Project acronym DECOR
Project Dynamic assembly and exchange of RNA polymerase II CTD factors
Researcher (PI) Richard Stefl
Host Institution (HI) Masarykova univerzita
Call Details Consolidator Grant (CoG), LS1, ERC-2014-CoG
Summary The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) largest subunit coordinates co-transcriptional processing and it is decorated by many processing factors throughout the transcription cycle. The composition of this supramolecular assembly is diverse and highly dynamic. Many of the factors associate with RNAPII weakly and transiently, and the association is dictated by different post-translational modification patterns and conformational changes of the CTD. To determine how these accessory factors assemble and exchange on the CTD of RNAPII has remained a major challenge. Here, we aim to unravel the structural and mechanistic bases for the dynamic assembly of RNAPII CTD with its processing factors.
Using NMR, we will determine high-resolution structures of several protein factors bound to the CTD carrying specific modifications. This will enable to decode how CTD modification patterns stimulate or prevent binding of a given processing factor. We will also establish the structural and mechanistic bases of proline isomerisation in the CTD that control the timing of isomer-specific protein-protein interactions. Next, we will combine NMR and SAXS approaches to unravel how the overall CTD structure is remodelled by binding of multiple copies of processing factors and how these factors cross-talk with each other. Finally, we will elucidate a mechanistic basis for the exchange of processing factors on the CTD.
Our study will answer the long-standing questions of how the overall CTD structure is modulated on binding to processing factors, and whether these factors cross-talk and compete with each other. The level of detail that we aim to achieve is currently not available for any transient molecular assemblies of such complexity. In this respect, the project will also provide knowledge and methodology for further studies of large and highly flexible molecular assemblies that still remain poorly understood.
Summary
The C-terminal domain (CTD) of the RNA polymerase II (RNAPII) largest subunit coordinates co-transcriptional processing and it is decorated by many processing factors throughout the transcription cycle. The composition of this supramolecular assembly is diverse and highly dynamic. Many of the factors associate with RNAPII weakly and transiently, and the association is dictated by different post-translational modification patterns and conformational changes of the CTD. To determine how these accessory factors assemble and exchange on the CTD of RNAPII has remained a major challenge. Here, we aim to unravel the structural and mechanistic bases for the dynamic assembly of RNAPII CTD with its processing factors.
Using NMR, we will determine high-resolution structures of several protein factors bound to the CTD carrying specific modifications. This will enable to decode how CTD modification patterns stimulate or prevent binding of a given processing factor. We will also establish the structural and mechanistic bases of proline isomerisation in the CTD that control the timing of isomer-specific protein-protein interactions. Next, we will combine NMR and SAXS approaches to unravel how the overall CTD structure is remodelled by binding of multiple copies of processing factors and how these factors cross-talk with each other. Finally, we will elucidate a mechanistic basis for the exchange of processing factors on the CTD.
Our study will answer the long-standing questions of how the overall CTD structure is modulated on binding to processing factors, and whether these factors cross-talk and compete with each other. The level of detail that we aim to achieve is currently not available for any transient molecular assemblies of such complexity. In this respect, the project will also provide knowledge and methodology for further studies of large and highly flexible molecular assemblies that still remain poorly understood.
