Project acronym XXDNAM
Project How does the X chromosome regulate DNA methylation in pluripotent stem cells?
Researcher (PI) Steen Kian Thye Ooi
Host Institution (HI) UNIVERSITY COLLEGE LONDON
Call Details Starting Grant (StG), LS2, ERC-2011-StG_20101109
Summary Epigenetic processes regulate gene transcription states during cellular differentiation, playing key roles in the maintenance of pluripotency and differentiation. Epigenetic alterations are common in diseases such as in cancer and cognitive disorders. Understanding the mechanisms by which epigenetic states are inherited and propagated is of fundamental importance, and will help in the development of biomarkers for screening as well identification of targets for disease treatment.
DNA methylation remains the best-characterized epigenetic process. XX pluripotent stem cells (Embryonic Stem (ES) and induced Pluripotent Stem (iPS) cells) display genome-wide hypomethylation relative to XY stem cells but the mechanisms are unknown. This proposal will elucidate the pathways responsible. Irradiation Microcell-Mediated Chromosome Transfer (XMMCT) will be used to identify the critical region(s) of the X chromosome involved. In parallel and as an alternative approach, candidate X-linked genes will be over-expressed in XY ES cells to identify the factors responsible for global hypomethylation. Further insight will be provided using protein interaction screens using epitope-tagged versions of all active Dnmts as well as the known regulators URHF1 and Dnmt3L in XX and XY ES cells. The role of XX-induced hypomethylation in cellular reprogramming will be investigated by using different cell types from Oct4-GFP transgenic mice to examine whether iPS efficiency is affected by cells with a greater propensity to lose DNA methylation. Together these aims will elucidate the signals necessary to maintain global genomic DNA methylation. Aberrant loss is an important hallmark and contributor of disease that could be used for disease diagnosis and treatment. It could also be exploited to help improve the efficiency of cellular reprogramming for regenerative medicine.
Summary
Epigenetic processes regulate gene transcription states during cellular differentiation, playing key roles in the maintenance of pluripotency and differentiation. Epigenetic alterations are common in diseases such as in cancer and cognitive disorders. Understanding the mechanisms by which epigenetic states are inherited and propagated is of fundamental importance, and will help in the development of biomarkers for screening as well identification of targets for disease treatment.
DNA methylation remains the best-characterized epigenetic process. XX pluripotent stem cells (Embryonic Stem (ES) and induced Pluripotent Stem (iPS) cells) display genome-wide hypomethylation relative to XY stem cells but the mechanisms are unknown. This proposal will elucidate the pathways responsible. Irradiation Microcell-Mediated Chromosome Transfer (XMMCT) will be used to identify the critical region(s) of the X chromosome involved. In parallel and as an alternative approach, candidate X-linked genes will be over-expressed in XY ES cells to identify the factors responsible for global hypomethylation. Further insight will be provided using protein interaction screens using epitope-tagged versions of all active Dnmts as well as the known regulators URHF1 and Dnmt3L in XX and XY ES cells. The role of XX-induced hypomethylation in cellular reprogramming will be investigated by using different cell types from Oct4-GFP transgenic mice to examine whether iPS efficiency is affected by cells with a greater propensity to lose DNA methylation. Together these aims will elucidate the signals necessary to maintain global genomic DNA methylation. Aberrant loss is an important hallmark and contributor of disease that could be used for disease diagnosis and treatment. It could also be exploited to help improve the efficiency of cellular reprogramming for regenerative medicine.
