Project acronym ACCENT
Project Unravelling the architecture and the cartography of the human centriole
Researcher (PI) Paul, Philippe, Desiré GUICHARD
Host Institution (HI) UNIVERSITE DE GENEVE
Call Details Starting Grant (StG), LS1, ERC-2016-STG
Summary The centriole is the largest evolutionary conserved macromolecular structure responsible for building centrosomes and cilia or flagella in many eukaryotes. Centrioles are critical for the proper execution of important biological processes ranging from cell division to cell signaling. Moreover, centriolar defects have been associated to several human pathologies including ciliopathies and cancer. This state of facts emphasizes the importance of understanding centriole biogenesis. The study of centriole formation is a deep-rooted question, however our current knowledge on its molecular organization at high resolution remains fragmented and limited. In particular, exquisite details of the overall molecular architecture of the human centriole and in particular of its central core region are lacking to understand the basis of centriole organization and function. Resolving this important question represents a challenge that needs to be undertaken and will undoubtedly lead to groundbreaking advances. Another important question to tackle next is to develop innovative methods to enable the nanometric molecular mapping of centriolar proteins within distinct architectural elements of the centriole. This missing information will be key to unravel the molecular mechanisms behind centriolar organization.
This research proposal aims at building a cartography of the human centriole by elucidating its molecular composition and architecture. To this end, we will combine the use of innovative and multidisciplinary techniques encompassing spatial proteomics, cryo-electron tomography, state-of-the-art microscopy and in vitro assays and to achieve a comprehensive molecular and structural view of the human centriole. All together, we expect that these advances will help understand basic principles underlying centriole and cilia formation as well as might have further relevance for human health.
Summary
The centriole is the largest evolutionary conserved macromolecular structure responsible for building centrosomes and cilia or flagella in many eukaryotes. Centrioles are critical for the proper execution of important biological processes ranging from cell division to cell signaling. Moreover, centriolar defects have been associated to several human pathologies including ciliopathies and cancer. This state of facts emphasizes the importance of understanding centriole biogenesis. The study of centriole formation is a deep-rooted question, however our current knowledge on its molecular organization at high resolution remains fragmented and limited. In particular, exquisite details of the overall molecular architecture of the human centriole and in particular of its central core region are lacking to understand the basis of centriole organization and function. Resolving this important question represents a challenge that needs to be undertaken and will undoubtedly lead to groundbreaking advances. Another important question to tackle next is to develop innovative methods to enable the nanometric molecular mapping of centriolar proteins within distinct architectural elements of the centriole. This missing information will be key to unravel the molecular mechanisms behind centriolar organization.
This research proposal aims at building a cartography of the human centriole by elucidating its molecular composition and architecture. To this end, we will combine the use of innovative and multidisciplinary techniques encompassing spatial proteomics, cryo-electron tomography, state-of-the-art microscopy and in vitro assays and to achieve a comprehensive molecular and structural view of the human centriole. All together, we expect that these advances will help understand basic principles underlying centriole and cilia formation as well as might have further relevance for human health.
Max ERC Funding
1 498 965 €
Duration
Start date: 2017-01-01, End date: 2021-12-31
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 ANTIVIRNA
Project Structural and mechanistic studies of RNA-guided and RNA-targeting antiviral defense pathways
Researcher (PI) Martin Jinek
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Starting Grant (StG), LS1, ERC-2013-StG
Summary The evolutionary pressures exerted by viruses on their host cells constitute a major force that drives the evolution of cellular antiviral mechanisms. The proposed research is motivated by our interest in the roles of protein-RNA interactions in both prokaryotic and eukaryotic antiviral pathways and will proceed in two directions. The first project stems from our current work on the CRISPR pathway, a recently discovered RNA-guided adaptive defense mechanism in bacteria and archaea that silences mobile genetic elements such as viruses (bacteriophages) and plasmids. CRISPR systems rely on short RNAs (crRNAs) that associate with CRISPR-associated (Cas) proteins and function as sequence-specific guides in the detection and destruction of invading nucleic acids. To obtain molecular insights into the mechanisms of crRNA-guided interference, we will pursue structural and functional studies of DNA-targeting ribonuceoprotein complexes from type II and III CRISPR systems. Our work will shed light on the function of these systems in microbial pathogenesis and provide a framework for the informed engineering of RNA-guided gene targeting technologies. The second proposed research direction centres on RNA-targeting antiviral strategies employed by the human innate immune system. Here, our work will focus on structural studies of major interferon-induced effector proteins, initially examining the allosteric activation mechanism of RNase L and subsequently focusing on other antiviral nucleases and RNA helicases, as well as mechanisms by which RNA viruses evade the innate immune response of the host. In our investigations, we plan to approach these questions using an integrated strategy combining structural biology, biochemistry and biophysics with cell-based functional studies. Together, our studies will provide fundamental molecular insights into RNA-centred antiviral mechanisms and their impact on human health and disease.
