Epigenetics and hidden heritability in tomato
Lead Research Organisation:
University of Cambridge
Department Name: Plant Sciences
Abstract
The information content of eukaryotic genomes is based primarily on the DNA sequence but there are additional layers that are dependent on methylation of cytosine residues in the DNA and modification of the histone proteins that bind to DNA in the chromosomes. A primary function of this epigenetic information is to protect against DNA parasites - transposable elements - that colonise genomes and have mutagenic properties.
Epigenetics, therefore, is part of the arms race between the host genomes and these DNA parasites but, like all arms races,the dynamics are not simple. The host may even benefit if a parasite has features or effects that increase the host's fitness and, conversely, the parasite may also benefit if the defense systems of the host reduce the damage caused by parasitism (the parasite is dependent on the well-being of the host).
Evidence of this complex dynamic relationship is from the phenotype of DNA methylation mutants in plants in which DNA methylation is lost throughout the genome. There is not only loss of transposon defense but additionally disrupted growth and development of the plant and sterility. Loss of the DNA methylation in localized parts of the genome, however, may have more specific effects including some that are beneficial. These observations prompt the hypothesis that epigenetics may account for the hidden heritability that is well known to breeders of crops. Hidden heritability is best illustrated with quantitative traits for which much of the heritable variation between plants cannot be linked to genetic markers. In some instances this hidden heritability or missing inheritance can account for a substantial part of the variation.
Much of the molecular understanding of epigenetics in plants is from the model plant Arabidopsis. Extensive resources that have facilitated experimentation with this plant and it has led to good understanding of the various epigenetic mechanisms. The rapid progress with this model species, however, is partly because the genome is small and has many fewer transposable elements than most other species, including crops. Perturbation of this reduced epigenome, therefore, has rather mild effects on the growth and development of the plant.
In plants with larger, transposon-rich genomes there are more profound effects on growth and development consistent with the epigenetic transposon defense overlapping with the normal function of the genome. Unfortunately, with the standard set of DNA methylation mutants, the plants may be sterile and be difficult to use experimentally.
To address this problem we have developed an approach in tomato with a DNA methylation mutant that causes limited disruption of the epigenome. In our preparatory work we have isolated and characterized such a mutant in the DNA methylation gene CMT3a. The partial effect allows recovery of fertile mutant plants so that we can implement a detailed analysis of the CMT3a-dependent epigenome and its effects.
In the first part of the proposed project we will fully characterize the CMT3a-dependent epigenome and its influence on gene expression. In our preliminary evidence we show that some regions of the genome lose their DNA methylation in the mutant plans and that they retain this hypomethylated state in backcrossed progeny to wild type plants whereas other regions regain the normal levels of DNA methylation. Integrating these findings with analyses of gene expression in cmt3a and its progeny will identify features in the genome that correlate with heritability of the epigenome and its effects on gene expression. We will then test these correlations by targeted modification of the epigenome using a modified CRISPR system. This system for targeted modification of the epigenome will also be explored as a system for epigenetic modification in crop plant improvement.
Epigenetics, therefore, is part of the arms race between the host genomes and these DNA parasites but, like all arms races,the dynamics are not simple. The host may even benefit if a parasite has features or effects that increase the host's fitness and, conversely, the parasite may also benefit if the defense systems of the host reduce the damage caused by parasitism (the parasite is dependent on the well-being of the host).
Evidence of this complex dynamic relationship is from the phenotype of DNA methylation mutants in plants in which DNA methylation is lost throughout the genome. There is not only loss of transposon defense but additionally disrupted growth and development of the plant and sterility. Loss of the DNA methylation in localized parts of the genome, however, may have more specific effects including some that are beneficial. These observations prompt the hypothesis that epigenetics may account for the hidden heritability that is well known to breeders of crops. Hidden heritability is best illustrated with quantitative traits for which much of the heritable variation between plants cannot be linked to genetic markers. In some instances this hidden heritability or missing inheritance can account for a substantial part of the variation.
Much of the molecular understanding of epigenetics in plants is from the model plant Arabidopsis. Extensive resources that have facilitated experimentation with this plant and it has led to good understanding of the various epigenetic mechanisms. The rapid progress with this model species, however, is partly because the genome is small and has many fewer transposable elements than most other species, including crops. Perturbation of this reduced epigenome, therefore, has rather mild effects on the growth and development of the plant.
In plants with larger, transposon-rich genomes there are more profound effects on growth and development consistent with the epigenetic transposon defense overlapping with the normal function of the genome. Unfortunately, with the standard set of DNA methylation mutants, the plants may be sterile and be difficult to use experimentally.
