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Title:
MOBILISATION OF TRANSPOSABLE ELEMENTS TO ENHANCE GENETIC AND EPIGENETIC VARIABILITY IN A POPULATION
Document Type and Number:
WIPO Patent Application WO/2017/093317
Kind Code:
A1
Abstract:
A method for the mobilization of a transposable element is provided. The method comprises the steps of a) providing an inhibitor of DNA methylation, and/or an inhibitor of transcription, and b) contacting the inhibitor(s) with a cell comprising inactivated transposable elements, yielding a cell with mobilized transposable elements. In a second aspect of the invention a method for increasing the genetic and/or epigenetic variation in a plurality of eukaryotic organisms is provided. The method comprises the steps of i. providing an inhibitor of DNA methylation and/or an inhibitor of transcription, ii. contacting the organism with the inhibitor(s) and iii. propagating the organism.

Inventors:
BUCHER ETIENNE (CH)
THIEME MICHAEL (CH)
Application Number:
PCT/EP2016/079276
Publication Date:
June 08, 2017
Filing Date:
November 30, 2016
Export Citation:
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Assignee:
UNIV BASEL (CH)
International Classes:
C12N15/01; A01H1/06; A01H3/04; A61K31/7068; A61L31/16; C07H19/06
Other References:
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TUNCAY BAUBEC ET AL: "Effective, homogeneous and transient interference with cytosine methylation in plant genomic DNA by zebularine", THE PLANT JOURNAL, vol. 57, no. 3, 1 February 2009 (2009-02-01), GB, pages 542 - 554, XP055271538, ISSN: 0960-7412, DOI: 10.1111/j.1365-313X.2008.03699.x
VLADIMIR V. CAVRAK ET AL: "How a Retrotransposon Exploits the Plant's Heat Stress Response for Its Activation", PLOS GENETICS, vol. 10, no. 1, 30 January 2014 (2014-01-30), pages e1004115, XP055271558, DOI: 10.1371/journal.pgen.1004115
W. MATSUNAGA ET AL: "The effects of heat induction and the siRNA biogenesis pathway on the transgenerational transposition of ONSEN, a copia-like retrotransposon in Arabidopsis thaliana", PLANT AND CELL PHYSIOLOGY, vol. 53, no. 5, 14 December 2011 (2011-12-14), UK, pages 824 - 833, XP055271560, ISSN: 0032-0781, DOI: 10.1093/pcp/pcr179
MARJORI A. MATZKE ET AL: "RNA-directed DNA methylation: an epigenetic pathway of increasing complexity", NATURE REVIEWS GENETICS, vol. 15, no. 6, 8 May 2014 (2014-05-08), GB, pages 394 - 408, XP055271562, ISSN: 1471-0056, DOI: 10.1038/nrg3683
WATARU MATSUNAGA ET AL: "Role of RNA polymerase IV in plant small RNA metabolism.", FRONTIERS IN PLANT SCIENCE, vol. 104, no. e129, 9 February 2015 (2015-02-09), pages 4536, XP055271567, DOI: 10.1073/pnas.0611456104
BUCHER ETIENNE ET AL: "Epigenetic control of transposon transcription and mobility inArabidopsis", CURRENT OPINION IN PLANT BIOLOGY, vol. 15, no. 5, 2012, pages 503 - 510, XP028958933, ISSN: 1369-5266, DOI: 10.1016/J.PBI.2012.08.006
S. VISPE ET AL: "Triptolide is an inhibitor of RNA polymerase I and II-dependent transcription leading predominantly to down-regulation of short-lived mRNA", MOLECULAR CANCER THERAPEUTICS, vol. 8, no. 10, 1 October 2009 (2009-10-01), US, pages 2780 - 2790, XP055272275, ISSN: 1535-7163, DOI: 10.1158/1535-7163.MCT-09-0549
H. SAZE ET AL: "DNA Methylation in Plants: Relationship to Small RNAs and Histone Modifications, and Functions in Transposon Inactivation", PLANT AND CELL PHYSIOLOGY, vol. 53, no. 5, 1 February 2012 (2012-02-01), UK, pages 766 - 784, XP055272283, ISSN: 0032-0781, DOI: 10.1093/pcp/pcs008
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Attorney, Agent or Firm:
SCHULZ JUNGHANS PATENTANWÄLTE PARTGMBB (DE)
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Claims:
Claims

1. A method for the mobilization of a transposable element in a eukaryotic cell, said method comprising the steps of:

a) providing a eukaryotic cell comprising a transposable element

b) contacting said cell with an an inhibitor of transcription,

thereby yielding a eukaryotic cell with a mobilized transposable element.

2. The method according to claim 1 , wherein the cell is additionally contacted with an inibitor of DNA methylation in step b).

3. The method according to claim 1 or 2, wherein the inhibitor of DNA methylation and/or the inhibitor of transcription is an exogenous compound, particularly an exogenous small molecule compound.

4. The method according to any one of the preceding claims, additionally comprising a step c):

c) exposing said cell to an abiotic stress, biotic stress or chemical stress.

5. The method according to any one of the preceding claims, wherein the method comprises a subsequent step of isolating said cell and determining whether a phenotype of said cell has been changed.

6. The method according to any one of claims 4 to 5, wherein said eukaryotic cell is part of a multicellular organism, particularly a non-human organism, particularly a plant, and wherein subsequent to exposure of said cell to step c), the cell is cultivated to render a multicellular organism, and the phenotype of said multicellular organism, in particular a resistance to a stressor, wherein the stressor causes the stress applied in step c), is determined.

