Project acronym CNIDARIAMICRORNA
Project Elucidation of the evolution of post-transcriptional regulation by characterizing the cnidarian microRNA pathway
Researcher (PI) Yehu Moran
Host Institution (HI) THE HEBREW UNIVERSITY OF JERUSALEM
Call Details Starting Grant (StG), LS2, ERC-2014-STG
Summary Over the past decade small RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been shown to carry pivotal roles in post-transcriptional regulation and genome protection and to play an important part in various physiological processes in animals. miRNAs can be found in a very wide range of animals yet their functions were studied almost exclusively in members of the Bilateria such as insects, nematodes and vertebrates. Hence studying their function in representatives of non-bilaterian phyla such as Cnidaria (sea anemones, corals, hydras and jellyfish) is crucial for understanding the evolution of miRNAs in animals and can provide important insights into their roles in the ancient ancestor of Cnidaria and Bilateria. The sea anemone Nematostella vectensis is an excellent model for such a study since it can be grown in large numbers throughout its life cycle in the lab and because well-established genetic manipulation techniques are available for this species. Our preliminary results indicate that miRNAs in Nematostella frequently have a nearly perfect match to their messenger RNA (mRNA) targets, resulting in cleavage of the target. This mode of action is common for plant miRNAs, but is very rare in Bilateria. This finding together with my recent discovery of a Nematostella homolog of HYL1, a protein involved in miRNA biogenesis in plants, raises the exciting possibility that the miRNA pathway existed in the common ancestor of plants and animals. Here I suggest to bring together an array of advanced biochemical and genetic methods such as gene knockdown, transgenesis, high throughput sequencing and immunoprecipitation in order to obtain - for the first time - a deep understanding of the biogenesis and mechanism of action of small RNAs in Cnidaria. This will provide a novel way to understand the evolution of this important molecular pathway and to evaluate its age and ancestral form.
Summary
Over the past decade small RNAs such as microRNAs (miRNAs) and small interfering RNAs (siRNAs) have been shown to carry pivotal roles in post-transcriptional regulation and genome protection and to play an important part in various physiological processes in animals. miRNAs can be found in a very wide range of animals yet their functions were studied almost exclusively in members of the Bilateria such as insects, nematodes and vertebrates. Hence studying their function in representatives of non-bilaterian phyla such as Cnidaria (sea anemones, corals, hydras and jellyfish) is crucial for understanding the evolution of miRNAs in animals and can provide important insights into their roles in the ancient ancestor of Cnidaria and Bilateria. The sea anemone Nematostella vectensis is an excellent model for such a study since it can be grown in large numbers throughout its life cycle in the lab and because well-established genetic manipulation techniques are available for this species. Our preliminary results indicate that miRNAs in Nematostella frequently have a nearly perfect match to their messenger RNA (mRNA) targets, resulting in cleavage of the target. This mode of action is common for plant miRNAs, but is very rare in Bilateria. This finding together with my recent discovery of a Nematostella homolog of HYL1, a protein involved in miRNA biogenesis in plants, raises the exciting possibility that the miRNA pathway existed in the common ancestor of plants and animals. Here I suggest to bring together an array of advanced biochemical and genetic methods such as gene knockdown, transgenesis, high throughput sequencing and immunoprecipitation in order to obtain - for the first time - a deep understanding of the biogenesis and mechanism of action of small RNAs in Cnidaria. This will provide a novel way to understand the evolution of this important molecular pathway and to evaluate its age and ancestral form.
Max ERC Funding
1 499 587 €
Duration
Start date: 2015-05-01, End date: 2020-04-30
Project acronym CohesinLooping
Project Cohesin-mediated chromosomal looping: From linear paths to 3D effects
Researcher (PI) Benjamin Rowland
Host Institution (HI) STICHTING HET NEDERLANDS KANKER INSTITUUT-ANTONI VAN LEEUWENHOEK ZIEKENHUIS
Call Details Consolidator Grant (CoG), LS2, ERC-2017-COG
Summary The 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.
