Project acronym ANOBEST
Project Structure function and pharmacology of calcium-activated chloride channels: Anoctamins and Bestrophins
Researcher (PI) Raimund Dutzler
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Advanced Grant (AdG), LS1, ERC-2013-ADG
Summary Calcium-activated chloride channels (CaCCs) play key roles in a range of physiological processes such as the control of membrane excitability, photoreception and epithelial secretion. Although the importance of these channels has been recognized for more than 30 years their molecular identity remained obscure. The recent discovery of two protein families encoding for CaCCs, Anoctamins and Bestrophins, was a scientific breakthrough that has provided first insight into two novel ion channel architectures. Within this proposal we aim to determine the first high resolution structures of members of both families and study their functional behavior by an interdisciplinary approach combining biochemistry, X-ray crystallography and electrophysiology. The structural investigation of eukaryotic membrane proteins is extremely challenging and will require us to investigate large numbers of candidates to single out family members with superior biochemical properties. During the last year we have made large progress in this direction. By screening numerous eukaryotic Anoctamins and prokaryotic Bestrophins we have identified well-behaved proteins for both families, which were successfully scaled-up and purified. Additional family members will be identified within the course of the project. For these stable proteins we plan to grow crystals diffracting to high resolution and to proceed with structure determination. With first structural information in hand we will perform detailed functional studies using electrophysiology and complementary biophysical techniques to gain mechanistic insight into ion permeation and gating. As the pharmacology of both families is still in its infancy we will in later stages also engage in the identification and characterization of inhibitors and activators of Anoctamins and Bestrophins to open up a field that may ultimately lead to the discovery of novel therapeutic strategies targeting calcium-activated chloride channels.
Summary
Calcium-activated chloride channels (CaCCs) play key roles in a range of physiological processes such as the control of membrane excitability, photoreception and epithelial secretion. Although the importance of these channels has been recognized for more than 30 years their molecular identity remained obscure. The recent discovery of two protein families encoding for CaCCs, Anoctamins and Bestrophins, was a scientific breakthrough that has provided first insight into two novel ion channel architectures. Within this proposal we aim to determine the first high resolution structures of members of both families and study their functional behavior by an interdisciplinary approach combining biochemistry, X-ray crystallography and electrophysiology. The structural investigation of eukaryotic membrane proteins is extremely challenging and will require us to investigate large numbers of candidates to single out family members with superior biochemical properties. During the last year we have made large progress in this direction. By screening numerous eukaryotic Anoctamins and prokaryotic Bestrophins we have identified well-behaved proteins for both families, which were successfully scaled-up and purified. Additional family members will be identified within the course of the project. For these stable proteins we plan to grow crystals diffracting to high resolution and to proceed with structure determination. With first structural information in hand we will perform detailed functional studies using electrophysiology and complementary biophysical techniques to gain mechanistic insight into ion permeation and gating. As the pharmacology of both families is still in its infancy we will in later stages also engage in the identification and characterization of inhibitors and activators of Anoctamins and Bestrophins to open up a field that may ultimately lead to the discovery of novel therapeutic strategies targeting calcium-activated chloride channels.
Max ERC Funding
2 176 000 €
Duration
Start date: 2014-02-01, End date: 2020-01-31
Project acronym CFRFSS
Project Chromatin Fiber and Remodeling Factor Structural Studies
Researcher (PI) Timothy John Richmond
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Advanced Grant (AdG), LS1, ERC-2012-ADG_20120314
Summary "DNA in higher organisms is organized in a nucleoprotein complex called chromatin. The structure of chromatin is responsible for compacting DNA to fit within the nucleus and for governing its access in nuclear processes. Epigenetic information is encoded chiefly via chromatin modifications. Readout of the genetic code depends on chromatin remodeling, a process actively altering chromatin structure. An understanding of the hierarchical structure of chromatin and of structurally based, remodeling mechanisms will have enormous impact for developments in medicine.
Following our high resolution structure of the nucleosome core particle, the fundamental repeating unit of chromatin, we have endeavored to determine the structure of the chromatin fiber. We showed with our X-ray structure of a tetranucleosome how nucleosomes could be organized in the fiber. Further progress has been limited by structural polymorphism and crystal disorder, but new evidence on the in vivo spacing of nucleosomes in chromatin should stimulate more advances. Part A of this application describes how we would apply these new findings to our cryo-electron microscopy study of the chromatin fiber and to our crystallographic study of a tetranucleosome containing linker histone.