7. The method according to claim 6, wherein said plant cell is part of crop plants, particularly rice, sugar cane, maize, wheat, rye, barley, oat or millet.

8. A method for increasing genetic and/or epigenetic variation in a population of eukaryotic organisms comprising:

i. providing an eukaryotic organism,

ii. contacting said eukaryotic organism with

an inhibitor of DNA methylation, and/or

an inhibitor of transcription,

iii. propagating said eukaryotic organism, yielding a population of eukaryotic organisms with increased genetic and/or epigenetic variation.

9. The method according to claim 8, additionally comprising a step ii.a following step ii.: ii.a exposing said eukaryotic organism to an abiotic stress, biotic stress or chemical stress.

10. The method according to any one of claims 7 or 8, wherein the method comprises the subsequent step iv. comprising:

a. determining any genetic changes in said organism, and/or

b. determining any changes in the phenotype of said organism, particularly an increased resistance to a stressor applied in step ii.a

wherein said genetic or phenotypical changes are determined in the individual constituent eukaryotic organisms or for a representative sample of the population of eukaryotic organisms, or for all of the constituent eukaryotic organisms of the population.

1 1 . The method according to any one of the preceding claims 4 to 7, wherein

i. said abiotic stress is selected from heat, cold, drought, submergence/ water excess, wind, UV-radiation, nuclear radiation, salinity, heavy metals, soil pH, tissue culture cultivation and starvation of phosphorous, nitrogen, light or C02, and/or

ii. said biotic stress is selected from the negative impact of fungi, bacteria, viruses, insects, wounding by herbivores and biological competition, and/or iii. said chemical stress is selected from herbicides, herbicide safener, insecticides, fungicides, plant secondary metabolites and synthetic or natural compounds that induce plant defense, in particular flagellin, more particularly flg22.

12. The method according to any one of the preceding claims, wherein said inhibitor of DNA-methylation is selected from 5-azacytidine, 5-aza-2'-deoxycytidine, 5-fluoro-2'- deoxycytidine, 5,6-dihydro-5-azacytidine and zebularine.

13. The method according to any one of the preceding claims, wherein said inhibitor of transcription is an RNA polymerase inhibitor, in particular an RNA polymerase II inhibitor, an RNA polymerase IV inhibitor or an RNA polymerase V inhibitor, more particular an RNA polymerase II inhibitor.

14. The method according to claim 13, wherein said RNA polymerase II inhibitor is selected from

i. amatoxins, in particular alpha-amanitin,

ii. derivatives of amatoxins, in particular alpha-amanitin oleate, iii. nucleoside analogues, in particular 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole (DRB),

iv. actinomycin D,

v. flavopiridol,

vi. triptolide.

15. The method according to claim 14, wherein said amatoxin, in particular alpha- amanitin is used at a concentration of 0.0005 pg/ml to 50 pg/ml, in particular 0.001 pg/ml to 25 pg/ml, more particular 0.005 pg/ml to 20 pg/ml, even more particular 0.005 pg/ml to 5 pg/ml.

16. The method according to any one of the preceding claims, wherein said inhibitor of

DNA-methylation, in particular zebularine, is used at a concentration of 5 μΜ to 100 μΜ, in particular 10 μΜ to 80 μΜ, more particular 10 μΜ to 40 μΜ, even more particular 20 μΜ to 40 μΜ.

17. The method according to any one of claims 8 to 16, wherein said increased genetic and/or epigenetic variation in a plurality of eukaryotic organisms results in increased resistance of said organisms to said abiotic, biotic or chemical stress said organisms have been exposed to.

18. Use of a composition in a method according to claims 1 to 17, wherein the composition comprises an inhibitor of transcription and an inhibitor of DNA- methylation, and wherein the ratio of the molar concentrations of the inhibitor of transcription, in particular alpha-amanitin, to the inhibitor of DNA-methylation, in particular zebularine, is 0.000005 to 1 1 , more particular 0.000125 to 2, even more particular 0.000125 to 0.125.

19. The use of a composition according to claim 18, wherein

- said inhibitor of DNA-methylation, in particular zebularine, is used at a concentration of 5 μΜ to 100 μΜ, in particular 10 μΜ to 80 μΜ, more particular 10 μΜ to 40 μΜ, even more particular 20 μΜ to 40 μΜ

and / or

- said amatoxin, in particular alpha-amanitin is used at a concentration of 0.0005 pg/ml to 50 pg/ml, in particular 0.001 pg/ml to 25 pg/ml, more particular

0.005 pg/ml to 20 pg/ml, even more particular 0.005 pg/ml to 5 pg/ml.

Description:
Mobilisation of Transposable Elements to Enhance Genetic and Epigenetic Variability in a Population

Field of the invention

The present invention relates to the mobilisation of transposable elements and related uses thereof.

Background of the invention

Transposable elements (TEs) were initially discovered in the early 1950s by Barbara McClintock due to their mutagenic activity that could influence kernel pigmentation (variegation) in maize (McClintock, PNAS, 1950 36(6):344-55). Since their initial discovery numerous functions have been attributed to TEs. Indeed, TEs now tend to be viewed as natural molecular tools that can reshape the genome (Bire et al., Methods Mol Biol, 2012; 859:1 -28). TEs have been identified in playing important (if not major) roles in structuring host genomes; especially centromeric regions are rich in TEs. The copy number of long terminal repeats (LTR) retrotransposons has been found to strongly correlate with host genome size and mobilization of TEs can have an impact on genome organization by inducing chromosome breakage and by influencing homologous recombination. At the gene level, TEs can have multiple effects: Cause mutations by directly inserting into genes, move genes within the genome, duplicate and/or create novel genes, regulate gene expression, create novel regulatory pathways and bring genes under epigenetic control. Currently, TEs are considered as a mutagen that can accentuate the positive outcome of the mutagenesis to the host (Bennetzen et al, Annu Rev Plant Biol, 2014, 65:505-30).