Max ERC Funding
1 844 604 €
Duration
Start date: 2015-08-01, End date: 2020-07-31
Project acronym HCPO
Project Hormonal cross-talk in plant organogenesis
Researcher (PI) Eva Benkova
Host Institution (HI) INSTITUTE OF SCIENCE AND TECHNOLOGYAUSTRIA
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Growth and development of plants are regulated by signalling substances such as hormones. In plants, interactions between hormonal pathways represent crucial factors that govern their action. As the molecular basis for such hormonal cross-talk remains largely unknown, we will investigate the underlying mechanisms with a special focus on regulation of postembryonic organogenesis. We consider lateral root formation in Arabidopsis as an ideally suited model system, because it encompasses fundamental aspects of plant growth and development, such as dedifferentiation, re-entry into the cell cycle, coordinated cell divisions and differentiation. Furthermore, in lateral root formation, these processes are controlled by multiple hormonal pathways. In our proposal, we will focus on four main research directions. 1. Convergence of hormonal pathways on transport-dependent auxin distribution upstream of lateral root formation. Here, we want to identify key points in which auxin and other signalling pathways converge during lateral root formation and the molecular components involved in the process. 2. Role of auxin-cytokinin interaction in lateral root formation. Molecular events involved in auxin-cytokinin regulated lateral root formation will be studied by transcriptome analysis. 3. Identification of components of hormonal cross-talk by genetic approaches. Using lateral root formation as a model, we will perform mutant screens that will specifically target interactions between selected hormonal pathways. The spectrum of identified molecular components will be further expanded by a chemical genomics approach. 4. Formulation of general models for hormonal regulation of organogenesis. The acquired knowledge on molecular networks and their mutual interactions will be used to mathematically model lateral root development and to extrapolate them also on other developmental situations.
Summary
Growth and development of plants are regulated by signalling substances such as hormones. In plants, interactions between hormonal pathways represent crucial factors that govern their action. As the molecular basis for such hormonal cross-talk remains largely unknown, we will investigate the underlying mechanisms with a special focus on regulation of postembryonic organogenesis. We consider lateral root formation in Arabidopsis as an ideally suited model system, because it encompasses fundamental aspects of plant growth and development, such as dedifferentiation, re-entry into the cell cycle, coordinated cell divisions and differentiation. Furthermore, in lateral root formation, these processes are controlled by multiple hormonal pathways. In our proposal, we will focus on four main research directions. 1. Convergence of hormonal pathways on transport-dependent auxin distribution upstream of lateral root formation. Here, we want to identify key points in which auxin and other signalling pathways converge during lateral root formation and the molecular components involved in the process. 2. Role of auxin-cytokinin interaction in lateral root formation. Molecular events involved in auxin-cytokinin regulated lateral root formation will be studied by transcriptome analysis. 3. Identification of components of hormonal cross-talk by genetic approaches. Using lateral root formation as a model, we will perform mutant screens that will specifically target interactions between selected hormonal pathways. The spectrum of identified molecular components will be further expanded by a chemical genomics approach. 4. Formulation of general models for hormonal regulation of organogenesis. The acquired knowledge on molecular networks and their mutual interactions will be used to mathematically model lateral root development and to extrapolate them also on other developmental situations.
Max ERC Funding
1 300 000 €
Duration
Start date: 2008-07-01, End date: 2014-03-31
Project acronym KINETOCORE
Project Molecular Dissection of the Kinetochore – Microtubule Interface
Researcher (PI) Stefan Westermann
Host Institution (HI) FORSCHUNGSINSTITUT FUR MOLEKULARE PATHOLOGIE GESELLSCHAFT MBH
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary The ability of spindle microtubules of to interact dynamically with centromeric chromatin is a critical feature of chromosome segregation and ensures the faithful distribution of genetic material. Errors in this process lead to abnormal chromosome numbers and are a hallmark of cancer and birth defects. The kinetochore is the key cell division organelle that enables high fidelity transmission of genetic information by coupling chromosomes to the plus-ends of spindle microtubules during mitosis and meiosis. Despite its cytological description more than a century ago, little information is available on kinetochore function at a molecular level. Here, I propose to dissect the molecular mechanisms of kinetochore function using the budding yeast Saccharomyces cerevisiae as a model system. My previous work has demonstrated that fundamental aspects of kinetochore organization are conserved throughout evolution. I will use a combination of biochemistry, electron microscopy, in-vitro assays with static and dynamic microtubule substrates as well as yeast cell biology to address fundamental questions of kinetochore function. Specifically, my experiments aim to elucidate 1) the mechanism of phospho-regulation at the kinetochore-microtubule interface 2) the roles of plus-end tracking proteins in chromosome segregation 3) the roles of kinetochore subcomplexes in connecting microtubules and centromeres. Successful completion of the project will help to move the kinetochore field towards a detailed understanding of the molecular mechanisms of chromosome segregation and can open up new perspectives for analyzing the functions of this complex macromolecular machine.
