Plenary Sessions - Wiley Online Library

PLS-Plenary Sessions

Abstracts

PLS – PLENARY SESSIONS PLS-1 – Epithelial Plasticity in Health and Disease Sunday 31 August 16:30–18:00, Grand Auditorium PLS-1-1 Skin stem cells in silence, action and cancer E. Fuchs Rockerfeller University, New York, United States Stem cells (SCs) have the ability to self-renew long term and differentiate into one or more tissues. Typically, SCs are used sparingly to replenish cells during normal homeostasis. However, even SCs that are quiescent must be able to respond quickly to injury in order to fuel rapid tissue regeneration. How SCs balance self-renewal and differentiation is of fundamental importance to our understanding of normal tissue maintenance and wound repair. Increasing evidence suggests that the regulatory circuitry governing this balancing act is at the root of some types of tumors both in mice and in humans. The skin is an excellent model system to understand how SCs function in normal tissue generation and how this process goes awry in cancer. We’ve identified SC niches within the epidermis, hair follicle, sebaceous glands and sweat glands. We’ve learned that different niches become activated in response to different types of injury, and that during normal homeostasis, SC behavior is controlled not only through cues received from their microenvironment but also through signals emanating from their differentiating lineages. We’ve been dissecting how extrinsic signaling to SCs trigger a cascade of transcriptional changes that govern SC activation during tissue development, homeostasis and hair regeneration. Our findings provide new insights into our understanding of the process of SC activation, and in so doing have revealed mechanisms which are also deregulated in a variety of different human cancers. Our goal is to understand how SCs start and stop making tissue, and how this changes in cancer. Our recent discoveries on this topic have led us to the realm of identifying and characterizing cancer SCs (tumor-initiating cells) of squamous cell carcinomas (SCCs) of the skin. We developed a new method to knockdown genes specifically in skin and oral progenitors, enabling us to screen not only the differences between cancer and normal SCs, but also the myriad of gene alterations surfacing from the Human Cancer Sequencing project. Our screens have illuminated new oncogenes and tumor suppressors for SCCs, among the most prevalent and lifethreating cancers world-wide that include cancers of lung, esophagus, breast, cervix, prostate, throat and oral tissues. Our findings are unearthing new targets for cancer therapeutics.

PLS-1-2 Lgr5 stem cells in self-renewal and cancer H. Clevers Hubrecht Institute, Utrecht, The Netherlands The intestinal epithelium is the most rapidly self-renewing tissue in adult mammals. We originally defined Lgr5 as a Wnt target gene, transcribed in colon cancer cells. Two knock-in alleles

FEBS Journal 281 (Suppl. 1) (2014) 5–8 ª 2014 The Authors. FEBS Journal ª 2014 FEBS

revealed exclusive expression of Lgr5 in cycling, columnar cells at the crypt base. Using lineage tracing experiments in adult mice, we found that these Lgr5+ve crypt base columnar cells (CBC) generated all epithelial lineages throughout life, implying that they represent the stem cell of the small intestine and colon. Lgr5 was subsequently found to represent an exquisitely specific and almost ‘generic’ marker for stem cells, including in hair follicles, kidney, liver, mammary gland, inner ear tongue and stomach epithelium. Single sorted Lgr5+ve stem cells can initiate ever-expanding crypt-villus organoids, or so called ‘mini-guts’ in 3D culture. The technology is based on the observation that Lgr5 is the receptor for a potent stem cell growth factor, R-spondin. Similar 3D cultures systems have been developed for the Lgr5+ve stem cells of stomach, liver, pancreas and kidney. Intestinal cancer is initiated by Wnt pathway-activating mutations in genes such as APC. As in most cancers, the cell of origin has remained elusive. Deletion of APC in stem cells, but not in other crypt cells results in progressively growing neoplasia, identifying the stem cell as the cell-of-origin of adenomas. Moreover, a stem cell/ progenitor cell hierarchy is maintained in early stem cell-derived adenomas, lending support to the ‘cancer stem cell’-concept.

