, which allows us to uncover the evolutionary conservation of codon optimality across teneukaryotic organisms. This analysis reveals distinct and universal patterns of conserved optimal andnon-optimal codons, often in clusters, which associate with the secondary structure of the translatedpolypeptides independent of the levels of expression. Quantifying the substrates of molecularchaperones at the ribosome further suggests a link between the translational efficiency and thecoordinated binding and specificity of co-translationally acting chaperones. Our analysis thussuggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitateco-translational nascent chain folding, beyond its previously proposed role of adapting to the cost ofexpression. These findings establish how mRNA sequences are generally under selection to optimizethe co-translational folding of corresponding polypeptides.


Extensive transcriptional heterogeneity revealed by isoformprofiling

Vicent Pelechanoa,*, Wu Weib, Lars Steinmetzc

a Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg,Germany.b Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.c Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg,Germany.Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.

Transcript function is determined by sequence elements arranged on an individual RNA molecule.Variation in transcripts can affect mRNA stability, localization, and translation, or give rise totruncated proteins with differing subcellular localizations and functions. Given the existence ofoverlapping, variable transcript isoforms, determining the functional impact of the transcriptomerequires identification of full-length transcripts, rather than just the genomic regions that aretranscribed. Here, by jointly determining both transcript ends for millions of RNA molecules(TIF-Seq), we reveal an extensive layer of isoform diversity previously hidden among overlappingRNA molecules. Variation in transcript boundaries appears to be the rule rather than the exception,even within a single population of yeast cells. Hundreds of short coding RNAs and truncatedversions of proteins are concomitantly encoded by alternative transcript isoforms, increasing proteindiversity. In addition, most genes express alternative isoforms that vary in posttranscriptionalregulatory elements, and tandem genes frequently produce overlapping or even bicistronictranscripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genomewith limited splicing, and within a genetically homogeneous population of cells. Our findings haveimplications for genome compaction, evolution, and phenotypic diversity between single cells. Theyalso suggest that isoform diversity as well as RNA abundance should be considered when assessingthe functional repertoire of genomes.


Root system development and plasticity

Benjamin Pereta,*, Laurent Nussaumea, Malcolm Bennettb

a Unité Mixte de Recherche 7265, Commissariat à l’Energie Atomique et aux Energies Alternatives, Centre National de laRecherche Scientifique, Aix-Marseille Université, Laboratoire de Biologie du Développement des Plantes, Université d'Aix-Marseille, Franceb Division of Plant and Crop Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, LeicestershireLE12 5RD, UK

Plants have evolved extraordinary adaptive mechanisms to cope with a heterogeneous environmentand to compete for water and nutrient acquisition. One key aspect is the continuous production ofnew organs throughout the plants life to compensate for their inability to move. As a consequence,plant development is characteristically highly plastic both at the shoot and root level. In the pastyears, I have focussed my research on root development and more recently on its plasticity in thecontext of the EMBO fellowship.In my presentation, I will give an overview of the molecular and hormonal mechanisms controllingthe formation of roots. More precisely, I will explain how highly specific expression patterns can beachieved through positive feedback loops. Auxin is a plant hormone that triggers lateral rootformation, acting as a signal to induce and synchronize its development. At the tissue level, thehormone auxin is transported by influx and efflux transporters. The study of the influx carrier LAX3led to the identification of the mechanism that controls cell separation during this criticaldevelopmental process.Another key aspect of plant development is the hydraulic properties of tissue that largely controlcellular elongation and growth. Aquaporins are membrane channels that allow water transport inboth animals and plants. I will describe how aquaporins are regulated by auxin during lateral rootdevelopment and how the most abundant root aquaporin PIP2.1 controls lateral root growth.Finally, I will describe how the environment controls the lateral root formation process. Phosphorusis a major element that is present in most organic molecules (ATP, DNA, RNA, Phospholipids,Phosphorylated proteins…). It is absorbed by plants in the form of inorganic phosphate (Pi) and itsavailability strongly affects root architecture. I am now studying how Pi deficiency is perceived bythe plant to induce lateral root formation.


MicroRNA regulation of naïve and primed pluripotent states

Barbara Pernautea,*, Juan Miguel Sanchez-Nietoa, Thomas Spruceb, KimberleySmithc, Tristan Rodrigueza

a National Heart and Lung Institute, Imperial College London, UKb MRC National Institute for Medical Research, London, UKc Comparative and Biomedical Sciences, Royal Veterinary College London, UK

Understanding how cells exit the pluripotent state and initiate the path to differentiation is a keyquestion in developmental and stem cell biology. To date the first identified step of differentiation inthe mammalian embryo is the transit from a of naïve state of pluripotency, found in thepre-implantation epiblast and in embryonic stem cells (ESCs), to a pluripotent state that is primed fordifferentiation, and that can be found in the post-implantation epiblast and in epiblast stem cells(EpiSCs). These two different states of pluripoteny are thought to be regulated by differentmechanisms as different combinations of growth factors are required for their maintenance both invivo and in vitro. We have found that during the transition from the naïve to the primed state ofpluripotency there is a critical change in the regulation of the apoptotic machinery. MicroRNAs(miRNAs) are small non-coding RNAs that repress gene expression post-transcriptionally. We showthat with the onset of differentiation the apoptotic pathway becomes primed for cell death and is onlykept in balance by an increase in the expression levels of miRNAs. Furthermore, we find that key tothe priming of the apoptotic pathway is the pro-apoptotic protein Bim, that becomes competent toinduce cell death as cells initiate differentiation. Our work provides insight into how miRNAsregulate the different pluripotent states and contributes to the understanding of the regulatorynetworks involved in stem cell homeostasis, pluripotency and differentiation during early embryodevelopment.


Telomeric repeat-containing RNA (TERRA) promotes telomereshortening through Exonuclease 1-mediated resection ofchromosome ends

Verena Pfeiffera,*, Joachim Lingnera

EPFL-Ecole Polytechnique Fédérale de Lausanne, School of Life Sciences, ISREC-Swiss Institute for Experimental CancerResearch, 1015 Lausanne, Switzerland

Telomeres protect chromosome ends from unwanted DNA repair activities and they preventchromosome end shortening due to the end replication problem by engaging telomerase [1, 2].Telomeres from animals and fungi are transcribed into the long noncoding telomeric repeatcontaining RNA (TERRA) [3].To elucidate TERRA function in Saccharomyces cerevisiae, a doxycycline regulatable promoter wasinserted upstream of the transcriptional start site of TERRA at telomere 1L. Induction of TERRA ledto shortening of telomere 1L but not other chromosome ends. TERRA interacts with the Ku70/80complex which represses the 5’-3’ Exonuclease 1 (Exo1) at telomeres [4]. Deletion of EXO1 fullysuppressed the TERRA-mediated telomere shortening.Telomeric DNA in S. cerevisiae has a loose consensus sequence (TG1-3). Sequence alignment ofsister telomeres allows distinction of a centromere proximal zone that is faithfully replicated bysemiconservative DNA replication and a telomere distal zone at which sequences diverge due to newtelomeric sequences that are added by telomerase or DNA recombination. In addition toExo1-mediated telomere shortening, TERRA transcription increases the frequency of telomerehealing events involving telomerase and Rad52-dependent recombination. Therefore, this dataindicates that telomere transcription regulates cellular lifespan through modulation of chromosomeend processing activities.

[1] Jain D., Cooper J.P. (2010) Annual Review of Genetics 44: 243-269[2] de Lange T. (2009) Science 326: 948-952[3] Feuerhahn S. et al. (2010) FEBS letters 17: 3812-3818[4] Pfeiffer V., Lingner J. (2012) PloS Genet. 6: e1002747


Genetic Handicap Principle: Low Mutation Burden of DownSyndrome Population

Konstantin Popadina,*, Reza Sailania, Audrey Letourneaua, Federico Santonia,Stylianos E. Antonarakisa

Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel-Servet, 1211Geneva, Switzerland

A handicap is a circumstance that makes progress or success difficult (Oxford Dictionary). As agenetic handicap we refer to trisomy of chromosome 21, which is highly deleterious due to 1.5 foldincreased level of expression of 300 genes from this chromosome. Since selection eliminatesorganisms with increased mutation burden, Down Syndrome (DS) fetuses should be preferentiallyfiltered out due to severe effect of the trisomy. Indeed, the rate of spontaneous abortions issignificantly increased for DS fetuses (80% versus 8% in euploid fetuses), corroborating thisselection. Thus live-birth DS individuals have to possess initially low mutation burden, which helpedthem to tolerate the harmful effect of trisomy and survive. According to the handicap principle, weexpect that live-birth DS cohort should be initially higher fitted as compared to Control cohorts,where there was no so strong selection. Previously, we have observed a low expression variation in 8live-birth DS versus 8 Control individuals, that can be interpreted as an evidence of increased fitness(ASHG 2012: http://abstracts.ashg.org/cgi-bin/2012/ashg12s?author=Popadin&sort=ptimes&sbutton=Detail&absno=120122658&sid=564830). Now we support this hypothesis by genetic data.Genome-wide variation in frequencies of SNPs between 380 unrelated European DS individuals and215 controls demonstrated that DS population has a deficit of rare derived alleles as compared to ourcontrol population. This leads to deficit of both heterozygous and homozygous DS genotypes withrare derived alleles. It is noteworthy that trisomic SNPs, reconstructed for chromosome 21demonstrate even more pronounced deficit of rare derived alleles in DS. Overall, our results (moreoptimal expression pattern in live-birth DS and deficit of rare, potentially more deleterious, alleles inDS) support the hypothesis of highly-fitted genomes of DS and suggest that during embryogenesisthere is a preferential elimination of DS fetuses, highly contaminated by slightly-deleteriousmutations.