Max ERC Funding
1 497 710 €
Duration
Start date: 2011-10-01, End date: 2017-03-31
Project acronym YEAST-TRANS
Project Deciphering the transport mechanisms of small xenobiotic molecules in synthetic yeast cell factories
Researcher (PI) Irina BORODINA
Host Institution (HI) DANMARKS TEKNISKE UNIVERSITET
Call Details Starting Grant (StG), LS9, ERC-2017-STG
Summary Industrial biotechnology employs synthetic cell factories to create bulk and fine chemicals and fuels from renewable resources, laying the basis for the future bio-based economy. The major part of the wanted bio-based chemicals are not native to the host cell, such as yeast, i.e. they are xenobiotic. Some xenobiotic compounds are readily secreted by synthetic cells, some are poorly secreted and some are not secreted at all, but how does this transport occur? Or why does it not occur? These fundamental questions remain to be answered and this will have great implications on industrial biotechnology, because improved secretion would bring down the production costs and enable the emergence of novel bio-based products.
YEAST-TRANS will fill in this knowledge gap by carrying out the first systematic genome-scale transporter study to uncover the transport mechanisms of small xenobiotic molecules by synthetic yeast cells and to apply this knowledge for engineering more efficient cell factories for bio-based production of fuels and chemicals.
Summary
Industrial biotechnology employs synthetic cell factories to create bulk and fine chemicals and fuels from renewable resources, laying the basis for the future bio-based economy. The major part of the wanted bio-based chemicals are not native to the host cell, such as yeast, i.e. they are xenobiotic. Some xenobiotic compounds are readily secreted by synthetic cells, some are poorly secreted and some are not secreted at all, but how does this transport occur? Or why does it not occur? These fundamental questions remain to be answered and this will have great implications on industrial biotechnology, because improved secretion would bring down the production costs and enable the emergence of novel bio-based products.
YEAST-TRANS will fill in this knowledge gap by carrying out the first systematic genome-scale transporter study to uncover the transport mechanisms of small xenobiotic molecules by synthetic yeast cells and to apply this knowledge for engineering more efficient cell factories for bio-based production of fuels and chemicals.
Max ERC Funding
1 423 358 €
Duration
Start date: 2017-12-01, End date: 2022-11-30
Project acronym ZAUBERKUGEL
Project Fulfilling Paul Ehrlich’s Dream: therapeutics with activity on demand
Researcher (PI) Dario Antonio Ansano Neri
Host Institution (HI) EIDGENOESSISCHE TECHNISCHE HOCHSCHULE ZUERICH
Call Details Advanced Grant (AdG), LS7, ERC-2014-ADG
Summary "Paul Ehrlich was the first scientist to postulate that if a compound could be made that selectively targeted disease-causing cells, then this agent could be used for the delivery of a toxin, which would enable a pharmacotherapy of unprecedented potency and selectivity. With this procedure, a ""magic bullet"" (Zauberkugel, his term for an ideal therapeutic agent) would be created, that killed diseased cells while sparing normal tissues.
The concept of a ""magic bullet"" was to some extent realized by the invention of monoclonal antibodies, as these molecules provide a very specific binding affinity to their cognate target. However, monoclonal antibodies used as single agents are typically not able to induce cures for cancer or chronic inflammatory diseases. More recently, intense academic and industrial research activities have aimed at “arming” monoclonal antibodies with drugs or cytokines, in order to preferentially deliver these therapeutic payloads to the site of disease. Unfortunately, in most cases, ""armed"" antibody products still cause unacceptable toxicities, which prevent escalation to potentially curative dose regimens.
In this Project, I outline a therapeutic strategy, which relies on the use of extremely specific tumor targeting agents, for the selective delivery of payloads, which can be conditionally activated at the site of disease. Methodologies for the conditional generation of active payloads include the stepwise non-covalent assembly of cytokines and the controlled release of cytotoxic drugs at suitable time points after injection, when the concentration of therapeutic agent in normal organs is acceptably low. Response to therapy will be profiled using innovative proteomic methodologies, based on HLA-peptidome analysis.
Pharmaceutical agents with “activity on demand” hold a considerable potential not only for the therapy of cancer, but also for the treatment of other serious diseases, including certain highly debilitating chronic inflammatory condition"
Summary
"Paul Ehrlich was the first scientist to postulate that if a compound could be made that selectively targeted disease-causing cells, then this agent could be used for the delivery of a toxin, which would enable a pharmacotherapy of unprecedented potency and selectivity. With this procedure, a ""magic bullet"" (Zauberkugel, his term for an ideal therapeutic agent) would be created, that killed diseased cells while sparing normal tissues.
