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 CIRCOMMUNICATION
Project Deciphering molecular pathways of circadian clock communication
Researcher (PI) gad ASHER
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Consolidator Grant (CoG), LS1, ERC-2017-COG
Summary The overarching objective of this interdisciplinary project is to elucidate mechanisms through which billions of individual clocks in the body communicate with each other and tick in harmony. The mammalian circadian timing system consists of a master clock in the brain and subsidiary oscillators in almost every cell of the body. Since these clocks anticipate environmental changes and function together to orchestrate daily physiology and behavior their temporal synchronization is critical.
Our recent finding that oxygen serves as a resetting cue for circadian clocks points towards the unprecedented involvement of blood gases as time signals. We will apply cutting edge continuous physiological measurements in freely moving animals, alongside biochemical/molecular biology approaches and advanced cell culture setup to determine the molecular role of oxygen, carbon dioxide and pH in circadian clock communication and function.
The intricate nature of the mammalian circadian system demands the presence of communication mechanisms between clocks throughout the body at multiple levels. While previous studies primarily addressed the role of the master clock in resetting peripheral clocks, our knowledge regarding the communication among clocks between and within peripheral organs is rudimentary. We will reconstruct the mammalian circadian system from the bottom up, sequentially restoring clocks in peripheral tissues of a non-rhythmic animal to (i) obtain a system-view of the peripheral circadian communication network; and (ii) study novel tissue-derived circadian communication mechanisms.
This integrative proposal addresses fundamental aspects of circadian biology. It is expected to unravel the circadian communication network and shed light on how billions of clocks in the body function in unison. Its impact extends beyond circadian rhythms and bears great potential for research on communication between cells/tissues in various fields of biology.
Summary
The overarching objective of this interdisciplinary project is to elucidate mechanisms through which billions of individual clocks in the body communicate with each other and tick in harmony. The mammalian circadian timing system consists of a master clock in the brain and subsidiary oscillators in almost every cell of the body. Since these clocks anticipate environmental changes and function together to orchestrate daily physiology and behavior their temporal synchronization is critical.
Our recent finding that oxygen serves as a resetting cue for circadian clocks points towards the unprecedented involvement of blood gases as time signals. We will apply cutting edge continuous physiological measurements in freely moving animals, alongside biochemical/molecular biology approaches and advanced cell culture setup to determine the molecular role of oxygen, carbon dioxide and pH in circadian clock communication and function.
The intricate nature of the mammalian circadian system demands the presence of communication mechanisms between clocks throughout the body at multiple levels. While previous studies primarily addressed the role of the master clock in resetting peripheral clocks, our knowledge regarding the communication among clocks between and within peripheral organs is rudimentary. We will reconstruct the mammalian circadian system from the bottom up, sequentially restoring clocks in peripheral tissues of a non-rhythmic animal to (i) obtain a system-view of the peripheral circadian communication network; and (ii) study novel tissue-derived circadian communication mechanisms.
This integrative proposal addresses fundamental aspects of circadian biology. It is expected to unravel the circadian communication network and shed light on how billions of clocks in the body function in unison. Its impact extends beyond circadian rhythms and bears great potential for research on communication between cells/tissues in various fields of biology.
Max ERC Funding
1 999 945 €
Duration
Start date: 2018-03-01, End date: 2023-02-28
Project acronym CLIP
Project Mapping functional protein-RNA interactions to identify new targets for oligonucleotide-based therapy
Researcher (PI) Jernej Ule
Host Institution (HI) UNIVERSITY COLLEGE LONDON
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary An important question of modern neurobiology is how neurons regulate synaptic function in response to excitation. In particular, the roles of alternative pre-mRNA splicing and mRNA translation regulation in this response are poorly understood. We will study the RNA-binding proteins (RBPs) that control these post-transcriptional changes using a UV crosslinking-based purification method (CLIP) and ultra-high throughput sequencing. Computational analysis of the resulting data will define the sequence and structural features of RNA motifs recognized by each RBP. Splicing microarrays and translation reporter assays will then allow us to examine the regulatory functions of RBPs and RNA motifs. By integrating the biochemical and functional datasets, we will relate the position of RNA motifs to the activity of bound RBPs, and predict the interactions that act as central nodes in the regulatory network. The physiological role of these core RBP-RNA interactions will then be tested using antisense RNAs. Together, these projects will provide insights to the regulatory mechanisms underlying neuronal activity-dependent changes, and provide new opportunities for future treatments of neurodegenerative disorders.