Summary
The 3D organization of chromosomes within the nucleus is of great importance to control gene expression. The cohesin complex plays a key role in such higher-order chromosome organization by looping together regulatory elements in cis. How these often megabase-sized looped structures are formed is one of the main open questions in chromosome biology. Cohesin is a ring-shaped complex that can entrap DNA inside its lumen. However, cohesin’s default behaviour is that it only transiently entraps and then releases DNA. Our recent findings indicate that chromosomes are structured through the processive enlargement of chromatin loops, and that the duration with which cohesin embraces DNA determines the degree to which loops are enlarged. The goal of this proposal is two-fold. First, we plan to investigate the mechanism by which chromatin loops are formed, and secondly we wish to dissect how looped structures are maintained. We will use a multi-disciplinary approach that includes refined genetic screens in haploid human cells, chromosome conformation capture techniques, the tracing in vivo of cohesin on individual DNA molecules, and visualization of chromosome organization by super-resolution imaging. With unbiased genetic screens, we have identified chromatin regulators involved in the formation of chromosomal loops. We will investigate how they drive loop formation, and also whether cohesin’s own enzymatic activity plays a role in the enlargement of loops. We will study whether and how these factors control the movement of cohesin along individual DNA molecules, and whether chromatin loops pass through cohesin rings during their formation. Ultimately, we plan to couple cohesin’s linear trajectory along chromatin to the 3D consequences for chromosomal architecture. Together our experiments will provide vital insight into how cohesin structures chromosomes.
Max ERC Funding
1 998 375 €
Duration
Start date: 2018-04-01, End date: 2023-03-31
Project acronym COMMOTS
Project Communication Motifs: Principles of bacterial communication in non-genetically diversified populations
Researcher (PI) Ilka Bischofs-Pfeifer
Host Institution (HI) RUPRECHT-KARLS-UNIVERSITAET HEIDELBERG
Call Details Starting Grant (StG), LS2, ERC-2010-StG_20091118
Summary Cell-to-cell communication is a central aspect for understanding how cells form and organize multi-cellular communities involving progressive cell specialization. Multi-cellularity cell specialization cell communication those keywords are frequently used to distinguish metazoans from bacteria. Yet bacteria can form morphologically complex multi-cellular communities, they can non-genetically diversify and they can communicate. This implies that even prokaryotic networks must possess the properties to facilitate these complex functions. Thus basic network features ( motifs ) determining these functions can be discovered and characterized from studying simpler bacterial networks. We want to focus on communication motifs that are present in the gene-regulatory network of Bacillus subtilis. Our proposed methodology involves a combination of quantitative fluorescence microscopy techniques (QFTLM, FRET), developmental assays, signal transduction studies in controlled micro-environments and information theory to quantitatively characterize communication motifs..
Summary
Cell-to-cell communication is a central aspect for understanding how cells form and organize multi-cellular communities involving progressive cell specialization. Multi-cellularity cell specialization cell communication those keywords are frequently used to distinguish metazoans from bacteria. Yet bacteria can form morphologically complex multi-cellular communities, they can non-genetically diversify and they can communicate. This implies that even prokaryotic networks must possess the properties to facilitate these complex functions. Thus basic network features ( motifs ) determining these functions can be discovered and characterized from studying simpler bacterial networks. We want to focus on communication motifs that are present in the gene-regulatory network of Bacillus subtilis. Our proposed methodology involves a combination of quantitative fluorescence microscopy techniques (QFTLM, FRET), developmental assays, signal transduction studies in controlled micro-environments and information theory to quantitatively characterize communication motifs..
Max ERC Funding
1 496 840 €
Duration
Start date: 2011-09-01, End date: 2016-08-31
Project acronym COMPLEX_TRAITS
Project High-throughput dissection of the genetics underlying complex traits
Researcher (PI) Lars Steinmetz
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Advanced Grant (AdG), LS2, ERC-2011-ADG_20110310
Summary The vast majority of genetic diseases are complex traits, conditioned by multiple genetic and environmental factors. Yet our understanding of the genetics underlying such traits in humans remains extremely limited, due largely to the statistical complexity of inferring the effects of allelic variants in a genetically diverse population. Novel tools for the dissection of the genetic architecture of complex traits, therefore, can be most effectively developed in model organisms, where the contribution of individual alleles can be quantitatively determined in controlled genetic backgrounds. We have previously established the yeast Saccharomyces cerevisiae as a model for complex traits by unravelling complex genetic architectures that govern quantitative phenotypes in this organism. We achieved this by pioneering approaches that have revealed crucial information about the complexity of the underlying genetics. Here we propose to advance to the next level of complex trait dissection by developing systematic, genome-wide technologies that aim to identify all of the variants underlying a complex trait in a single step. In particular, we will investigate traits involved in mitochondrial function, which are both clinically relevant and highly conserved in yeast. Our combination of genomic technologies will allow us to: 1) systematically detect, with maximal sensitivity, the majority of genetic variants (coding and non-coding) that condition these traits; 2) quantify the contributions of these variants and their interactions; and 3) evaluate the strengths and limitations of current methods for dissecting complex traits. Taken together, our research will yield fundamental insights into the genetic complexity of multifactorial traits, providing valuable lessons and establishing novel genomic tools that will facilitate the investigation of complex diseases.