Recently, my laboratory succeeded in providing the first structurally based mechanism for nucleosome spacing by a chromatin remodeling factor. We combined the X-ray structure of ISW1a(ATPase) bound to DNA with cryo-EM structures of the factor bound to two different nucleosomes to build a model showing how this remodeler uses a dinucleosome, not a mononucleosome, as its substrate. Our results from a functional assay using ISW1a further justified this model. Part B of this application describes how we would proceed to the relevant cryo-EM and X-ray structures incorporating dinucleosomes. Our recombinant ISW1a allows us to study in addition the interaction of the ATPase domain with nucleosome substrates."
Summary
"DNA in higher organisms is organized in a nucleoprotein complex called chromatin. The structure of chromatin is responsible for compacting DNA to fit within the nucleus and for governing its access in nuclear processes. Epigenetic information is encoded chiefly via chromatin modifications. Readout of the genetic code depends on chromatin remodeling, a process actively altering chromatin structure. An understanding of the hierarchical structure of chromatin and of structurally based, remodeling mechanisms will have enormous impact for developments in medicine.
Following our high resolution structure of the nucleosome core particle, the fundamental repeating unit of chromatin, we have endeavored to determine the structure of the chromatin fiber. We showed with our X-ray structure of a tetranucleosome how nucleosomes could be organized in the fiber. Further progress has been limited by structural polymorphism and crystal disorder, but new evidence on the in vivo spacing of nucleosomes in chromatin should stimulate more advances. Part A of this application describes how we would apply these new findings to our cryo-electron microscopy study of the chromatin fiber and to our crystallographic study of a tetranucleosome containing linker histone.
Recently, my laboratory succeeded in providing the first structurally based mechanism for nucleosome spacing by a chromatin remodeling factor. We combined the X-ray structure of ISW1a(ATPase) bound to DNA with cryo-EM structures of the factor bound to two different nucleosomes to build a model showing how this remodeler uses a dinucleosome, not a mononucleosome, as its substrate. Our results from a functional assay using ISW1a further justified this model. Part B of this application describes how we would proceed to the relevant cryo-EM and X-ray structures incorporating dinucleosomes. Our recombinant ISW1a allows us to study in addition the interaction of the ATPase domain with nucleosome substrates."
Max ERC Funding
2 500 000 €
Duration
Start date: 2013-01-01, End date: 2017-12-31
Project acronym CsnCRL
Project The molecular basis of CULLIN E3 ligase regulation by the COP9 signalosome
Researcher (PI) Nicolas Thoma
Host Institution (HI) FRIEDRICH MIESCHER INSTITUTE FOR BIOMEDICAL RESEARCH FONDATION
Call Details Advanced Grant (AdG), LS1, ERC-2014-ADG
Summary Specificity in the ubiquitin-proteasome system is largely conferred by ubiquitin E3 ligases (E3s). Cullin-RING ligases (CRLs), constituting ~30% of all E3s in humans, mediate the ubiquitination of ~20% of the proteins degraded by the proteasome. CRLs are divided into seven families based on their cullin constituent. Each cullin binds a RING domain protein, and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 200 distinct CRLs. All CRLs are regulated by the COP9 signalosome (CSN), an eight-protein isopeptidase that removes the covalently attached activator, NEDD8, from the cullin. Independent of NEDD8 cleavage, CSN forms protective complexes with CRLs, which prevents destructive auto-ubiquitination.
The integrity of the CSN-CRL system is crucially important for the normal cell physiology. Based on our previous work on CRL structures (Fischer, et al., Nature 2014; Fischer, et al., Cell 2011) and that of isolated CSN (Lingaraju et al., Nature 2014), We now intend to provide the underlying molecular mechanism of CRL regulation by CSN. Structural insights at atomic resolution, combined with in vitro and in vivo functional studies are designed to reveal (i) how the signalosome deneddylates and maintains the bound ligases in an inactive state, (ii) how the multiple CSN subunits bind to structurally diverse CRLs, and (iii) how CSN is itself subject to regulation by post-translational modifications or additional further factors.
