Project acronym BIZEB
Project Bio-Imaging of Zoonotic and Emerging Bunyaviruses
Researcher (PI) Juha Huiskonen
Host Institution (HI) HELSINGIN YLIOPISTO
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary We aim to understand host cell entry of enveloped viruses at molecular level. A crucial step in this process is when the viral membrane fuses with the cell membrane. Similarly to cell–cell fusion, this step is mediated by fusion proteins (classes I–III). Several medically important viruses, notably dengue and many bunyaviruses, harbour a class II fusion protein. Class II fusion protein structures have been solved in pre- and post-fusion conformation and in some cases different factors promoting fusion have been determined. However, questions about the most important steps of this key process remain unanswered. I will focus on the entry mechanism of bunyaviruses by using cutting-edge, high spatial and temporal resolution bio-imaging techniques. These viruses have been chosen as a model system to maximise the significance of the project: they form an emerging viral threat to humans and animals, no approved vaccines or antivirals exist for human use and they are less studied than other class II fusion protein systems. Cryo-electron microscopy and tomography will be used to solve high-resolution structures (up to ~3 Å) of viruses, in addition to virus–receptor and virus–membrane complexes. Advanced fluorescence microscopy techniques will be used to probe the dynamics of virus entry and fusion in vivo and in vitro. Deciphering key steps in virus entry is expected to contribute to rational vaccine and drug design. During this project I aim to establish a world-class laboratory in structural and cellular biology of emerging viruses. The project greatly benefits from our unique biosafety level 3 laboratory offering advanced bio-imaging techniques. Furthermore it will also pave way for similar projects on other infectious viruses. Finally the novel computational image processing methods developed in this project will be broadly applicable for the analysis of flexible biological structures, which often pose the most challenging yet interesting questions in structural biology.
Summary
We aim to understand host cell entry of enveloped viruses at molecular level. A crucial step in this process is when the viral membrane fuses with the cell membrane. Similarly to cell–cell fusion, this step is mediated by fusion proteins (classes I–III). Several medically important viruses, notably dengue and many bunyaviruses, harbour a class II fusion protein. Class II fusion protein structures have been solved in pre- and post-fusion conformation and in some cases different factors promoting fusion have been determined. However, questions about the most important steps of this key process remain unanswered. I will focus on the entry mechanism of bunyaviruses by using cutting-edge, high spatial and temporal resolution bio-imaging techniques. These viruses have been chosen as a model system to maximise the significance of the project: they form an emerging viral threat to humans and animals, no approved vaccines or antivirals exist for human use and they are less studied than other class II fusion protein systems. Cryo-electron microscopy and tomography will be used to solve high-resolution structures (up to ~3 Å) of viruses, in addition to virus–receptor and virus–membrane complexes. Advanced fluorescence microscopy techniques will be used to probe the dynamics of virus entry and fusion in vivo and in vitro. Deciphering key steps in virus entry is expected to contribute to rational vaccine and drug design. During this project I aim to establish a world-class laboratory in structural and cellular biology of emerging viruses. The project greatly benefits from our unique biosafety level 3 laboratory offering advanced bio-imaging techniques. Furthermore it will also pave way for similar projects on other infectious viruses. Finally the novel computational image processing methods developed in this project will be broadly applicable for the analysis of flexible biological structures, which often pose the most challenging yet interesting questions in structural biology.
Max ERC Funding
1 998 375 €
Duration
Start date: 2015-04-01, End date: 2020-03-31
Project acronym BrokenGenome
Project Breaking and rebuilding the genome: mechanistic rules for the dangerous game of sex.