The concept of a ""magic bullet"" was to some extent realized by the invention of monoclonal antibodies, as these molecules provide a very specific binding affinity to their cognate target. However, monoclonal antibodies used as single agents are typically not able to induce cures for cancer or chronic inflammatory diseases. More recently, intense academic and industrial research activities have aimed at “arming” monoclonal antibodies with drugs or cytokines, in order to preferentially deliver these therapeutic payloads to the site of disease. Unfortunately, in most cases, ""armed"" antibody products still cause unacceptable toxicities, which prevent escalation to potentially curative dose regimens.
In this Project, I outline a therapeutic strategy, which relies on the use of extremely specific tumor targeting agents, for the selective delivery of payloads, which can be conditionally activated at the site of disease. Methodologies for the conditional generation of active payloads include the stepwise non-covalent assembly of cytokines and the controlled release of cytotoxic drugs at suitable time points after injection, when the concentration of therapeutic agent in normal organs is acceptably low. Response to therapy will be profiled using innovative proteomic methodologies, based on HLA-peptidome analysis.
Pharmaceutical agents with “activity on demand” hold a considerable potential not only for the therapy of cancer, but also for the treatment of other serious diseases, including certain highly debilitating chronic inflammatory condition"
Max ERC Funding
2 000 000 €
Duration
Start date: 2015-09-01, End date: 2020-08-31
Project acronym zebraHeart
Project Novel insights into cardiac regeneration through studies in the zebrafish
Researcher (PI) Nadia Isabel Mercader Huber
Host Institution (HI) UNIVERSITAET BERN
Call Details Starting Grant (StG), LS4, ERC-2013-StG
Summary Myocardial infarction (MI) leads to cardiomyocyte death and accumulation of myofibroblasts (MFs) at the site of injury, which produce large amounts of extracellular matrix (ECM), generating a scar. Initially, cardiac fibrosis protects from ventricular wall rupture, but subsequent myocardial remodelling causes heart failure, representing a leading cause of death in Europe. While MFs play a central role in cardiac fibrosis, there is confusion on their origin, a lack of specific markers and the existence of a unique MF type is debatable. Different MF might reveal distinct characteristics regarding ECM production, contractility, and autophagy, making them more or less pernicious. While in humans cardiac fibrosis is irreversible, other vertebrates have a remarkable capacity to regenerate damaged tissue. We recently established a zebrafish MI model and found that cardiac fibrosis is reversible and occurs as an intermediate step during regeneration. Here, the endogenous mechanisms of MFs and ECM regression will be explored. In addition, MF origin, types and fate will be characterized and manipulated to improve regeneration. As in mammals, cardiac injury elicits an inflammatory response in the zebrafish. The regenerative capacity of a species has been directly linked to features of its immune system, but surprisingly little is known on zebrafish leukocyte subtypes. We will study the role of macrophages and particularly analyse a subtype, which accumulates in the outer mesothelial layer of the heart, the epicardium. Epicardial derived cells play a key role as a trophic factor and progenitor cell source, and a first step towards regeneration includes the reestablishment of the epicardial layer. The zebrafish will offer a screening platform for small molecules triggering its activation. In sum, the project will increase the knowledge on the molecular and cellular basis of fibrosis regression, provide novel MF markers and identify new drugs to enhance cardiac regeneration.