The ERC funding would allow my lab to pursue an ambitious interdisciplinary approach combining X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology. This is expected to provide a unique molecular understanding of CSN action. Beyond ubiquitination, insight into this >13- subunit CSN-CRL assembly will allow examining general principles of multi-subunit complex action and reveal how the numerous, often essential, subunits contribute to complex function.
Summary
Specificity in the ubiquitin-proteasome system is largely conferred by ubiquitin E3 ligases (E3s). Cullin-RING ligases (CRLs), constituting ~30% of all E3s in humans, mediate the ubiquitination of ~20% of the proteins degraded by the proteasome. CRLs are divided into seven families based on their cullin constituent. Each cullin binds a RING domain protein, and a vast repertoire of adaptor/substrate receptor modules, collectively creating more than 200 distinct CRLs. All CRLs are regulated by the COP9 signalosome (CSN), an eight-protein isopeptidase that removes the covalently attached activator, NEDD8, from the cullin. Independent of NEDD8 cleavage, CSN forms protective complexes with CRLs, which prevents destructive auto-ubiquitination.
The integrity of the CSN-CRL system is crucially important for the normal cell physiology. Based on our previous work on CRL structures (Fischer, et al., Nature 2014; Fischer, et al., Cell 2011) and that of isolated CSN (Lingaraju et al., Nature 2014), We now intend to provide the underlying molecular mechanism of CRL regulation by CSN. Structural insights at atomic resolution, combined with in vitro and in vivo functional studies are designed to reveal (i) how the signalosome deneddylates and maintains the bound ligases in an inactive state, (ii) how the multiple CSN subunits bind to structurally diverse CRLs, and (iii) how CSN is itself subject to regulation by post-translational modifications or additional further factors.
The ERC funding would allow my lab to pursue an ambitious interdisciplinary approach combining X-ray crystallography, cryo-electron microscopy, biochemistry and cell biology. This is expected to provide a unique molecular understanding of CSN action. Beyond ubiquitination, insight into this >13- subunit CSN-CRL assembly will allow examining general principles of multi-subunit complex action and reveal how the numerous, often essential, subunits contribute to complex function.
Max ERC Funding
2 200 677 €
Duration
Start date: 2016-01-01, End date: 2020-12-31
Project acronym EUKARYOTIC RIBOSOME
Project Structural studies of the eukaryotic ribosome by X-ray crystallography
Researcher (PI) Nenad Ban
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary The ribosome is a large cellular organelle that plays a central role in the process of protein synthesis in all organisms. Currently, structural information at atomic resolution exists only for bacterial ribosomes and some of their functional complexes. Eukaryotic ribosomes are larger and significantly more complex than their bacterial counterparts. They consist of two unequal subunits with a combined molecular weight of approximately 4 million Daltons and contain 70-80 different protein molecules and four different RNAs. Currently the only structural information on eukaryotic ribosomes is available from cryo electron microscopic reconstructions in the nanometer resolution range, which is insufficient to derive information about the function of the eukaryotic ribosome at the atomic level. The aim of this proposal is to use X-ray crystallography to obtain structural and functional information on the eukaryotic ribosome and its functional complexes at high resolution. The key targets of the structural work will be: i) the structure of the small ribosomal subunit, ii) the structure of the large ribosomal subunit, and iii) structures of complexes involved in the initiation of protein synthesis. Besides the obvious fundamental importance of this research for understanding protein synthesis in eukaryotes the proposed studies will also be the prerequisite for understanding the structural basis of the regulation of protein synthesis in normal cells and how it is perturbed in various diseases. Finally, comparing the structures of bacterial and eukaryotic ribosomes is important for understanding the specificity of various clinically used antibiotics for the bacterial ribosome.