Researcher (PI) Corentin CLAEYS BOUUAERT
Host Institution (HI) UNIVERSITE CATHOLIQUE DE LOUVAIN
Call Details Starting Grant (StG), LS1, ERC-2018-STG
Summary Sexual reproduction depends on the programmed induction of DNA double-strand breaks (DSBs) and their ensuing repair by homologous recombination. This complex process is essential for sexual reproduction because it ultimately allows the pairing and separation of homologous chromosomes during formation of haploid gametes. Although meiotic recombination has been investigated for decades, many of the underlying molecular processes remain unclear, largely due to the lack of biochemical studies. I have recently made important progress by, for the first time, successfully purifying proteins involved in two aspects of meiotic recombination: DSB formation and the final stage of formation of the crossovers that are a central raison-d’être of meiotic recombination. This has opened new avenues for future research that I intend to pursue in my own laboratory. Here, I propose a set of biochemical approaches, complemented by molecular genetics methods, to gain insights into four central problems: (i) How meiotic proteins collaborate to induce DSBs; (ii) How DSB proteins interact with components that form the axes of meiotic chromosomes; (iii) How proteins involved at later stages of recombination form crossovers; and (iv) How crossover proteins interact with components of synapsed chromosomes. For each problem, I will set up in vitro systems to probe the activities of the players involved, their interactions with DNA, and their assembly into macromolecular complexes. In addition, I propose to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis. My goal is to gain insights into the mechanisms that govern meiotic recombination. Importantly, these mechanisms are intimately linked not only to gamete formation, but also to the general recombination pathways that all cells use to maintain genome stability. In both contexts, our findings will be relevant to the development and avoidance of disease states.
Summary
Sexual reproduction depends on the programmed induction of DNA double-strand breaks (DSBs) and their ensuing repair by homologous recombination. This complex process is essential for sexual reproduction because it ultimately allows the pairing and separation of homologous chromosomes during formation of haploid gametes. Although meiotic recombination has been investigated for decades, many of the underlying molecular processes remain unclear, largely due to the lack of biochemical studies. I have recently made important progress by, for the first time, successfully purifying proteins involved in two aspects of meiotic recombination: DSB formation and the final stage of formation of the crossovers that are a central raison-d’être of meiotic recombination. This has opened new avenues for future research that I intend to pursue in my own laboratory. Here, I propose a set of biochemical approaches, complemented by molecular genetics methods, to gain insights into four central problems: (i) How meiotic proteins collaborate to induce DSBs; (ii) How DSB proteins interact with components that form the axes of meiotic chromosomes; (iii) How proteins involved at later stages of recombination form crossovers; and (iv) How crossover proteins interact with components of synapsed chromosomes. For each problem, I will set up in vitro systems to probe the activities of the players involved, their interactions with DNA, and their assembly into macromolecular complexes. In addition, I propose to develop new methodology for identifying proteins that are associated with DNA that has undergone recombination-related DNA synthesis. My goal is to gain insights into the mechanisms that govern meiotic recombination. Importantly, these mechanisms are intimately linked not only to gamete formation, but also to the general recombination pathways that all cells use to maintain genome stability. In both contexts, our findings will be relevant to the development and avoidance of disease states.
Max ERC Funding
1 499 075 €
Duration
Start date: 2019-07-01, End date: 2024-06-30
Project acronym BUNDLEFORCE
Project Unravelling the Mechanosensitivity of Actin Bundles in Filopodia
Researcher (PI) Antoine Guillaume Jegou
Host Institution (HI) CENTRE NATIONAL DE LA RECHERCHE SCIENTIFIQUE CNRS
Call Details Starting Grant (StG), LS1, ERC-2015-STG
Summary Eukaryotic cells constantly convert signals between biochemical energy and mechanical work to timely accomplish many key functions such as migration, division or development. Filopodia are essential finger-like structures that emerge at the cell front to orient the cell in response to its chemical and mechanical environment. Yet, the molecular interactions that make the filopodia mechanosensitive are not known. To tackle this challenge we propose unique biophysical in vitro and in vivo experiments of increasing complexity. Here we will focus on how the underlying actin filament bundle regulates filopodium growth and retraction cycles at the micrometer and seconds scales. These parallel actin filaments are mainly elongated at their barbed-end by formins and cross-linked by bundling proteins such as fascins.
We aim to:
1) Elucidate how formin and fascin functions are regulated by mechanics at the single filament level. We will investigate how formin partners and competitors present in filopodia affect formin processivity; how fascin affinity for the side of filaments is modified by filament tension and formin presence at the barbed-end.
2) Reconstitute filopodium-like actin bundles in vitro to understand how actin bundle size and fate are regulated down to the molecular scale. Using a unique experimental setup that combines microfluidics and optical tweezers, we will uncover for the first time actin bundles mechanosensitive capabilities, both in tension and compression.
3) Decipher in vivo the mechanics of actin bundles in filopodia, using fascins and formins with integrated fluorescent tension sensors.
This framework spanning from in vitro single filament to in vivo meso-scale actin networks will bring unprecedented insights into the role of actin bundles in filopodia mechanosensitivity.