Analysis of the yeast plasma membrane using soluble fluorescentreporters

Dušan Popov-Čeleketića,*, Frans Bianchia, Gemma Moiset Colla, FebrinaMeutiawatia, Bert Poolmana

Biochemistry Department, University of Groningen, The Netherlands

In budding yeast S. cerevisiae proteins and lipids of the plasma membrane (PM) are heterogeneouslydistributed. Yeast PM exhibits high complexity and prominent patterns of membranecompartmentalization that have been extensively studied. Most studies on the PM organization invivo largely rely on the localization of integral membrane proteins. These proteins, however, diffusevery slowly, are prone to aggregation, low expression and non-specific localization. The aim of thisstudy is to develop new tools for the analysis of architecture and dynamics of the yeast PM.As model proteins we used amino acid permeases (AAP) residing in the yeast PM. Using analysis insilico followed by a series of mutational and truncational analyses we have found that the C-terminalsequences derived from nine amino acid transporters (Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3,Agp1, and Gnp1) were associated with the PM. Two reporters derived from Gap1 and Hip1 wereselected for further studies based on their contrasting behaviour; the former being homogenouslydistributed along the membrane and very mobile and the latter present in patches in the membraneand static. Using super-resolution microscopy (photoactivated light microscopy, PALM) ourreporters have enabled us to analyze the PM at the resolution of 50 nm. In parallel, we have beenobserving changes in the dynamics of our reporters under various conditions using FluorescenceRecovery After Photobleaching (FRAP).These short and soluble sequences (48-62 aa residues) are less prone to aggregation and exhibithigher expression than the full-length proteins they are derived from. Importantly, the reportersexhibit different patterns of localization and move along the membrane with various speeds makingthem excellent tools to study the factors that contribute to the organization of the yeast plasmamembrane.


Magnetic resonance imaging of tumor glycolysis usinghyperpolarized 13C-labeled glucose

Tiago Rodriguesa,*, Eva Serraoa, Brett Kennedya, De-en Hua, Mikko Kettunena, et al.CRUK Cambridge Institute and Department of Biochemistry, University of Cambridge, Li Ka Shing Centre, Robinson Way,Cambridge CB2 0RE, UK

High rate of aerobic glycolysis in tumors provides opportunities for tumor detection and treatmentmonitoring using metabolic imaging such as FDG-PET and hyperpolarized [1-13C]pyruvate. In thecurrent study we investigated the possibility to use hyperpolarized [U-2H,U-13C]glucose to detectglycolysis in vivo.

EL4 tumor-bearing mice were imaged before and 24h after treatment with etoposide (67mg/kg).Hyperpolarized [U-2H,U-13C]glucose sample (3.55M) was dissolved with 3ml of 2H2O-saline. MRdata were collected at 7 T using a surface coil placed over the tissue of interest (tumor, heart, kidney)15s after the start of the glucose injection. A series of 13C spectra were collected: 4 spectra collectedfrom the lactate region followed by 1 collected from the glucose region every second for 40s. Insome animals, a glucose and lactate 13C chemical shift selective images were collected. The ratios ofthe signal integrals from lactate (183-187ppm) and glucose (60-100ppm) were calculated.

Signal from labeled [U-13C]lactate was observed in EL4 or LL2 tumor spectra but was not observedin other tissues. Consistent with this, lactate was predominantly located tumor in 13C chemical-shiftimage. Low levels of dihydroxyacetone phosphate, 6-phosphogluconate and bicarbonate , likelyproduced by 6-phosphogluconate dehydrogenase, were also observed in the tumor spectra. Thisexperiment could therefore give simultaneous information on both glycolysis and Pentose PhosphatePathway activity. The flux of 13C label from glucose to lactate was decreased at 24 h after treatmentwith the labelled lactate/glucose ratio decreasing from 1.82±0.42 to 0.69±0.11 after treatment (n=6,p=0.026).The current results suggest that despite relatively short T1 (~9s), lifetime of hyperpolarized glucoseis long enough for real-time in vivo imaging of glycolysis. The capacity to non-invasively measurethe glycolytic pathway may provide more profound understanding of the altered metabolism ofcancer and empower the identification of targets for diagnostic imaging, prognosis or therapy.


Suppression of protophloem differentiation and root meristemgrowth in Arabidopsis by CLE45 requires the receptor-likekinase BAM3

Antia Rodriguez-Villalona,*, Stephen Depuydta, Luca Santuaria, Christian Hardtkea

University of Lausanne, Switzerland

Peptide signaling occupies central role in plant development, yet only few concrete examples ofreceptor-ligand pairs that act in the context of specific differentiation processes have been described.In this work, we report that second-site null mutations in the Arabidopsis leucine-rich repeatreceptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic rootmeristem growth defect and the associated perturbed protophloem development of the brevis radix(brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA3/endosperm surrounding region 45 (CLE45) peptide ligand. Wild-type plants transformed with aconstruct for ectopic overexpression of CLE45 could not be recovered, with the exception of a singleseverely dwarfed and sterile plant that eventually died. By contrast, we obtained numeroustransgenic bam3 mutants transformed with the same construct. These transgenic plants displayed awild type phenotype however, supporting the notion that CLE45 is the likely BAM3 ligand. Theresults correlate with the observation that external CLE45 application represses protophloemdifferentiation in wild type, but not in bam3 mutants. BAM3, BRX and CLE45 are expressed in asimilar spatio-temporal trend along the developing protophloem, up to the end of the transition zone.Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brxroot meristems and laser ablation experiments suggest that signaling homeostasis throughintertwined regulatory activity of BRX, BAM3 and CLE45 guides the proper transition ofprotophloem cells from proliferation todifferentiation, which in turn determines post-embryonic growth capacity of the root meristem.


Deep sequencing the RNA encapsidated by viruses: mapping ofRNA recombination; discovery of functional motifs; andpackaging of host RNAs including retrotransposons

Andrew Routha,*, Johnson Johna

The Scripps Research Institute, La Jolla, USA

RNA recombination within viral genomes is a powerful driving force behind the evolution andadaption of RNA viruses and has been attributed to be the source of outbreaks of new virus strainsincluding Echovirus and Poliovirus as well as the emergence of entirely new viruses such asSARS-CoV. Using Deep Sequencing (RNAseq) we have set out to analyse the genomicpolymorphism and quasi-species present within an array of eukaryotic +ssRNA viruses, includingFlock House Virus, Cricket Paralysis Virus, Sindbis Virus and Human Rhinovirus.

We present a method to map viral RNA recombination events with single-nucleotide precision andhave applied this to reveal prolific viral RNA recombination providing a detailed and quantitativedescription of the complex mutational landscape of the transmissible viral genome. Moreover, asrecombination events that remove functional motifs are negatively selected, the distribution ofrecombination events reveals the genomic motifs required for viral replication and encapsidation.Consequently, functional viral motifs can be discovered de novo without the requirement for priorcharacterisation of the viral lifecycle.

In addition to their viral genomes, we found that some viruses also package host mRNAs, rRNAs,non-coding RNAs and retrotransposons. The packaging of these host RNAs elicits the possibility ofhorizontal gene transfer between hosts that share a viral pathogen.


Generation and modulation of B-cell memory in humanvolunteers subjected to experimental malaria infection

Anja Scholzena,*, Anne C. Teirlincka, Wiebke Nahrendorfa, Else M. Bijkera, RobertW. Sauerweina, et al.Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Naturally-acquired immunity to the malaria parasite Plasmodium falciparum (Pf) is thought to beinefficient and short-lived. Impaired immunological memory is partly due to immune evasionstrategies of Pf, but there is also evidence that the parasite actively modulates the immune system. Astriking feature in individuals from malaria-endemic areas is the expansion of a rare, novel B-cellsubset, termed atypical memory B-cells (MBCs). The mechanisms underlying their expansion andtheir function in malaria-infection remain unknown. We analyzed the acquisition of antigen-specificMBCs and the modulation of B-cells in the unique setting of controlled malaria-infection in humanvolunteers.

Project 1: We analyzed the human B-cell compartment in blood samples collected prior to, duringand after a primary and low-grade malaria- infection. Atypical MBCs were expanded andproliferating immediately after parasite exposure. Their proliferation associated with elevated levelsof B-cell activating factor, a key cytokine driving B-cell differentiation. After resolved infection,however, atypical MBC proliferation and proportions were comparable to pre-infection levels.