Summary
The ribosome is a large cellular organelle that plays a central role in the process of protein synthesis in all organisms. Currently, structural information at atomic resolution exists only for bacterial ribosomes and some of their functional complexes. Eukaryotic ribosomes are larger and significantly more complex than their bacterial counterparts. They consist of two unequal subunits with a combined molecular weight of approximately 4 million Daltons and contain 70-80 different protein molecules and four different RNAs. Currently the only structural information on eukaryotic ribosomes is available from cryo electron microscopic reconstructions in the nanometer resolution range, which is insufficient to derive information about the function of the eukaryotic ribosome at the atomic level. The aim of this proposal is to use X-ray crystallography to obtain structural and functional information on the eukaryotic ribosome and its functional complexes at high resolution. The key targets of the structural work will be: i) the structure of the small ribosomal subunit, ii) the structure of the large ribosomal subunit, and iii) structures of complexes involved in the initiation of protein synthesis. Besides the obvious fundamental importance of this research for understanding protein synthesis in eukaryotes the proposed studies will also be the prerequisite for understanding the structural basis of the regulation of protein synthesis in normal cells and how it is perturbed in various diseases. Finally, comparing the structures of bacterial and eukaryotic ribosomes is important for understanding the specificity of various clinically used antibiotics for the bacterial ribosome.
Max ERC Funding
2 446 725 €
Duration
Start date: 2010-07-01, End date: 2015-06-30
Project acronym MIRIAM
Project Mismatch repair interactome and mutagenesis
Researcher (PI) Josef Jiricny
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Advanced Grant (AdG), LS1, ERC-2011-ADG_20110310
Summary "The mismatch repair (MMR) system has evolved to correct errors of DNA replication and prevent recombination between non-homologous sequences. Correspondingly, MMR malfunction leads to increased mutation rates and illegitimate recombination, and individuals with inherited MMR gene mutations are predisposed to cancer of the colon and other organs. However, MMR has recently been implicated in processes ranging from DNA damage signaling and drug sensitivity to antibody maturation, some of which contradict and even subvert the classical role of MMR as a guardian of genomic integrity. We suspect that the latter processes are linked to a new, non-canonical MMR (ncMMR) pathway that can be activated outside of the S- and G2 phases of the cell cycle by a variety of lesions and structures. ncMMR lacks strand directionality, involves long stretches of DNA degradation, and our preliminary in vitro evidence suggests that resynthesis of these repair tracts can be mediated not only by high-fidelity, replicative polymerases, but also by error-prone enzymes. In this scenario, ncMMR would actually contribute to mutagenesis. I plan to deploy proteomic, genomic and imaging technologies to identify the components of the ncMMR “mutasome” and to reconstitute the system from purified recombinant components. Furthermore, I wish to study the “action radius” of MMR proteins by characterizing their interactome and analyze its dependence on endogenous states (e.g. cell cycle stages) and exogenous stimuli (e.g. drug treatments) in human and chicken (DT40) cells, and Xenopus laevis egg extracts. I intend to exploit a new system of inducible protein replacement that was recently developed in my laboratory, to stably express MMR, replication, repair and recombination proteins (both wild type and variants). This program should increase our understanding of the pivotal role of MMR in DNA metabolism and its involvement in human disease and cancer."
Summary
"The mismatch repair (MMR) system has evolved to correct errors of DNA replication and prevent recombination between non-homologous sequences. Correspondingly, MMR malfunction leads to increased mutation rates and illegitimate recombination, and individuals with inherited MMR gene mutations are predisposed to cancer of the colon and other organs. However, MMR has recently been implicated in processes ranging from DNA damage signaling and drug sensitivity to antibody maturation, some of which contradict and even subvert the classical role of MMR as a guardian of genomic integrity. We suspect that the latter processes are linked to a new, non-canonical MMR (ncMMR) pathway that can be activated outside of the S- and G2 phases of the cell cycle by a variety of lesions and structures. ncMMR lacks strand directionality, involves long stretches of DNA degradation, and our preliminary in vitro evidence suggests that resynthesis of these repair tracts can be mediated not only by high-fidelity, replicative polymerases, but also by error-prone enzymes. In this scenario, ncMMR would actually contribute to mutagenesis. I plan to deploy proteomic, genomic and imaging technologies to identify the components of the ncMMR “mutasome” and to reconstitute the system from purified recombinant components. Furthermore, I wish to study the “action radius” of MMR proteins by characterizing their interactome and analyze its dependence on endogenous states (e.g. cell cycle stages) and exogenous stimuli (e.g. drug treatments) in human and chicken (DT40) cells, and Xenopus laevis egg extracts. I intend to exploit a new system of inducible protein replacement that was recently developed in my laboratory, to stably express MMR, replication, repair and recombination proteins (both wild type and variants). This program should increase our understanding of the pivotal role of MMR in DNA metabolism and its involvement in human disease and cancer."