PLS-1-3 Cell Plasticity in health and disease A. Nieto Instituto de Neurociencias, UMH - CSIC, Alicante, Spain The epithelial to mesenchymal transition (EMT) is required in the embryo for the formation of tissues which cells originate far from their final destination. This programme endows cells with migratory and invasive properties. Interestingly, the reverse process, known as mesenchymal to epithelial transition (MET), is also essential in embryogenesis to allow the differentiation of tissues and organs once the embryonic migratory cells reach their final destination. This epithelial plasticity also operates after the pathological activation of the EMT that promotes the delamination of cancer cells from the primary tumor in their way to form metastasis. Similar to the situation in embryos, a reversion of the EMT (MET) is necessary for metastatic colonization once malignant cells extravasate and find their niche in distant organs. The epithelial plasticity has important implications in the design of therapeutic strategies. On the one hand, fully preventing delamination from the primary tumor will impede metastasis, which is the principle that has inspired efforts by academia and pharmaceutical companies to block the EMT. However, in the light of these recent data on the need for cancer cells to revert to the epithelial phenotype for metastasis formation, inhibiting EMT could favour the formation of secondary tumors from already disseminated cells. By contrast, the EMT associated to the development of fibrosis can be considered as an end stage. Renal epithelial cells undergo a partial EMT in response to a chronic insult that leads to organ degeneration and failure. These dedifferentiated cells become non-functional and never revert to the epithelial phenotype. Therefore, in contrast to the situation in cancer, therapeutic intervention inhibiting EMT can be envisioned as a promising strategy to amelliorate fibrosis.

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Abstracts PLS-2 – Epigenetics and Gene Expression Monday 1 September 16:30–18:00, Grand Auditorium PLS-2-1 When genomes meet – RNA, epigenetics and phenotypes of hybrid plant D. Baulcombe, P. Shivaprasad, Q. Gouil and D. Bond Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, United Kingdom Eukaryotes contain small regulatory RNAs that have been referred to as the dark matter of genetics. They are typically 21–24 nucleotides long, associated with Argonaut or Piwi proteins. Some of these small RNAs guide the Argonaut/Piwi protein to a complementary RNA and they are negative regulators of gene expression acting at the level of messenger RNA turnover or translation. Others participate in more complex epigenetic systems affecting chromatin or they act as part of an RNA signal that moves between cells. In plants the posttranscriptional mechanism is involved in defense against RNA viruses. The chromatin effects play a role in defense against DNA viruses and transposable elements and it is associated with the establishment of heritable epigenetic marks. However the importance of this defense system goes beyond suppression of transposons and viruses. There are secondary effects of the epigenetic marks that may influence the expression of adjacent genes in the sense of McClintocks ‘controlling elements’. In most instances the effect is gene silencing and in some instances the effect may influence the biology of the affected plant. I will describe how RNA silencing may be particularly important following wide cross hybridisation and how it may influence hybrid vigour and transgressive segregation.

PLS-2-2 The role of DNA modifications in epigenetic reprogramming and signalling W. Reik Babraham Institute, Cambridge, United Kingdom Epigenetic information is relatively stable in somatic cells but is reprogrammed on a genome wide level in germ cells and early embryos. This reprogramming is essential for imprinting, and important for the return to pluripotency including the generation of iPS cells, the erasure of epimutations, and perhaps for the control of transposons in the genome. Following reprogramming, epigenetic marking occurs during lineage commitment in the embryo in order to ensure the stability of the differentiated state in adult tissues. Signalling and cell interactions that occur during these sensitive periods in development may have an impact on the epigenome with potentially long lasting effects. A key component of reprogramming is the erasure of DNA methylation, which may occur by passive (replication dependent) or active mechanisms. We are using genome wide profiling methods of methylation as well as the transcriptome in order to understand better the dynamics of erasure of DNA methylation and its biological outcomes. We have carried out genome-wide BS-seq (bisulfite sequencing) and RNA-seq in primordial germ cells and in early embryos, which has revealed the extent of reprogramming on an unprecedented scale, but also identifies potential mechanisms of transgenerational epigenetic inheritance. Reprogramming appears to be inextricably linked with expression of the pluripotency transcription factor network. The mechanisms leading to loss of DNA methylation probably involve an intricate combination of passive (DNA replication without maintaining methylation) and active mechanisms, in which

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PLS – Plenary Sessions deamination (by AID) and hydroxylation (by TET1-3) as well as base excision repair is implicated. We have identified signalling events which regulate DNA methylation dynamics during early development, and which connect reprogramming firmly with na€ıve pluripotency. We are investigating the roles of these pathways in natural and in experimental reprogramming. Recent work has identified proteins that may interpret different DNA modifications in the genome to regulate transcription and chromatin.