Project 2: The acquisition of antigen-specific MBCs was analyzed in volunteers subjected to multiplePf exposures under curative drug cover, which efficiently induces protection from challengeinfection. Pf-specific MBCs were efficiently and step-wise generated by this immunization regimen,and stably maintained. In this setting, we also observed repeated expansion of atypical MBCs, butthey did not accumulate over time.

Project 3: To get further insights into the role and function of atypical MBCs, we analyzed theirphenotype in human blood and spleen. They were phenotypically clearly distinct from other B-cellsubsets, expressing a unique pattern of receptors mediating B-cell survival, activation, migration andT-cell interaction. The strong similarity of blood and spleen atypical MBCs and the much greaterabsolute number of this rare subset in spleen now enables us to perform more in-depths functionalanalysis of these cells, which is currently underway.


A steroid receptor-microRNA switch regulates longevity inresponse to germline ablation

Yidong Shena,*, Adam Antebia, et al.Max Planck Institute for Biology of Ageing, Cologne, Germany

While the gonad primarily functions in procreation, it also affects animal longevity. Ablation ofgermline stem cells in C. elegans or D. melanogaster significantly extends lifespan, suggesting thatgonadal longevity could be evolutionarily conserved. This longevity depends on the presence ofsomatic gonad and is therefore not a simple tradeoff between fertility and survival but under thecontrol of careful signaling. Our results show that germline ablation actively triggers the productionof dafachronic acids (DAs) production, which switches on a molecular module of DAF-12/NHRsteroid receptor and two microRNAs of let-7 family. The microRNA targets, AKT-1 and LIN-14, aresubsequently inhibited to extend lifespan through enhanced DAF-16/FOXO activity. Notably, thecore components of this steroid receptor-microRNA switch are evolutionarily conserved and alsoregulate developmental progression in worm larvae. Therefore, these studies suggest that metazoanlifespan is coupled to the gonad through elements of an early-life developmental timing switch.


Polymerase epsilon nonessential subunits prevent toxicrecombination during DNA replication

Jennifer Svendsena,*, Jillian Youdsa, Simon Boultona

DNA Damage Response Laboratory, Clare Hall, London Research Institute, South Mimms, EN6 3LD, UK

Of paramount importance to organism survival is the faithful duplication of a cells genetic materialthrough the concerted action of DNA replication and repair factors. Using a genome-wide RNAiscreen we identified a genetic interaction between the DNA repair helicase RTEL-1 and thenonessential subunit of the leading strand replicative polymerase epsilon, POLE-4. RTEL-1 is ananti-recombinase that prevents inappropriate recombination by unwinding D-loop intermediatesduring DNA repair, meiosis, and at telomeres. POLE-4 is a nonessential subunit of polymeraseepsilon that promotes polymerase processivity. Our studies reveal that C.elegans pole-4 mutants are100% embryonic lethal when combined with the rtel-1 mutation. One possibility is that the absenceof POLE-4 decreases the processivity of polymerase epsilon leading to an increased number ofaberrant fork structures that must be counteracted by RTEL-1. In this case, loss of both POLE-4 andRTEL-1 would lead to persistent aberrant replication fork structures that may be processed bystructure-specific nucleases. Consistent with this hypothesis pole-4; rtel-1 lethality is associated witha significant decrease in DNA replication within the mitotic zone of the worm germline. Coincidentwith the decrease in DNA replication is activation of the replication checkpoint and the accumulationof spontaneous double strand breaks, DSBs, marked by RAD-51 and RPA. Furthermore theaccumulation of DSBs in pole-4; rtel-1 worms is dependent on the structure-specific nucleaseMUS-81 and the RAD-51 paralog RFS-1 suggesting that DSBs occur at stalled replication forksfollowing processing by MUS-81. These data suggest that POLE-4 and RTEL-1 function to preventthe accumulation of toxic recombination intermediates during DNA replication. Recent generation ofa pole-4-/- mouse model has revealed insight into the function of POLE-4 in development andageing. Future plans will be aimed at further elucidating the relationship between POLE-4 anddisease and the genetic interaction between pole-4 and rtel-1 in mice.


Chromatin proteins and sequence evolution in eukaryotes andprokaryotes

Tobias Warneckea,*, Fran Supeka, Ben Lehnera, Marc Facciottib, Corey Nislowc, etal.a Centre for Genomic Regulation, Barcelona, Spainb UC Davis Genom Center, CA, USAc University of British Columbia, Vancouver, Canada

What is the relationship between genetic and epigenetic change? Do epigenetic changes simplyreflect changes that occurred at the genetic level? Or may epigenetic features prompt such changes inthe first place (e.g. by affecting mutation dynamics and DNA repair)?

The presence of nucleosomes, for example, can promote or impede the formation of DNA lesions orinterfere with efficient DNA repair, so that some mutations may occur more, others less frequently ina nucleosomal context. Conversely, evolution at the sequence level can affect nucleosomearchitecture. Notably, as nucleosomes form preferentially on more bendable DNA, mutations thatrender the DNA template less bendable can prompt the nucleosome to shift to a position that is moreconducive to nucleosome formation.

In short, nucleosomes (and other DNA-binding proteins) can both affect and reflect sequenceevolution. But what is the dominant direction of causality in genetic versus epigenetic change? Andwhat are the implications of chromatin architecture for mutability and longer-term patterns sequenceevolution?

Drawing on data from multiple eukaryotes but also bacteria and archaea, which encode their ownunique complements of structural DNA-binding proteins, and summarizing results obtained over thelast three years, I will dissect the complex relationship between sequence evolution and chromatinarchitecture and argue that the way genomes are packaged fundamentally biases trajectories ofsequence evolution.


Dispersed PSM oscillator assay reveals the cell autonomousnature of the segmentation clock

Alexis Webba,*, Annelie Oswalda, Laurel Rohdeb, Daniele Soroldonib, AndrewOatesb

a MRC National Institute for Medical Research, London, UKb MRC National Institute for Medical Research, London, UKUniversity College London, UK

Most current models of the segmentation clock postulate that the molecular oscillations drivingsomite formation are autonomous at the level of single cells, without the input of their neighbors orother tissues. But this assumption has not been explicitly tested and theories exist in the literature thatcan reproduce the observed tissue-level patterning without autonomous oscillators. We havedeveloped a series of assays to systematically test whether the zebrafish segmentation clock containsan autonomous oscillator at the level of a single PSM cell. Using our cyclic gene reporter line,her1-YFP, we are able to monitor oscillations in gene expression in transplanted cells within theembryo, PSM explants, tailbud pieces, and dispersed and fully isolated cells. We have quantifiedproperties that characterize oscillators, such as period, amplitude, number of cycles, and signalquality factor, from individual PSM cells in culture. We are also using single cells to directly addressquestions of molecular clock function that were previously inaccessible in the intact embryo. Ourstudies reveal the ability of single zebrafish PSM cells to oscillate autonomously in culture,independent of neighboring cells or tissues. They also demonstrate the tractability of the single cellmodel system for investigating the segmentation clock.


The Cotranslational Function of Ribosome-Associated Hsp70 inEukaryotic Protein Homeostasis

Felix Willmunda,*, Marta DelAlamob, Sebastian Pechmannb, Taotao Chenb, JudithFrydmanb, et al.a Stanford University, USA/University of Kaiserslautern, Germanyb Stanford University, USA

During protein biosynthesis, the processing and maturation of newly made polypeptides is performedby a highly complex machinery. As nascent chains emerge from the ribosome, they require a lineupof specific factors for further maturation and distribution throughout the cell. In contrast to theunderstanding of de novo folding in prokaryotes, little is know about the principles and selectivity ofnascent chain binding factors in the cytosol of eukaryotes. We applied a system-level approach toanalyze the role of the Hsp70 molecular chaperones in co-translational nascent chain binding. Thisenabled us to identify and to define the broad substrate set of the ubiquitous yeast Hsp70 homologue,Ssb1/2. We could determine for the first time, the cellular distribution and sequence characteristics ofHsp70-bound nascent chains. Our analysis revealed an important function of Hsp70 in protectingthose emerging nascent polypeptides threatened for misfolding and aggregation by their slowco-translational folding kinetics. Thus, our system-level approach provides a powerful tool tointerrogate protein homeostasis networks as they occur at the eukaryotic ribosome.