Summary
Eukaryotic cells constantly convert signals between biochemical energy and mechanical work to timely accomplish many key functions such as migration, division or development. Filopodia are essential finger-like structures that emerge at the cell front to orient the cell in response to its chemical and mechanical environment. Yet, the molecular interactions that make the filopodia mechanosensitive are not known. To tackle this challenge we propose unique biophysical in vitro and in vivo experiments of increasing complexity. Here we will focus on how the underlying actin filament bundle regulates filopodium growth and retraction cycles at the micrometer and seconds scales. These parallel actin filaments are mainly elongated at their barbed-end by formins and cross-linked by bundling proteins such as fascins.
We aim to:
1) Elucidate how formin and fascin functions are regulated by mechanics at the single filament level. We will investigate how formin partners and competitors present in filopodia affect formin processivity; how fascin affinity for the side of filaments is modified by filament tension and formin presence at the barbed-end.
2) Reconstitute filopodium-like actin bundles in vitro to understand how actin bundle size and fate are regulated down to the molecular scale. Using a unique experimental setup that combines microfluidics and optical tweezers, we will uncover for the first time actin bundles mechanosensitive capabilities, both in tension and compression.
3) Decipher in vivo the mechanics of actin bundles in filopodia, using fascins and formins with integrated fluorescent tension sensors.
This framework spanning from in vitro single filament to in vivo meso-scale actin networks will bring unprecedented insights into the role of actin bundles in filopodia mechanosensitivity.
Max ERC Funding
1 499 190 €
Duration
Start date: 2016-03-01, End date: 2021-02-28
Project acronym BURSTREG
Project Single-molecule visualization of transcription dynamics to understand regulatory mechanisms of transcriptional bursting and its effects on cellular fitness
Researcher (PI) Tineke LENSTRA
Host Institution (HI) STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS
Call Details Starting Grant (StG), LS1, ERC-2017-STG
Summary Transcription in single cells is a stochastic process that arises from the random collision of molecules, resulting in heterogeneity in gene expression in cell populations. This heterogeneity in gene expression influences cell fate decisions and disease progression. Interestingly, gene expression variability is not the same for every gene: noise can vary by several orders of magnitude across transcriptomes. The reason for this transcript-specific behavior is that genes are not transcribed in a continuous fashion, but can show transcriptional bursting, with periods of gene activity followed by periods of inactivity. The noisiness of a gene can be tuned by changing the duration and the rate of switching between periods of activity and inactivity. Even though transcriptional bursting is conserved from bacteria to yeast to human cells, the origin and regulators of bursting remain largely unknown. Here, I will use cutting-edge single-molecule RNA imaging techniques to directly observe and measure transcriptional bursting in living yeast cells. First, bursting properties will be quantified at different endogenous and mutated genes to evaluate the contribution of cis-regulatory promoter elements on bursting. Second, the role of trans-regulatory complexes will be characterized by dynamic depletion or gene-specific targeting of transcription regulatory proteins and observing changes in RNA synthesis in real-time. Third, I will develop a new technology to visualize the binding dynamics of single transcription factor molecules at the transcription site, so that the stability of upstream regulatory factors and the RNA output can directly be compared in the same cell. Finally, I will examine the phenotypic effect of different bursting patterns on organismal fitness. Overall, these approaches will reveal how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.
Summary
Transcription in single cells is a stochastic process that arises from the random collision of molecules, resulting in heterogeneity in gene expression in cell populations. This heterogeneity in gene expression influences cell fate decisions and disease progression. Interestingly, gene expression variability is not the same for every gene: noise can vary by several orders of magnitude across transcriptomes. The reason for this transcript-specific behavior is that genes are not transcribed in a continuous fashion, but can show transcriptional bursting, with periods of gene activity followed by periods of inactivity. The noisiness of a gene can be tuned by changing the duration and the rate of switching between periods of activity and inactivity. Even though transcriptional bursting is conserved from bacteria to yeast to human cells, the origin and regulators of bursting remain largely unknown. Here, I will use cutting-edge single-molecule RNA imaging techniques to directly observe and measure transcriptional bursting in living yeast cells. First, bursting properties will be quantified at different endogenous and mutated genes to evaluate the contribution of cis-regulatory promoter elements on bursting. Second, the role of trans-regulatory complexes will be characterized by dynamic depletion or gene-specific targeting of transcription regulatory proteins and observing changes in RNA synthesis in real-time. Third, I will develop a new technology to visualize the binding dynamics of single transcription factor molecules at the transcription site, so that the stability of upstream regulatory factors and the RNA output can directly be compared in the same cell. Finally, I will examine the phenotypic effect of different bursting patterns on organismal fitness. Overall, these approaches will reveal how bursting is regulated at the molecular level and how different bursting patterns affect the heterogeneity and fitness of the organism.