Summary
The vast majority of genetic diseases are complex traits, conditioned by multiple genetic and environmental factors. Yet our understanding of the genetics underlying such traits in humans remains extremely limited, due largely to the statistical complexity of inferring the effects of allelic variants in a genetically diverse population. Novel tools for the dissection of the genetic architecture of complex traits, therefore, can be most effectively developed in model organisms, where the contribution of individual alleles can be quantitatively determined in controlled genetic backgrounds. We have previously established the yeast Saccharomyces cerevisiae as a model for complex traits by unravelling complex genetic architectures that govern quantitative phenotypes in this organism. We achieved this by pioneering approaches that have revealed crucial information about the complexity of the underlying genetics. Here we propose to advance to the next level of complex trait dissection by developing systematic, genome-wide technologies that aim to identify all of the variants underlying a complex trait in a single step. In particular, we will investigate traits involved in mitochondrial function, which are both clinically relevant and highly conserved in yeast. Our combination of genomic technologies will allow us to: 1) systematically detect, with maximal sensitivity, the majority of genetic variants (coding and non-coding) that condition these traits; 2) quantify the contributions of these variants and their interactions; and 3) evaluate the strengths and limitations of current methods for dissecting complex traits. Taken together, our research will yield fundamental insights into the genetic complexity of multifactorial traits, providing valuable lessons and establishing novel genomic tools that will facilitate the investigation of complex diseases.
Max ERC Funding
2 499 821 €
Duration
Start date: 2012-11-01, End date: 2017-10-31
Project acronym ComplexAssembly
Project The birth of protein complexes
Researcher (PI) Martin BECK
Host Institution (HI) EUROPEAN MOLECULAR BIOLOGY LABORATORY
Call Details Consolidator Grant (CoG), LS2, ERC-2016-COG
Summary Protein complexes are central to many cellular functions but our knowledge of how cells assemble protein complexes remains very sparse. Biophysical and structural data of assembly intermediates are extremely rare. Particularly in higher eukaryotes, it has become clear that complex assembly by random collision of subunits cannot cope with the spatial and temporal complexity of the intricate architecture of many cellular machines. Here I propose to combine systems biology approaches with in situ structural biology methods to visualize protein complex assembly. I want to investigate experimentally in which order the interfaces of protein complexes are formed and to which extent structures of assembly intermediates resemble those observed in fully assembled complexes. I want develop methods to systematically screen for additional factors involved in assembly pathways. I furthermore want to test the hypothesis that mechanisms must exist in eukaryotes that coordinate local mRNA translation with the ordered formation of protein complex interfaces. I believe that in order to understand assembly pathways, these processes, that so far are often studied autonomously, need to be considered jointly and in a protein complex centric manner. The research proposed here will bridge across these different scientific disciplines. In the long term, a better mechanistic understanding of protein complex assembly and the structural characterization of critical intermediates will be of high relevance for scenarios under which a cell’s protein quality control system has to cope with stress, such as aging and neurodegenerative diseases. It might also facilitate the more efficient industrial production of therapeutically relevant proteins.