Summary
An important question of modern neurobiology is how neurons regulate synaptic function in response to excitation. In particular, the roles of alternative pre-mRNA splicing and mRNA translation regulation in this response are poorly understood. We will study the RNA-binding proteins (RBPs) that control these post-transcriptional changes using a UV crosslinking-based purification method (CLIP) and ultra-high throughput sequencing. Computational analysis of the resulting data will define the sequence and structural features of RNA motifs recognized by each RBP. Splicing microarrays and translation reporter assays will then allow us to examine the regulatory functions of RBPs and RNA motifs. By integrating the biochemical and functional datasets, we will relate the position of RNA motifs to the activity of bound RBPs, and predict the interactions that act as central nodes in the regulatory network. The physiological role of these core RBP-RNA interactions will then be tested using antisense RNAs. Together, these projects will provide insights to the regulatory mechanisms underlying neuronal activity-dependent changes, and provide new opportunities for future treatments of neurodegenerative disorders.
Max ERC Funding
900 000 €
Duration
Start date: 2008-09-01, End date: 2013-08-31
Project acronym COCO
Project The molecular complexity of the complement system
Researcher (PI) Piet Gros
Host Institution (HI) UNIVERSITEIT UTRECHT
Call Details Advanced Grant (AdG), LS1, ERC-2008-AdG
Summary The complement system is a regulatory pathway in mammalian plasma that enables the host to recognize and clear invading pathogens and altered host cells, while protecting healthy host tissue. This regulatory system consists of ~30 large multi-domain plasma and cell-surface proteins, that act in concert through an interplay of proteolysis and complex formations on target membranes. We study the molecular events on membranes that ensure initiation and amplification of the response, protection of host cells and activation of immune responses leading to cell lysis, phagocytosis and B-cell stimulation.
In the past few years, we have resolved the structural details of the large complement proteins involved in the central, aspecific labelling and amplification step; with recent data we revealed the structural basis of the assembly and activity of the protease complex associated with this step. These insights into the central aspecific reaction, and the experiences gained on working with these large multi-domain proteins and complexes, give us an excellent starting point to addres the questions of specificity, protection and activation of immune cells.
The goal of the proposal is to elucidate the multivalent molecular mechanisms of recognition, regulation and immune cell activation of the complement system on target membranes. We will use protein crystallography and electron microscopy to study the interactions and conformational changes involved in protein complex formation, and (single-molecule) fluorescence to resolve the multivalent molecular events, the conformational states and transitions that occur on the membrane. The combined data will provide mechanistic insights into the specifity of immune clearance by the complement system.
Understanding the molecular mechanisms of complement activation and regulation will be instrumental in developing more potent therapeutics to control infections, prevent tissue damage and fight tumours by immunotherapies.
Summary
The complement system is a regulatory pathway in mammalian plasma that enables the host to recognize and clear invading pathogens and altered host cells, while protecting healthy host tissue. This regulatory system consists of ~30 large multi-domain plasma and cell-surface proteins, that act in concert through an interplay of proteolysis and complex formations on target membranes. We study the molecular events on membranes that ensure initiation and amplification of the response, protection of host cells and activation of immune responses leading to cell lysis, phagocytosis and B-cell stimulation.
In the past few years, we have resolved the structural details of the large complement proteins involved in the central, aspecific labelling and amplification step; with recent data we revealed the structural basis of the assembly and activity of the protease complex associated with this step. These insights into the central aspecific reaction, and the experiences gained on working with these large multi-domain proteins and complexes, give us an excellent starting point to addres the questions of specificity, protection and activation of immune cells.
The goal of the proposal is to elucidate the multivalent molecular mechanisms of recognition, regulation and immune cell activation of the complement system on target membranes. We will use protein crystallography and electron microscopy to study the interactions and conformational changes involved in protein complex formation, and (single-molecule) fluorescence to resolve the multivalent molecular events, the conformational states and transitions that occur on the membrane. The combined data will provide mechanistic insights into the specifity of immune clearance by the complement system.