Max ERC Funding
1 950 775 €
Duration
Start date: 2018-01-01, End date: 2022-12-31
Project acronym C-CLEAR
Project Complement: to clear or not to clear
Researcher (PI) Piet Gros
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Advanced Grant (AdG), LS1, ERC-2017-ADG
Summary Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear.
My lab has a long-standing track record in elucidating molecular mechanisms underlying key complement reactions. We have revealed, for example, how the interplay between assembly and proteolysis of these large multi-domain protein complexes achieves elementary regulatory functions, such as localization, amplification and inhibition, in the central (so-called alternative) pathway of complement. Results from my lab underpin research programs for the development of novel therapeutic approaches in academia and industry.
Here the goal is to understand how the molecular mechanisms of complement attack and defense on cell membranes determine clearance of a cell. Enabled by new mechanistic insights and preliminary data we can now address both long-standing and novel questions. In particular, we will address the role of membrane organization and dynamics in complement attack and defense. Facilitated by recent technological developments, we will combine crystallography, cryo-EM, cryo-ET and high-resolution microscopy to resolve complement complex formations and reactions on membranes.
Thus, this project aims to provide an integrative understanding of the molecular complement mechanisms that determine cell fate. Results will likely be of immediate importance for novel therapeutic approaches for a range of complement-related diseases. Furthermore, it will provide clarity into the general, and possibly fundamental, role of complement in tissue maintenance in mammals.
Summary
Mammalian complement recognizes a variety of cell-surface danger and damage signals to clear invading microbes and injured host cells, while protecting healthy host cells. Improper complement responses contribute to diverse pathologies, ranging from bacterial infections up to paralyzing Guillain-Barré syndrome and schizophrenia. What determines the balance between complement attack reactions and host-cell defense measures and, thus, what drives cell fate is unclear.
My lab has a long-standing track record in elucidating molecular mechanisms underlying key complement reactions. We have revealed, for example, how the interplay between assembly and proteolysis of these large multi-domain protein complexes achieves elementary regulatory functions, such as localization, amplification and inhibition, in the central (so-called alternative) pathway of complement. Results from my lab underpin research programs for the development of novel therapeutic approaches in academia and industry.
Here the goal is to understand how the molecular mechanisms of complement attack and defense on cell membranes determine clearance of a cell. Enabled by new mechanistic insights and preliminary data we can now address both long-standing and novel questions. In particular, we will address the role of membrane organization and dynamics in complement attack and defense. Facilitated by recent technological developments, we will combine crystallography, cryo-EM, cryo-ET and high-resolution microscopy to resolve complement complex formations and reactions on membranes.
Thus, this project aims to provide an integrative understanding of the molecular complement mechanisms that determine cell fate. Results will likely be of immediate importance for novel therapeutic approaches for a range of complement-related diseases. Furthermore, it will provide clarity into the general, and possibly fundamental, role of complement in tissue maintenance in mammals.
Max ERC Funding
2 332 500 €
Duration
Start date: 2018-07-01, End date: 2023-06-30
Project acronym CaBiS
Project Chemistry and Biology in Synergy - Studies of hydrogenases using a combination of synthetic chemistry and biological tools
Researcher (PI) Gustav Oskar BERGGREN
Host Institution (HI) UPPSALA UNIVERSITET
Call Details Starting Grant (StG), LS1, ERC-2016-STG
Summary My proposal aims to take advantage of my ground-breaking finding that it is possible to mature, or activate, the [FeFe] hydrogenase enzyme (HydA) using synthetic mimics of its catalytic [2Fe] cofactor. (Berggren et al, Nature, 2013) We will now explore the chemistry and (bio-)technological potential of the enzyme using an interdisciplinary approach ranging from in vivo biochemical studies all the way to synthetic model chemistry. Hydrogenases catalyse the interconversion between protons and H2 with remarkable efficiency. Consequently, they are intensively studied as alternatives to Pt-catalysts for these reactions, and are arguably of high (bio-) technological importance in the light of a future “hydrogen society”.