Summary
Myocardial infarction (MI) leads to cardiomyocyte death and accumulation of myofibroblasts (MFs) at the site of injury, which produce large amounts of extracellular matrix (ECM), generating a scar. Initially, cardiac fibrosis protects from ventricular wall rupture, but subsequent myocardial remodelling causes heart failure, representing a leading cause of death in Europe. While MFs play a central role in cardiac fibrosis, there is confusion on their origin, a lack of specific markers and the existence of a unique MF type is debatable. Different MF might reveal distinct characteristics regarding ECM production, contractility, and autophagy, making them more or less pernicious. While in humans cardiac fibrosis is irreversible, other vertebrates have a remarkable capacity to regenerate damaged tissue. We recently established a zebrafish MI model and found that cardiac fibrosis is reversible and occurs as an intermediate step during regeneration. Here, the endogenous mechanisms of MFs and ECM regression will be explored. In addition, MF origin, types and fate will be characterized and manipulated to improve regeneration. As in mammals, cardiac injury elicits an inflammatory response in the zebrafish. The regenerative capacity of a species has been directly linked to features of its immune system, but surprisingly little is known on zebrafish leukocyte subtypes. We will study the role of macrophages and particularly analyse a subtype, which accumulates in the outer mesothelial layer of the heart, the epicardium. Epicardial derived cells play a key role as a trophic factor and progenitor cell source, and a first step towards regeneration includes the reestablishment of the epicardial layer. The zebrafish will offer a screening platform for small molecules triggering its activation. In sum, the project will increase the knowledge on the molecular and cellular basis of fibrosis regression, provide novel MF markers and identify new drugs to enhance cardiac regeneration.
Max ERC Funding
1 499 215 €
Duration
Start date: 2014-02-01, End date: 2019-01-31
Project acronym ZF-MEL-CHEMBIO
Project Chemical Biology in Zebrafish: Drug-Leads and New Targets in the Melanocyte Lineage and Melanoma
Researcher (PI) Eleanor Elizabeth Patton
Host Institution (HI) THE UNIVERSITY OF EDINBURGH
Call Details Consolidator Grant (CoG), LS4, ERC-2014-CoG
Summary Melanoma (cancer of the melanocyte) kills over 20,000 Europeans each year and incidence continues to rise rapidly. BRAF(V600E) inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance, or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance.
An important and new therapeutic concept for melanoma is to target the melanocyte lineage. Recent evidence reveals that a melanocyte lineage specific programme maintains melanoma survival, and we have engineered the first animal model in zebrafish to demonstrate that targeting the master melanocyte lineage transcription factor MITF leads to rapid melanoma regression. Thus, understanding and targeting the melanocyte lineage is directly relevant to melanoma, and reveals therapeutically targetable processes.
Our vision is to use live-imaging of the melanocyte lineage as the basis for phenotypic chemical screens in zebrafish to find drugs/leads and identify targetable processes that might elucidate pathways for cancer therapy. Screening for targets of the melanocyte lineage is highly relevant to melanoma because melanocytes are the melanoma cell of origin, and genes that specify the melanocyte stem cells and the lineage during embryogenesis are the same genes that play fundamental roles in cancer. We will use innovative chemical-biology to capture and validate targets in vivo, and perform chemo-preventative and -therapeutic trials in zebrafish melanoma models using known and novel drug-delivery methods.
Ultimately, we aim to translate our most promising drug/leads and targets into the mammalian system, to establish the basis for patent applications and clinical trials.
Summary
Melanoma (cancer of the melanocyte) kills over 20,000 Europeans each year and incidence continues to rise rapidly. BRAF(V600E) inhibitors have led to clinically significant improvements in outcomes for melanoma patients, yet many patients with metastatic melanoma rapidly succumb to the disease due to eventual chemoresistance, or insensitivity to the drug. Thus, it is critical to identify new therapies that can act alone, or be combined with available treatments for enhanced efficacy and/or to overcome drug resistance.
An important and new therapeutic concept for melanoma is to target the melanocyte lineage. Recent evidence reveals that a melanocyte lineage specific programme maintains melanoma survival, and we have engineered the first animal model in zebrafish to demonstrate that targeting the master melanocyte lineage transcription factor MITF leads to rapid melanoma regression. Thus, understanding and targeting the melanocyte lineage is directly relevant to melanoma, and reveals therapeutically targetable processes.