Summary
The evolutionary pressures exerted by viruses on their host cells constitute a major force that drives the evolution of cellular antiviral mechanisms. The proposed research is motivated by our interest in the roles of protein-RNA interactions in both prokaryotic and eukaryotic antiviral pathways and will proceed in two directions. The first project stems from our current work on the CRISPR pathway, a recently discovered RNA-guided adaptive defense mechanism in bacteria and archaea that silences mobile genetic elements such as viruses (bacteriophages) and plasmids. CRISPR systems rely on short RNAs (crRNAs) that associate with CRISPR-associated (Cas) proteins and function as sequence-specific guides in the detection and destruction of invading nucleic acids. To obtain molecular insights into the mechanisms of crRNA-guided interference, we will pursue structural and functional studies of DNA-targeting ribonuceoprotein complexes from type II and III CRISPR systems. Our work will shed light on the function of these systems in microbial pathogenesis and provide a framework for the informed engineering of RNA-guided gene targeting technologies. The second proposed research direction centres on RNA-targeting antiviral strategies employed by the human innate immune system. Here, our work will focus on structural studies of major interferon-induced effector proteins, initially examining the allosteric activation mechanism of RNase L and subsequently focusing on other antiviral nucleases and RNA helicases, as well as mechanisms by which RNA viruses evade the innate immune response of the host. In our investigations, we plan to approach these questions using an integrated strategy combining structural biology, biochemistry and biophysics with cell-based functional studies. Together, our studies will provide fundamental molecular insights into RNA-centred antiviral mechanisms and their impact on human health and disease.
Max ERC Funding
1 467 180 €
Duration
Start date: 2013-11-01, End date: 2018-10-31
Project acronym BFTERRA
Project Biogenesis and Functions of Telomeric Repeat-containing RNA
Researcher (PI) Claus Maria Azzalin
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Starting Grant (StG), LS1, ERC-2009-StG
Summary Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.
Summary
Telomeres are heterochromatic nucleoprotein complexes located at the end of linear eukaryotic chromosomes. Contrarily to a longstanding dogma, we have recently demonstrated that mammalian telomeres are transcribed into TElomeric Repeat containing RNA (TERRA) molecules. TERRA transcripts contain telomeric RNA repeats and are produced at least in part by DNA-dependent RNA polymerase II-mediated transcription of telomeric DNA. TERRA molecules form discrete nuclear foci that co-localize with telomeric heterochromatin in both interphase and transcriptionally inactive metaphase cells. This indicates that TERRA is an integral component of telomeres and suggests that TERRA might participate in maintaining proper telomere heterochromatin. We will use a variety of biochemistry, cell biology, molecular biology and microscopy based approaches applied to cultured mammalian cells and to the yeast Schizosaccharomyces pombe, to achieve four distinct major goals: i) We will over-express or deplete TERRA in mammalian cells in order to characterize the molecular details of putative TERRA-associated functions in maintaining normal telomere structure and function; ii) We will locate TERRA promoter regions on different human chromosome ends; iii) We will generate mammalian cellular systems in which to study artificially seeded telomeres that can be transcribed in an inducible fashion; iv) We will identify physiological regulators of TERRA by analyzing it in mammalian cultured cells where the functions of candidate factors are compromised. In parallel, taking advantage of the recent discovery of TERRA also in fission yeast, we will systematically analyze TERRA levels in fission yeast mutants derived from a complete gene knockout collection. The study of TERRA regulation and function at chromosome ends will strongly contribute to our understanding of how telomeres are maintained and will help to clarify the general functions of mammalian non-coding RNAs.