The project involves the preparation of novel “artificial” hydrogenases with the primary aim of designing spectroscopic model systems via modification(s) of the organometallic [2Fe] subsite. In parallel we will prepare in vitro loaded forms of the maturase HydF and study its interaction with apo-HydA in order to further elucidate the maturation process of HydA. Moreover we will develop the techniques necessary for in vivo application of the artificial activation concept, thereby paving the way for a multitude of studies including the reactivity of artificial hydrogenases inside a living cell, but also e.g. gain-of-function studies in combination with metabolomics and proteomics. Inspired by our work on the artificial maturation system we will also draw from our knowledge of Nature’s [FeS] cluster proteins in order to prepare a novel class of “miniaturized hydrogenases” combining synthetic [4Fe4S] binding oligopeptides with [2Fe] cofactor model compounds.
Our interdisciplinary approach is particularly appealing as it not only provides further insight into hydrogenase chemistry and the maturation of metalloproteins, but also involves the development of novel tools and concepts applicable to the wider field of bioinorganic chemistry.
Summary
My proposal aims to take advantage of my ground-breaking finding that it is possible to mature, or activate, the [FeFe] hydrogenase enzyme (HydA) using synthetic mimics of its catalytic [2Fe] cofactor. (Berggren et al, Nature, 2013) We will now explore the chemistry and (bio-)technological potential of the enzyme using an interdisciplinary approach ranging from in vivo biochemical studies all the way to synthetic model chemistry. Hydrogenases catalyse the interconversion between protons and H2 with remarkable efficiency. Consequently, they are intensively studied as alternatives to Pt-catalysts for these reactions, and are arguably of high (bio-) technological importance in the light of a future “hydrogen society”.
The project involves the preparation of novel “artificial” hydrogenases with the primary aim of designing spectroscopic model systems via modification(s) of the organometallic [2Fe] subsite. In parallel we will prepare in vitro loaded forms of the maturase HydF and study its interaction with apo-HydA in order to further elucidate the maturation process of HydA. Moreover we will develop the techniques necessary for in vivo application of the artificial activation concept, thereby paving the way for a multitude of studies including the reactivity of artificial hydrogenases inside a living cell, but also e.g. gain-of-function studies in combination with metabolomics and proteomics. Inspired by our work on the artificial maturation system we will also draw from our knowledge of Nature’s [FeS] cluster proteins in order to prepare a novel class of “miniaturized hydrogenases” combining synthetic [4Fe4S] binding oligopeptides with [2Fe] cofactor model compounds.
Our interdisciplinary approach is particularly appealing as it not only provides further insight into hydrogenase chemistry and the maturation of metalloproteins, but also involves the development of novel tools and concepts applicable to the wider field of bioinorganic chemistry.
Max ERC Funding
1 494 880 €
Duration
Start date: 2017-02-01, End date: 2022-01-31
Project acronym CANCER SIGNALOSOMES
Project Spatially and temporally regulated membrane complexes in cancer cell invasion and cytokinesis
Researcher (PI) Johanna Ivaska
Host Institution (HI) TEKNOLOGIAN TUTKIMUSKESKUS VTT
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Cancer progression, characterized by uncontrolled proliferation and motility of cells, is a complex and deadly process. Integrins, a major cell surface adhesion receptor family, are transmembrane proteins known to regulate cell behaviour by transducing extracellular signals to cytoplasmic protein complexes. We and others have shown that recruitment of specific protein complexes by the cytoplasmic domains of integrins is important in tumorigenesis. Here our aim is to study three interrelated processes in cancer progression which involve integrin signalling, but which have not been elucidated earlier at all. 1) Integrins in cell division (cytokinesis). Since coordinated action of the cytoskeleton and membranes is needed both for cell division and motility, shared integrin functions can regulate both events. 2) Dynamic integrin signalosomes at the leading edge of invading cells. Spatially and temporally regulated, integrin-protein complexes at the front of infiltrating cells are likely to dictate the movement of cancer cells in tissues. 3) Transmembrane segments of integrins as scaffolds for integrin signalling. In addition to cytosolic proteins, integrins most likely interact with proteins within the membrane resulting into new signalling modalities. In this proposal we will use innovative, modern and even unconventional techniques (such as RNAi and live-cell arrays detecting integrin traffic, cell motility and multiplication, laser-microdissection, proteomics and bacterial-two-hybrid screens) to unravel these new integrin functions, for which we have preliminary evidence. Each project will give fundamentally novel mechanistic insight into the role of integrins in cancer. Moreover, these interdisciplinary new openings will increase our understanding in cancer progression in general and will open new possibilities for therapeutic intervention targeting both cancer proliferation and dissemination in the body.