PLS-2-3 Loss of heterochromatin: effects on development and genome stability S. M. Gasser Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland Over 60% of the human genome consists of repeat DNA and only a few per cent of our genome actually codes for proteins. As cells differentiate into specific cells types, basically all of the repeat DNA and most of our non-repeat sequences are packaged into a nontranscribed structure called heterochromatin. The histones bound to heterochroamtin bear specific modifications, which themselves attract transcription-repressing nonhistone proteins. In most cells heterochromatin is sequestered away from active chromatin by interaction with the nuclear envelope or the nucleolus. We have used the simple threadworm C. elegans to examine the importance of heterochromatin sequestration during the development of a multicellular organism. Genetic screens have identified the readers and writers of heterochromatin marks, and we have been able to eliminate these in worm embryos. Intriguing are the developmental aberrations that occur in cells that lack the characteristic heterochromatin marks. What happens when repeat sequences are no longer silenced ? Is development misprogrammed. C. elegans has offered a unique and elegant model system to explore these questions, since centromere function is not dependent on H3K9 methylation. We find that genome stability is one of the primary functions achieved by packaging the genome into heterochromatin: repeat DNA including transposons are transcribed and meiotic maturation of germline cells becomes perturbed. This suggests that the changes in heterochromatin that accompany oncogenic transformation may cause and not only result from transformation.

PLS-3 – Bioinformatics and Systems Biology Tuesday 2 September 16:30–18:00, Grand Auditorium PLS-3-1 Human genetic diversity and functional variation R. Durbin Wellcome Trust Sanger Institute, Cambridge, United Kingdom Fourteen years after the initial publication of the draft human genome sequence, we can now sequence an individual human genome for approximately 1000 euros. This decrease in cost and increase in throughput has allowed us to identify genetic variants both at the population and individual level, supporting new insights both into our genetic history and functional information in the genome. I will talk about results from the final phase of the 1000 Genomes Project, obtained from sequencing 2,500 people from 26 populations from around the world, from the UK10K project sequencing 3,600 people with 61 measured biomedical traits alongside ~6,000 exomes from disease patients, and initial results from the British Autozygosity Population Gene Function Study looking at rare functional homozygous variants.


PLS – Plenary Sessions PLS-3-2 Genetic variability and the quantitative proteome R. Aebersold Department of Biology, Institute of Molecular Systems Biology, ETH, Zurich, Switzerland The question how genetic variability is translated into phenotypes is fundamental in biology and medicine. Powerful genomic technologies now determine genetic variability at a genomic level and at unprecedented speed, accuracy and rather low cost. To date, the effects of genomic variability on the expressed information of the cell has been mainly studied by transcript profiling. In this presentation we will discuss emerging computational and quantitative proteomic technologies to relate genotypic variation to the proteome. Proteomic data to support such correlations need to be quantitatively accurate, highly reproducible across multiple measurements and samples, and generable at high throughput. Data with these qualities can now be generated by the targeted proteomic methods selected reaction monitoring (SRM) and, at higher throughput, by SWATH-MS. We will discuss the principles of these mass spectrometric methods, discuss the computational challenges they pose for data analysis, and demonstrate with selected applications their ability to determine the effect of genetic variability on the quantitative proteome, thus functionally connecting the genome to the proteom.

PLS-3-3 RNA architectural modules and the RNAPuzzles modeling contest E. Westhof Institut de Biologie Mol eculaire et Cellulaire (IBMC), Strasbourg, France RNA architecture is thus viewed as the hierarchical assembly of preformed double-stranded helices defined by Watson-Crick base pairs and RNA modules maintained by non-Watson-Crick base pairs. RNA modules are recurrent ensemble of ordered non-Watson-Crick base pairs. Such RNA modules constitute a signal for detecting structured non-coding RNAs with specific biological functions. It is, therefore, important to be able to recognize such elements within genomes. Through systematic comparisons between homologous sequences and X-ray structures, followed by automatic clustering, the whole range of sequence diversity in recurrent RNA modules has been characterized. These data permitted the construction of a computational pipeline for identifying known 3D structural modules in single and multiple RNA sequences in the absence of any other information. Any module can in principle be searched, but four can be searched automatically: the G-bulged loop, the Kink-turn, the C-loop and the tandem GA loop. The present pipeline can be used for RNA 2D structure refinement, 3D model assembly, and for searching and annotating structured RNAs. RNA-Puzzles are collective and blind experiments in RNA three-dimensional structure prediction. The goals are to assess the leading edge of RNA structure prediction techniques, compare existing methods and tools, and evaluate their relative strengths, weaknesses, and limitations in terms of


Abstracts sequence length and structural complexity. The results should give potential users insight into the suitability of available methods for different applications and facilitate efforts in the RNA structure prediction community in their efforts to improve their tools. Generally, the less well-predicted models always had worse non-Watson-Crick scores, demonstrating the importance of identifying non-Watson-Crick pairs and RNA modules.