Impact of miRNAs on bone development

Nadine Wittkoppa,*, Suzanne W. Hoogstrateb, Lucas J. Kaaijc, Anneloes Dummera,Rene F. Kettinga, et al.a IMB, Mainz, Germanyb Wageningen University, The Netherlandsc Hubrecht Institute, Utrecht, The Netherlands

Bones give stability and at the same time flexibility to our bodies. To achieve those properties, aspatial and temporal developmental balancing is needed between mainly two cell types shaping thebone: the osteoblasts that secrete the extracellular matrix, which will later on mineralise and theosteoclasts, which resorb this matrix. The process of bone remodelling is active not only duringdevelopment, but also during an organism’s adult lifespan.miRNAs are well-known players in development and fine-tune gene expression on apost-transcriptional level. Their role in bone formation has been investigated, but only on a piece-by-piece (bit by bit) basis and mainly in cell culture. We have aimed to unravel this gene regulatorynetwork in a more systematic approach. Therefore, we have isolated early and terminal-differentiated/ mineralised osteoblasts during development. Employing a set of reporter lines wewere able to distinguish and label the various stages and isolate those cells from whole zebrafishlarvae (Danio rerio). We assessed the miRNA content of early osteoblasts as well as their mRNAexpression profile. To complete the picture we generated Maternal Zygotic dicer mutants, which aredeprived of almost all mature miRNAs and compared their expression profile to early andmineralised wildtype osteoblasts. This will help us to determine those mRNAs that are targeted bythe identified miRNAsWe found almost 100 annotated miRNAs enriched in early stage osteoblasts. From those we verifiedthe expression of a couple in the bone forming tissue using In Situ hybridisation and knocked downtheir expression using morpholinos. So far we were able to observe a phenotype for two miRNAknockdowns; one of the phenotypes is indeed related to bone formation, the other one can besurprisingly observed in the lymphatic system. Currently, we are systematically testing candidategenes that are likely targeted by these miRNAs.


Ribosomal oxygenases (ROXs) - a new enzyme family ofribosomal regulators

Alexander Wolfa,*, Wei Gea, Tianshu Fengb, Peter J Ratcliffeb, Christopher JSchofielda

a Chemistry Research Laboratory, University of Oxford, UKb Henry Wellcome Building for Molecular Physiology, University of Oxford, UK

Oxygen (O2) supply is crucial for most cells but hypoxia (low oxygen levels) occurs underphysiological as well as various pathological conditions, like tumour growth, stroke andinflammation. A well-known cellular adaption to low O2-levels is the up-regulated expression ofhypoxia response genes, mediated by the transcription factor HIF (hypoxia-inducible factor). In orderto prevent HIF up-regulation under normoxic conditions cells have developed control mechanisms,including enzymes of the 2-oxoglutarate (2OG) dependent oxygenase superfamily. 2OG oxygenasescatalyse hydroxylation reactions by transferring molecular O2 to a substrate.

We have identified a new set of 2OG oxygenases, the ROXs (ribosomal oxygenases), which targetthe ribosome. These ROXs modify ribosomal proteins posttranslationally by hydroxylating specificamino acids in the center of the ribosome. We have found ROX-catalysed modifications in human,yeast and E.coli ribosomes, indicating that this is a well-conserved process of protein synthesisregulation.

Ribosome action translates mRNA code into the amino acid sequence of a protein. In the decodingcenter (DC) of the ribosome mRNA and the appropriate aminoacyl-tRNA molecule bind via codon-anticodon recognition. Subsequently the amino acid is transferred to the growing peptide chain in thepeptidyl transferase center (PTC) of the ribosome. ROX-catalysed modifications occur on Arg-, His-and Pro-residues facing either the PTC or the DC and can thereby affect translation. Alteredtranslational accuracy in hypoxia for instance is due to a lack of prolyl hydroxylation in the DC.

The ribosomal oxygenases are a new subgroup of 2OG oxygenases having the potential fortranslational regulation. In E.coli the ycfD protein catalyses arginyl hydroxylation of the ribosomalprotein Rpl16. Deletion of ycfD slows growth. Reduced expression of the human ycfD-homologsMinA53 and NO66 inhibits cell proliferation and both proteins are highly expressed in severalcancers.


abstracts | posters

Making sense of sensory stimuli on millisecond time scale

Nixon Abrahama,*, Alan Carletona

Laboratory of Sensory Perception and Plasticity, Geneva, Switzerland

The behavioral response to a sensory stimulus is preceded by the detection and discriminationprocesses. The temporal evolution of discrimination process of different odorants has been shown tobe depending on the representation of stimuli in the olfactory bulb (OB) (Abraham et al., 2004) andthe refinement of stimulus by the inhibitory circuits of OB (Abraham et al., 2010). Theseobservations clearly show that discrimination is a complex process involving the evaluation ofdifferent properties of stimuli. The question if this detailed stimulus evaluation is essential for thedetection process remains to be addressed. Here we investigate the evolution of temporal scales ofdetection and discrimination processes while the animal is in learning process of novel olfactorystimuli. Mice were trained, under head restrained conditions, on a go/no-go operant conditioning taskto learn different odorants of varying complexity. Based on the performance levels of animals weclassified the learning period as different stages, poor (≤ 60%), average (>60 and <80%) and good(≥80%) learning and analyzed the evolution of detection and discrimination times during thesedifferent epochs of learning. Even though animals learned all the odorants including enantiomerswith very high accuracy, they took longer time periods to discriminate complex odorants comparedto simple odorants, establishing the stimulus dependent discrimination times using a behavior readout towards positively rewarded odorants. We then asked if this reward value is analyzed by theanimal before responding to a stimulus by measuring the detection times towards rewarded andnon-rewarded trials. While monitoring the mice’ behavioral response on millisecond time scale wefound out that the detection of both simple and complex stimuli precedes the real discriminationprocess even at highly learned stages. Therefore we conclude that mice need additional tens ofmilliseconds to evaluate the reward value of the stimuli after the detection process.


The cellular basis of visual motion detection in the fly optic lobe

Alexander Arenza,*, Alexander Borsta

Max-Planck-Institute for Neurobiology, Martinsried, Germany

Visual motion as a result of ego-motion (optic flow) is a crucial sensory stimulus for flight control inflies. While photoreceptors only respond to changes in brightness within their receptive fieldsirrespective of visual motion, large-field tangential cells in the lobula plate (LPTCs) of the fly opticlobe respond primarily to visual motion in a direction specific way. This feature extraction of visualmotion direction appears to be achieved along processing lines comprising only a handful of steps.Based on anatomical evidence and genetic inactivation studies several neuronal cell types requiredhave been identified, and it has emerged that two parallel processing streams exist, splitting visualinformation in an ON and an OFF pathway of visual motion detection.

However, how the computation and feature extraction is performed on a cellular basis remainslargely unexplored, mainly due to the small size of the columnar elements.Here we address this question at the level of the presumed input elements of LPTCs, T4 and T5 cells.We performed whole-cell patch-clamp recordings from T4 and T5 cells in the optic lobes ofDrosophila melanogaster and Calliphora vicina in order to determine the cellular and synapticmechanisms involved in the processing of visual motion information.


Unlimited in vitro expansion of adult bi-potential pancreasprogenitors

Paola Bonfantia,*, Meritxell Huchb, Sylvia F. Bojb, Harry Heimberga, Hans Cleversb,et al.a Diabetes Research Center, Vrije Universiteit Brussel, Belgiumb Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, Netherlands

Novel cell therapy strategies in diabetes would greatly benefit from the availability of beta cellstem/progenitor cells. Lgr5 marks adult stem cells in multiple adult organs and is a receptor for theWnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3Dcultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signaling isinactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. We reporthere that the Wnt pathway is robustly activated upon injury by Partial Duct Ligation (PDL),concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, ductfragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop intobudding cyst-like structures (organoids) which expand extensively for >40 weeks. Prospectivelyisolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitorcells that can be clonally expanded. Pancreas organoids can be induced to differentiate into duct aswell as endocrine cells, thus proving their bi-potentiality. Adult pancreas organoid cultures mayrepresent a novel approach in regenerative medicine.


High resolution genome-wide analysis of small RNA mediatedtranslational regulation in Chlamydomonas reinhardtii

Betty Chunga,*, Baulcombe Davida

Department of Plant Sciences, University of Cambridge, UK

The diverse natural roles for small RNAs in eukaryotes range from defense against viruses to theregulation of gene expression and chromosome structure via mechanisms affecting transcription,RNA stability and translational control. These processes are primarily guided by the evolutionarilyconserved protein Argonaute. In plants, evidence for small-RNA-mediated translational control hasemerged recently but little is known about the full complement of mRNAs that are affected orwhether the mechanisms are the same as those that operate in animal systems. To address theseissues, I am implementing ribosome profiling and HITS-CLIP on Argonaute-associated mRNAs inthe green alga Chlamydomonas reinhardtii. It is not only the most established unicellular modelorganism for studying small RNA mediated pathways, but it can also be cultured synchronously andhas simple genetics in the haploid vegetative phase, thus aiding follow-up genetic studies.