Max ERC Funding
2 208 084 €
Duration
Start date: 2012-04-01, End date: 2016-08-31
Project acronym NEXTBINDERS
Project General light triggered switches and sensors for studying proteins inside the cell
Researcher (PI) Andreas Georg Plückthun
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Advanced Grant (AdG), LS1, ERC-2010-AdG_20100317
Summary The overall objective is to develop general strategies for creating sensors and switches that work inside cells. They will be engineered from Designed Ankyrin Repeat Proteins (DARPins) as general binding proteins that function inside cells and can be generated against any target by selection and directed evolution. Light-triggered switches will be engineered by placing a LOV domain from phototropin in such a way across the DARPin that it is blocked, and only the light-triggered conformational change makes the DARPin accessible. As a second strategy, DARPins specifically recognizing each one of the two isomers of azobenzene, which can be interconverted by light, will be used within light-dependent cross-linkers. Third, DARPins selected to tightly bind fluorogens, by which these small molecules increase their fluorescence by several orders of magnitude, will be converted into general sensors of the conformation of a target protein working within the cell: large DARPins will be created with overlapping binding sites for the protein of interest and the fluorogen. By using DARPins which can selectively distinguish conformations of the target protein, the conformational changes are made visible in a spatiotemporal manner in an individual cell. As proof of principle, we will generate sensors and switches for the kinase domains of the four ErbB receptors, pivotal in signal transduction in human cancers. To increase the impact of this research further, these novel switches and sensors have to be efficiently brought into cells. For this purpose, adenovirus will be engineered for novel cell tropism, also by using DARPins, to homogenously infect tumor cells to study ErbB signaling and the effect of therapeutics in real time in a receptor-differentiating manner. While tested for the ErbB family as a prototype, the strategies to be developed will be totally general and should open up novel ways of studying signaling within cells in real time and with high spatial resolution.
Summary
The overall objective is to develop general strategies for creating sensors and switches that work inside cells. They will be engineered from Designed Ankyrin Repeat Proteins (DARPins) as general binding proteins that function inside cells and can be generated against any target by selection and directed evolution. Light-triggered switches will be engineered by placing a LOV domain from phototropin in such a way across the DARPin that it is blocked, and only the light-triggered conformational change makes the DARPin accessible. As a second strategy, DARPins specifically recognizing each one of the two isomers of azobenzene, which can be interconverted by light, will be used within light-dependent cross-linkers. Third, DARPins selected to tightly bind fluorogens, by which these small molecules increase their fluorescence by several orders of magnitude, will be converted into general sensors of the conformation of a target protein working within the cell: large DARPins will be created with overlapping binding sites for the protein of interest and the fluorogen. By using DARPins which can selectively distinguish conformations of the target protein, the conformational changes are made visible in a spatiotemporal manner in an individual cell. As proof of principle, we will generate sensors and switches for the kinase domains of the four ErbB receptors, pivotal in signal transduction in human cancers. To increase the impact of this research further, these novel switches and sensors have to be efficiently brought into cells. For this purpose, adenovirus will be engineered for novel cell tropism, also by using DARPins, to homogenously infect tumor cells to study ErbB signaling and the effect of therapeutics in real time in a receptor-differentiating manner. While tested for the ErbB family as a prototype, the strategies to be developed will be totally general and should open up novel ways of studying signaling within cells in real time and with high spatial resolution.