Understanding the molecular mechanisms of complement activation and regulation will be instrumental in developing more potent therapeutics to control infections, prevent tissue damage and fight tumours by immunotherapies.
Max ERC Funding
1 700 000 €
Duration
Start date: 2009-04-01, End date: 2014-03-31
Project acronym CohesinMolMech
Project Molecular mechanisms of cohesin-mediated sister chromatid cohesion and chromatin organization
Researcher (PI) Jan-Michael Peters
Host Institution (HI) FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
Call Details Advanced Grant (AdG), LS1, ERC-2015-AdG
Summary During S-phase newly synthesized “sister” DNA molecules become physically connected. This sister chromatid cohesion resists the pulling forces of the mitotic spindle and thereby enables the bi-orientation and subsequent symmetrical segregation of chromosomes. Cohesion is mediated by ring-shaped cohesin complexes, which are thought to entrap sister DNA molecules topologically. In mammalian cells, cohesin is loaded onto DNA at the end of mitosis by the Scc2-Scc4 complex, becomes acetylated during S-phase, and is stably “locked” on DNA during S- and G2-phase by sororin. Sororin stabilizes cohesin on DNA by inhibiting Wapl, which can otherwise release cohesin from DNA again. In addition to mediating cohesion, cohesin also has important roles in organizing higher-order chromatin structures and in gene regulation. Cohesin performs the latter functions in both proliferating and post-mitotic cells and mediates at least some of these together with the sequence-specific DNA-binding protein CTCF, which co-localizes with cohesin at many genomic sites. Although cohesin and CTCF perform essential functions in mammalian cells, it is poorly understood how cohesin is loaded onto DNA by Scc2-Scc4, how cohesin is positioned in the genome, how cohesin is released from DNA again by Wapl, and how Wapl is inhibited by sororin. Likewise, it is not known how cohesin establishes cohesion during DNA replication and how cohesin cooperates with CTCF to organize chromatin structure. Here we propose to address these questions by combining biochemical reconstitution, single-molecule TIRF microscopy, genetic and cell biological approaches. We expect that the results of these studies will advance our understanding of cell division, chromatin structure and gene regulation, and may also provide insight into the etiology of disorders that are caused by cohesin dysfunction, such as Down syndrome and “cohesinopathies” or cancers, in which cohesin mutations have been found to occur frequently.
Summary
During S-phase newly synthesized “sister” DNA molecules become physically connected. This sister chromatid cohesion resists the pulling forces of the mitotic spindle and thereby enables the bi-orientation and subsequent symmetrical segregation of chromosomes. Cohesion is mediated by ring-shaped cohesin complexes, which are thought to entrap sister DNA molecules topologically. In mammalian cells, cohesin is loaded onto DNA at the end of mitosis by the Scc2-Scc4 complex, becomes acetylated during S-phase, and is stably “locked” on DNA during S- and G2-phase by sororin. Sororin stabilizes cohesin on DNA by inhibiting Wapl, which can otherwise release cohesin from DNA again. In addition to mediating cohesion, cohesin also has important roles in organizing higher-order chromatin structures and in gene regulation. Cohesin performs the latter functions in both proliferating and post-mitotic cells and mediates at least some of these together with the sequence-specific DNA-binding protein CTCF, which co-localizes with cohesin at many genomic sites. Although cohesin and CTCF perform essential functions in mammalian cells, it is poorly understood how cohesin is loaded onto DNA by Scc2-Scc4, how cohesin is positioned in the genome, how cohesin is released from DNA again by Wapl, and how Wapl is inhibited by sororin. Likewise, it is not known how cohesin establishes cohesion during DNA replication and how cohesin cooperates with CTCF to organize chromatin structure. Here we propose to address these questions by combining biochemical reconstitution, single-molecule TIRF microscopy, genetic and cell biological approaches. We expect that the results of these studies will advance our understanding of cell division, chromatin structure and gene regulation, and may also provide insight into the etiology of disorders that are caused by cohesin dysfunction, such as Down syndrome and “cohesinopathies” or cancers, in which cohesin mutations have been found to occur frequently.