Co-transcriptional Drosha cleavage in terminal exon of liverspecific lncRNA couples transcriptional termination with highmir-122 expression

Ashish Dhira,*, Nick Proudfootb, Catherine Joplingc

a Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UKb Sir William Dunn School of Pathology, University of Oxford,South Parks Road, Oxford, OX1 3RE, UKc RNA Biology Group, Centre for Biomolecular Sciences,University of Nottingham, University Park, Nottingham NG7 2RD, UK

Long non coding RNA (lncRNA) that contain pre-miRNA are of obvious functional importance.Although only a minority of miRNA derive from exons of lncRNA host genes, highly expressedmiRNA often derive from lncRNA terminal exons. Here, we present data for the liver specificmiRNA, mir-122 that represents ~50% of the total miRNA population in liver. This highly expressedmiRNA is located in the terminal exon of a ~5kb lncRNA. Nucleo-cytoplasmic fractionationfollowed by RT-PCR demonstrated the retention of this lncRNA in the nucleus. Furthermore,northern blot analysis revealed the presence of both spliced and unspliced transcripts that havenon-canonical 3’ends defined by Drosha cleavage at the pre-miRNA stem loop region, as validatedby RNase protection analysis. Consequently, these lncRNAs are non-polyadenylated and rapidlyturned over as demonstrated by transcription block experiments with actinomycin D treatment.Interestingly, upon Drosha and DGCR8 knock down, mir-122 primary transcripts were extended attheir 3’ ends and 3’ end processed at a canonical poly (A) signal (PAS). Deletion of the mir-122 stemregion in cis confirmed the microprocessor knock down effect. Finally, ChIP analysis demonstratedlow RNA polymerase II CTD Ser2P levels across the mir-122 lncRNA gene as compared to Ser5Plevels. This suggests a non-productive environment for PAS recognition and illustrates thatprocessing of terminal exonic pre-miRNAs can facilitate transcription termination. Altogether, ourstudies show that this lncRNA skews the RNA processing machinery towards the dedicatedproduction of mir-122 resulting in very high levels of expression.


Illuminating the role of inhibitory microcircuits in higher-orderolfactory processing in zebrafish

Thomas Franka,*, Yan-Ping Zhang Schaerera, Rainer W Friedricha

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

The brain creates dynamic representations of the sensory environment by extracting stimulus featuresat early processing stages and synthesizing more abstract object representations in higher brain areas.However, the nature of the underlying neuronal circuits and their strategies to perform this task arestill poorly understood. This research project aims at identifying neuronal computationsrepresentative for basic cortical circuits and uncovering the underlying cellular mechanisms. To thisend, we visualize and manipulate different types of interneurons (INs) in the posterior zone of thedorsal telencephalon (Dp) of zebrafish, which is homologous to olfactory cortex in mammals andassumed to be involved in olfactory object representations and odor memory. Using transgenicmarker lines, we measure odor responses of different IN types by 2-photon Ca2+ imaging andtargeted whole-cell recordings. We analyze the synaptic architecture of Dp using electrophysiologyand optogenetics, and derive hypotheses for the function of different IN types in the context of theneural circuit. These hypotheses are subsequently tested by reversibly silencing INs in transgeniclines, and the resulting cell-type specific effects are examined at the level of individual neurons andlarge-scale activity patterns. The results provide insights into canonical computations performed bybasic cortical circuits.


RNA binding and RNA remodeling activities of the half-atetratricopeptide (HAT) protein HCF107 underlie its effects ongene expression

Kamel Hammania,*, William B. Cookb, Alice Barkanc

a CNRS, Institut de Biologie Moléculaire des Plantes, University of Strasbourg, Franceb Department of Biology, Midwestern State University, Wichita Falls, TX 76308c Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat motif found in proteins thatinfluence various aspects of RNA metabolism, including rRNA biogenesis, RNA splicing, andpolyadenylation. This functional association with RNA suggested that HAT repeat tracts might bindRNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recentliterature has emphasized a protein binding role. In this study, we show that a chloroplast-localizedHAT protein, HCF107, is a sequence-specific RNA binding protein. HCF107 consists of 11 tandemHAT repeats and short flanking regions that are also predicted to form helical hairpins. The minimalHCF107 binding site spans ∼11 nt, consistent with the possibility that HAT repeats bind RNAthrough a modular one repeat–1 nt mechanism. Binding of HCF107 to its native RNA ligand in thepsbH 5’ UTR remodels local RNA structure and protects the adjacent RNA from exonucleases invitro. These activities can account for the RNA stabilizing, RNA processing, and translationalactivation functions attributed to HCF107 based on genetic data. We suggest that analogous activitiescontribute to the functions of HAT domains found in ribonucleoprotein complexes in the nuclear–cytosolic compartment.


Epithelial polarity factors are essential for the organization ofmuscle fibers

Aynur Kaya-Copura,*, Frank Schnorrera

Max Planck Institute of Biochemistry, Martinsried, Germany

Higher animals owe all their movements to their muscles. Muscle fibers achieve their contractileproperties by subdividing their cytoplasm into a highly organised array of myofibrils, T-tubules andnuclei. Disorganisation of any of these components leads to many skeletal and cardiac myopathies.Similar to vertebrates adult Drosophila possess various muscle types such as heart, striated bodymuscles, and specialized indirect flight muscles enabling fast wing oscillations. Most structuralcomponents important for formation or function of myofibrils and sarcomeres are conserved fromflies to vertebrates. Therefore we chose Drosophila for a systematic genetic analysis of muscle fibermorphogenesis in vivo, with a particular focus on myofibrillogenesis. To identify key regulators, webioinformatically selected all genes with putative cytoskeletal or sarcomere related functions, andcombined these with genome-wide RNAi data for muscle function [Schnorrer F et al 2010]. Weanalyzed muscle fiber phenotypes after muscle-specific gene knockdown of 195 candidate genesusing fluorescently tagged sarcomeric markers. We focused on fibrillar indirect flight muscles andthe morphologically distinct tubular leg and abdominal muscles. This screen identified many novelfactors required for proper organisation of muscle fibers with some of them being exclusivelyimportant for formation of either tubular or fibrillar muscles.

Muscle-specific knockdown of an uncharacterized gene exclusively affects indirect flight musclesresulting in fewer flight muscles and flightless animals. The closest vertebrate homolog of this geneis required for establishment of polarity in epithelia by regulating vesicular trafficking. We find thatour protein of interest also localizes to vesicular structures pointing to a similar function in musclefiber organisation. Strikingly we discovered a role for various other genes implicated in epithelialpolarity for correct flight muscle morphogenesis. Therefore, we propose a novel role for epithelialpolarity proteins in the development and organization of muscle fibers.


Inter-species variation in transcription factor occupancy foressential developmental regulators

Pierre Khoueirya,*, Eileen Furlonga

European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Gene regulatory networks and environmental inputs determine cell fate choices duringembryogenesis. Cis-Regulatory Modules (CRMs), the docking platforms for transcription factors, areat the heart of regulatory networks. They regulate gene expression at early developmental stages andlater during differentiation processes.Genome-wide identification of in-vivo functional CRMs remains a challenge. Using Drosophilamelanogaster as a model organism, our lab lately showed the ability to enhance spatio-temporalCRM activity prediction using in vivo transcription factor binding data for a set of 5 myogenicfactors (Twist, Mef2, Tinman, Bapipe and Biniou) implicated in mesoderm specification (1). Thisapproach unveiled a large number of CRMs linked to a wide range of putative mesodermal genes.

My project aims are 1) to filter the mesoderm network from likely non-functional CRMs and 2) toinvestigate the degree of evolutionary divergence in the targets and combinatorial binding of the fivemesodermal factors in two Drosophila species.

For this, I am performing ChIP-seq analysis for the five myogenic factors on Drosophila virilis, adistantly related species to D. melanogaster (60 Million years). These experiments are carried out onfive consecutive time points spanning mesoderm specification. Prior to ChIP, we generated D. virilisspecific antibodies that showed the expected spatio-temporal activity in immunostaining assays. Inparallel, I expanded and staged D. virilis embryos to match the corresponding stages in D.melanogaster. I then collected and fixed embryos, extracted chromatin and performed ChIPexperiments. IP-purified DNA from different samples as well as input controls are then multiplexedfor library preparation and subjected to sequencing with Illumina HiSeq. In parallel, I have begun toanalyze the data obtained using a combination of in-house scripts and available tools for nextgeneration sequencing analysis.

1: Zinzen, R.P., Girardot, C., Gagneur, J., Braun, M. & Furlong, E.E.M. Nature 462, 65-70 (2009).


Polarization of the Drosophila oocyte by a Cdc42-regulated actincytoskeleton

Andrea Leibfrieda,*, Sandra Müllera, Anne Ephrussia

European Molecular Biology Laboratory, Heidelberg

The Drosophila oocyte is a polarized cell. Polarity establishment and maintenance is essential forcorrect development of the egg and future embryo. Microtubules are prominent regulators of polarityand were believed to mediate the establishment of polarity during oogenesis.The Par proteins Par-6, aPKC and Bazooka are needed to maintain oocyte polarity and localize tospecific domains early in oocyte development. No upstream regulator or mechanism for localizationof the Par proteins in the oocyte has been identified so far. We have analyzed the role of the smallGTPase Cdc42 in oocyte polarity. We show that Cdc42 is required to maintain oocyte fate, which itachieves by mediating localization of Par proteins at distinct sites within the oocyte. We establishthat Cdc42 localization itself is polarized to the antero-lateral cortex of the oocyte and that Cdc42ensures the integrity of the oocyte actin network. Disrupting this network with Latrunculin Aphenocopies loss of Cdc42 or Par protein function in early stages of oogenesis. Finally, we show thatCdc42 and aPKC interact genetically with regard to oocyte polarity and that loss of Par proteinsreciprocally affects Cdc42 localization and the actin network. These results reveal a mutualdependence between Par proteins and Cdc42 for their localization, regulation of the actincytoskeleton and, consequently, for the establishment of oocyte polarity. Our data allow ordering thecomplex sequence of microtubule and actin dependent events required for early oocyte polarity andits maintenance. Cdc42 and actin dominate in the establishment and maintenance of polarity in theyoung oocyte, whereas microtubules act in oocyte specification and late polarity events.