Max ERC Funding
1 602 600 €
Duration
Start date: 2009-10-01, End date: 2014-09-30
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 CHROCODYLE
Project Chromosomal Condensin Dynamics: From Local Loading to Global Architecture
Researcher (PI) Stephan GRUBER
Host Institution (HI) UNIVERSITE DE LAUSANNE
Call Details Consolidator Grant (CoG), LS1, ERC-2016-COG
Summary Striking morphological transformations are a hallmark of any cell division cycle. During nuclear division chromatin is compacted into distinctive rod-shaped chromatids in preparation of chromosome segregation by the spindle apparatus. Multi-subunit SMC protein complexes and a large number of regulatory factors are at the heart of this elementary process. SMC complexes also play key roles during other aspects of genome function such as the control of gene expression and the repair of damaged DNA. They are thought to act as chromatin linkers with exquisite specificity for certain pairs of DNA fibres. However, the underlying molecular mechanisms are not understood. Active extrusion of DNA loops by the SMC complex has been proposed to be the mechanistic basis for the establishment of long-range, intra-chromatid DNA bridges.
Here, I put forward a multi-pronged research programme that aims to elucidate fundamentally conserved features of SMC protein function and action using the prokaryotic SMC condensin complex in Bacillus subtilis as a tractable model system. We will conduct a combined structural, biochemical and cell biology approach (including crystallography, electron paramagnetic resonance, ChIP-Seq and ‘native’ HiC) to uncover how the SMC complex acts at the higher levels of organization of the bacterial chromosome to promote the efficient individualization of sister DNA molecules. We will reveal the molecular and structural bases for the association between the SMC complex and the bacterial chromosome at different stages of the loading reaction – each representing a crucial intermediate in a sophisticated chromosome organization process. For the first time, we will be able to map the paths of chromosomal DNA through an SMC complex.
Our in-depth mechanistic insights will likely have implications for the understanding of various pathological conditions and have the potential to contribute to the development of novel antibacterial compounds.
Summary
Striking morphological transformations are a hallmark of any cell division cycle. During nuclear division chromatin is compacted into distinctive rod-shaped chromatids in preparation of chromosome segregation by the spindle apparatus. Multi-subunit SMC protein complexes and a large number of regulatory factors are at the heart of this elementary process. SMC complexes also play key roles during other aspects of genome function such as the control of gene expression and the repair of damaged DNA. They are thought to act as chromatin linkers with exquisite specificity for certain pairs of DNA fibres. However, the underlying molecular mechanisms are not understood. Active extrusion of DNA loops by the SMC complex has been proposed to be the mechanistic basis for the establishment of long-range, intra-chromatid DNA bridges.
Here, I put forward a multi-pronged research programme that aims to elucidate fundamentally conserved features of SMC protein function and action using the prokaryotic SMC condensin complex in Bacillus subtilis as a tractable model system. We will conduct a combined structural, biochemical and cell biology approach (including crystallography, electron paramagnetic resonance, ChIP-Seq and ‘native’ HiC) to uncover how the SMC complex acts at the higher levels of organization of the bacterial chromosome to promote the efficient individualization of sister DNA molecules. We will reveal the molecular and structural bases for the association between the SMC complex and the bacterial chromosome at different stages of the loading reaction – each representing a crucial intermediate in a sophisticated chromosome organization process. For the first time, we will be able to map the paths of chromosomal DNA through an SMC complex.
Our in-depth mechanistic insights will likely have implications for the understanding of various pathological conditions and have the potential to contribute to the development of novel antibacterial compounds.
Max ERC Funding
1 999 599 €
Duration
Start date: 2017-06-01, End date: 2022-05-31
Project acronym CIRCATRANS
Project Control of mouse metabolism by circadian clock-coordinated mRNA translation
Researcher (PI) Frédéric Bruno Martin Gachon
Host Institution (HI) NESTEC SA
Call Details Starting Grant (StG), LS1, ERC-2010-StG_20091118
Summary The mammalian circadian clock plays a fundamental role in the liver by regulating fatty acid, glucose and xenobiotic metabolism. Impairment of this rhythm has been show to lead to diverse pathologies including metabolic syndrome. At present, it is supposed that the circadian clock regulates metabolism mostly by regulating the expression of liver enzymes at the transcriptional level. We have now collected evidence that post-transcriptional regulations play also an important role in this regulation. Particularly, recent results from our laboratory show that the circadian clock can synchronize mRNA translation in mouse liver through rhythmic activation of the Target Of Rapamycin Complex 1 (TORC1) with a 12-hours period. Based on this unexpected observation, we plan to identify the genes rhythmically translated in the mouse liver as well as the mechanisms involved in this translation. Indeed, our initial observations suggest a cap-independent translation during the day and a cap-dependent translation during the night. Identification of the different complexes involved in translation at this two different times and their correlation with the sequence, structure, and/or function of the translated genes will provide new insight into the action of the circadian clock on animal metabolism. In parallel, we will identify the signalling pathways involved in the rhythmic activation of TORC1 in mouse liver. Finally, we will study the consequences of a deregulated rhythmic translation in circadian clock-deficient mice on the metabolism and the longevity of these animals. Perturbations of the circadian clock have been linked to numerous pathologies, including obesity, type 2 diabetes and cancer. Our project on the importance of circadian clock-coordinated translation will likely reveal new findings in the field of regulation of animal metabolism by the circadian clock.