Max ERC Funding
2 500 000 €
Duration
Start date: 2016-10-01, End date: 2021-09-30
Project acronym COMPLEXNMD
Project NMD Complexes: Eukaryotic mRNA Quality Control
Researcher (PI) Christiane Helene Berger-Schaffitzel
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Nonsense-mediated mRNA decay (NMD) is an essential mechanism controlling translation in the eukaryotic cell. NMD ascertains accurate expression of the genetic information by quality controlling messenger RNA (mRNA). During translation, NMD factors recognize and target to degradation aberrant mRNAs that have a premature stop codon (PTC) and that would otherwise lead to the production of truncated proteins which could be harmful for the cell. A wide range of genetic diseases have their origin in the mechanisms of NMD. Discrimination of a PTC from a correct termination codon depends on splicing and translation, and it is the first and foremost step in human NMD. The molecular mechanism of this process remains elusive to date.
In the research proposed, I will undertake to elucidate the molecular basis of translation termination and induction of NMD. I will study complexes involved in human translation termination at a normal stop codon and involved in NMD. I will employ an array of innovative techniques including recombinant production of human protein complexes by the MultiBac system, mammalian in vitro translation, mass spectrometry for detecting relevant protein modifications, biophysical techniques, mutational analyses and RNA-interference experiments. Stable ribosomal complexes with termination factors and complexes of NMD factors will be used for structure determination by cryo-electron microscopy. State-of-the-art image processing will be applied to address the inherent heterogeneity of the complexes. Hybrid approaches will allow the combination of cryo-EM structures with existing high-resolution structures of factors involved for generation of quasi-atomic models thereby visualizing molecular mechanisms of NMD action. This interdisciplinary work will foster our understanding at a molecular level of a paramount step in mRNA quality control, which is a vital prerequisite for the development of new treatment strategies in NMD-related diseases.
Summary
Nonsense-mediated mRNA decay (NMD) is an essential mechanism controlling translation in the eukaryotic cell. NMD ascertains accurate expression of the genetic information by quality controlling messenger RNA (mRNA). During translation, NMD factors recognize and target to degradation aberrant mRNAs that have a premature stop codon (PTC) and that would otherwise lead to the production of truncated proteins which could be harmful for the cell. A wide range of genetic diseases have their origin in the mechanisms of NMD. Discrimination of a PTC from a correct termination codon depends on splicing and translation, and it is the first and foremost step in human NMD. The molecular mechanism of this process remains elusive to date.
In the research proposed, I will undertake to elucidate the molecular basis of translation termination and induction of NMD. I will study complexes involved in human translation termination at a normal stop codon and involved in NMD. I will employ an array of innovative techniques including recombinant production of human protein complexes by the MultiBac system, mammalian in vitro translation, mass spectrometry for detecting relevant protein modifications, biophysical techniques, mutational analyses and RNA-interference experiments. Stable ribosomal complexes with termination factors and complexes of NMD factors will be used for structure determination by cryo-electron microscopy. State-of-the-art image processing will be applied to address the inherent heterogeneity of the complexes. Hybrid approaches will allow the combination of cryo-EM structures with existing high-resolution structures of factors involved for generation of quasi-atomic models thereby visualizing molecular mechanisms of NMD action. This interdisciplinary work will foster our understanding at a molecular level of a paramount step in mRNA quality control, which is a vital prerequisite for the development of new treatment strategies in NMD-related diseases.
Max ERC Funding
1 176 825 €
Duration
Start date: 2012-02-01, End date: 2016-11-30
Project acronym CondStruct
Project Structural basis for the coordination of chromosome architecture by condensin complexes
Researcher (PI) Christian Helmut Haering
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Consolidator Grant (CoG), LS1, ERC-2015-CoG
Summary Chromosomes undergo dramatic changes in their three-dimensional organisation during all aspects of genome function, ranging from the regulation of gene expression during cellular differentiation to chromosome duplication and partitioning over the course of a cell division cycle. The multi-subunit condensin protein complex plays major roles for these changes in DNA topology. Despite its fundamental importance, the mechanisms of condensin’s action are not understood.