Our vision is to use live-imaging of the melanocyte lineage as the basis for phenotypic chemical screens in zebrafish to find drugs/leads and identify targetable processes that might elucidate pathways for cancer therapy. Screening for targets of the melanocyte lineage is highly relevant to melanoma because melanocytes are the melanoma cell of origin, and genes that specify the melanocyte stem cells and the lineage during embryogenesis are the same genes that play fundamental roles in cancer. We will use innovative chemical-biology to capture and validate targets in vivo, and perform chemo-preventative and -therapeutic trials in zebrafish melanoma models using known and novel drug-delivery methods.
Ultimately, we aim to translate our most promising drug/leads and targets into the mammalian system, to establish the basis for patent applications and clinical trials.
Max ERC Funding
1 865 345 €
Duration
Start date: 2015-09-01, End date: 2020-08-31
Project acronym ZF_Blood
Project Less is more: Single Cell Analysis of Zebrafish Blood Development
Researcher (PI) Ana Cvejic
Host Institution (HI) THE CHANCELLOR MASTERS AND SCHOLARS OF THE UNIVERSITY OF CAMBRIDGE
Call Details Starting Grant (StG), LS3, ERC-2015-STG
Summary Blood stem cells need to both perpetuate themselves (self-renew) and differentiate into all mature blood cells to maintain blood formation throughout life. However, it is unclear how the underlying gene regulatory network maintains this population of self-renewing and differentiating stem cells, and how it accommodates the transition from a stem cell to a mature blood cell. Our current knowledge of transcriptomes of various blood cell types has mainly been advanced by population-level analysis. However, the population of seemingly homogenous blood cells may include many distinct cell types with substantially different transcriptomes and abilities to make diverse fate decisions. To overcome these limitations, I will use single-cell transcriptome sequencing of zebrafish blood cells. I will apply an integrative strategy, combining genetic perturbation with computational sequence and network analysis methods, to reconstruct the regulatory networks that maintain the dynamic balance between different blood cell types. This will be achieved by pursuing two main aims:
1) I will create a comprehensive atlas of single cell gene expression in adult zebrafish blood cells and computationally reconstruct the blood lineage tree. I will order cells according to their most likely developmental chronology and identify genes and gene regulatory networks that define distinct cell types. The completion of the first aim will be followed by a more ambitious long-term one that is based on:
2) The in-depth functional characterisation of a subset of novel key regulators of blood formation and identified cell types in vivo. To achieve this I will generate a number of loss-of-function and transgenic zebrafish lines.
By sequencing thousands of single cells, this study is poised to go beyond traditional approaches in examining the complex relationships between the continuous spectra of blood cells, and will provide unprecedented insight into the regulation of blood cell formation.
Summary
Blood stem cells need to both perpetuate themselves (self-renew) and differentiate into all mature blood cells to maintain blood formation throughout life. However, it is unclear how the underlying gene regulatory network maintains this population of self-renewing and differentiating stem cells, and how it accommodates the transition from a stem cell to a mature blood cell. Our current knowledge of transcriptomes of various blood cell types has mainly been advanced by population-level analysis. However, the population of seemingly homogenous blood cells may include many distinct cell types with substantially different transcriptomes and abilities to make diverse fate decisions. To overcome these limitations, I will use single-cell transcriptome sequencing of zebrafish blood cells. I will apply an integrative strategy, combining genetic perturbation with computational sequence and network analysis methods, to reconstruct the regulatory networks that maintain the dynamic balance between different blood cell types. This will be achieved by pursuing two main aims:
1) I will create a comprehensive atlas of single cell gene expression in adult zebrafish blood cells and computationally reconstruct the blood lineage tree. I will order cells according to their most likely developmental chronology and identify genes and gene regulatory networks that define distinct cell types. The completion of the first aim will be followed by a more ambitious long-term one that is based on:
2) The in-depth functional characterisation of a subset of novel key regulators of blood formation and identified cell types in vivo. To achieve this I will generate a number of loss-of-function and transgenic zebrafish lines.