Summary
Protein complexes are central to many cellular functions but our knowledge of how cells assemble protein complexes remains very sparse. Biophysical and structural data of assembly intermediates are extremely rare. Particularly in higher eukaryotes, it has become clear that complex assembly by random collision of subunits cannot cope with the spatial and temporal complexity of the intricate architecture of many cellular machines. Here I propose to combine systems biology approaches with in situ structural biology methods to visualize protein complex assembly. I want to investigate experimentally in which order the interfaces of protein complexes are formed and to which extent structures of assembly intermediates resemble those observed in fully assembled complexes. I want develop methods to systematically screen for additional factors involved in assembly pathways. I furthermore want to test the hypothesis that mechanisms must exist in eukaryotes that coordinate local mRNA translation with the ordered formation of protein complex interfaces. I believe that in order to understand assembly pathways, these processes, that so far are often studied autonomously, need to be considered jointly and in a protein complex centric manner. The research proposed here will bridge across these different scientific disciplines. In the long term, a better mechanistic understanding of protein complex assembly and the structural characterization of critical intermediates will be of high relevance for scenarios under which a cell’s protein quality control system has to cope with stress, such as aging and neurodegenerative diseases. It might also facilitate the more efficient industrial production of therapeutically relevant proteins.
Max ERC Funding
1 957 717 €
Duration
Start date: 2018-02-01, End date: 2023-01-31
Project acronym CONSERVREGCIRCUITRY
Project Conservation and Divergence of Tissue-Specific Transcriptional Regulation
Researcher (PI) Duncan Odom
Host Institution (HI) THE CHANCELLOR MASTERS AND SCHOLARSOF THE UNIVERSITY OF CAMBRIDGE
Call Details Starting Grant (StG), LS2, ERC-2007-StG
Summary Vertebrates contain hundreds of different cell types which maintain phenotypic identity by a combination of epigenetic programming and genomic regulation. Systems biology approaches are now used in a number of laboratories to determine how transcription factors and chromatin marks pattern the human genome. Despite high conservation of the cellular and molecular function of many mammalian transcription factors, our recent experiments in matched mouse and human tissues indicates that most transcription factor binding events to DNA are very poorly conserved. A hypothesis that could account for this apparent divergence is that the larger regional pattern of transcription factor binding may be conserved. To test this, (1) we are characterizing the global transcriptional profile, chromatin state, and complete genomic occupancy of a set of tissue-specific transcription factors in hepatocytes of strategically chosen mammals; (2) to further identify the precise mechanistic contribution of cis and trans effects, we are comparing transcription factor binding at homologous regions of human and mouse DNA in a mouse line that carries human chromosome 21. Together, these projects will provide insight into the general principles of how transcriptional networks are evolutionarily conserved to regulate cell fate specification and function using a clinically important cell type as a model.
Summary
Vertebrates contain hundreds of different cell types which maintain phenotypic identity by a combination of epigenetic programming and genomic regulation. Systems biology approaches are now used in a number of laboratories to determine how transcription factors and chromatin marks pattern the human genome. Despite high conservation of the cellular and molecular function of many mammalian transcription factors, our recent experiments in matched mouse and human tissues indicates that most transcription factor binding events to DNA are very poorly conserved. A hypothesis that could account for this apparent divergence is that the larger regional pattern of transcription factor binding may be conserved. To test this, (1) we are characterizing the global transcriptional profile, chromatin state, and complete genomic occupancy of a set of tissue-specific transcription factors in hepatocytes of strategically chosen mammals; (2) to further identify the precise mechanistic contribution of cis and trans effects, we are comparing transcription factor binding at homologous regions of human and mouse DNA in a mouse line that carries human chromosome 21. Together, these projects will provide insight into the general principles of how transcriptional networks are evolutionarily conserved to regulate cell fate specification and function using a clinically important cell type as a model.
Max ERC Funding
960 000 €
Duration
Start date: 2008-10-01, End date: 2013-09-30
Project acronym Coupled gene circuit
Project Dynamics, noise, and coupling in gene circuit modules
Researcher (PI) James Charles Wallace Locke
Host Institution (HI) THE CHANCELLOR MASTERS AND SCHOLARSOF THE UNIVERSITY OF CAMBRIDGE
Call Details Starting Grant (StG), LS2, ERC-2013-StG
Summary Cells must integrate output from multiple genetic circuits in order to correctly control cellular processes. Despite much work characterizing regulation in these circuits, how circuits interact to control global cellular programs remains unclear. This is particularly true given that recent research at the single cell level has revealed that genetic circuits often generate variable or stochastic regulation dynamics. In this proposal we will use a multi-disciplinary approach, combining modelling and time-lapse microscopy, to investigate how cells can robustly integrate signals from multiple dynamic genetic circuits. In particular we will answer the following questions: 1) What types of dynamic signal encoding strategies are available for the cell? 2) What are the benefits of dynamic gene activation, whether stochastic or oscillatory, to the cell? 3) How do cells couple and integrate output from diverse gene modules despite the noise and variability observed in gene circuit dynamics?