Here, I propose a comprehensive research program that aims to reveal the elusive mechanisms behind the functions of the condensin complex. We intend to unravel how the condensin complex engages DNA, how this interaction activates large-scale ATPase-dependent conformational rearrangements within the complex, and how condensin eventually encircles chromatin fibres within its ring-shaped architecture. Insights from these mechanistic studies will be invaluable for understanding how networks of condensin-mediated linkages can shape linear DNA helices into higher-order chromosome structures. To achieve this ambitious and timely goal, we will combine an integrative structural biology approach with biochemical and cell biological methods. By applying complementary technologies, including X-ray protein crystallography, electron microscopy, cross-linking mass spectrometry, single molecule fluorescence microscopy and reconstitution experiments, we anticipate to build the first model of the entire condensin complex at near-atomic resolution and explain how dynamic conformational changes confer function.
The insights gained from this research program will provide an in-depth mechanistic comprehension of the core molecular machinery that determines the architecture of our genomes and will have major implications for understanding how genomic integrity is affected in various disease conditions.
Summary
Chromosomes undergo dramatic changes in their three-dimensional organisation during all aspects of genome function, ranging from the regulation of gene expression during cellular differentiation to chromosome duplication and partitioning over the course of a cell division cycle. The multi-subunit condensin protein complex plays major roles for these changes in DNA topology. Despite its fundamental importance, the mechanisms of condensin’s action are not understood.
Here, I propose a comprehensive research program that aims to reveal the elusive mechanisms behind the functions of the condensin complex. We intend to unravel how the condensin complex engages DNA, how this interaction activates large-scale ATPase-dependent conformational rearrangements within the complex, and how condensin eventually encircles chromatin fibres within its ring-shaped architecture. Insights from these mechanistic studies will be invaluable for understanding how networks of condensin-mediated linkages can shape linear DNA helices into higher-order chromosome structures. To achieve this ambitious and timely goal, we will combine an integrative structural biology approach with biochemical and cell biological methods. By applying complementary technologies, including X-ray protein crystallography, electron microscopy, cross-linking mass spectrometry, single molecule fluorescence microscopy and reconstitution experiments, we anticipate to build the first model of the entire condensin complex at near-atomic resolution and explain how dynamic conformational changes confer function.
The insights gained from this research program will provide an in-depth mechanistic comprehension of the core molecular machinery that determines the architecture of our genomes and will have major implications for understanding how genomic integrity is affected in various disease conditions.
Max ERC Funding
1 982 479 €
Duration
Start date: 2016-07-01, End date: 2021-06-30
Project acronym ConflictResolution
Project Transcription-replication conflicts in disease and development
Researcher (PI) Stephan Hamperl
Host Institution (HI) HELMHOLTZ ZENTRUM MUENCHEN DEUTSCHES FORSCHUNGSZENTRUM FUER GESUNDHEIT UND UMWELT GMBH
Call Details Starting Grant (StG), LS1, ERC-2019-STG
Summary Genetic and epigenetic instability contribute to cancers, aging, developmental disorders, and neurological diseases, so in-depth understanding how this instability arises is an important question affecting millions in Europe. Physical conflicts between the transcription and DNA replication machineries are a potent endogenous source of this instability.
My preliminary data indicate that a single collision can trigger long-term epigenetic changes and affect the normal expression state of genes. I hypothesize that collisions can rewire gene expression networks and lead to cellular transformations relevant to disease and development. Unfortunately, this mechanism is largely understudied owing to the lack of suitable cellular systems to characterize collisions in molecular detail. My proposal will address this key gap in knowledge.
I recently pioneered a unique human cell-based episomal system to analyse collisions in an inducible and localized fashion. Using this highly tractable system, we will molecularly characterize the (epi)genetic consequences and identify novel factors that prevent or resolve collisions (Aim 1).
To address the relevance of collisions in disease, we will establish a novel proximity-labelling system (Split-APEX2) to map collision sites and identify their associated genetic and chromatin changes in a breast cancer cell model. This cutting-edge technology will decipher their role in pathological transformations observed in breast cancer genomes (Aim 2).