Regulation of pol III transcription by Id2 and E47 proteins

Dritan Likoa,*, Nikiforos Spandidosa, Robert Whitea, Owen Sansoma

Beatson Institute for Cancer Research, Glasgow, UK

RNA polymerase III (pol III) is responsible for transcribing short untranslated sequences, such astRNAs and 5S rRNA. Transcription by pol III is vital for cellular maintenance and involves a numberof intrinsic transcription factors, which recruit the polymerase and others such as c-myc, and HDACsthat interact with members of the pol III machinery or modulate chromatin marks in order toinfluence pol III trancription. Furthermore a number of growth related kinases, oncoproteins andtumor suppressors directly influence pol III. Indeed a number of pol III transcripts areover-represented in cancer samples.Id2 is part of the Id family of proteins that are HLH motif-containing transcription factors. Idsinteracting with factors such as MyoD and E47, inhibit differentiation and promote cell proliferation.They are highly expressed in rapidly proliferating cells, including tumour cells. A protein inhibitedby Id dimerization is E47. It contains a bHLH domain and binds DNA to promote tissue specificdifferentiation.To date, all targets of Id2 and E47 regulation are genes encoding protein. Work presented belowshows that attenuating levels of these proteins influences expression of short non-coding RNAs suchas tRNA and 5S rRNA. Ids stimulate expression of tRNA and 5S rRNA, while E47 represses theirexpression. ChIP experiments position Id2 and E47 at tDNA and 5S rRNA genes arguing for a directlink between Id2/E47 and pol III transcription. Moreover, upon Id2 depletion promoter occupancy ofpol III and factors at tDNAs genes is reduced. The opposite effect is seen upon E47 depletion.Taken together, these observations unearth a possible direct link between Id2, E47 proteins and polIII transcription machinery. Given that Id2, E47 and pol III are involved in tumorigenesis, it isconceivable that part of the effects Id2/E47 have on tumorigenesis are dependent on pol III and viceversa.


PknG mediated signalling pathways in Mycobacteriumtuberculosis

Maria Natalia Lisaa,*, Nathalie Barilonea, Pedro Alzaria

Unité de Microbiologie Structurale, Institut Pasteur, Paris, France.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis and is still a major worldhealth problem. During its life cycle Mtb goes through distinct replicative stages and is also capableof lying in a dormant state for several years. Then, Mtb bacilli must possess efficient signallingsystems to sense the environment and to adapt the bacterial physiology accordingly. Structuralinformation about these mechanisms will thus contribute to the development of new drugs againstMtb.

Mtb possess eukaryotic-like serine/threonine protein kinases that regulate metabolic processes andthe interaction with the host (Cole ST et al, 1998, Nature; Sassetti CM et al, 2003, Mol Microbiol).Among them, protein kinase G (PknG) is essential for mycobacterial survival inside macrophages(Walburger A et al, 2004, Science) and also controls the glutamate metabolism by regulatingdownstream partners (O'Hare HM et al, 2008, Mol Microbiol). Then, PknG is an attractive candidatefor drug target against Mtb, and we are conducting structural studies to better understand themolecular determinants of the enzyme activity. PknG has a unique multidomain topology, with acentral kinase domain flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeatdomains, respectively. Several high resolution X-ray crystal structures have been obtained ofMtb-PknG and also the homologous kinase from Corynebacterium glutamicum. All the obtainedstructures reveal most enzyme active-site signature elements adopting conformations that arecharacteristic of an active protein kinase (Huse M and Kuriyan J, 2002, Cell). However, a tilted androtated orientation of helix alpha-C (a known hotspot for regulation) precludes the formation of aconserved and essential Lys-Glu ionic at the active site of PknG.

After analysis of the data we conclude that the different crystal structures obtained for the Ser/Thrprotein kinase PknG represent an auto-inhibited latent conformation of the enzyme. A hypothesisabout how a conformational switch may occur will also be provided as part of the discussion.


tRNA wobble uridine hypomodification decreases the decodingefficiency of cognate codons in vivo

Danny Nedialkovaa,*, Sebastian Leidela

Max Planck Institute for Molecular Biomedicine, Max Planck Research Group for RNA Biology, Münster, 48149, Germany

Nucleotide modifications in tRNA are ubiquitous in all domains of life and those in the anticodon areimportant for accurate codon recognition during translation. Thiolation at the 2-carbon (s2) of thewobble uridine (U34) base is universally conserved in three tRNA species - tE(UUC), tK(UUU), andtQ(UUG). In the cytoplasm of eukaryotes, U34 also carries a 5-methoxycarbonylmethyl group(mcm5). Aberrant U34 modification is associated with increased stress sensitivity in manyorganisms. In yeast, the phenotypes can be suppressed by overexpressing hypomodified tK(UUU)and tQ(UUG), but the underlying molecular events are unknown. To delineate them, we analyzed thein vivo roles of U34 modification in translation by deep sequencing of ribosome-protected mRNAfragments (ribosome profiling) in Saccharomyces cerevisiae. This approach allowed us toquantitatively compare transcriptome-wide ribosome occupancy in wild type and U34 thiolation-deficient yeast (ncs2Δ) grown in rich medium or after diamide-induced oxidative stress. Diamidetreatment led to widespread changes in gene expression of a similar magnitude in both strains. Lackof U34 thiolation thus does not seem to compromise the cellular response to diamide, despite thehypersensitivity of ncs2Δ yeast to this compound. The number of transcripts with altered ribosomeloading in ncs2Δ cells, however, was larger in the expression response elicited by diamide.Strikingly, we detected significantly higher ribosome occupancy for codons read by thehypomodified tRNAs in the putative ribosomal A site within ribosome-protected fragments fromncs2Δ. This effect was discernible both in cells grown in rich medium or subjected to diamide-induced oxidative stress and was accompanied by smaller increases of cognate codon occupancy alsoupstream of the A site. Our data provides the first in vivo evidence that U34 hypomodification isassociated with translational slowdown at cognate codons in endogenous transcripts. We arecurrently investigating whether this phenomenon is conserved in C. elegans and what its impact is onprotein abundance and function.


Silver birch (Betula pendula): a novel model tree for moleculargenetics

Kaisa Nieminena,*, Juha Immanenb, Risto Hagqvista, Katri Kärkkäinena, YkäHelariuttab

a Finnish Forest Research Institute, Vantaa Research Unit, Vantaa, Finlandb University of Helsinki, Department of Biosciences, Helsinki, Finland

The aim of our research is to understand molecular mechanisms controlling tree development; wewill explore natural variation in forest trees to identify novel genetic regulators of this process. Ourmodel organism is an important forestry tree, silver birch, whose small diploid genome is currentlybeing sequenced. This tree is monoecious, and already young seedlings can be induced to flowerunder greenhouse conditions. Birch brings the power of inbreeding and short generation times intotree genetics, enabling exploitation of advanced crossing schemes for genetic analyses.The main focus of our study is a collection of naturally occurring tree mutants: a diverse set of treeswith atypical wood quality, cambial activity, branching pattern, or secondary metabolite content. Weare currently most advanced in characterisation of a “swirly birch” mutant, which has a strikinggrowth pattern. Its stem and branches grow initially normally, but when they get longer they are notanymore able to stay upright, and instead begin to bend downwards. Excitingly, this tree has anintriguing stem phenotype, where the tension wood formation is mutated into a rotating, “swirly”pattern. We aim, through a whole genome sequencing approach, map the causative gene behind thisphenotype, and study its function in transgenic trees.Besides its fascinating basic science aspect (what makes a tree a tree?), understanding molecularmechanisms regulating tree development has immense applied value. Historically, due to their largesize and long generation times, trees have not been easily accessible for traditional breeding.Detailed knowledge about regulatory mechanisms controlling tree traits will provide us with tools fortheir domestication. Boosting efficient production of woody biomass in commercial forests isessential for sustainable management of natural resources. With birch as our model, our projectrepresents a novel approach of tree genetics with potential for ground-breaking insights into treedevelopment and breeding.