We will study these questions using 2 key model systems. In Aim 1, we will examine stochastic pulse regulation dynamics and coupling between alternative sigma factors in B. subtilis. Our preliminary data has revealed that multiple B. subtilis sigma factors stochastically pulse under stress. We will look for evidence of any coupling or interactions between these stochastic pulse circuits. This system will serve as a model for how a cell uses stochastic pulsing to control diverse cellular processes. In Aim 2, we will examine coupling between a deterministic oscillator, the circadian clock, and multiple other key pathways in Cyanobacteria. We will examine how the cell can dynamically couple multiple cellular processes using an oscillating signal. This work will provide an excellent base for Aim 3, in which we will use synthetic biology approaches to develop ‘bottom up’ tests of generation of novel dynamic coupling strategies.
Summary
Cells must integrate output from multiple genetic circuits in order to correctly control cellular processes. Despite much work characterizing regulation in these circuits, how circuits interact to control global cellular programs remains unclear. This is particularly true given that recent research at the single cell level has revealed that genetic circuits often generate variable or stochastic regulation dynamics. In this proposal we will use a multi-disciplinary approach, combining modelling and time-lapse microscopy, to investigate how cells can robustly integrate signals from multiple dynamic genetic circuits. In particular we will answer the following questions: 1) What types of dynamic signal encoding strategies are available for the cell? 2) What are the benefits of dynamic gene activation, whether stochastic or oscillatory, to the cell? 3) How do cells couple and integrate output from diverse gene modules despite the noise and variability observed in gene circuit dynamics?
We will study these questions using 2 key model systems. In Aim 1, we will examine stochastic pulse regulation dynamics and coupling between alternative sigma factors in B. subtilis. Our preliminary data has revealed that multiple B. subtilis sigma factors stochastically pulse under stress. We will look for evidence of any coupling or interactions between these stochastic pulse circuits. This system will serve as a model for how a cell uses stochastic pulsing to control diverse cellular processes. In Aim 2, we will examine coupling between a deterministic oscillator, the circadian clock, and multiple other key pathways in Cyanobacteria. We will examine how the cell can dynamically couple multiple cellular processes using an oscillating signal. This work will provide an excellent base for Aim 3, in which we will use synthetic biology approaches to develop ‘bottom up’ tests of generation of novel dynamic coupling strategies.
Max ERC Funding
1 499 571 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
Project acronym CrackEpitranscriptom
Project Cracking the epitranscriptome
Researcher (PI) Schraga SCHWARTZ
Host Institution (HI) WEIZMANN INSTITUTE OF SCIENCE
Call Details Starting Grant (StG), LS2, ERC-2016-STG
Summary Over 100 types of distinct modifications are catalyzed on RNA molecules post-transcriptionally. In an analogous manner to well-studied chemical modifications on proteins or DNA, modifications on RNA - and particularly on mRNA - harbor the exciting potential of regulating the complex and interlinked life cycle of these molecules. The most abundant modification in mammalian and yeast mRNA is N6-methyladenosine (m6A). We have pioneered approaches for mapping m6A in a transcriptome wide manner, and we and others have identified factors involved in encoding and decoding m6A. While experimental disruption of these factors is associated with severe phenotypes, the role of m6A remains enigmatic. No single methylated site has been shown to causally underlie any physiological or molecular function. This proposal aims to establish a framework for systematically deciphering the molecular function of a modification and its underlying mechanisms and to uncover the physiological role of the modification in regulation of a cellular response. We will apply this framework to m6A in the context of meiosis in budding yeast, as m6A dynamically accumulates on meiotic mRNAs and as the methyltransferase catalyzing m6A is essential for meiosis. We will (1) aim to elucidate the physiological targets of methylation governing entry into meiosis (2) seek to elucidate the function of m6A at the molecular level, and understand its impact on the various steps of the mRNA life cycle, (3) seek to understand the mechanisms underlying its effects. These aims will provide a comprehensive framework for understanding how the epitranscriptome, an emerging post-transcriptional layer of regulation, fine-tunes gene regulation and impacts cellular decision making in a dynamic response, and will set the stage towards dissecting the roles of m6A and of an expanding set of mRNA modifications in more complex and disease related systems.