Max ERC Funding
2 158 800 €
Duration
Start date: 2011-05-01, End date: 2016-04-30
Project acronym NucEnv
Project Nuclear Envelope Biogenesis, Function and Dynamics
Researcher (PI) Ulrike Kutay
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Advanced Grant (AdG), LS1, ERC-2012-ADG_20120314
Summary The nuclear envelope (NE) harbors key roles in cellular and organismal homeostasis, reflected by a variety of diseases caused by mutations in NE proteins. Despite the fundamental role of the NE in protecting and organizing the genome, still little is known about the molecular mechanisms underlying NE biogenesis, dynamics and functionality. We will address 3 open questions in NE biology, using vertebrate cells as model system. (1) To understand how the functional specification of the NE by transmembrane proteins is generated, we will decipher how membrane proteins are sorted to the inner nuclear membrane (INM). To reach this goal, we will define targeting signals and the mode of NPC passage of INM proteins, and identify the molecular requirements for transport. (2) Based on structural analysis, we will investigate how the molecular organization of LINC complexes, which are formed by interacting pairs of SUN and KASH proteins spanning the NE, determines their role in NE architecture and as tethers of the NE to the cytoskeleton. (3) We will study dynamic changes of the NE that occur at the onset of ’open’ mitosis, when the nuclear compartment is disintegrated to allow for the formation of a cytoplasmic mitotic spindle. NE breakdown (NEBD) presents a dramatic change of cellular architecture and comprises a series of events including disassembly of nuclear pore complexes, the nuclear lamina and retraction of NE membranes into the endoplasmic reticulum. To elucidate the molecular mechanisms controlling and executing these steps of nuclear disassembly, we will characterize the cellular machinery involved in NEBD and unravel the molecular function of identified components. All these questions will be addressed by a blend of in vivo approaches relying on high-end fluorescence imaging linked to computational image analysis, RNAi screening, as well as powerful in vitro systems recapitulating protein transport to the INM or NEBD that we have developed, and biochemical methods.
Summary
The nuclear envelope (NE) harbors key roles in cellular and organismal homeostasis, reflected by a variety of diseases caused by mutations in NE proteins. Despite the fundamental role of the NE in protecting and organizing the genome, still little is known about the molecular mechanisms underlying NE biogenesis, dynamics and functionality. We will address 3 open questions in NE biology, using vertebrate cells as model system. (1) To understand how the functional specification of the NE by transmembrane proteins is generated, we will decipher how membrane proteins are sorted to the inner nuclear membrane (INM). To reach this goal, we will define targeting signals and the mode of NPC passage of INM proteins, and identify the molecular requirements for transport. (2) Based on structural analysis, we will investigate how the molecular organization of LINC complexes, which are formed by interacting pairs of SUN and KASH proteins spanning the NE, determines their role in NE architecture and as tethers of the NE to the cytoskeleton. (3) We will study dynamic changes of the NE that occur at the onset of ’open’ mitosis, when the nuclear compartment is disintegrated to allow for the formation of a cytoplasmic mitotic spindle. NE breakdown (NEBD) presents a dramatic change of cellular architecture and comprises a series of events including disassembly of nuclear pore complexes, the nuclear lamina and retraction of NE membranes into the endoplasmic reticulum. To elucidate the molecular mechanisms controlling and executing these steps of nuclear disassembly, we will characterize the cellular machinery involved in NEBD and unravel the molecular function of identified components. All these questions will be addressed by a blend of in vivo approaches relying on high-end fluorescence imaging linked to computational image analysis, RNAi screening, as well as powerful in vitro systems recapitulating protein transport to the INM or NEBD that we have developed, and biochemical methods.
Max ERC Funding
2 500 000 €
Duration
Start date: 2013-04-01, End date: 2018-03-31
Project acronym REPLISTRESS
Project DNA Replication: From Physiology to Replication Stress in Human Cancer
Researcher (PI) Athanassios Dimitrios HALAZONETIS
Host Institution (HI) UNIVERSITE DE GENEVE
Call Details Advanced Grant (AdG), LS1, ERC-2017-ADG
Summary Replication of the genome is of critical importance for cell proliferation and organismal development. To ensure accurate and complete replication of their genome, eukaryotes have hundreds to thousands of replication origins. In budding yeast, the genomic positions of all the origins are known, as is the order in which they fire. In contrast, in human cells, the mapping of origins is controversial and origin firing may be stochastic and plastic. Furthermore, while normal cells replicate their genomes with high fidelity; in cancer cells, the presence of activated oncogenes leads to collapse of DNA replication forks (DNA replication stress), DNA damage and genomic instability.
My laboratory has recently elucidated key differences in DNA replication after oncogene induction. We mapped replication origins on the human genome and found that, in addition to the origins present before oncogene induction, a new class of “oncogene-induced” origins was observed upon activation of the CCNE1 (Cyclin E) or MYC (c-Myc) genes. Only forks from the oncogene-induced origins were prone to collapse, leading to the genomic instability patterns observed in the common human cancers.