By sequencing thousands of single cells, this study is poised to go beyond traditional approaches in examining the complex relationships between the continuous spectra of blood cells, and will provide unprecedented insight into the regulation of blood cell formation.
Max ERC Funding
1 500 000 €
Duration
Start date: 2016-05-01, End date: 2021-04-30
Project acronym ZFISHSLEEP
Project Resolving the Neuropharmacology and Genetics of Zebrafish Sleep
Researcher (PI) Jason Rihel
Host Institution (HI) UNIVERSITY COLLEGE LONDON
Call Details Starting Grant (StG), LS5, ERC-2011-StG_20101109
Summary Sleep is a fundamental process, yet the genetic and neural mechanisms that regulate sleep are largely unknown. We have developed the zebrafish as a model system to study the regulation of sleep because it combines the genetics of invertebrates with the basic brain structures that regulate sleep in humans. We previously designed high throughput behavioural assays to measure sleep behaviours in the fish and used genetic tools to demonstrate that the wake-regulating hypocretin/orexin (Hcrt) system is functionally conserved in the zebrafish. We have also used our assays to perform a small molecule screen and identified both conserved and novel candidate regulators of sleep in zebrafish.
In Aim 1, we will observe the behaviour of wild type and Hcrt receptor mutants to a panel of small molecules known to alter zebrafish sleep. This aim tests the hypothesis that these compounds exert their effects on sleep and wake through the Hcrt system. In Aim 2, we will follow-up on the compounds that had differential effects in the mutants. We will monitor the activity of Hcrt neurons in response to drugs using a new neuroluminescent technique to observe the activity of neurons in freely behaving zebrafish larvae. This Aim will extend the behavioural data to the level of neural circuits. In Aim 3, we will use new methods to globally observe neuronal activity in the zebrafish brain to extend our analysis to neurons thought to interact with the Hcrt system. By observing activity across the sleep/wake cycle, we may also uncover novel sleep regulating neurons.
Overall, this project takes a multidisciplinary approach to the study of sleep and the Hcrt system, leveraging new methods from chemical biology, molecular genetics, and behavioural neuroscience in the zebrafish. As little is known about the mechanisms and sites of action for most sleep-altering compounds, any progress would advance the sleep field and could have clinical relevance to the treatment of sleep disorders.
Summary
Sleep is a fundamental process, yet the genetic and neural mechanisms that regulate sleep are largely unknown. We have developed the zebrafish as a model system to study the regulation of sleep because it combines the genetics of invertebrates with the basic brain structures that regulate sleep in humans. We previously designed high throughput behavioural assays to measure sleep behaviours in the fish and used genetic tools to demonstrate that the wake-regulating hypocretin/orexin (Hcrt) system is functionally conserved in the zebrafish. We have also used our assays to perform a small molecule screen and identified both conserved and novel candidate regulators of sleep in zebrafish.
In Aim 1, we will observe the behaviour of wild type and Hcrt receptor mutants to a panel of small molecules known to alter zebrafish sleep. This aim tests the hypothesis that these compounds exert their effects on sleep and wake through the Hcrt system. In Aim 2, we will follow-up on the compounds that had differential effects in the mutants. We will monitor the activity of Hcrt neurons in response to drugs using a new neuroluminescent technique to observe the activity of neurons in freely behaving zebrafish larvae. This Aim will extend the behavioural data to the level of neural circuits. In Aim 3, we will use new methods to globally observe neuronal activity in the zebrafish brain to extend our analysis to neurons thought to interact with the Hcrt system. By observing activity across the sleep/wake cycle, we may also uncover novel sleep regulating neurons.