Summary
Over 100 types of distinct modifications are catalyzed on RNA molecules post-transcriptionally. In an analogous manner to well-studied chemical modifications on proteins or DNA, modifications on RNA - and particularly on mRNA - harbor the exciting potential of regulating the complex and interlinked life cycle of these molecules. The most abundant modification in mammalian and yeast mRNA is N6-methyladenosine (m6A). We have pioneered approaches for mapping m6A in a transcriptome wide manner, and we and others have identified factors involved in encoding and decoding m6A. While experimental disruption of these factors is associated with severe phenotypes, the role of m6A remains enigmatic. No single methylated site has been shown to causally underlie any physiological or molecular function. This proposal aims to establish a framework for systematically deciphering the molecular function of a modification and its underlying mechanisms and to uncover the physiological role of the modification in regulation of a cellular response. We will apply this framework to m6A in the context of meiosis in budding yeast, as m6A dynamically accumulates on meiotic mRNAs and as the methyltransferase catalyzing m6A is essential for meiosis. We will (1) aim to elucidate the physiological targets of methylation governing entry into meiosis (2) seek to elucidate the function of m6A at the molecular level, and understand its impact on the various steps of the mRNA life cycle, (3) seek to understand the mechanisms underlying its effects. These aims will provide a comprehensive framework for understanding how the epitranscriptome, an emerging post-transcriptional layer of regulation, fine-tunes gene regulation and impacts cellular decision making in a dynamic response, and will set the stage towards dissecting the roles of m6A and of an expanding set of mRNA modifications in more complex and disease related systems.
Max ERC Funding
1 402 666 €
Duration
Start date: 2016-11-01, End date: 2021-10-31
Project acronym CRISPRsition
Project Developing CRISPR adaptation platforms for basic and applied research
Researcher (PI) Ehud Itzhak Qimron
Host Institution (HI) TEL AVIV UNIVERSITY
Call Details Consolidator Grant (CoG), LS2, ERC-2018-COG
Summary The CRISPR-Cas system has been extensively studied for its ability to cleave DNA. In contrast, studies of the ability of the system to acquire and integrate new DNA from invaders as a form of prokaryotic adaptive immunity, have lagged behind. This delay reflects the extreme enthusiasm surrounding the potential of using the system’s cleavage capabilities as a genome editing tool. However, the enormous potential of the adaptation process can and should arouse a similar degree of enthusiasm. My lab has pioneered studies on the CRISPR adaptation process by establishing new methodologies, and applying them to demonstrate the essential role of the proteins and DNA elements, as well as the molecular mechanisms, operating in this process. In this project, I will establish novel platforms for studying adaptation and develop them into biotechnological applications and research tools. These tools will allow me to identify the first natural and synthetic inhibitors of the adaptation process. This, in turn, will provide genetic tools to control adaptation, as well as advance the understanding of the arms race between bacteria and their invaders. I will also harness the adaptation process as a platform for diversifying genetic elements for phage display, and for extending phage recognition of a wide range of hosts. Lastly, I will provide the first evidence for an association between the CRISPR adaptation system and gene repression. This linkage will form the basis of a molecular scanner and recorder platform that I will develop and that can be used to identify crucial genetic elements in phage genomes as well as novel regulatory circuits in the bacterial genome. Together, my findings will represent a considerable leap in the understanding of CRISPR adaptation with respect to the process, potential applications, and the intriguing evolutionary significance.