TEs have proven to be very useful genetic tools and have been broadly exploited for gene disruption and transgenesis in a wide variety of organisms. However, because TEs naturally very rarely get activated under normal growth conditions only few active TEs are currently known. Thus only a very limited number of TEs are actively being used for genetic modification. Some examples include P elements (Drosophila), PiggyBac (insects, human cell lines), L1 LINE elements (mouse), Mariner (vertebrates), SleepingBeauty (animals). In order to create genetic diversity, these TEs are introduced into the organisms of interest via transgenesis. However, this limits use of organisms modified in such a manner because they are considered as genetically modified organisms (GMOs) by current legislation.

In plants, it has been demonstrated that the mobility of transposable elements is limited by DNA methylation and certain histone marks (Miura et al., Nature, 2001 , 41 1 (6834):212-4; Mirouze et al., Nature, 2009, 461 (7262):427-30). Suppression of DNA methylation in genetic mutants can therefore result in the mobilization of transposable elements. It was also shown that drugs that reduce DNA methylation (e.g. 5-aza-2'-deoxycytidine) can mobilize certain DNA TEs (Scortecci et al., Plant Cell Physiol, 1997, 38(3):336-43). Furthermore, it has been reported that stresses on plants defective in RNA-directed DNA methylation (RdDM) activate transposable elements (Ito, H. et al., Nature, 201 1 , 472:1 15-19). However, the requirement of genetic mutants in components involved in the defense against TEs is limiting the possibility to activate TEs in non-model organisms or organisms that are difficult to transform. Therefore, the exploitation of endogenous TEs to obtain genetic and epigenetic diversity is currently very limited.

Under normal growth conditions, TEs are very rarely mobilized and different treatments to activate TEs have so far been very inefficient in eukaryotes. Treatments with drugs that reduce genomic DNA methylation levels have been shown to allow mild activation of TEs (Baubec, T. et al., Plant J, 2009 57:542-54), but without resulting in novel insertions of those TEs. It has been shown in plants that mutations in factors involved in the RNA-directed DNA methylation pathway could mobilize TEs at a high frequency. An important limitation in these approaches is that they are either inefficient (aforementioned drug treatment) or they require genetic mutations that are difficult to obtain, especially in non-model organisms. These technical problems therefore limit the use and the study of transposable elements in most organisms.

The problem underlying the present invention is to provide the means for efficient mobilization of transposable elements. This problem is solved by the subject-matter of the independent claims.

Specific description of the invention

The inventors provide herein a drug-based treatment that can mobilize transposable elements in eukaryotes. Additionally, the combination of this treatment with specific stresses leads to the mobilization of specific TEs that respond to this particular stress. The treatment leads to a high accumulation of extrachromosomal DNA of the activated TEs in the treated organism. Furthermore, the progeny of the treated organism shows stable integration of a high number of TE copies in the genome and increased resistance to the stress that is part of the treatment. Therefore, the method of the invention overcomes the necessity of genetic mutations to inactivate TE defense, thus allowing transposable elements to be efficiently activated in virtually any eukaryote. This invention enables the induction of TE mediated changes in genome size and structure, modulation of endogenous gene expression, gene transduplication, heterosis, homologous recombination and stress adaptation. Furthermore, this invention allows the identification of novel functional TEs. According to a first aspect of the invention a method for the mobilization of a transposable element, particularly within the genome, of a eukaryotic cell is provided. The method comprises:

a) providing a eukaryotic cell comprising one or several dormant, i.e. inactive, transposable elements, and

b) contacting the cell(s) with an inhibitor of transcription,

and optionally, contacting the cell addtionally with an inhibitor of DNA- methylation,

thereby yielding a eukaryotic cell with one or several mobilized transposable elements.

In the context of the present specification the terms transposable element or transposons are used in their meaning known in the art of molecular genetics; they refer to DNA sequences in the genome of an organism that are able to change their position within the genome (cut and paste mechanism) or being able to produce novel copies of themselves that integrate into the genome (copy and paste mechanism). Transposition can result in multiplication of the element thereby influencing the size of the genome. There are two classes of transposons, class 1 transposons also referred to as retrotransposons and class 2 transposons also referred to as DNA transposons. Retrotransposons are first transcribed into RNA by the molecular apparatus provided by the host cell, and are then reverse transcribed into a double stranded DNA copy of the RNA, termed complementary DNA (cDNA) before they are inserted at a new position into the genome. They share some characteristics such as the dependency on a reverse transcriptase with retroviruses. DNA transposons do not have a RNA intermediate and are transferred to their new position in the genome by a transposase. The majority of transposons in the genome are inactive and will not duplicate or change position. The activation of transposons is therefore also referred to as mobilization of transposons. Examples of transposons that are responsive to certain stresses are provided in Table 1 . These transposons are activated by the indicated stress up to a certain degree. However, use of the method of the invention mobilizes these transposons to a much larger extent as can be seen in the examples provided.