To link collisions to developmental transformations, we will determine their potential to induce local epigenetic changes during zygotic genome activation in mouse embryonic cells. This approach can shift the paradigm how cells in development first start to differ from each other and reprogram their genome into different cell types (Aim 3).
Uncovering the key principles of collisions may implement highly innovative approaches to avoid or establish cellular transformations in disease and development.
Summary
Genetic and epigenetic instability contribute to cancers, aging, developmental disorders, and neurological diseases, so in-depth understanding how this instability arises is an important question affecting millions in Europe. Physical conflicts between the transcription and DNA replication machineries are a potent endogenous source of this instability.
My preliminary data indicate that a single collision can trigger long-term epigenetic changes and affect the normal expression state of genes. I hypothesize that collisions can rewire gene expression networks and lead to cellular transformations relevant to disease and development. Unfortunately, this mechanism is largely understudied owing to the lack of suitable cellular systems to characterize collisions in molecular detail. My proposal will address this key gap in knowledge.
I recently pioneered a unique human cell-based episomal system to analyse collisions in an inducible and localized fashion. Using this highly tractable system, we will molecularly characterize the (epi)genetic consequences and identify novel factors that prevent or resolve collisions (Aim 1).
To address the relevance of collisions in disease, we will establish a novel proximity-labelling system (Split-APEX2) to map collision sites and identify their associated genetic and chromatin changes in a breast cancer cell model. This cutting-edge technology will decipher their role in pathological transformations observed in breast cancer genomes (Aim 2).
To link collisions to developmental transformations, we will determine their potential to induce local epigenetic changes during zygotic genome activation in mouse embryonic cells. This approach can shift the paradigm how cells in development first start to differ from each other and reprogram their genome into different cell types (Aim 3).
Uncovering the key principles of collisions may implement highly innovative approaches to avoid or establish cellular transformations in disease and development.
Max ERC Funding
1 497 530 €
Duration
Start date: 2020-02-01, End date: 2025-01-31
Project acronym Controlling MAC
Project Structural basis of controlling the membrane attack complex
Researcher (PI) Doryen Bubeck
Host Institution (HI) IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICINE
Call Details Consolidator Grant (CoG), LS1, ERC-2019-COG
Summary Structural basis of controlling the membrane attack complex
Complement is a fundamental component of the human immune system; central to the battle between hosts and pathogens. The membrane attack complex (MAC) is the direct killing arm of complement that acts by forming large pores in target cell membranes. Uncontrolled activation results in by-stander damage, which can have devastating consequences for host cells and impact inflammatory pathologies, thrombosis and cancer. Understanding how MAC activity is controlled on human cells during an immune response is a major unresolved question.
My lab has pioneered the use of cryo electron microscopy (cryoEM) to investigate the molecular mechanism underpinning MAC assembly. We have defined the stoichiometry of the complex and identified interaction interfaces that determine its sequential assembly mechanism. Recent data from my lab has now revealed atomic resolution information for the complete transmembrane pore. Results from my lab have provided a molecular and biophysical basis for MAC pore formation, which has led to a general mechanism for how proteins cross lipid bilayers.
Here, the goal is to understand the structural basis for how MAC activity is controlled by (i) cell surface receptor CD59, (ii) removal of pores from the plasma membrane, and (iii) clearance of assembly by-products from the plasma. MAC interacts with a defined set of cellular proteins through these three pathways. In this proposal, we will integrate structural information that spans cellular to molecular length scales. Recent technical advances in cryoEM, cryo soft X-ray tomography (cryoSXT) and correlated fluorescence imaging make it now possible to address how MAC activity is controlled in and around the plasma membrane. In doing so, we will answer a longstanding question in immunology and open new research directions exploring fundamental cellular processes. These results will provide a foundation for the development of novel therapeutics.
Summary
Structural basis of controlling the membrane attack complex
Complement is a fundamental component of the human immune system; central to the battle between hosts and pathogens. The membrane attack complex (MAC) is the direct killing arm of complement that acts by forming large pores in target cell membranes. Uncontrolled activation results in by-stander damage, which can have devastating consequences for host cells and impact inflammatory pathologies, thrombosis and cancer. Understanding how MAC activity is controlled on human cells during an immune response is a major unresolved question.
