Abstract
BACKGROUND: DNA methylation ensures genome integrity and regulates gene expression in diverse eukaryotes. In Arabidopsis, methylation occurs in three sequence contexts: CG, CHG and CHH. The initial establishment of DNA methylation at all three sequence contexts occurs through a process known as RNA-directed DNA methylation (RdDM), in which small RNAs bound by Argonaute4 (AGO4) guide DNA methylation at homologous loci through the de novo methyltransferase DRM2. Once established, DNA methylation at each of the three sequence contexts is maintained through different mechanisms. Although some players involved in RdDM and maintenance methylation have been identified, the underlying molecular mechanisms are not fully understood. To aid the comprehensive identification of players in DNA methylation, we generated a transgenic reporter system that permits genetic and chemical genetic screens in Arabidopsis. RESULTS: A dual 35S promoter (d35S) driven luciferase (LUC) reporter was introduced into Arabidopsis and LUCL, a line with a low basal level of luciferase activity, was obtained. LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context. Methylation was present throughout the promoter and LUC coding region. Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity. A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression. Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1. Using LUCL as a reporter line, we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor. CONCLUSION: We developed a luciferase-based reporter system, LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis. The low basal level of LUCL expression provides an easy readout in genetic and chemical genetic screens that will dissect the mechanisms of RdDM and methylation maintenance.
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Claims 7
Claim · model-written summary
LUCL is a multi-copy, single-insertion transgene with methylated cytosines in CG, CHG and CHH contexts.
- Taxon
- Arabidopsis thaliana
Arabidopsis thalianat0 - Material
- LUCLe0
- Outcome
- methylated cytosinese0
- Comparator
- CHG and CHH contextse0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e0“LUCL was found to be a multi-copy, single-insertion transgene that contains methylated cytosines in CG, CHG and CHH contexts, with the highest methylation in the CG context”
Claim · model-written summary
Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity in LUCL.
- Taxon
- Arabidopsis thalianaArabidopsis thalianat0
- Treatment
- inhibitor of cytosine methylatione1
- Outcome
- luciferase activitye1
- Direction
- de-repressede1
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e1“Treatment with an inhibitor of cytosine methylation de-repressed luciferase activity”
Claim · model-written summary
A mutation in MET1 drastically reduced CG methylation in LUCL.
- Taxon
- Arabidopsis thalianaArabidopsis thalianat0
- Treatment
- mutation in MET1e2
- Gene
- MET1e2
- Outcome
- CG methylatione2
- Direction
- drastically reducede2
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e2“A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression”
Claim · model-written summary
A mutation in MET1 de-repressed LUC expression in LUCL.
- Taxon
- Arabidopsis thalianaArabidopsis thalianat0
- Treatment
- mutation in MET1e2
- Gene
- MET1e2
- Outcome
- LUC expressione2
- Direction
- de-repressede2
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e2“A mutation in MET1, which encodes the CG maintenance methyltransferase, drastically reduced CG methylation and de-repressed LUC expression”
Claim · model-written summary
Mutations in AGO4 and DRM2 de-repressed LUC expression, to a smaller extent than loss of MET1.
- Taxon
- Arabidopsis thalianaArabidopsis thalianat0
- Treatment
- Mutations in AGO4 and DRM2e3
- Gene
- AGO4 and DRM2e3
- Outcome
- LUC expressione3
- Direction
- de-repressede3
- Qualifier
- to a smaller extent than loss of MET1e3
- Comparator
- loss of MET1e3
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e3“Mutations in AGO4 and DRM2 also de-repressed LUC expression, albeit to a smaller extent than loss of MET1”
Claim · model-written summary
A chemical screen with LUCL identified methotrexate as a compound that de-represses LUC expression.
- Taxon
- Arabidopsis thalianaArabidopsis thalianat0
- Treatment
- chemical screen for compoundse4
- Compound
- methotrexatee4
- Outcome
- LUC expressione4
- Direction
- de-represse4
Not reportedComparator
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e4“we performed a chemical screen for compounds that de-repress LUC expression, and identified a chemical, methotrexate, known to be involved in biogenesis of the methyl donor”
Claim · model-written summary
LUCL reports both RdDM and CG maintenance methylation, with a low basal level of luciferase activity.
- Taxon
- Arabidopsis thalianaArabidopsis thalianat0
- Material
- LUCLe6
- Outcome
- RdDM and CG maintenance methylatione6
- Value
- low basal level of luciferase activitye7
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
t0“Generation of a luciferase-based reporter for CHH and CG DNA methylation inArabidopsis thaliana”
e6“LUCL, which reports both RdDM and CG maintenance methylation in Arabidopsis”
e7“LUCL, a line with a low basal level of luciferase activity, was obtained”
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