Summary
The CRISPR-Cas system has been extensively studied for its ability to cleave DNA. In contrast, studies of the ability of the system to acquire and integrate new DNA from invaders as a form of prokaryotic adaptive immunity, have lagged behind. This delay reflects the extreme enthusiasm surrounding the potential of using the system’s cleavage capabilities as a genome editing tool. However, the enormous potential of the adaptation process can and should arouse a similar degree of enthusiasm. My lab has pioneered studies on the CRISPR adaptation process by establishing new methodologies, and applying them to demonstrate the essential role of the proteins and DNA elements, as well as the molecular mechanisms, operating in this process. In this project, I will establish novel platforms for studying adaptation and develop them into biotechnological applications and research tools. These tools will allow me to identify the first natural and synthetic inhibitors of the adaptation process. This, in turn, will provide genetic tools to control adaptation, as well as advance the understanding of the arms race between bacteria and their invaders. I will also harness the adaptation process as a platform for diversifying genetic elements for phage display, and for extending phage recognition of a wide range of hosts. Lastly, I will provide the first evidence for an association between the CRISPR adaptation system and gene repression. This linkage will form the basis of a molecular scanner and recorder platform that I will develop and that can be used to identify crucial genetic elements in phage genomes as well as novel regulatory circuits in the bacterial genome. Together, my findings will represent a considerable leap in the understanding of CRISPR adaptation with respect to the process, potential applications, and the intriguing evolutionary significance.
Max ERC Funding
2 000 000 €
Duration
Start date: 2019-12-01, End date: 2024-11-30
Project acronym CTCFStableGenome
Project CTCF control of genome stability in ageing
Researcher (PI) Duncan ODOM
Host Institution (HI) DEUTSCHES KREBSFORSCHUNGSZENTRUM HEIDELBERG
Call Details Advanced Grant (AdG), LS2, ERC-2017-ADG
Summary . Genome stability is one of the most important features in maintaining tissue homeostasis throughout the human lifespan. The research presented here will dissect how the insulator protein CCCTC-binding factor (CTCF), a ubiquitous 11 zinc finger transcription factor, controls the stability of the mammalian genome during ageing.
. In Aim 1, we will elucidate how CTCF and tissue-specific master regulators maintain the functional stability of the genome during healthy ageing by developing a novel protocol to map simultaneously transcription and open chromatin in isolated hepatocyte nuclei. Using this protocol, we will explore how CTCF binding stabilizes cellular homeostasis during ageing by knocking down CTCF in vivo, both in isolation and simultaneously with knock down of liver-specific master regulators.
. In Aim 2, we will reveal the molecular mechanisms underlying CTCF binding sites as susceptibility loci for somatic mutations. We will profile the mutations in open chromatin of single nuclei immediately following acute exposure to a chemical mutagen; comparing how the pattern of mutations in CTCF bound regions changes across an allelic series of CTCF knockdown mice will reveal how CTCF binding shapes the stability of the genome towards mutations.
. These integrated strategies develop and deploy powerful, cutting-edge experimental approaches to reveal novel aspects of how CTCF binding stabilises the mammalian genome during healthy ageing as well as during mutagenesis.
Summary
. Genome stability is one of the most important features in maintaining tissue homeostasis throughout the human lifespan. The research presented here will dissect how the insulator protein CCCTC-binding factor (CTCF), a ubiquitous 11 zinc finger transcription factor, controls the stability of the mammalian genome during ageing.
. In Aim 1, we will elucidate how CTCF and tissue-specific master regulators maintain the functional stability of the genome during healthy ageing by developing a novel protocol to map simultaneously transcription and open chromatin in isolated hepatocyte nuclei. Using this protocol, we will explore how CTCF binding stabilizes cellular homeostasis during ageing by knocking down CTCF in vivo, both in isolation and simultaneously with knock down of liver-specific master regulators.
. In Aim 2, we will reveal the molecular mechanisms underlying CTCF binding sites as susceptibility loci for somatic mutations. We will profile the mutations in open chromatin of single nuclei immediately following acute exposure to a chemical mutagen; comparing how the pattern of mutations in CTCF bound regions changes across an allelic series of CTCF knockdown mice will reveal how CTCF binding shapes the stability of the genome towards mutations.
. These integrated strategies develop and deploy powerful, cutting-edge experimental approaches to reveal novel aspects of how CTCF binding stabilises the mammalian genome during healthy ageing as well as during mutagenesis.
Max ERC Funding
2 488 251 €
Duration
Start date: 2019-09-01, End date: 2024-08-31