My lab has pioneered the use of cryo electron microscopy (cryoEM) to investigate the molecular mechanism underpinning MAC assembly. We have defined the stoichiometry of the complex and identified interaction interfaces that determine its sequential assembly mechanism. Recent data from my lab has now revealed atomic resolution information for the complete transmembrane pore. Results from my lab have provided a molecular and biophysical basis for MAC pore formation, which has led to a general mechanism for how proteins cross lipid bilayers.
Here, the goal is to understand the structural basis for how MAC activity is controlled by (i) cell surface receptor CD59, (ii) removal of pores from the plasma membrane, and (iii) clearance of assembly by-products from the plasma. MAC interacts with a defined set of cellular proteins through these three pathways. In this proposal, we will integrate structural information that spans cellular to molecular length scales. Recent technical advances in cryoEM, cryo soft X-ray tomography (cryoSXT) and correlated fluorescence imaging make it now possible to address how MAC activity is controlled in and around the plasma membrane. In doing so, we will answer a longstanding question in immunology and open new research directions exploring fundamental cellular processes. These results will provide a foundation for the development of novel therapeutics.
Max ERC Funding
1 999 990 €
Duration
Start date: 2020-07-01, End date: 2025-06-30
Project acronym COSMIC
Project Complex Synthetic Mimics of the Cell Membrane
Researcher (PI) Mark Ian Wallace
Host Institution (HI) KING'S COLLEGE LONDON
Call Details Starting Grant (StG), LS1, ERC-2012-StG_20111109
Summary I propose to bridge the gap between simple in vitro measurements of biological processes, and the complexities of the cellular environment. This requires reduced in vitro systems that are sufficiently complex to reproduce the subtleties of the in vivo biological phenomenon, but sufficiently controllable to test how quantitative changes in a particular property affects function. The challenge is to step beyond the most simple and straightforward in vitro mimics of the cell membrane, and create model systems that more closely reproduce the conditions in vivo.
I propose to tackle two specific, but interrelated membrane phenomena, that are currently not captured in artificial bilayers and create new complex mimics of the cell membrane capable of tackling these systems; namely (1) protein crowding and the cytoskeleton, and (2) lateral forces and membrane curvature. Testing our synthetic mimics with models that we understand in vivo is vital. This benchmarking will ensure that the mimics we create are relevant and will help ensure the more ambitious later goals of the this proposal are successful.We will then take these tools to go on and aim to create a synthetic mimic of the bacterial membrane.
However we are not limited to creating purely natural duplicates, and we can exploit a much wider range of building material than nature. In addition to creating complex mimics, we will also create totally new synthetic systems inspired by the properties of the cell membrane, but possessing unique properties.
Summary
I propose to bridge the gap between simple in vitro measurements of biological processes, and the complexities of the cellular environment. This requires reduced in vitro systems that are sufficiently complex to reproduce the subtleties of the in vivo biological phenomenon, but sufficiently controllable to test how quantitative changes in a particular property affects function. The challenge is to step beyond the most simple and straightforward in vitro mimics of the cell membrane, and create model systems that more closely reproduce the conditions in vivo.
I propose to tackle two specific, but interrelated membrane phenomena, that are currently not captured in artificial bilayers and create new complex mimics of the cell membrane capable of tackling these systems; namely (1) protein crowding and the cytoskeleton, and (2) lateral forces and membrane curvature. Testing our synthetic mimics with models that we understand in vivo is vital. This benchmarking will ensure that the mimics we create are relevant and will help ensure the more ambitious later goals of the this proposal are successful.We will then take these tools to go on and aim to create a synthetic mimic of the bacterial membrane.
However we are not limited to creating purely natural duplicates, and we can exploit a much wider range of building material than nature. In addition to creating complex mimics, we will also create totally new synthetic systems inspired by the properties of the cell membrane, but possessing unique properties.
Max ERC Funding
1 498 523 €
Duration
Start date: 2013-02-01, End date: 2018-10-31