To address this problem we have developed an approach in tomato with a DNA methylation mutant that causes limited disruption of the epigenome. In our preparatory work we have isolated and characterized such a mutant in the DNA methylation gene CMT3a. The partial effect allows recovery of fertile mutant plants so that we can implement a detailed analysis of the CMT3a-dependent epigenome and its effects.
In the first part of the proposed project we will fully characterize the CMT3a-dependent epigenome and its influence on gene expression. In our preliminary evidence we show that some regions of the genome lose their DNA methylation in the mutant plans and that they retain this hypomethylated state in backcrossed progeny to wild type plants whereas other regions regain the normal levels of DNA methylation. Integrating these findings with analyses of gene expression in cmt3a and its progeny will identify features in the genome that correlate with heritability of the epigenome and its effects on gene expression. We will then test these correlations by targeted modification of the epigenome using a modified CRISPR system. This system for targeted modification of the epigenome will also be explored as a system for epigenetic modification in crop plant improvement.
Technical Summary
In this project we will use a cmt3a DNA (methyl transferase) mutant to study epigenetic effects on gene expression and heritability in tomato. We will use cmt3a because other DNA methylation mutants (nrpd1, nrpe1, met1) are completely sterile and they cannot be used for studies of heritability.
Approximately 5% of the hypomethylated regions in cmt3a persist in backcrossed progeny with a wild type CMT3a - these regions lack the genomic information for reestablishment of the DNA methylation. The other 95% regain their methylation indicating that they have genomic features that guide the recruitment of CMT3a.
In the first subproject we will characterize the heritability and gene expression characteristics of CMT3a targets using bisulphite sequencing and RNAseq. The methylation patterns will be followed over at least five generations and in controlled environment and glasshouse environments.
The second subproject will involve a multiple correspondence analysis of epigenetic properties (heritability, gene expression) with intrinsic (repetition, base composition etc ) and extrinsic (gene, type of transposon) genomic features. From this activity we expect to identify different classes of DNA methylation loci. The robustness of this classification model will be tested in mutant lines for mutants other than cmt3a that will influence the pattern of DNA methylation or demethylation.
Finally we will develop a dCas9 SunTag system in which the TET1 DNA hydroxylase will target DNA demethylation to specific regions of the genomes so that we can further test the classification model and explore the extent to which heritable epigenetic changes to gene can be engineered into the tomato genome.
The project is basic science addressing a fundamental question in epigenetics: why is DNA methylation attracted to some regions of the genome and not others. In the longer term the project will inform approaches to crop improvement by epigenetic modification.
Approximately 5% of the hypomethylated regions in cmt3a persist in backcrossed progeny with a wild type CMT3a - these regions lack the genomic information for reestablishment of the DNA methylation. The other 95% regain their methylation indicating that they have genomic features that guide the recruitment of CMT3a.
In the first subproject we will characterize the heritability and gene expression characteristics of CMT3a targets using bisulphite sequencing and RNAseq. The methylation patterns will be followed over at least five generations and in controlled environment and glasshouse environments.
The second subproject will involve a multiple correspondence analysis of epigenetic properties (heritability, gene expression) with intrinsic (repetition, base composition etc ) and extrinsic (gene, type of transposon) genomic features. From this activity we expect to identify different classes of DNA methylation loci. The robustness of this classification model will be tested in mutant lines for mutants other than cmt3a that will influence the pattern of DNA methylation or demethylation.
Finally we will develop a dCas9 SunTag system in which the TET1 DNA hydroxylase will target DNA demethylation to specific regions of the genomes so that we can further test the classification model and explore the extent to which heritable epigenetic changes to gene can be engineered into the tomato genome.
The project is basic science addressing a fundamental question in epigenetics: why is DNA methylation attracted to some regions of the genome and not others. In the longer term the project will inform approaches to crop improvement by epigenetic modification.
Planned Impact
This project will defuse some of the academic controversy associated with epigenetics and heritability (see Academic beneficiaries). Outside the academic community our findings will provide a framework for engagement with the public about what epigenetics really means. Our public engagement work will complement the excellent 'Eva Jablonka and Marion J. Lamb. Evolution in four dimensions: Genetic, epigenetic, behavioral, and symbolic variation in the history of life. 2005.The MIT Press'
The better understanding of epigenetics will also inform future technology development based on epigenetic modification of crop plant genomes. To that end I will engage with industry and regulators to inform them about progress in this project and the ways that the findings could be developed into technology.
The targets of our impact activities will be:
-the general public and educators
-regulators who will need good understanding of the basic concepts associated with epigenetics if any new regulatory framework is to have a rational and evidence base.