Summary
Cancer progression, characterized by uncontrolled proliferation and motility of cells, is a complex and deadly process. Integrins, a major cell surface adhesion receptor family, are transmembrane proteins known to regulate cell behaviour by transducing extracellular signals to cytoplasmic protein complexes. We and others have shown that recruitment of specific protein complexes by the cytoplasmic domains of integrins is important in tumorigenesis. Here our aim is to study three interrelated processes in cancer progression which involve integrin signalling, but which have not been elucidated earlier at all. 1) Integrins in cell division (cytokinesis). Since coordinated action of the cytoskeleton and membranes is needed both for cell division and motility, shared integrin functions can regulate both events. 2) Dynamic integrin signalosomes at the leading edge of invading cells. Spatially and temporally regulated, integrin-protein complexes at the front of infiltrating cells are likely to dictate the movement of cancer cells in tissues. 3) Transmembrane segments of integrins as scaffolds for integrin signalling. In addition to cytosolic proteins, integrins most likely interact with proteins within the membrane resulting into new signalling modalities. In this proposal we will use innovative, modern and even unconventional techniques (such as RNAi and live-cell arrays detecting integrin traffic, cell motility and multiplication, laser-microdissection, proteomics and bacterial-two-hybrid screens) to unravel these new integrin functions, for which we have preliminary evidence. Each project will give fundamentally novel mechanistic insight into the role of integrins in cancer. Moreover, these interdisciplinary new openings will increase our understanding in cancer progression in general and will open new possibilities for therapeutic intervention targeting both cancer proliferation and dissemination in the body.
Max ERC Funding
1 529 369 €
Duration
Start date: 2008-08-01, End date: 2013-07-31
Project acronym CANCER&AGEING
Project COMMOM MECHANISMS UNDERLYING CANCER AND AGEING
Researcher (PI) Manuel Serrano
Host Institution (HI) FUNDACION CENTRO NACIONAL DE INVESTIGACIONES ONCOLOGICAS CARLOS III
Call Details Advanced Grant (AdG), LS1, ERC-2008-AdG
Summary "In recent years, we have made significant contributions to the understanding of the tumour suppressors p53, p16INK4a, and ARF, particularly in relation with cellular senescence and aging. The current project is motivated by two hypothesis: 1) that the INK4/ARF locus is a sensor of epigenetic damage and this is at the basis of its activation by oncogenes and aging; and, 2) that the accumulation of cellular damage and stress is at the basis of both cancer and aging, and consequently ""anti-damage genes"", such as tumour suppressors, simultaneously counteract both cancer and aging. With regard to the INK4/ARF locus, the project includes: 1.1) the generation of null mice for the Regulatory Domain (RD) thought to be essential for the proper regulation of the locus; 1.2) the study of the INK4/ARF anti-sense transcription and its importance for the assembly of Polycomb repressive complexes; 1.3) the generation of mice carrying the human INK4/ARF locus to analyze, among other aspects, whether the known differences between the human and murine loci are ""locus autonomous""; and, 1.4) to analyze the INK4/ARF locus in the process of epigenetic reprogramming both from ES cells to differentiated cells and, conversely, from differentiated cells to induced-pluripotent stem (iPS) cells. With regard to the impact of ""anti-damage genes"" on cancer and aging, the project includes: 2.1) the analysis of the aging of super-INK4/ARF mice and super-p53 mice; 2.2) we have generated super-PTEN mice and we will examine whether PTEN not only confers cancer resistance but also anti-aging activity; and, finally, 2.3) we have generated super-SIRT1 mice, which is among the best-characterized anti-aging genes in non-mammalian model systems (where it is named Sir2) involved in protection from metabolic damage, and we will study the cancer and aging of these mice. Together, this project will significantly advance our understanding of the molecular mechanisms underlying cancer and aging."
