Final Report Summary - CAPMEM (Comparative analysis of plant and mammalian DNA methylation functions in epigenetic Arabidopsis mutants)
PUBLISHABLE SUMMARY
• A summary description of project objectives
DNA methylation is a key epigenetic change in eukaryotic genomes, defined as the covalent addition of a methyl group to the cytosine DNA nucleotide to form 5-methylcytosine. This epigenetic modification plays an important role in diverse biological processes including cell differentiation, organismal development, stress and disease. Despite the evolutionary diversification and different morphology, plants and mammals share similarities in many aspects of genome and epigenome organization and functions, such as genome size, the ratio of heterochromatin to euchromatin, use of DNA methylation and histone posttranslational modifications for gene regulation. Mammalian DNA methylation is mostly restricted to the symmetric CG context, while in plants it occurs in all sequence contexts: CG, CHG and CHH (where H is any nucleotide except for C). DNA methylation patterns are established by a highly conserved family of enzymes. There are two principal types of DNA methyltransferase activities: de novo methylation targets previously unmethylated double-stranded DNA sequences, maintenance methylation occurs when pre-existing methylation marks are copied onto new DNA strands during replication. In contrast to mammals, plants tolerate the absence of methylation and can be used as an experimental system to study the methylation machinery.
The main objective of the Marie Curie IEF-project CAPMEM is to evaluate the extent of conservation and diversification of the plant MET1/DDM1 and mammalian DNMT1/LSH enzymes that control maintenance DNA methylation, using the plant model Arabidopsis thaliana as an experimental system. A. thaliana offers some unique advantages to study DNA methylation: 1) Knockout mutants for essential DNA methylation functions, including MET1 and DDM1, are viable and fertile, which allows to erase CG methylation patterns and analyse complementation effects of recombinant constructs; 2) Unlike animals, methylation patterns in plants are not erased via developmental demethylation/remethylation cycles. Several genomic loci where DNA methylation is re-established after restoring MET1 activity have been identified. Thus, de novo methylation, methylation spreading and maintenance fidelity of methylation can be followed over several generations; 3) Some of these targets only require MET1 activity, others require additional DDM1 activity. This permits studying methylation effects at loci that are dependent or independent of ATPase functions. Another important objective of CAPMEM was to investigate how methylation patterns affect plant adaptation to environmental challenges, comparing the efficiency of stress adaptation in wild-type plants and methylation mutants. Maintenance functions of DNA methylation are essential to preserve and transmit epigenetic changes to the next generation, which could improve long-term plant adaptation to adverse environment, and determine the potential of a gene to become active or inactive without changing its DNA sequence.
• Description of the work performed since the beginning of the project
Work on the project CAPMEM was organised into four work packages (WP), each divided into monitorable tasks. During the project period, the experimental work was performed according to the project management plan and schedule, following several research lines that are summarised in the following points: 1) Development of research tools to determine the extent of conservational overlap between mammalian DNMT1/LSH and plant MET1/DDM1 – during the reported period ten different constructs were designed, and used for transformation of wild-type plants and met1 and ddm1 mutants; 2) Analysis of re-methylation efficiency of the plant and mammalian constructs in the DNA methylation met1 and ddm1 mutants – different target loci with varying requirement for MET1 and DDM1 were examined for remethylation: FWA, ATENSPM5, CACTA, At3g01345, ncRNA, EVD; 3) Evaluation of quantitative and developmental effects of methyltransferase overexpression - overexpression effects of MET1 were analysed in three Arabidopsis lines constitutively overexpressing MET1 under 35S CaMV promoter, and the observed phenotypic changes were linked to expression of individual genes; 4) Evaluation of the importance of stress-induced epigenetic modifications for plant adaptation to environmental changes – the work was mainly focused on the stress responses of the methylation mutants to short-term abiotic stress factors, such as osmotic stress, and the importance of DNA methylation for the control of genes involved in stress tolerance; 5) investigation of the quantitative effects of MET1 and an examination of the heritability of these effects.
• Description of the main results achieved so far
The CAPMEM project was successfully completed in August 2015 after an overall term of two years. The main results are summarised bellow:
-The research tools for determining the extent of conservational overlap between mammalian DNMT1/LSH and plant MET1/DDM1 were constructed, and persistence of stable expression of the mammalian DNMT1/LSH in transgenic Arabidopsis plants was achieved.