-investors and industry so that they can be helped to identify and support new epigenetic technologies as they emerge.
t-he postdoctoral researcher so that they can develop their career with experience of driving impact in science
1. Public engagement in evolution and epigenetics
The initial engagement will involve the internet and a display in the Cambridge University Botanic Garden. The Garden has a large audience (around 250000 pa) who will receive passive exposure to the display.
In the later stages of the project we will use publications from our work as an opportunity to broaden the public engagement.
Milestone I1 - 12 months - website presentation of basic concepts in heritability in epigenetics.
Milestone I2 - 24 months - demonstration plot in Cambridge University Botanic Garden illustrating Mendelian inheritance due to epigenetics . The "meet the scientist" sessions will be included as part of this milestone.
Milestone I3 - 36 months - presentation to popular media of the concepts in heritability and epigenetics.
2. Industry engagement in the importance of epigenetics in crop plant breeding.
Engagement with industry will be through contacts and meetings at conferences. I will also approach industry directly towards the end of the project to discuss the possibility of follow on projects.
Milestone I4 - 36 months - at least ten meetings with relevant industry contacts to discuss the implications of this project
3. Regulatory engagement in the development rational, risk-based regulatory framework for epigenetics.
As part of the engagement with regulatory bodies we will apply to ACRE for permission to "field-test" BC.F2(CMT3a) that have been generated in connection with SP1.3. This plants are descended from the transgenic plants with the Casa9 mutagenic transgene but they will have been subsequently backcrossed through three generations (twice to generate the cmt3a mutant) and this activity is intended to clarify the need to have then covered by ACRE.
Milestone I5 - 24 months - decision from ACRE over field testing of and one year of trials. If successful I will apply for a second year of trials by month 36..
3. Training of a postdoctoral researcher on a project that will introduce involvement in commercialization and regulatory aspects of plant biotechnology.
In this project, with the unusually prominent opportunities for engagement with the public, industry and regulators, there is a corresponding opportunity for training of the PDRA. The PI will involve this individual fully in the impact activity in the expectation that she or he will substitute for the PI on many occasions.
Milestone I6- 36 months - postdoctoral researcher with skills in experimental and computational epigenetics and familiarity with regulatory, commercialization and public engagement aspects of a research project.
The better understanding of epigenetics will also inform future technology development based on epigenetic modification of crop plant genomes. To that end I will engage with industry and regulators to inform them about progress in this project and the ways that the findings could be developed into technology.
The targets of our impact activities will be:
-the general public and educators
-regulators who will need good understanding of the basic concepts associated with epigenetics if any new regulatory framework is to have a rational and evidence base.
-investors and industry so that they can be helped to identify and support new epigenetic technologies as they emerge.
t-he postdoctoral researcher so that they can develop their career with experience of driving impact in science
1. Public engagement in evolution and epigenetics
The initial engagement will involve the internet and a display in the Cambridge University Botanic Garden. The Garden has a large audience (around 250000 pa) who will receive passive exposure to the display.
In the later stages of the project we will use publications from our work as an opportunity to broaden the public engagement.
Milestone I1 - 12 months - website presentation of basic concepts in heritability in epigenetics.
Milestone I2 - 24 months - demonstration plot in Cambridge University Botanic Garden illustrating Mendelian inheritance due to epigenetics . The "meet the scientist" sessions will be included as part of this milestone.
Milestone I3 - 36 months - presentation to popular media of the concepts in heritability and epigenetics.
2. Industry engagement in the importance of epigenetics in crop plant breeding.
Engagement with industry will be through contacts and meetings at conferences. I will also approach industry directly towards the end of the project to discuss the possibility of follow on projects.
Milestone I4 - 36 months - at least ten meetings with relevant industry contacts to discuss the implications of this project
3. Regulatory engagement in the development rational, risk-based regulatory framework for epigenetics.
As part of the engagement with regulatory bodies we will apply to ACRE for permission to "field-test" BC.F2(CMT3a) that have been generated in connection with SP1.3. This plants are descended from the transgenic plants with the Casa9 mutagenic transgene but they will have been subsequently backcrossed through three generations (twice to generate the cmt3a mutant) and this activity is intended to clarify the need to have then covered by ACRE.
Milestone I5 - 24 months - decision from ACRE over field testing of and one year of trials. If successful I will apply for a second year of trials by month 36..
3. Training of a postdoctoral researcher on a project that will introduce involvement in commercialization and regulatory aspects of plant biotechnology.
In this project, with the unusually prominent opportunities for engagement with the public, industry and regulators, there is a corresponding opportunity for training of the PDRA. The PI will involve this individual fully in the impact activity in the expectation that she or he will substitute for the PI on many occasions.
Milestone I6- 36 months - postdoctoral researcher with skills in experimental and computational epigenetics and familiarity with regulatory, commercialization and public engagement aspects of a research project.