Summary
The ability of spindle microtubules of to interact dynamically with centromeric chromatin is a critical feature of chromosome segregation and ensures the faithful distribution of genetic material. Errors in this process lead to abnormal chromosome numbers and are a hallmark of cancer and birth defects. The kinetochore is the key cell division organelle that enables high fidelity transmission of genetic information by coupling chromosomes to the plus-ends of spindle microtubules during mitosis and meiosis. Despite its cytological description more than a century ago, little information is available on kinetochore function at a molecular level. Here, I propose to dissect the molecular mechanisms of kinetochore function using the budding yeast Saccharomyces cerevisiae as a model system. My previous work has demonstrated that fundamental aspects of kinetochore organization are conserved throughout evolution. I will use a combination of biochemistry, electron microscopy, in-vitro assays with static and dynamic microtubule substrates as well as yeast cell biology to address fundamental questions of kinetochore function. Specifically, my experiments aim to elucidate 1) the mechanism of phospho-regulation at the kinetochore-microtubule interface 2) the roles of plus-end tracking proteins in chromosome segregation 3) the roles of kinetochore subcomplexes in connecting microtubules and centromeres. Successful completion of the project will help to move the kinetochore field towards a detailed understanding of the molecular mechanisms of chromosome segregation and can open up new perspectives for analyzing the functions of this complex macromolecular machine.
Max ERC Funding
900 000 €
Duration
Start date: 2008-10-01, End date: 2013-09-30
Project acronym NPC GENEXPRESS
Project The nuclear pore connection: adaptor complexes bridging genome regulation and nuclear transport
Researcher (PI) Alwin Köhler
Host Institution (HI) MEDIZINISCHE UNIVERSITAET WIEN
Call Details Starting Grant (StG), LS1, ERC-2011-StG_20101109
Summary Nuclear pore complexes (NPCs) form macromolecular assemblies in the nuclear envelope and mediate bidirectional cargo movement between the nucleus and cytoplasm. Recent evidence suggests that NPCs are not merely transport channels but act as gene regulators. Studies in yeast demonstrate that inducible genes can reposition from the nuclear interior to the nuclear periphery upon activation. At the periphery activated genes engage in physical contacts with nuclear pores. Targeting and tethering of genes to nuclear pores involves multifunctional adaptor complexes, which are thought to couple chromatin modification, transcription and mRNA export. Knowledge of the structure, dynamics and evolution of these adaptor complexes is key to understanding how NPCs control nuclear gene positioning and gene expression. I propose to systematically dissect the architecture and function of NPC-associated adaptor complexes. Our studies will be a unique combination of biochemical and structural approaches in three different model organisms. I anticipate, that this line of research will create a powerful basis to address a number of key questions in the field.
Summary
Nuclear pore complexes (NPCs) form macromolecular assemblies in the nuclear envelope and mediate bidirectional cargo movement between the nucleus and cytoplasm. Recent evidence suggests that NPCs are not merely transport channels but act as gene regulators. Studies in yeast demonstrate that inducible genes can reposition from the nuclear interior to the nuclear periphery upon activation. At the periphery activated genes engage in physical contacts with nuclear pores. Targeting and tethering of genes to nuclear pores involves multifunctional adaptor complexes, which are thought to couple chromatin modification, transcription and mRNA export. Knowledge of the structure, dynamics and evolution of these adaptor complexes is key to understanding how NPCs control nuclear gene positioning and gene expression. I propose to systematically dissect the architecture and function of NPC-associated adaptor complexes. Our studies will be a unique combination of biochemical and structural approaches in three different model organisms. I anticipate, that this line of research will create a powerful basis to address a number of key questions in the field.