-Analysis of re-methylation efficiency after transfer of MET1/DDM1 and DNMT1/LSH transgene into homozygous met1 and ddm1 mutants was performed. Although some changes in the expression of the selected target loci in DNMT1 transformants, homozygous for met1-1, were observed, the transgenes could not induce complete silencing in the selected epigenetically regulated target loci, suggesting that full remethylation did not occur. Transformation with DNMT1 did not lead to reversion of met1-1 phenotype, and caused sterility in all transformants, homozygous for met1-1. Transfer of MET1 into the wild-type Col-0 led to an increased shoot branching and changed the expression level of the MAX family of genes. Transformation of homozygous ddm1-10 with 35S-LSH resulted in obvious phenotypic variations in the transformants expressing Lsh, but the tested target loci remained activated.
-Examination of three lines constitutively overexpressing MET1 under 35S CaMV promoter showed that overexpression of MET1 induced a number of different shoot and root phenotypes. The most severe phenotype changes, such as delayed flowering and sterility were observed in the line with the highest expression of a gene causing late flowering.
-Comparison of stress responses in different DNA methylation mutants showed that DNA methylation is involved in the ability of plant roots to optimise their growth direction in stress environment. The roots of ddm1-10 and the wild-type Col-0 reduced their exposure to high salinity by growing away from the salt media, whereas met1-1 seedlings did not modify their growth direction following the gravity vector. High salinity led to changes in the expression of the genes from the Salt sensitive (SOS) pathway, which was induced by a change in DNA methylation status. The reduced methylation in met1-1 correlated with a higher expression of the tested SOS genes, which was exactly opposite for ddm1-10 and Col-0. Their methylation status and expression levels did not change.
-Mutation in the MET1 methyltransferase gene caused pleiotropic root phenotypes in homozygous met1-1 seedlings. These phenotypes persisted in F1 heterozygous met1-1 progeny and could be attributed to the quantitative effect of MET1. In F2 progeny, only 15% of the wild-type segregants restored their root morphology to the wild-type, showing that the observed root phenotypes are reversible, but this reversibility is rather limited.
-In homozygous and heterozygous met1-1 mutants, the generation and maintenance of auxin gradients is disturbed, which negatively affects primary root growth and lateral root development. These developmental problems could be related, at least partly, to the internalisation of PIN1. The MET1 methyltransferase, either directly or indirectly, is involved in the regulation of genes responsible for the generation of consistent auxin gradients, and proper DNA methylation patterns are required for a number of root-related developmental processes during Arabidopsis post-embryogenesis.
• Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
This interdisciplinary research uses Arabidopsis as an expression system for recombinant heterologous proteins providing a valuable research tool for the functional characterisation of DNA methylation regulators. CAPMEM contributes to understanding of the evolution of DNA methylation, especially maintenance methylation functions, which preserve epigenetic changes and transmit them to the next generation. Such preservation can increase genomic flexibility and enable stress-treated plants and their offspring to undergo stress-adaptive evolution. Maintenance functions of DNA methylation determine the potential of a gene to become active or inactive without changing its DNA sequence. Alteration in DNA methylation results in a unique gene expression pattern that may play a critical role in plant development and stress adaptation. So far, studies of plant populations displaying epigenetic variation are rather preliminary and they have not provided firm evidence of epigenetic gene variants contributing to heritable changes in stress responses. The CAPMEM results can have far-reaching impacts not only on understanding the fundamental epigenetic mechanisms but also on practical issues related to agriculture, human biology and disease. The information gained initially in the model plant A. thaliana is easily transmittable to crop plants and to the medical labs. Therefore, this research has a great potential for understanding of plant and mammalian DNA methylation phenomena as major determinants of gene expression and integrity, and can contribute to the efforts directed towards enhancing abiotic stress tolerance both in theory and practice.
• If applicable, explain the reasons for deviations from Annex I and their impact on other tasks as well as on available resources and planning;
There were no deviations from Annex I. All critical objectives have been achieved during the project period and no deviations have been observed between the actual and planned research months.
• If applicable, explain the reasons for failing to achieve critical objectives and/or not being on schedule and explain the impact on other tasks as well as on available resources and planning (the explanations should be coherent with the declaration by the project coordinator);
Not applicable
• A statement on the use of resources, in particular highlighting and explaining deviations between actual and planned researcher-months in Annex 1 (Description of Work)
There were no deviations between actual and planned researcher-months
• If applicable, propose corrective actions.