Summary
"In recent years, we have made significant contributions to the understanding of the tumour suppressors p53, p16INK4a, and ARF, particularly in relation with cellular senescence and aging. The current project is motivated by two hypothesis: 1) that the INK4/ARF locus is a sensor of epigenetic damage and this is at the basis of its activation by oncogenes and aging; and, 2) that the accumulation of cellular damage and stress is at the basis of both cancer and aging, and consequently ""anti-damage genes"", such as tumour suppressors, simultaneously counteract both cancer and aging. With regard to the INK4/ARF locus, the project includes: 1.1) the generation of null mice for the Regulatory Domain (RD) thought to be essential for the proper regulation of the locus; 1.2) the study of the INK4/ARF anti-sense transcription and its importance for the assembly of Polycomb repressive complexes; 1.3) the generation of mice carrying the human INK4/ARF locus to analyze, among other aspects, whether the known differences between the human and murine loci are ""locus autonomous""; and, 1.4) to analyze the INK4/ARF locus in the process of epigenetic reprogramming both from ES cells to differentiated cells and, conversely, from differentiated cells to induced-pluripotent stem (iPS) cells. With regard to the impact of ""anti-damage genes"" on cancer and aging, the project includes: 2.1) the analysis of the aging of super-INK4/ARF mice and super-p53 mice; 2.2) we have generated super-PTEN mice and we will examine whether PTEN not only confers cancer resistance but also anti-aging activity; and, finally, 2.3) we have generated super-SIRT1 mice, which is among the best-characterized anti-aging genes in non-mammalian model systems (where it is named Sir2) involved in protection from metabolic damage, and we will study the cancer and aging of these mice. Together, this project will significantly advance our understanding of the molecular mechanisms underlying cancer and aging."
Max ERC Funding
2 000 000 €
Duration
Start date: 2009-04-01, End date: 2015-03-31
Project acronym CANCERLINC
Project Functional and Mecahnistic Roles of Large Intergenic Non-coding RNAs in Cancer
Researcher (PI) Maite Huarte Martinez
Host Institution (HI) FUNDACION PARA LA INVESTIGACION MEDICA APLICADA FIMA
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Mammalian cells express thousands of RNA molecules structurally similar to protein coding genes –they are large, spliced, poly-adenylated, transcribed by RNA Pol II, with conserved promoters and exonic structures –however lack coding capacity. Although thousands exist, only few of these large intergenic non-coding RNAs (lincRNAs) have been characterized. The few that have, show powerful biological roles as regulators of gene expression by diverse epigenetic and non-epigenetic mechanisms. Significantly, their expression patterns suggest that some lincRNAs are involved in cellular pathways critical in cancer, like the p53 pathway. I explored this association demonstrating that p53 induces the expression of many lincRNAs. One them, named lincRNA-p21, is directly induced by p53 to play a critical role in the p53 response, being required for the global repression of genes that interfere with p53 induction of apoptosis. My results, together with the emerging evidence in the field, suggest that lincRNAs may play key roles in numerous tumor-suppressor and oncogenic pathways, representing an unknown paradigm in cellular transformation. However, their mechanisms of function and biological roles remain largely unexplored.
The goal of this project is to decipher the functional and biological roles of lincRNAs in the context of oncogenic pathways to better understand the cellular mechanisms of gene regulation at the epigenetic and non-epigenetic levels, and be able to implement lincRNA use for diagnostics and therapies. In order to accomplish these goals we will integrate molecular and cell biology techniques with functional genomics approaches and in vivo studies. Importantly, the profiling of patient samples will reveal the relevance of our findings in human disease. Together, the functional study of lincRNAs will not only be crucial for developing improved diagnostics and therapies, but also will help a better understanding of the mechanisms that govern cellular network.
Summary
Mammalian cells express thousands of RNA molecules structurally similar to protein coding genes –they are large, spliced, poly-adenylated, transcribed by RNA Pol II, with conserved promoters and exonic structures –however lack coding capacity. Although thousands exist, only few of these large intergenic non-coding RNAs (lincRNAs) have been characterized. The few that have, show powerful biological roles as regulators of gene expression by diverse epigenetic and non-epigenetic mechanisms. Significantly, their expression patterns suggest that some lincRNAs are involved in cellular pathways critical in cancer, like the p53 pathway. I explored this association demonstrating that p53 induces the expression of many lincRNAs. One them, named lincRNA-p21, is directly induced by p53 to play a critical role in the p53 response, being required for the global repression of genes that interfere with p53 induction of apoptosis. My results, together with the emerging evidence in the field, suggest that lincRNAs may play key roles in numerous tumor-suppressor and oncogenic pathways, representing an unknown paradigm in cellular transformation. However, their mechanisms of function and biological roles remain largely unexplored.