In certain embodiments of any aspect of the invention, a class 1 transposon is mobilized. In certain embodiments of any aspect of the invention, a class 2 transposon is mobilized.

In the context of the present specification the term DNA methylation is used in its meaning known in the art of molecular biology and molecular genetics; it refers to the addition of methyl groups to the DNA, which in eukaryotes occurs mainly on cytosines. Methylation of DNA is catalyzed by DNA methyltransferases (DNMT) and can be divided into maintenance methylation, which is necessary to transfer methylation patterns on newly synthesized DNA strands, and de novo methylation. DNA-methylation is associated with the inactivation of gene expression and the silencing of transposons. DNA methylation can be passed on to following generations and therefore represents a common form of epigenetic modification.

Table 1 : Examples of transposons

Transposable Activating stress Organism Reference element heat, flagellin Ito et al., 201 1 ,

ONSEN Arabidopsis thaliana Nature; Yu et al., 2012, PNAS salicylic acid, abscisic acid, methyl Salazar et al.,

TLC1.1 jasmonate, hydrogen peroxide Solanum chilense 2007, Plant Cell and the synthetic auxin 2,4-D. wounding, biotic elicitors and Melayah et al.,

TntIA pathogen attacks of fungal Nicotiana tabacum 2001 , Plant extracts Journal

Erikal heat, drought and wounding Alzohairy, et al.,

Hordeum vulgare 2012; Life

Sabrina cell culture

Science Journal

Butelli et al.,

Tcs1 cold Citrus sinensis

2012; Plant Cell In certain embodiments, the inhibitor of DNA methylation is an exogenous compound.

In certain embodiments, the inhibitor of transcription is an exogenous compound.

In certain embodiments, the exogenous compound is a small molecule compound having a molecular mass of <1000u, particularly <920u.

In the context of the present specification the term exogenous compound refers to molecules that are not present in the cell under physiological conditions unless added technically.

In certain embodiments, the inhibitor of DNA methylation might be present in at least some of the cells under at least some particular physiological conditions in trace amounts, but is added in the method of the invention at much higher concentrations to exert a significant impact on cell physiology. To achieve this, the compound is present in the cell's medium at a concentration being selected to be at least 10 times higher than the concentration of the inhibitor of DNA methylation found in the interior of the cell.

In certain embodiments, the inhibitor of transcription is present in the cell under physiological conditions and present in a medium at a concentration being selected to be at least 10 times, 100 times, 1000 times, or even 10.000 times higher than the concentration of the inhibitor of transcription found in the interior of the cell.

In certain embodiments, the method of the invention as specified in any aspect or embodiment disclosed herein additionally comprises a step c):

c) exposing the cell to an abiotic stress, biotic stress or chemical stress.

In the context of the present specification the term abiotic stress refers to the negative impact of non-living factors on a living organism in a specific environment. The non-living variable influences the environment beyond its normal range of variation. Non-limiting examples of abiotic stress are heat, cold, drought, submergence/ water excess, wind, UV-radiation, nuclear radiation, salinity, heavy metals, soil pH, tissue culture cultivation and starvation of phosphorous, nitrogen, light, C0 2 etc.. In contrast the term biotic stress refers to the negative impact of fungi, bacteria, viruses, insects, wounding by herbivores and biological competition etc..

The term chemical stress refers to the negative impact of chemical substances ("stressors") on a living organism. These substances may also comprise substances that are stress- mimicking substances that mimic an abiotic or biotic stress. Non-limiting examples of chemical stressors are herbicides, herbicide safener, insecticides, fungicides, plant secondary metabolites, synthetic or natural compounds that induce plant defense.

The term herbicide safener refers to a compound that selectively protects monocotyledonous plants from herbicide damage whereas dicotyledonous plants are still affected by the herbicides. The common crop plants such as rice, wheat, maize etc but also forage grass, sugar cane and bamboo are monocotyledonous plants whereas most weed species are dicotyledonous plants. Herbicide safeners can be applied as a dressing for the seeds before sowing, to prepare the soil of agricultures or be applied to the foils of grown plants. In the two latter cases herbicide safeners can be applied together with the herbicides. Examples of common herbicide safeners are: Benoxacor (CAS 98730-04-2), Cloquintocet-mexyl (CAS 99607-70-2), Cyometrinil (CAS 63278-33-1 ), Dichlormid (CAS 37764-25-3), Fenchlorazole- ethyl (CAS 1031 12-35-2), Fenclorim (CAS 3740-92-9), Flurazole (CAS 72850-64-7), Fluxofenim (CAS 88485-37-4), Furilazole (CAS 121776-33-8), Mefenpyr-diethyl (CAS 135590-91-9), MG 191 (CAS 96420-72-3), Naphthalic anhydride (CAS 81 -84-5), MON-13900 (CAS 121776-33-8), LAB 145138 (CAS 79260-71 -2) and Oxabetrinil (CAS 74782-23-3). In certain embodiments, the transposable element is a retrotransposon.

In certain embodiment, the cell is part of a multicellular organism. In certain embodiments, the eukaryotic cell is part of a non-human organism.

In certain embodiments, the eukaryotic cell is a plant cell. In certain embodiments, the plant cell is a cell from Arabidopsis, particularly Arabidopsis thaliana.

In certain embodiments, the plant cell is part of crop plants, particularly of the family of Poaceae that comprises plants such as rice, sugar cane, maize, wheat, rye, barley, oat or millet. In certain embodiments, the method comprises a subsequent step of isolating said cell and determining whether a phenotype of the cell has been changed.