Max ERC Funding
1 481 556 €
Duration
Start date: 2011-11-01, End date: 2016-10-31
Project acronym TETRAHYMENA
Project RNA directed DNA elimination in Tetrahymena
Researcher (PI) Kazufumi Mochizuki
Host Institution (HI) INSTITUT FUER MOLEKULARE BIOTECHNOLOGIE GMBH
Call Details Starting Grant (StG), LS1, ERC-2007-StG
Summary Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction (DNA elimination). It is believed that the DNA to be eliminated is identified by their homology with ~28 nt small RNAs (scnRNAs) which are made by a RNAi-related mechanism. Detailed mechanisms as well as exact biological functions of the RNA-directed DNA elimination remain obscure. The goal of this specific project is to understand: (i) How a RNAi-related mechanism directs DNA elimination. (ii) How the mating types are determined as a consequence of DNA elimination. We recently revealed that scnRNAs are processed by Dicer protein Dcl1p and form complex with Argonaute protein Twi1p. The RNA helicase Ema1p facilitates interaction of scnRNA and chromatin and this interaction induces H3K9 and K27 methylations that are catalyzed by the histone methyltransferase Ezl1p. To understand how these proteins are connected to each other, their detailed functions and novel proteins interacting with them will be analyzed. One of the possible biological functions of the DNA elimination is mating type determination. Tetrahymena possesses seven mating types and the different mating types are thought to be determined by alternative DNA elimination of a single locus. But how mating types are determined is totally unknown. We aim to identify mating type determinants using microarray screening, proteomics and mating type transformation with a cDNA library. Then, we will analyze how expression of those molecules is controlled by DNA elimination. In diverse eukaryotes, RNA silencing mechanisms mediate heterochromatin formation and regulate many chromatin functions. Through our study, we could gain deeper insights not only into DNA elimination in a curious microbe, but also into how chromatin functions are epigenetically controlled by small RNAs in most of the eukaryotes. We are confident that this work will provide a big impact on RNA silencing and chromatin studies.
Summary
Tetrahymena eliminates micronuclear-limited sequences from the developing macronucleus during sexual reproduction (DNA elimination). It is believed that the DNA to be eliminated is identified by their homology with ~28 nt small RNAs (scnRNAs) which are made by a RNAi-related mechanism. Detailed mechanisms as well as exact biological functions of the RNA-directed DNA elimination remain obscure. The goal of this specific project is to understand: (i) How a RNAi-related mechanism directs DNA elimination. (ii) How the mating types are determined as a consequence of DNA elimination. We recently revealed that scnRNAs are processed by Dicer protein Dcl1p and form complex with Argonaute protein Twi1p. The RNA helicase Ema1p facilitates interaction of scnRNA and chromatin and this interaction induces H3K9 and K27 methylations that are catalyzed by the histone methyltransferase Ezl1p. To understand how these proteins are connected to each other, their detailed functions and novel proteins interacting with them will be analyzed. One of the possible biological functions of the DNA elimination is mating type determination. Tetrahymena possesses seven mating types and the different mating types are thought to be determined by alternative DNA elimination of a single locus. But how mating types are determined is totally unknown. We aim to identify mating type determinants using microarray screening, proteomics and mating type transformation with a cDNA library. Then, we will analyze how expression of those molecules is controlled by DNA elimination. In diverse eukaryotes, RNA silencing mechanisms mediate heterochromatin formation and regulate many chromatin functions. Through our study, we could gain deeper insights not only into DNA elimination in a curious microbe, but also into how chromatin functions are epigenetically controlled by small RNAs in most of the eukaryotes. We are confident that this work will provide a big impact on RNA silencing and chromatin studies.
Max ERC Funding
900 000 €
Duration
Start date: 2008-09-01, End date: 2013-12-31