No corrective actions are required
• A summary description of project objectives
DNA methylation is a key epigenetic change in eukaryotic genomes, defined as the covalent addition of a methyl group to the cytosine DNA nucleotide to form 5-methylcytosine. This epigenetic modification plays an important role in diverse biological processes including cell differentiation, organismal development, stress and disease. Despite the evolutionary diversification and different morphology, plants and mammals share similarities in many aspects of genome and epigenome organization and functions, such as genome size, the ratio of heterochromatin to euchromatin, use of DNA methylation and histone posttranslational modifications for gene regulation. Mammalian DNA methylation is mostly restricted to the symmetric CG context, while in plants it occurs in all sequence contexts: CG, CHG and CHH (where H is any nucleotide except for C). DNA methylation patterns are established by a highly conserved family of enzymes. There are two principal types of DNA methyltransferase activities: de novo methylation targets previously unmethylated double-stranded DNA sequences, maintenance methylation occurs when pre-existing methylation marks are copied onto new DNA strands during replication. In contrast to mammals, plants tolerate the absence of methylation and can be used as an experimental system to study the methylation machinery.
The main objective of the Marie Curie IEF-project CAPMEM is to evaluate the extent of conservation and diversification of the plant MET1/DDM1 and mammalian DNMT1/LSH enzymes that control maintenance DNA methylation, using the plant model Arabidopsis thaliana as an experimental system. A. thaliana offers some unique advantages to study DNA methylation: 1) Knockout mutants for essential DNA methylation functions, including MET1 and DDM1, are viable and fertile, which allows to erase CG methylation patterns and analyse complementation effects of recombinant constructs; 2) Unlike animals, methylation patterns in plants are not erased via developmental demethylation/remethylation cycles. Several genomic loci where DNA methylation is re-established after restoring MET1 activity have been identified. Thus, de novo methylation, methylation spreading and maintenance fidelity of methylation can be followed over several generations; 3) Some of these targets only require MET1 activity, others require additional DDM1 activity. This permits studying methylation effects at loci that are dependent or independent of ATPase functions. Another important objective of CAPMEM was to investigate how methylation patterns affect plant adaptation to environmental challenges, comparing the efficiency of stress adaptation in wild-type plants and methylation mutants. Maintenance functions of DNA methylation are essential to preserve and transmit epigenetic changes to the next generation, which could improve long-term plant adaptation to adverse environment, and determine the potential of a gene to become active or inactive without changing its DNA sequence.
• Description of the work performed since the beginning of the project
Work on the project CAPMEM was organised into four work packages (WP), each divided into monitorable tasks. During the project period, the experimental work was performed according to the project management plan and schedule, following several research lines that are summarised in the following points: 1) Development of research tools to determine the extent of conservational overlap between mammalian DNMT1/LSH and plant MET1/DDM1 – during the reported period ten different constructs were designed, and used for transformation of wild-type plants and met1 and ddm1 mutants; 2) Analysis of re-methylation efficiency of the plant and mammalian constructs in the DNA methylation met1 and ddm1 mutants – different target loci with varying requirement for MET1 and DDM1 were examined for remethylation: FWA, ATENSPM5, CACTA, At3g01345, ncRNA, EVD; 3) Evaluation of quantitative and developmental effects of methyltransferase overexpression - overexpression effects of MET1 were analysed in three Arabidopsis lines constitutively overexpressing MET1 under 35S CaMV promoter, and the observed phenotypic changes were linked to expression of individual genes; 4) Evaluation of the importance of stress-induced epigenetic modifications for plant adaptation to environmental changes – the work was mainly focused on the stress responses of the methylation mutants to short-term abiotic stress factors, such as osmotic stress, and the importance of DNA methylation for the control of genes involved in stress tolerance; 5) investigation of the quantitative effects of MET1 and an examination of the heritability of these effects.
• Description of the main results achieved so far
The CAPMEM project was successfully completed in August 2015 after an overall term of two years. The main results are summarised bellow:
-The research tools for determining the extent of conservational overlap between mammalian DNMT1/LSH and plant MET1/DDM1 were constructed, and persistence of stable expression of the mammalian DNMT1/LSH in transgenic Arabidopsis plants was achieved.