The goal of this project is to decipher the functional and biological roles of lincRNAs in the context of oncogenic pathways to better understand the cellular mechanisms of gene regulation at the epigenetic and non-epigenetic levels, and be able to implement lincRNA use for diagnostics and therapies. In order to accomplish these goals we will integrate molecular and cell biology techniques with functional genomics approaches and in vivo studies. Importantly, the profiling of patient samples will reveal the relevance of our findings in human disease. Together, the functional study of lincRNAs will not only be crucial for developing improved diagnostics and therapies, but also will help a better understanding of the mechanisms that govern cellular network.
Max ERC Funding
1 500 000 €
Duration
Start date: 2012-01-01, End date: 2017-12-31
Project acronym Celcelfus
Project Cell-Cell fusion in fertilization and developmental biology: a structural biology approach
Researcher (PI) Félix A. Rey
Host Institution (HI) INSTITUT PASTEUR
Call Details Advanced Grant (AdG), LS1, ERC-2013-ADG
Summary My group has made seminal contributions in the past toward understanding the mechanism of membrane fusion used by enveloped viruses to infect a cell. This aim of this ERC grant proposal is to achieve similar breakthroughs in understanding fusion between cells, both during fertilization and organogenesis. This proposal is based in recent important results not yet published.
We have determined the crystal structure of the C. elegans protein EFF-1, a member of the “fusion family” (FF). EFF-1 is responsible for a cell-cell fusion event during skin formation in the nematode. Strikingly, the crystal structure shows that EFF-1 is homologous to the “Class II” viral protein fusogens, thus indicating that they have diverged from a common ancestor. The observed homology could not be identified by other means because the proteins have diverged to the point where no remnants of sequence similarity are left, yet the tertiary and quaternary organization is the same. However, the homotypic fusion mechanism of EFF-1 is clearly different to that of viral fusion proteins.
This proposal intends to build on the momentum generated by this exciting discovery, in an attempt to cast light into the fusion mechanism of FF proteins. We will reconstitute them in artificial liposomes and will also follow them within cells with the use of light microscopy. We will also focus in determining the crystal structure of the monomeric pre-fusion form of EFF-1,and of the intact trans-membrane post fusion trimer. In parallel, we want to make use the experience accumulated over the years in crystallizing viral glycoproteins, to apply it to the conserved family of HAP2/GSC1 proteins involved in fusion of gametes during fertilization. These proteins exhibit a similar pattern of secondary structure elements in the ectodomain as class II proteins, but only a crystallographic analysis can identify a possible structural homology and provide the basis to understand the molecular mechanisms of cell-cell fusion.
Summary
My group has made seminal contributions in the past toward understanding the mechanism of membrane fusion used by enveloped viruses to infect a cell. This aim of this ERC grant proposal is to achieve similar breakthroughs in understanding fusion between cells, both during fertilization and organogenesis. This proposal is based in recent important results not yet published.
We have determined the crystal structure of the C. elegans protein EFF-1, a member of the “fusion family” (FF). EFF-1 is responsible for a cell-cell fusion event during skin formation in the nematode. Strikingly, the crystal structure shows that EFF-1 is homologous to the “Class II” viral protein fusogens, thus indicating that they have diverged from a common ancestor. The observed homology could not be identified by other means because the proteins have diverged to the point where no remnants of sequence similarity are left, yet the tertiary and quaternary organization is the same. However, the homotypic fusion mechanism of EFF-1 is clearly different to that of viral fusion proteins.
This proposal intends to build on the momentum generated by this exciting discovery, in an attempt to cast light into the fusion mechanism of FF proteins. We will reconstitute them in artificial liposomes and will also follow them within cells with the use of light microscopy. We will also focus in determining the crystal structure of the monomeric pre-fusion form of EFF-1,and of the intact trans-membrane post fusion trimer. In parallel, we want to make use the experience accumulated over the years in crystallizing viral glycoproteins, to apply it to the conserved family of HAP2/GSC1 proteins involved in fusion of gametes during fertilization. These proteins exhibit a similar pattern of secondary structure elements in the ectodomain as class II proteins, but only a crystallographic analysis can identify a possible structural homology and provide the basis to understand the molecular mechanisms of cell-cell fusion.
Max ERC Funding
2 478 800 €
Duration
Start date: 2014-03-01, End date: 2019-02-28