In certain embodiments, the eukaryotic cell is part of a multicellular organism, particularly a plant, and wherein subsequent to exposure of the cell to step c), the cell is cultivated to render a multicellular organism, and the phenotype of the multicellular organism is determined.

In certain embodiments, the phenotype of the organism comprises determining resistance to the stressor, wherein the stressor causes the stress applied in step c).

In certain embodiments, the resistance to the stressor that causes the stress applied in step c) is increased after application of the method of the invention.

According to a second aspect of the invention a method for increasing genetic and/or epigenetic variation in a population of eukaryotic organisms is provided. The method comprises:

i. providing an eukaryotic organism,

ii. contacting the eukaryotic organism with

o an inhibitor of DNA methylation, and/or

o an inhibitor of transcription,

iii. propagating the eukaryotic organism, yielding the eukaryotic population with increased genetic and/or epigenetic variation.

The method mobilizes dormant, i.e. inactive, not currently transcribed or reverse transcribed, transposable elements within the eukaryotic organism. Since to the knowledge of the inventors, all eukaryotic organisms comprise dormant transposable elements within their genome, the element "eukaryotic organism" is synonymous with "eukaryotic organism comprising a dormant transposable element".

In certain embodiments, the method is employed on a eukaryotic organism comprising any one of the specific transposable elements recited in the current specification. In certain embodiments of the second aspect of the invention, the method additionally comprises a step ii.a, which is following step ii.:

ii.a exposing the eukaryotic organism to an abiotic stress, biotic stress or chemical stress.

In certain embodiments, the inhibitor of DNA methylation and/or the inhibitor of transcription are provided as a solution in a polar solvent, in particular a polar aprotic solvent, more particularly Dimethyl sulfoxide (DMSO).

In certain embodiments, the inhibitor of DNA methylation and/or the inhibitor of transcription are provided as a solution in a polar solvent, in particular water.

In certain embodiments, the method comprises the subsequent step iv. comprising:

a. Determining any genetic and/or epigenetic changes or

b. Determining any changes in the phenotype, particularly the resistance to any stressors applied in step ii.a

wherein these changes are determined in the individual constituent eukaryotic organisms or for a representative sample of the population of eukaryotic organisms, or for all of the constituent eukaryotic organisms of the population.

In certain embodiments of the first and the second aspect of the invention, the abiotic stress is selected from heat, cold, drought, submergence/ water excess, wind, UV-radiation, nuclear radiation, salinity, heavy metals, soil pH, tissue culture cultivation and starvation (phosphorous, nitrogen, light, C0 2 etc.).

In certain embodiments of the first and the second aspect of the invention, the biotic stress is selected from the negative impact of fungi, bacteria, viruses, insects, wounding by herbivores and biological competition. Non-limiting examples of fungi having a negative impact would be Phytophthora infestans (potato blight) and Magnaporthe grisea (rice blast). Non-limiting examples for bacteria having a negative impact are Botrytis cinerea (gray mold), Xylella fastidiosa (Olive Quick Decline Syndrome) and Puccinia spp. (wheat rust). Non-limiting examples of viruses having a negative impact are Tobacco mosaic virus and Tomato spotted wilt virus. Non-limiting examples for insects having a negative impact are Mamestra brassicae (Cabbage moth), Helicoverpa zea (corn earworm) and Ostrinia nubilalis (European corn borer). Non limiting examples of other organisms that can have a negative impact due to biological competition are Orobanche (broomrape) and Ambrosia trifida (giant ragweed).

In certain embodiments of the first and second aspect of the invention, the chemical stress is selected from herbicides, herbicide safener, insecticides, fungicides, plant secondary metabolites, synthetic or natural compounds that induce plant defense. Non-limiting examples of compounds that induce plant defense are flagellin (natural compound, bacterial elicitor; Felix et al., 1999, Plant J.), a 22-amino acid sequence of the conserved N-terminal part of flagellin (flg22), salicylic acid and analogues e.g. Bion® (natural compound with synthetic analogues; (Vlot et al., 2009, Annu. Rev. Phytopathol.;Friedrich et al., 1996, Plant J.)), jasmonic acid and jasmonic methyl ester (natural compounds; Cohen et al., 1993, Phytopathology), ethylene (natural compound; van Loon et al., 2006, Trends Plant Sci.), abscisic acid (natural compound; Mauch-Mani and Mauch, 2005, Curr. Opin. Plant Biol.) and volatiles such as terpenes and green leaf volatiles (natural compounds; reviewed by Unsicker et al., 2009, Curr Opin Plant Biol).

In certain embodiments of the first and the second aspect of the invention, the DNA- methylation inhibitor is a nucleoside analogue.

In certain embodiments of the first and the second aspect of the invention, the DNA- methylation inhibitor is selected from 5-azacytidine, 5-aza-2'-deoxycytidine, 5-fluoro-2'- deoxycytidine, 5,6-dihydro-5-azacytidine and zebularine.

In certain embodiments of the first and the second aspect of the invention, the inhibitor of transcription is a RNA polymerase inhibitor, in particular a RNA polymerase II inhibitor, a RNA polymerase IV inhibitor or a RNA polymerase V inhibitor, more particularly a RNA polymerase II inhibitor.

In certain embodiments of the first and the second aspect of the invention, the RNA polymerase II inhibitor is selected from

- amatoxins, in particular alpha-amanitin (CAS 23109-05-9),

- derivatives of amatoxins, in particular alpha-amanitin oleate,

- nucleoside analogues, in particular 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole (DRB; CAS 53-85-0),

- actinomycin D (CAS 50-76-0),

- flavopiridol (CAS 146426-40-6),

- triptolide (CAS 38748-32-2).