In this proposal, we aim to map with high precision the human replication origins, determine if their firing is stochastic or deterministic and identify sequence motifs that are important for origin firing (Aim 1). We further aim to explore how transcription in the G1 phase of the cell cycle regulates origin firing (Aim 2). This endeavour is motivated by our observation that transcription in G1 inactivates intragenic origins. Finally, we aim to understand how transcription, replication and repair are coordinated to avoid DNA replication stress in normal cells (Aim 3).
The proposed experiments will help us understand how normal cells replicate their genome with high fidelity and how oncogenes interfere with this process.
Summary
Replication of the genome is of critical importance for cell proliferation and organismal development. To ensure accurate and complete replication of their genome, eukaryotes have hundreds to thousands of replication origins. In budding yeast, the genomic positions of all the origins are known, as is the order in which they fire. In contrast, in human cells, the mapping of origins is controversial and origin firing may be stochastic and plastic. Furthermore, while normal cells replicate their genomes with high fidelity; in cancer cells, the presence of activated oncogenes leads to collapse of DNA replication forks (DNA replication stress), DNA damage and genomic instability.
My laboratory has recently elucidated key differences in DNA replication after oncogene induction. We mapped replication origins on the human genome and found that, in addition to the origins present before oncogene induction, a new class of “oncogene-induced” origins was observed upon activation of the CCNE1 (Cyclin E) or MYC (c-Myc) genes. Only forks from the oncogene-induced origins were prone to collapse, leading to the genomic instability patterns observed in the common human cancers.
In this proposal, we aim to map with high precision the human replication origins, determine if their firing is stochastic or deterministic and identify sequence motifs that are important for origin firing (Aim 1). We further aim to explore how transcription in the G1 phase of the cell cycle regulates origin firing (Aim 2). This endeavour is motivated by our observation that transcription in G1 inactivates intragenic origins. Finally, we aim to understand how transcription, replication and repair are coordinated to avoid DNA replication stress in normal cells (Aim 3).
The proposed experiments will help us understand how normal cells replicate their genome with high fidelity and how oncogenes interfere with this process.
Max ERC Funding
2 231 378 €
Duration
Start date: 2018-10-01, End date: 2023-09-30
Project acronym TERRA
Project Telomeric Repeat Containing RNA: Biogenesis, Composition and Function
Researcher (PI) Joachim Lingner
Host Institution (HI) ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Call Details Advanced Grant (AdG), LS1, ERC-2008-AdG
Summary The ends of eukaryotic chromosomes, known as telomeres, play crucial roles as guardians of genome stability and tumor suppressors. Telomeric DNA is maintained by the ribonucleoprotein enzyme telomerase. Most normal human somatic cells express only very low levels of telomerase and telomeres shorten with continuous cell division cycles. Ultimately, short telomeres activate a DNA damage response that leads to a permanent cell cycle arrest or apoptosis. Reactivation of telomerase is a key requisite for human cancer cells to attain unlimited proliferation potential. The key questions that need to be tackled in the telomere field are (1) how telomeres protect from DNA repair activities, (2) how recruitment and regulation of telomerase is mediated by telomere structure, (3) how cell cycle arrest occurs upon telomere shortening, and (4) how telomeres regulate their heterochromatic state. In all four areas, important progress is expected in the near future. We recently made the unexpected discovery that telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) and that this RNA is an integral part of telomeric heterochromatin. Our working hypothesis is that the telomere is an RNA-dependent machine, and that several if not most of its crucial functions are regulated by TERRA. In this proposal we will explore TERRA functions, by elucidating its biogenesis, by identifying its protein partners and by genetic manipulation of the expression of TERRA and TERRA binding proteins. This work should provide fundamental insight into how our chromosome ends function. The gained knowledge may also provide novel avenues on how to manipulate telomere function and dysfunction in cancer cells and other diseased tissue.