Summary
The mammalian circadian clock plays a fundamental role in the liver by regulating fatty acid, glucose and xenobiotic metabolism. Impairment of this rhythm has been show to lead to diverse pathologies including metabolic syndrome. At present, it is supposed that the circadian clock regulates metabolism mostly by regulating the expression of liver enzymes at the transcriptional level. We have now collected evidence that post-transcriptional regulations play also an important role in this regulation. Particularly, recent results from our laboratory show that the circadian clock can synchronize mRNA translation in mouse liver through rhythmic activation of the Target Of Rapamycin Complex 1 (TORC1) with a 12-hours period. Based on this unexpected observation, we plan to identify the genes rhythmically translated in the mouse liver as well as the mechanisms involved in this translation. Indeed, our initial observations suggest a cap-independent translation during the day and a cap-dependent translation during the night. Identification of the different complexes involved in translation at this two different times and their correlation with the sequence, structure, and/or function of the translated genes will provide new insight into the action of the circadian clock on animal metabolism. In parallel, we will identify the signalling pathways involved in the rhythmic activation of TORC1 in mouse liver. Finally, we will study the consequences of a deregulated rhythmic translation in circadian clock-deficient mice on the metabolism and the longevity of these animals. Perturbations of the circadian clock have been linked to numerous pathologies, including obesity, type 2 diabetes and cancer. Our project on the importance of circadian clock-coordinated translation will likely reveal new findings in the field of regulation of animal metabolism by the circadian clock.
Max ERC Funding
1 475 831 €
Duration
Start date: 2011-03-01, End date: 2016-02-29
Project acronym CRISPR2.0
Project Microbial genome defence pathways: from molecular mechanisms to next-generation molecular tools
Researcher (PI) Martin JINEK
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Consolidator Grant (CoG), LS1, ERC-2018-COG
Summary The constant arms race between prokaryotic microbes and their molecular parasites such as viruses, plasmids and transposons has driven the evolution of complex genome defence mechanisms. The CRISPR-Cas defence systems provide adaptive RNA-guided immunity against invasive nucleic acid elements. CRISPR-associated effector nucleases such as Cas9, Cas12a and Cas13 have emerged as powerful tools for precision genome editing, gene expression control and nucleic acid detection. However, these technologies suffer from drawbacks that limit their efficacy and versatility, necessitating the search for additional exploitable molecular activities. Building on our recent structural and biochemical studies, the goal of this project is to investigate the molecular architectures and mechanisms of CRISPR-associated systems and other genome defence mechanisms, aiming not only to shed light on their biological roles but also inform their technological development. Specifically, the proposed studies will examine (i) the molecular basis of cyclic oligoadenylate signalling in type III CRISPR-Cas systems, (ii) the mechanism of transposon-associated type I CRISPR-Cas systems and their putative function in RNA-guided DNA transposition, and (iii) molecular activities associated with recently described non-CRISPR defence systems. Collectively, the proposed studies will advance our understanding of the molecular functions of genome defence mechanisms in shaping the evolution of prokaryotic genomes and make critical contributions to their development as novel genetic engineering tools.