In certain embodiments of any aspect of the invention disclosed herein, the amatoxin, in particular alpha-amanitin is used with a concentration of 0.0005pg/ml to 50pg/ml, in particular 0.001 pg/ml to 25pg/ml, more particular 0.005pg/ml to 20pg/ml, even more particular 0.005 pg/ml to 5 pg/ml.

In certain embodiments of any aspect of the invention disclosed herein, the inhibitor of DNA methylation, in particular zebularine, is used at a concentration of 5 μΜ to 100 μΜ, in particular 10 μΜ to 80 μΜ, more particular 10 μΜ to 40 μΜ, even more particular 20 μΜ to 40 μΜ.

In certain embodiments of the second aspect of the invention, the increased genetic and/or epigenetic variation in a plurality of eukaryotic organisms results in increased resistance of the organisms to the abiotic or biotic stress the organisms have been exposed to. In other words the increase in genetic and/or epigenetic variation is not random as for example would be expected from a chemical mutagen. The increase is directed toward resistance against the stress used in the method. For example using the abiotic stress heat would preferentially result in heat-resistant organisms. Without wishing to be bound by theory the inventors assume that transposons are preferentially integrated into the genome in the vicinity of genes thereby creating novel gene regulatory pathways that are able to respond to the previously applied stress. This may lead to genetic variety in genes activated by the respective stress and thereby confers increased resistance to the respective stress.

According to a third aspect of the invention, the use of a composition in a method according to the first and second aspect of the invention is provided. The composition comprises an inhibitor of DNA-methylation and an inhibitor of transcription.

In certain embodiments, the DNA-methylation inhibitor is a nucleoside analogue.

In certain embodiments, the DNA-methylation inhibitor is selected from 5-azacytidine (CAS 320-67-2), 5-aza-2'-deoxycytidine (CAS 2353-33-5), 5-fluoro-2'-deoxycytidine (CAS 10356- 76-0), 5,6-dihydro-5-azacytidine (CAS 62488-57-7) and zebularine (CAS 3690-10-6).

In certain embodiments, the inhibitor of transcription is a RNA polymerase inhibitor, in particular a RNA polymerase II inhibitor, a RNA polymerase IV inhibitor or a RNA polymerase V inhibitor, more particularly a RNA polymerase II inhibitor.

In certain embodiments, the RNA polymerase II inhibitor is selected from

- amatoxins, in particular alpha-amanitin (CAS 23109-05-9),

- derivatives of amatoxins, in particular alpha-amanitin oleate,

- nucleoside analogues, in particular 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole (DRB; CAS 53-85-0),

- actinomycin D (CAS 50-76-0),

- flavopiridol (CAS 146426-40-6),

- triptolide (CAS 38748-32-2).

In certain embodiments, the amatoxin, in particular alpha-amanitin is used with a concentration of 0.5 nM to 55 μΜ, in particular 1 nM to 27.5 μΜ, more particular 5 nM to 20 μΜ, even more particular 5 nM to 5 μΜ.

In certain embodiments of any of the aspects of the invention disclosed herein, the ratio of the molar concentrations of the inhibitor of transcription, in particular alpha-amanitin, to the inhibitor of DNA-methylation, in particular zebularine, is 0.000005 to 1 1 , more particular 0.000125 to 2, even more particular 0.000125 to 0.125. In certain embodiments, the ratio of the molar concentration depends on the concentrations a and b, which are as follows:

a) the inhibitor of DNA-methylation, in particular zebularine, is used at a concentration of 5 μΜ to 100 μΜ, in particular 10 μΜ to 80 μΜ, more particular 10 μΜ to 40 μΜ, even more particular 20 μΜ to 40 μΜ

b) amatoxin, in particular alpha-amanitin is used at a concentration of 0.0005 pg/ml to 50 pg/ml, in particular 0.001 pg/ml to 25 pg/ml, more particular 0.005 pg/ml to 20 pg/ml, even more particular 0.005 pg/ml to 5 pg/ml.

A fourth aspect of the invention provides a kit of parts for use in the method according to the first and second aspect of the invention. The kit of parts comprises an inhibitor of DNA- methylation and an inhibitor of transcription.

In certain embodiments, the DNA-methylation inhibitor is a nucleoside analogue.

In certain embodiments, the DNA-methylation inhibitor is selected from 5-azacytidine (CAS 320-67-2), 5-aza-2'-deoxycytidine (CAS 2353-33-5), 5-fluoro-2'-deoxycytidine (CAS 10356- 76-0), 5,6-dihydro-5-azacytidine (CAS 62488-57-7) and zebularine (CAS 3690-10-6).

In certain embodiments, the inhibitor of transcription is a RNA polymerase inhibitor, in particular a RNA polymerase II inhibitor, a RNA polymerase IV inhibitor or a RNA polymerase V inhibitor, more particularly a RNA polymerase II inhibitor.

In certain embodiments the RNA polymerase II inhibitor is selected from

- amatoxins, in particular alpha-amanitin (CAS 23109-05-9),

- derivatives of amatoxins, in particular alpha-amanitin oleate,

- nucleoside analogues, in particular 5,6-dichloro-1-beta-D- ribofuranosylbenzimidazole (DRB; CAS 53-85-0),

- actinomycin D (CAS 50-76-0),

- flavopiridol (CAS 146426-40-6),

- triptolide (CAS 38748-32-2).