Bacteriophage ΦCbK hijacks the Caulobacter crescentus cellcycle machinery

Gaël Panisa,*, Laurence Théraulaza, Patrick Violliera

Institute of Genetics & Genomics in Geneva (iGE3), Switzerland

Bacteriophages engage in a Trojan horse-style biological warfare with their host: destroying from theinside out by appropriating the host’s major biosynthetic machineries. Caulobacter dividesasymmetrically at each cell cycle, giving two progeny cells with distinct morphologies and fates: thesessile replicating stalked (St) cell and the adventurous quiescent swarmer (Sw) cell that resides inG1-like non-replicative state and must differentiate into the St cell before proceeding to division.Consequently, differentiation and chromosomal replication are coordinated temporally and spatiallyby successive transcriptional waves of master regulators as CtrA and GcrA that are active in Sw andSt cell stages, respectively, that might be targeted by phages. Phage ΦCbK is a virulent double-stranded DNA phage with a flexible noncontractile tail (Order Caudovirales, Family Siphoviridae)and a prolate cylindrical head encasing a 205 kb genome. As ΦCbK infection is restricted to the Swphase the lytic cycle of ΦCbK might be directly coordinated with the Caulobacter cell cycle.Characterizing the lytic cycle revealed a burst size of 13 phage progeny per cell and that the integrityof the host chromosome is maintained. Using ChIP-Seq and immunoblotting approaches, weidentified that ΦCbK phage infection reprograms the host transcriptional pattern and preventsexpression of the host master regulator GcrA. Interestingly, the ΦCbK genome encodes a GcrAortholog suggesting that host GcrA is replaced with phage variant. By contrast, CtrA is still presentin Sw cells during ΦCbK infection and disappears concomitantly with the beginning of phagereplication. ChIP-seq revealed that CtrA binds multiple promoters genes thought to encode phageDNA replication factors such as DNApolI, DNApolIII, TerSL …).This finding overturns the current dogma that in the lytic cycle that phage only manipulates the host.Instead, the phage also exploits intrinsic regulatory cues of the host cell cycle for its own replication.


Molecular mechanisms of chromosome segregation duringmeiosis, a genome-wide approach

Silvia Polakovaa,*, Zsigmond Benkoa, Juraj Gregana

Max F. Perutz Laboratories, Department of Chromosome Biology, University of Vienna, Austria

Sexual reproduction depends on meiosis, the process which produces eggs and sperm cells. Duringmeiosis, the number of chromosomes is halved. Although we understand certain aspects of meiosis,some of the key regulators of meiotic chromosome segregation are still missing and only now we dohave tools to identify these proteins and to determine their function. I will present our systematicscreen of S. pombe knock-out library to identify genes involved in meiotic chromosome segregation.I believe that it is important to understand how the halving of the chromosome copy number duringmeiosis is achieved, because defects in this process cause miscarriages, infertility and geneticdiseases such as Down syndrome.


Serotonin promotes neurogenesis following spinal cord injury inadult zebrafish

Angela Scotta,*, Thomas Beckerb, Catherina Beckerb

a Department of Biology, McMaster University, Hamilton, Canadab Centre for Neuroregeneration, University of Edinburgh, Edinburgh, UK

Neurogenesis, the birth of neurons, occurs within central nervous system (CNS) of all adultvertebrates. Until recently, adult neurogenesis was believed to only take place in specific regions ofthe vertebrate brain. It is now known that, contrary to mammals, adult neurogenesis occurs within thespinal cord of adult zebrafish following a spinal lesion. Lessons from these natural ‘regenerators’will significantly help to uncover signals important for successful endogenous neural regenerationthat are not present in mammals, and identify potential courses of therapy for spinal cord injury. Todate, the molecular factors responsible for spinal neurogenesis within the injured spinal cord arelargely unknown. Previous work has shown that, following spinal cord lesion, serotonergic neuronsare able to regenerate through the lesion site and re-innervate the tissue below the injury. Given this,we investigated the possible neurogenic role serotonin may play within the injured spinal cord. Ourstudy demonstrated that serotonin treatment significantly enhanced neurogenesis of spinal motorneurons following complete spinal cord transection in adult zebrafish. A pharmacological screenhighlighted the potential role of the serotonin receptor 5-HT1A in the generation and differentiationof spinal motor neurons in zebrafish embryos. We confirmed, in adult zebrafish, that the expressionof 5-HT1A was upregulated at the level of the lesion site and the receptor was expressed on theneural progenitor cells within the spinal cord. In addition, the enhanced expression of this receptorwas significantly correlated to the successful recovery of swimming performance at 2 weeks and 6weeks following spinal cord injury in these fish. All together, these findings offer significant insightinto the molecular mechanisms involved in the stimulation of adult neurogenesis and functionalrecovery in vertebrates following spinal cord injury.


Community genomics reveals novel members and complextime-series dynamics in the human microbiome

Itai Sharona,*, Michael J. Morowitzb, Brian C. Thomasa, Elizabeth K. Costelloc,David A. Relmanc, et al.a Department of Earth and Planetary Science, University of California, Berkeley, USAb School of Medicine, University of Pittsburgh, USAc Department of Microbiology & Immunology, School of Medicine, Stanford University, USA

The human microbiome has been associated with various medical conditions and physiologicalprocesses critically important to human health. However, the microbial communities that make upthe human microbiome consist of both closely related and novel species of bacteria, neither of whichcan be resolved or studied using existing methods. Here, I will present the results of our two recentstudies achieved using novel methods developed for the reconstruction of complete genomes frommixed microbial community DNA sequencing (metagenomics), an approach we call communitygenomics. These methods were successfully used to study gut colonization in a premature infant,where we tracked community dynamics over time and revealed strain-level differences between keymembers of the community. Furthermore, in the adult gut microbiome we were able to recover andcharacterize complete genomes from several members of an uncultivated clade ofnon-photosynthetic, phylogenetically diverse Cyanobacteria. These genomes shed light on theevolution of Cyanobacteria, an important microbial phylum responsible for the oxygenation of theEarth’s atmosphere.


Deletion of cavin genes reveals tissue-specific mechanisms formorphogenesis of endothelial caveolae

Carsten Gram Hansena, Elena Shvetsb,*, Gillian Howardb, Kirsi Rientob, BenjaminNicholsb

a Sanford Consortium for Regenerative Medicine and UCSD, 2880 Torrey Pines Scenic Drive, La Jolla, California 92037,USAb MRC Laboratory of Molecular Biology, Cambridge, UK

Caveolae, flask-shaped invaginations of the plasma membrane, are especially abundant inendothelial cells and among their other proposed functions, are thought to play important roles inendothelial permeability and transcytosis. The cavin proteins represent a family of novel keycomponents of caveolae. Importantly, cavins have differential tissue distributions and ability to bindeach other. Thus, we hypothesize that the ability to form complexes is crucial for their functionin-vivo. We find that cavins form high molecular weight complexes in tissues and that apparent sizeof these complexes varies between tissues, when lung and fat contain smaller size complexes, whileheart and kidney tissues contain larger size complexes. We next analyzed the formation of caveolinor cavins complexes in caveolin 1, cavin 1, cavin 2 and cavin 3 gene knockout mice. Caveolin 1 wasnot responsible for cavins complex formation, while cavin 1 was essential for normal oligomerisationof caveolin 1. Deletion of cavin 3 was found to slightly reduce the size of cavin1 complex. Deletionof cavin 2, however, resulted in a marked increase in the size of cavin 1-containing complexes.Importantly, deletion of cavin 2, but not cavin 3 caused loss of endothelial caveolae in lung andadipose tissue, but has no effect on the abundance of endothelial caveolae in the heart and othertissues. This correlated well with tissue-specific changes of endothelial function in cavin 2 null mice.To gain further insight into the role of cavin 2 specifically in lung endothelial cells, we establishedcultures of endothelial cells and further confirmed that cavin 2 controls the size of cavin complexes,and acts to shape caveolae. Our data reveal that endothelial caveolae are unexpectedlyheterogeneous, and identify cavin 2 as a key molecular determinant of this heterogeneity.


The role of core RNA splicing factors in spindle assembly

Magdalena Strzeleckaa,*, Rebecca Healda

University of California, Berkeley, USA

The mitotic spindle is an intricate macromolecular structure required for accurate distribution ofgenetic material during cell division. Most research in the past couple of decades has focused on theprotein components of the spindle. Interestingly, our lab has shown that an RNA component is alsoessential for spindle integrity. Furthermore, a number of genome-wide screens have implicated RNAprocessing factors in regulation of mitotic events. Interestingly, TPX2, a spindle assembly factor hasbeen found to co-purify with active spliceosome, a multicomponent complex catalyzing RNAsplicing reactions. In order to investigate whether spliceosome components play a direct role inmitosis I have been using transcriptionally silent and metaphase-arrested Xenopus egg extracts toreconstitute spindle assembly and study mitotic RNAs. Strikingly, molecular and biochemicalperturbations of spliceosome assembly and function led to defects in spindle integrity. Moreover,next generation sequencing of RNAs from Xenopus tropicalis egg extract and of RNAsco-immunoprecipitated with spliceosomal small nuclear ribonucleoproteins (snRNPs) revealed thatintron-containing pre-mRNAs are present at metaphase, suggesting that their processing might berequired for mitotic progression. However, translation inhibition does not recapitulate spindleintegrity phenotype caused by spliceosome perturbation, indicating that spliceosome plays a role inmitosis in the context of non-coding RNAs. Interestingly, we find that intron-containing non-codingRNAs are also associated with spliceosomal snRNPs in metaphase arrested extracts. Our datasupport the direct involvement of spliceosomal components in mitosis and suggest that it occursthrough non-coding RNAs in splicing dependent or independent manner.