Summary
The constant arms race between prokaryotic microbes and their molecular parasites such as viruses, plasmids and transposons has driven the evolution of complex genome defence mechanisms. The CRISPR-Cas defence systems provide adaptive RNA-guided immunity against invasive nucleic acid elements. CRISPR-associated effector nucleases such as Cas9, Cas12a and Cas13 have emerged as powerful tools for precision genome editing, gene expression control and nucleic acid detection. However, these technologies suffer from drawbacks that limit their efficacy and versatility, necessitating the search for additional exploitable molecular activities. Building on our recent structural and biochemical studies, the goal of this project is to investigate the molecular architectures and mechanisms of CRISPR-associated systems and other genome defence mechanisms, aiming not only to shed light on their biological roles but also inform their technological development. Specifically, the proposed studies will examine (i) the molecular basis of cyclic oligoadenylate signalling in type III CRISPR-Cas systems, (ii) the mechanism of transposon-associated type I CRISPR-Cas systems and their putative function in RNA-guided DNA transposition, and (iii) molecular activities associated with recently described non-CRISPR defence systems. Collectively, the proposed studies will advance our understanding of the molecular functions of genome defence mechanisms in shaping the evolution of prokaryotic genomes and make critical contributions to their development as novel genetic engineering tools.
Max ERC Funding
1 996 525 €
Duration
Start date: 2019-05-01, End date: 2024-04-30
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 DiVineGenoMe
Project Decoding cell-to-cell variation in genome integrity maintenance
Researcher (PI) Matthias Florian ALTMEYER
Host Institution (HI) UNIVERSITAT ZURICH
Call Details Starting Grant (StG), LS1, ERC-2016-STG
Summary Cells must ensure the integrity of their genome, and failure to do so can lead to mutations and cause disease. A sophisticated molecular network senses genomic lesions and coordinates their faithful repair with other DNA transactions, including transcription and DNA replication. Research over the last years has significantly advanced our understanding of the DNA damage response and continues to provide crucial insights that explain how cells deal with genotoxic stress to avoid malignant transformation.
More recently, the intriguing phenomenon of cellular heterogeneity reached into the limelight as it became increasingly clear that significant variability exists between individual cells, even of the same genetic background and cell type. Single cells matter, for instance during cellular transformation or tumor relapse, and cellular variability thus impacts disease development and therapeutic outcome. Its determinants are surprisingly unexplored, however, and have not been studied in context of genome integrity maintenance.
The main objective of this project is to systematically assess cellular heterogeneity in genome integrity maintenance and characterize its causes and consequences. Quantitative automated high-content imaging of large cell cohorts will be used to identify hitherto unknown determinants of variability in the cellular responses to genotoxic stress and dissect at the single cell level the variability in (1) the chromatin response to DNA double-strand breaks, (2) the cellular response to replication stress, and (3) the cellular capacity to trigger phase transitions, a newly emerging mechanism of dynamic compartmentalization, at sites of genomic lesions. This project will bridge two thus far independently developed research fields (genome stability and cellular heterogeneity), reveal how cell-to-cell variation impacts cell fate and survival in response to genotoxic stress, and may uncover ways to homogenize this response for improved cancer therapies.
Summary
Cells must ensure the integrity of their genome, and failure to do so can lead to mutations and cause disease. A sophisticated molecular network senses genomic lesions and coordinates their faithful repair with other DNA transactions, including transcription and DNA replication. Research over the last years has significantly advanced our understanding of the DNA damage response and continues to provide crucial insights that explain how cells deal with genotoxic stress to avoid malignant transformation.
More recently, the intriguing phenomenon of cellular heterogeneity reached into the limelight as it became increasingly clear that significant variability exists between individual cells, even of the same genetic background and cell type. Single cells matter, for instance during cellular transformation or tumor relapse, and cellular variability thus impacts disease development and therapeutic outcome. Its determinants are surprisingly unexplored, however, and have not been studied in context of genome integrity maintenance.
The main objective of this project is to systematically assess cellular heterogeneity in genome integrity maintenance and characterize its causes and consequences. Quantitative automated high-content imaging of large cell cohorts will be used to identify hitherto unknown determinants of variability in the cellular responses to genotoxic stress and dissect at the single cell level the variability in (1) the chromatin response to DNA double-strand breaks, (2) the cellular response to replication stress, and (3) the cellular capacity to trigger phase transitions, a newly emerging mechanism of dynamic compartmentalization, at sites of genomic lesions. This project will bridge two thus far independently developed research fields (genome stability and cellular heterogeneity), reveal how cell-to-cell variation impacts cell fate and survival in response to genotoxic stress, and may uncover ways to homogenize this response for improved cancer therapies.
Max ERC Funding
1 500 000 €
Duration
Start date: 2017-04-01, End date: 2022-09-30