Wherever alternatives for single separable features such as, for example, a type of inhibitor or organism are laid out herein as "embodiments", it is to be understood that such alternatives may be combined freely to form discrete embodiments of the invention disclosed herein.

The invention is further illustrated by the following examples and figures, from which further embodiments and advantages can be drawn. These examples are meant to illustrate the invention but not to limit its scope.

Short description of the figures Fig. 1 shows accumulation of ONSEN extrachromosomal DNA upon pharmacological treatment and heat stress, (a) ONSEN DNA accumulation measured by qPCR directly after control stress (CS) heat stress (HS)-treatment in wild-type (WT) and nrpb2-3 plants and treatments with alpha-amanitin (A, 5 pg/ml) or zebularine (Z, 10 μΜ) (mean ± s.e.m., n=6 biological repetitions, values relative to ACTIN2). (b) ONSEN copy number measured by quantitative PCR (qPCR) in seedlings of Columbia (Col) WT directly after control stress (CS; 24h 6°C), heat stress (HS; 24h 6°C and 24h 37°C) and a treatment with A (5 pg/ml), Z (40 μΜ) or a combination thereof (A&Z). (Mean ± s.e.m., n= 3 biological repetitions). The double treatment (A&Z) leads to a very strong heat-stress dependent activation of ONSEN resulting in up to 700 extrachromosomal ONSEN DNA copies.

Fig. 2 shows the stress-dependence of ONSEN mobilisation. The graph shows ONSEN copy numbers in A. thaliana seedlings after chemical treatment with A (5 pg/ml), Z (40 μΜ), the combinations of A and Z (A&Z) in WT, nrpb2-3 and nrpdl plants following the CS. ONSEN copy number measured by qPCR (mean s.e.m., n=3 biological replicates, values relative to ACTIN2). This result shows that the production of ONSEN extrachromosomal DNA is dependent on heat-stress.

Fig. 3 shows that simultaneous inhibition of methyltransferases and Pol II mimics the nrpdl- mutant. (a) Asymmetric methylation analysis of the ONSEN LTR and the soloLTR in untreated and A (5 pg/ml), Z (40 μΜ) or A&Z-treated seedlings of the WT and the nrpdl mutant after CS. PCR products obtained from genomic DNA that was used undigested (input) or after digestion with the CHH-methylation sensitive restriction enzyme Dde\. ACTIN2 is included as a control for complete Dde\ digestion. The A&Z double treatment with A (5 pg/ml) and Z (40 μΜ) resulted in a very strong reduction of DNA methylation at ONSEN and soloLTR comparable to the nrpdl mutant, (b) Northern blot indicating ONSEN- transcription directly after CS, HS and HS plus treatment with A (5 pg/ml), Z (40 μΜ) or a combination of A&Z in WT and nrpdl plants. A Midori-stained agarose-gel is shown as a loading control. The level of the full length ONSEN transcript after heat stress and the double treatment with A (5 pg/ml) and Z (40 μΜ) is comparable to the nrpc 7-mutant. (c) Accumulation of ONSEN DNA measured by qPCR in seedlings of WT, rdr6, dcl2/3/4 and nrpdl plants directly after CS, HS and HS plus treatment with A, Z or a combination of A&Z. This result shows that RNA pol II is active upstream of the DICER-like enzymes.

Fig. 4 shows the drug-induced mobilisation of ONSEN in wild-type Arabidopsis plants, (a) Transposon display confirming novel ONSEN insertions in the F2 generation of HS (HS control) and HS and A (5 pg/ml) and Z (40 μΜ) treated WT plants (HS+A&Z). Integrated ONSEN copies were measured by qPCR (upper part) and detected by transposon display (lower part). ONSEN copy numbers of seven selected individual, non-related plants are depicted. Copy numbers exceeding eight as measured by qPCR (upper part) and the observed additional bands on the transposon display (lower part) in the HS+A&Z treated Col WT plants indicate novel insertions of additional ONSEN copies. M is a 1 kb size marker, (b), ONSEN copy number in the F1 , F2 and F3 generation measured by qPCR (n=3 technical replicates, values relative to ACTIN2) Copy numbers >8 in lines 1 -7 indicate insertions of additional ONSEN copies, c, and d, photographs of F2-plants containing novel ONSEN insertions showing both homogeneous and stress-dependent phenotypic variability induced by the HS+A&Z treatment when grown under long (c) and short day conditions (d). qPCR- Data for the F3-generation of line 6 in (b) as well as pictures of phenotypes in (c) and (d) are missing due to severe infertility and extinction of this line. Examples for phenotypes observed in of lines with novel ONSEN insertions (lines1 -7) include higher biomass under short day conditions (line 3), early flowering under long day conditions (line 7) and reduced chlorophyll accumulation (line 1 ). In summary this dataset shows that A&Z-treatment leads to an efficient burst of ONSEN transposition. New ONSEN insertions are stably inherited over several generations and cause phenotypic changes.

Fig. 5 shows the increase in ONSEN copy numbers in F1 pools of heat-stressed and treated plants. Parental plants were treated and heat stressed in independent experiments (characters a-c) with a combination of A (5 pg/ml) and Z (40 μΜ). Pools with significantly increased ONSEN-copy numbers (>10) are highlighted in dark grey. ONSEN copy number measured by qPCR (mean ± s.e.m., n=3 technical repetitions, values relative to ACTIN2). Approximately 29.4% of tested F1 pools of heat stressed and A+Z treated wild type plants showed a significantly increased ONSEN copy number.