Overall, this project takes a multidisciplinary approach to the study of sleep and the Hcrt system, leveraging new methods from chemical biology, molecular genetics, and behavioural neuroscience in the zebrafish. As little is known about the mechanisms and sites of action for most sleep-altering compounds, any progress would advance the sleep field and could have clinical relevance to the treatment of sleep disorders.
Max ERC Funding
1 902 750 €
Duration
Start date: 2012-02-01, End date: 2017-01-31
Project acronym ZINC-HUBS
Project Engineering zinc fingers to target cancer hub genes
Researcher (PI) Mark Isalan
Host Institution (HI) IMPERIAL COLLEGE OF SCIENCE TECHNOLOGY AND MEDICINE
Call Details Starting Grant (StG), LS7, ERC-2007-StG
Summary For the last ten years, protein engineering technologies have been developed to make zinc finger peptides to recognise a wide variety of user-defined DNA sequences. This has enabled the construction of synthetic transcription factors that can upregulate or repress target genes at will. More recently, synthetic zinc fingers have been linked to nucleases to direct double stranded breaks at desired loci within genomes. These breaks increase the efficiency of homologous recombination so that, by providing an exogenous repair sequence, it is possible to repair or mutate endogenous genes. Although zinc finger engineering has reached a state of maturity, there are very few groups in the world who have the technical know-how to adopt this technology, and this has delayed general uptake. We will use the expertise we have developed, in both zinc finger engineering and gene repair, to construct zinc finger proteins to recognise some of the most highly-connected (and widely-studied) genes in biology. This will serve as a toolkit for the research community to target hub genes and either mutate or repair them. As a starting point we propose to target the following hub genes: TBP (TATA-binding protein), p53, p300, RXR, pRB, RelA, c-jun, c-myc, and c-fos. These genes are the most connected hubs in the human transcription factor network (TRANSFAC 8.2 database) and their mutants are associated with a variety of diseases. We will engineer and characterise zinc finger proteins that recognise these DNA sequences in vitro and induce gene repair in vivo. For example, this will allow cancer cell lines to have particular oncogenes repaired or mutated, within the context of all the other mutations that have been accrued during the process of oncogenesis. This will help to characterise the contribution of network nodes and hubs to the observed phenotypes. Ultimately, some of the gene repair peptides we create will have therapeutic potential, as well as providing tools for systems biology.
Summary
For the last ten years, protein engineering technologies have been developed to make zinc finger peptides to recognise a wide variety of user-defined DNA sequences. This has enabled the construction of synthetic transcription factors that can upregulate or repress target genes at will. More recently, synthetic zinc fingers have been linked to nucleases to direct double stranded breaks at desired loci within genomes. These breaks increase the efficiency of homologous recombination so that, by providing an exogenous repair sequence, it is possible to repair or mutate endogenous genes. Although zinc finger engineering has reached a state of maturity, there are very few groups in the world who have the technical know-how to adopt this technology, and this has delayed general uptake. We will use the expertise we have developed, in both zinc finger engineering and gene repair, to construct zinc finger proteins to recognise some of the most highly-connected (and widely-studied) genes in biology. This will serve as a toolkit for the research community to target hub genes and either mutate or repair them. As a starting point we propose to target the following hub genes: TBP (TATA-binding protein), p53, p300, RXR, pRB, RelA, c-jun, c-myc, and c-fos. These genes are the most connected hubs in the human transcription factor network (TRANSFAC 8.2 database) and their mutants are associated with a variety of diseases. We will engineer and characterise zinc finger proteins that recognise these DNA sequences in vitro and induce gene repair in vivo. For example, this will allow cancer cell lines to have particular oncogenes repaired or mutated, within the context of all the other mutations that have been accrued during the process of oncogenesis. This will help to characterise the contribution of network nodes and hubs to the observed phenotypes. Ultimately, some of the gene repair peptides we create will have therapeutic potential, as well as providing tools for systems biology.
Max ERC Funding
1 327 689 €
Duration
Start date: 2008-10-01, End date: 2014-09-30