PLS-4 – Gene Diversification and Immune Recognition Wednesday 4 September 16:30–18:00, Grand Auditorium PLS-4-1 Evolution of alternative adaptive immune systems in vertebrates M. D. Cooper Emory University School of Medicine, Atlanta, United States All living organisms have innate immune systems which can be used for self-defense. However, only vertebrate species have been shown to have an adaptive immune system that is based on clonally-diverse lymphocytes that are capable of recognizing specific pathogens and providing protective memory against a second encounter. Surprisingly, alternative adaptive immune systems have been found in jawed and jawless vertebrates. While both employ lymphocytes with unique anticipatory receptors, the lymphocytes in jawless vertebrates (lampreys and hagfish) use leucine-rich-repeat (LRR)-based variable lymphocyte receptors (VLR) to recognize antigens, whereas lymphocytes in jawed vertebrates use immunoglobulin-based T and B cell receptors (TCR/ BCR) for the same purpose. Three lamprey VLR loci, VLRA, VLRB and VLRC, undergo recombinatorial assembly to generate highly diverse VLRA, VLRB and VLRC receptors; these are differentially expressed by separate populations of lymphocytes that resemble our thymus-derived cd and ab T cells and bone marrow-derived B cells. The fundamentally similar genetic programs employed in jawless and jawed vertebrates for two major T-cell differentiation pathways and a B-cell differentiation pathway suggests they were already present in a common ancestor before the subsequent convergent evolution of the LRR-based VLR types and Ig-based TCR/BCR types of antigen receptors. Evolutionary and biomedical implications of these alternative adaptive immune systems will be discussed in this presentation.

PLS-4-2 Maintaining the integrity of the genome: gene conversion and the repair of double-strand breaks M. Jasin Memorial Sloan Kettering Cancer Center, New York, United States Abstract not available.

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Abstracts PLS-4-3 Sensing the unknown: how B cells are activated by a large set of different ligands M. Reth Max Planck Institute of Immunobiology and Epigenetics, Freiburg, Germany Since the discovery of antibodies (first in the form of antitoxins in 1890) immunologists have always wondered how our body can produce antibodies against so many different substances that they summarize with the word antigen. In 1956 Burnet proposed that a specific antibody response is established by a selection process that out of a large population of diverse B cells activates those with the right cognate B cell antigen receptor (BCR). The antigen-dependent stimulation of these cells results in their clonal outgrowth and differentiation to antibody producing plasma cells. How the BCR diversity is generated in the B cell population was explained by the finding of the V(D)J rearrangement and somatic hypermutation mechanisms of the immunoglobulin genes over the last decades. What is less well understood is how the many different antigens can activate the BCR in a way that is independent of the spatial organization of epitopes on the antigen molecules. This is quite different from most other receptors that have only one ligand whose structure helps to of bring or stabilize the receptor in an active conformation. As a solution for this problem, we have proposed the Dissociation Activation Model (DAM) according to which it is the opening of a pre-

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PLS – Plenary Sessions organized receptor cluster (oligomer) that activates the BCR independently of the structural input of the antigen. With the Fab base proximity ligation assay (Fab-PLA) we have developed a method which allows us to reliably monitor the conformation of the BCR on the surface of normal B cells at 10-20 nanometer distances. With this and other methods we found that the BCR exits indeed as an ordered oligomer on the surface of B cells and that in contact to antigen this oligomer is opened thus initiating the signaling process. Furthermore we discovered that the majority of the roughly 120.000 BCR on the surface of B cells are not opened by antigen but rather by the spleen tyrosine kinase (Syk) via an inside-out signaling mechanism. The details of this process will be discussed during my lecture. This study was supported by the Excellence Initiative of the German Federal and State Governments (EXC 294), by the Deutsche Forschungsgemeinschaft through SFB746 and TRR130 and by an ERC Advanced Grant on nano-Islands. References 1. Yang, J. and Reth, M. (2010). The dissociation activation model of B cell antigen receptor triggering. FEBS Letters 584: 4872. 2. Klaesener, K., P. Maity, et al. (2014). ‘B cell activation involve nanoscale receptor reorganizations and i out signaling by Syk’. eLife, on-line in June.