Summary
The ends of eukaryotic chromosomes, known as telomeres, play crucial roles as guardians of genome stability and tumor suppressors. Telomeric DNA is maintained by the ribonucleoprotein enzyme telomerase. Most normal human somatic cells express only very low levels of telomerase and telomeres shorten with continuous cell division cycles. Ultimately, short telomeres activate a DNA damage response that leads to a permanent cell cycle arrest or apoptosis. Reactivation of telomerase is a key requisite for human cancer cells to attain unlimited proliferation potential. The key questions that need to be tackled in the telomere field are (1) how telomeres protect from DNA repair activities, (2) how recruitment and regulation of telomerase is mediated by telomere structure, (3) how cell cycle arrest occurs upon telomere shortening, and (4) how telomeres regulate their heterochromatic state. In all four areas, important progress is expected in the near future. We recently made the unexpected discovery that telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) and that this RNA is an integral part of telomeric heterochromatin. Our working hypothesis is that the telomere is an RNA-dependent machine, and that several if not most of its crucial functions are regulated by TERRA. In this proposal we will explore TERRA functions, by elucidating its biogenesis, by identifying its protein partners and by genetic manipulation of the expression of TERRA and TERRA binding proteins. This work should provide fundamental insight into how our chromosome ends function. The gained knowledge may also provide novel avenues on how to manipulate telomere function and dysfunction in cancer cells and other diseased tissue.
Max ERC Funding
2 385 047 €
Duration
Start date: 2009-04-01, End date: 2014-03-31
Project acronym TIMESIGNAL
Project Signalling within the mammalian circadian timing system
Researcher (PI) Ulrich Schibler
Host Institution (HI) UNIVERSITE DE GENEVE
Call Details Advanced Grant (AdG), LS1, ERC-2009-AdG
Summary The main objective of this interdisciplinary research project is to elucidate regulatory mechanisms through which the circadian timing system coordinates temporal physiology. This system has a hierarchical architecture, in that a master clock in the brain s suprachiasmatic nucleus synchronizes subsidiary oscillators in nearly all body cells. The establishment of phase coherence is obviously of utmost importance in the coordination of circadian physiology. While recent studies have identified feeding cycles, hormone rhythms, and body temperature oscillations as timing cues for peripheral clocks, the molecular makeup of the involved signalling mechanisms is largely unknown. Using liver and cultured cells as model systems, we will employ two innovative strategies for the elucidation of relevant signalling pathways. (1) STAR-Prom (Synthetic TAndem Repeat-PROmoter display), a technique developed in our laboratory, will hopefully identify most if not all immediate early transcription factors activated in cultured cells by rhythmic blood-borne and temperature-dependent signals. (2) A transgenic mouse model with conditionally active liver clocks will be explored in the genome-wide identification of coding and non-coding transcripts whose rhythmic accumulation is system-driven. The in vivo significance of the components emerging from these approaches will be assessed via RNA interference. Thus, relevant siRNAs will be injected into the tail vein of mice, and their effect on the phase of circadian liver gene expression will be monitored in freely moving mice by using whole body fluorescence imaging. Physiologically important components will serve as entry points for the identification of upstream and downstream constituents in the corresponding signal transduction cascades.
Summary
The main objective of this interdisciplinary research project is to elucidate regulatory mechanisms through which the circadian timing system coordinates temporal physiology. This system has a hierarchical architecture, in that a master clock in the brain s suprachiasmatic nucleus synchronizes subsidiary oscillators in nearly all body cells. The establishment of phase coherence is obviously of utmost importance in the coordination of circadian physiology. While recent studies have identified feeding cycles, hormone rhythms, and body temperature oscillations as timing cues for peripheral clocks, the molecular makeup of the involved signalling mechanisms is largely unknown. Using liver and cultured cells as model systems, we will employ two innovative strategies for the elucidation of relevant signalling pathways. (1) STAR-Prom (Synthetic TAndem Repeat-PROmoter display), a technique developed in our laboratory, will hopefully identify most if not all immediate early transcription factors activated in cultured cells by rhythmic blood-borne and temperature-dependent signals. (2) A transgenic mouse model with conditionally active liver clocks will be explored in the genome-wide identification of coding and non-coding transcripts whose rhythmic accumulation is system-driven. The in vivo significance of the components emerging from these approaches will be assessed via RNA interference. Thus, relevant siRNAs will be injected into the tail vein of mice, and their effect on the phase of circadian liver gene expression will be monitored in freely moving mice by using whole body fluorescence imaging. Physiologically important components will serve as entry points for the identification of upstream and downstream constituents in the corresponding signal transduction cascades.
Max ERC Funding
2 360 136 €
Duration
Start date: 2010-04-01, End date: 2015-12-31