-Analysis of re-methylation efficiency after transfer of MET1/DDM1 and DNMT1/LSH transgene into homozygous met1 and ddm1 mutants was performed. Although some changes in the expression of the selected target loci in DNMT1 transformants, homozygous for met1-1, were observed, the transgenes could not induce complete silencing in the selected epigenetically regulated target loci, suggesting that full remethylation did not occur. Transformation with DNMT1 did not lead to reversion of met1-1 phenotype, and caused sterility in all transformants, homozygous for met1-1. Transfer of MET1 into the wild-type Col-0 led to an increased shoot branching and changed the expression level of the MAX family of genes. Transformation of homozygous ddm1-10 with 35S-LSH resulted in obvious phenotypic variations in the transformants expressing Lsh, but the tested target loci remained activated.
-Examination of three lines constitutively overexpressing MET1 under 35S CaMV promoter showed that overexpression of MET1 induced a number of different shoot and root phenotypes. The most severe phenotype changes, such as delayed flowering and sterility were observed in the line with the highest expression of a gene causing late flowering.
-Comparison of stress responses in different DNA methylation mutants showed that DNA methylation is involved in the ability of plant roots to optimise their growth direction in stress environment. The roots of ddm1-10 and the wild-type Col-0 reduced their exposure to high salinity by growing away from the salt media, whereas met1-1 seedlings did not modify their growth direction following the gravity vector. High salinity led to changes in the expression of the genes from the Salt sensitive (SOS) pathway, which was induced by a change in DNA methylation status. The reduced methylation in met1-1 correlated with a higher expression of the tested SOS genes, which was exactly opposite for ddm1-10 and Col-0. Their methylation status and expression levels did not change.
-Mutation in the MET1 methyltransferase gene caused pleiotropic root phenotypes in homozygous met1-1 seedlings. These phenotypes persisted in F1 heterozygous met1-1 progeny and could be attributed to the quantitative effect of MET1. In F2 progeny, only 15% of the wild-type segregants restored their root morphology to the wild-type, showing that the observed root phenotypes are reversible, but this reversibility is rather limited.
-In homozygous and heterozygous met1-1 mutants, the generation and maintenance of auxin gradients is disturbed, which negatively affects primary root growth and lateral root development. These developmental problems could be related, at least partly, to the internalisation of PIN1. The MET1 methyltransferase, either directly or indirectly, is involved in the regulation of genes responsible for the generation of consistent auxin gradients, and proper DNA methylation patterns are required for a number of root-related developmental processes during Arabidopsis post-embryogenesis.
• Expected final results and their potential impact and use (including the socio-economic impact and the wider societal implications of the project so far)
This interdisciplinary research uses Arabidopsis as an expression system for recombinant heterologous proteins providing a valuable research tool for the functional characterisation of DNA methylation regulators. CAPMEM contributes to understanding of the evolution of DNA methylation, especially maintenance methylation functions, which preserve epigenetic changes and transmit them to the next generation. Such preservation can increase genomic flexibility and enable stress-treated plants and their offspring to undergo stress-adaptive evolution. Maintenance functions of DNA methylation determine the potential of a gene to become active or inactive without changing its DNA sequence. Alteration in DNA methylation results in a unique gene expression pattern that may play a critical role in plant development and stress adaptation. So far, studies of plant populations displaying epigenetic variation are rather preliminary and they have not provided firm evidence of epigenetic gene variants contributing to heritable changes in stress responses. The CAPMEM results can have far-reaching impacts not only on understanding the fundamental epigenetic mechanisms but also on practical issues related to agriculture, human biology and disease. The information gained initially in the model plant A. thaliana is easily transmittable to crop plants and to the medical labs. Therefore, this research has a great potential for understanding of plant and mammalian DNA methylation phenomena as major determinants of gene expression and integrity, and can contribute to the efforts directed towards enhancing abiotic stress tolerance both in theory and practice.
• If applicable, explain the reasons for deviations from Annex I and their impact on other tasks as well as on available resources and planning;
There were no deviations from Annex I. All critical objectives have been achieved during the project period and no deviations have been observed between the actual and planned research months.
• If applicable, explain the reasons for failing to achieve critical objectives and/or not being on schedule and explain the impact on other tasks as well as on available resources and planning (the explanations should be coherent with the declaration by the project coordinator);
Not applicable
• A statement on the use of resources, in particular highlighting and explaining deviations between actual and planned researcher-months in Annex 1 (Description of Work)
There were no deviations between actual and planned researcher-months
• If applicable, propose corrective actions.
No corrective actions are required