Host-parasite interactions in an anciently asexual invertebrate

Christopher Wilsona,*, Lara Meadea, Grace Mastersb

a Imperial College London, Ascot, UKb Cornell University, Ithaca, NY, USA

Sex and recombination entail multiple evolutionary and genetic costs, yet they are nearly ubiquitousin plants and animals, whereas obligately asexual lineages rapidly become extinct. One explanationfor this puzzle is that recombination enables populations to keep pace with relentlessly coevolvingparasites and pathogens, by facilitating rapid adaptation. In contrast, asexual populations can onlychange their resistance phenotypes via sequential mutation. This 'Red Queen' hypothesis hasconsiderable empirical support, but is challenged by the persistence of at least one lineage for over30 million years without sex. Bdelloid rotifers are microscopic, filter-feeding invertebrates that havediversified into over 400 species and occupy almost every freshwater habitat worldwide. Like allanimals, they suffer from pathogens, including deadly endoparasitic fungi. If the Red Queenhypothesis is correct, how have these hosts successfully resisted their natural enemies for so longwithout sex?

To investigate this question, I isolated and cultured bdelloid rotifers from the genera Habrotrocha andAdineta, differentiating clonal haplotypes by sequencing mitochondrial cytochrome oxidase I. Clonalpopulations were inoculated under controlled conditions with spores from fungal parasites in thegenus Rotiferophthora, and mortality due to infection was quantified after 48 hours. Rotiferophthoraisolates were found to have broad host ranges, but some rotifer clones in both genera werecompletely immune to certain parasite isolates. In quantitative comparisons, even closely relatedrotifer clones differed in their resistance to the same parasite isolate. These infectivity patternssuggest genotype by genotype interactions between hosts and parasites, which are required forreciprocal coevolution. I extracted total RNA from bdelloid rotifers at two time points afterinoculation, in cases where hosts were either highly resistant or highly susceptible to the parasite.Transcriptomic data will help elucidate the genetic basis of resistance to parasites in hosts that havenot undergone sex for millions of years.


KRAB/KAP1 epigenetic regulation in hematopoietic stem cellshomeostasis and lineage commitment

Benyamin Yazdanpanaha,*, Isabelle Bardea, W. Benjamin Rauwela, Matthias Lutolfa,Didier Tronoa

École polytechnique fédérale de Lausanne EPFL, Switzerland

As multipotent progenitors, hematopoietic stem cells (HSC) undergo extensive epigenetic changeson the road towards terminal differentiation. Tightly regulated asymmetric cell divisions ensure theacquisition of lineage-commitment marks by one daughter cell and the maintenance of multipotencyof the other. KAP1 is known as a transcriptional corepressor serving as universal cofactor forKRAB-containing zinc finger proteins (KRAB-ZFPs), a large family of sequence-specific DNAbinding factors notably responsible for the early embryonic silencing of endogenous retroelements.We found that the hemato-restricted knockout of KAP1 in the mouse induced bone marrow failure,correlating with i) the immediate proliferation followed by the exhaustion of long-term repopulatingHSC (LT-HSC) in vivo, with premature upregulation of genes specific of differentiated blood cells;ii) a proliferation defect of LT-HSC ex vivo; iii) an in vivo homing and ex vivo differentiation defectof KAP1 knockdown Lin- Sca1+ cKit+ cells; and iv) the binding of KAP1 to HSC- and otherhemato-specific genes in human CD34+ cells. Furthermore, over 200 KRAB-ZFPs exhibitedlineage- and stage-specific patterns of expression in murine and human hematopoietic cells,consistent with a role for this regulatory system at multiples steps of hematopoiesis. Furtherinvestigation of KRAB/KAP1 mediated epigenetic modifications is on the way to unravel themolecular mechanisms that link KAP1 genomic recruitment and spatio-temporal control of geneexpression with blood differentiation.


Regulation and function of the TRPM6 Mg2+ channel – proteinkinase

Jenny van der Wijsta,*, Joost GJ Hoenderopb, René JM Bindelsb, Dario R Alessia

a The MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Scotlandb Department of Physiology, Radboud University Nijmegen Medical Centre, The Netherlands

IntroductionThe transient receptor potential melastatin subtype 6 (TRPM6) has been identified as the Mg2+ entrypathway in the distal convoluted tubule (DCT) of the kidney. Mutations in the TRPM6 gene arelinked to familial hypomagnesemia with secondary hypocalcemia, characterized by renal Mg2+wasting. This finding highlights the critical role of TRPM6 in controlling Mg2+ homeostasis.Together with its closest homologue TRPM7, TRPM6 is unique as it consists of an ion channelmerged with an alpha-kinase domain. It is classified as “atypical” as it has no sequence homology toany conventional protein kinase. To date, the function of this carboxyl-terminally fused alpha-kinasedomain is poorly understood. My project, therefore, aims to study the regulation of the TRPM6alpha-kinase.MethodsTo this end, in vitro kinase assays, peptide pull down studies and fluorescence polarization analyseswere used.ResultsWild type full length TRPM6 is able to phosphorylate MBP in an in vitro kinase assay, whiletruncating TRPM6 at M1719 blocked this kinase activity. This evidenced a so-called‘dimerization/activation’ region that has previously been identified in TRPM7. Subsequent alaninescanning mutagenesis of residues L1700 to V1730 identified two conserved residues (L1718 andL1721) that are essential for kinase activity and binding within TRPM6 itself. A wild type biotinpeptide, corresponding to residues 1700-1740, demonstrated binding to a region upstream of V1730,which is impossible using the equivalent mutant peptide (L1718/21AA). Reviewing the resolvedTRPM7 alpha-kinase structure predicts the involvement of four residues within the alpha-kinase, andone downstream of the domain. Mutating these residues (L1743A, Q1832K, A1836N, L1840A,L1919Q) resulted in abolished kinase activity due to hampered binding.ConclusionThe data generated in this project will lead to new fundamental understanding of the regulatorymechanisms of the TRPM6 alpha-kinase domain, contributing to development of novel therapies forhypomagnesemia and associated pathologies.


A TPR domain-containing N-terminal module of MPS1 isrequired for its kinetochore localization by Aurora B

WIlco Nijenhuisa, Eleonore von Castelmurb,*, Dene Littlerb, Anastassis Perrakisb,Geert JPL Kopsa, et al.a Molecular Cancer Research and Department of Medical Oncology, University Medical Centre Utrecht, 3584 CG,Utrecht, The Netherlandsb Netherlands Cancer Institute, Department of Biochemistry, 1066 CX, Amsterdam, The Netherlands

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progressionuntil all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitoticcheckpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminalextension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined thecrystal structure. Although the module was necessary for kinetochore localization of MPS1 andessential for the mitotic checkpoint, the predominant kinetochore binding activity resided within theNTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1localization to kinetochores depended on the calponin homology domain of HEC1 but not on AuroraB-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator ofAurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive toAurora B inhibition. These data are consistent with a model in which Aurora B activity relieves aTPR-dependent inhibitory constraint on MPS1 localization.


Structural and biochemical studies of the SLIP1-SLBP complex

Holger von Moellera,*, Rachel Lernerb, Adele Ricciardib, Claire Basquinc, William F.Marzluffb, et al.a Free University of Berlin, Germanyb Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USAc Structural Cell Biology Department, Max Planck Institute of Biochemistry, Munich, 82152 Germany

Messenger RNAs (mRNAs) are produced as precursors that undergo multiple maturation steps(splicing, addition of a 5’-cap structure, 3’-cleavage and, in most cases, polyadenylation) before theycan be exported from the nucleus to guide protein biosynthesis in the cytoplasm. These steps areinterconnected and linked to upstream (transcription) and downstream (export, surveillance andtranslation) processes and are mediated by multi-protein and RNA-protein (RNP) molecularmachines. Compared to the bulk of mRNAs the majority of metazoan replication-dependent histonemRNAs exhibit several distinct features: (1) Their genes are physically linked in clusters; (2) Theirexpression and translation is cell cycle regulated; (3) They are intronless and (4) They form a uniquestem loop (SL) structure in their 3’-untranslated regions (3’-UTR).This specialized 3’-end is bound by SLBP, a multifunctional protein that participates in multiplesteps of the histone gene expression pathway, including nuclear export and translation. Thetranslational activity of SLBP is mediated by interaction with SLIP1, a MIF4G-like protein thatconnects to translation initiation. We determined the 2.5 Å resolution crystal structure of SLIP1bound to the translation-activation domain of SLBP and identified the determinants of recognition.We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiationfactor eIF3g and the mRNA-export factor DBP5. Pull-down assays confirmed the binding of SLIP1to DBP5 and eIF3g and we determined the 3.25 Å resolution structure of SLIP1 bound to the SBMof DBP5. Our results suggest how the SLIP1 homodimer or a SLIP1-CTIF heterodimer can functionas platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNAmetabolism.


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