Fig. 6 shows a summary of confirmed novel ONSEN insertions, (a) Genome wide distribution and (b) Close-up of regions with new ONSEN insertions in the F2 generation of a selected HS+A&Z treated WT plant (line #3). Orientation of the ONSEN insertions is indicated central arrows.

Fig. 7 shows the drug-induced activation of the Houba retrotransposon in rice (O. sativa). Mobilome analysis of DNA extracted from seedlings after growth on control conditions (C), A (5 pg/ml), Z (40 μΜ), and the combination of A&Z. (a) Detail of the normalized depth of coverage compared to the untreated control plant obtained after aligning the sequenced reads on one Houba element, (b) Scheme of primers localization (black bar. Houba element, arrows: PCR primers, box: LTR). (c) extrachromosomal circular forms of Houba are specifically detected in plants treated with both A&Z using inverse PCR with primers shown in 4b. (d) Specific PCR on circular chloroplast DNA is shown as a loading control. Total DNA subjected to a rolling circle amplification was used as a template. These results demonstrate the efficient A&Z-dependent mobilization of the Houba transposon in rice. Fig. 8 shows increased heat tolerance in the F2 generation of treated Arabidopsis seedlings. Tolerance to repetitive heat stress (42°C) in the F2 progeny of wildtype plants that were either only heat stressed (control) or heat stressed and treated with A (5pg/ml) and Z (40 μΜ) (#1-3). (a) Two biological replicates (I and II) are depicted, (b) Percentage of vital seedlings (Mean ± s.e.m., n=2 biological repetitions). F2 seedlings originating from Heat stressed and A&Z-treated plants show a significantly increased heat tolerance (>60% vital seedlings) compared to the F2 of a plant that was only heat stressed (10% vital seedlings). This demonstrates that the A&Z-dependent mobilization of a heat-stress responsive transposon can produce plants that are better adapted to heat stress.

Fig. 9 shows dose dependent accumulation of ONSEN extrachromosomal DNA upon pharmacological treatment and heat stress. ONSEN copy number was measured by qPCR in seedlings of Columbia (Col) wildtype directly after control stress (CS), heat stress (HS) and a treatment with a-amanitin in different concentrations given in pg/ml. (Mean ± s.e.m., n=3 technical repetitions). This shows that the number of mobilized transposons can be regulated by the amount of A used for the treatment.

Fig. 10 shows epigenetic changes at the DNA methylation level induced by the treatment of plants and human cells with A. (a) Midori stained Agarose gel showing reduction of DNA- methylation at the ONSEN LTR upon pharmacological treatment and heat stress in WT (Col) seedlings directly after control stress (CS; 24h 6°C), heat stress (HS; 24h 6°C and 24h 37°C) and a treatment with A (20 pg/ml), Z (10 μΜ) or a combination thereof (A&Z). Undigested DNA was used as a PCR-template for the loading control (Input). PCR on Dc el-digested DNA shows reduction in DNA-methylation after treatment with A, Z or a combination of A&Z. (b) CHH methylation state at the ONSEN LTR assessed by bisulfite sequencing performed on CS plants grown on medium with or without A. (c) LINE-1 DNA methylation levels assessed in human A549 cancer cells grown in control medium and medium supplemented with 0.5 pg/ml A. This shows that A can be used as a potent DNA demethylating agent in plants and human cells.

Fig. 1 1 shows accumulation of ONSEN extrachromosomal DNA upon combined pharmacological and flagellin-treatment. ONSEN copy number measured by quantitative PCR (qPCR) in seedlings of Col wild type directly after control stress, 5h after treatment with flagellin (flg22) alone or in combination with A (5 pg/ml), Z (40 μΜ) or a combination thereof (A&Z). (Mean ± s.e.m., n=3 technical repetitions).

Fig. 12 shows activation of ATCOPIA 17 upon combined pharmacological and flagellin- treatment. A TCOPIA 17 fold change was measured by quantitative PCR (qPCR) on total DNA in seedlings of the Col wild type directly after control stress, 5h after treatment with flagellin (flg22) alone or in combination with A (5 pg/ml), Z (40 μΜ) or a combination thereof (A&Z). (Mean ± s.e.m., n=3 technical repetitions).

Fig. 13 shows accumulation of ONSEN extrachromosomal DNA upon pharmacological treatment and heat stress. ONSEN copy number was measured by quantitative PCR (qPCR) on total DNA in seedlings of WT directly after control stress (CS; 24h 6°C), heat stress (HS; 24h 6°C and 24h 37°C) and HS plus treatment with alpha-aminitin(A, 20 pg/ml), zebularine (Z, 10 μΜ) and HS plus the combination of A&Z. This result demonstrates the robustness of the treatments independent of the relative concentrations of A and Z.

Examples

The inventors have discovered a highly efficient method to activate and mobilize TEs in eukaryotes. The treatment involves drugs that target highly conserved eukaryotic mechanisms: DNA methylation and transcription.

Example 1

In order to investigate the role of Pol II on TE mobility the inventors chose the well- characterized heat-responsive copia-like ONSEN retrotransposon (Ito, H. et al., Nature,

201 1 , 472: 1 15-1 19) of Arabidopsis. The inventors first tested if Pol II deficient plants showed enhanced TE activity. For that purpose, the inventors took advantage of the hypomorphic nrpb2-3 mutant allele that accumulates reduced NRPB2 protein levels (Zeng, B. et al., Genes Dev, 2009, 23: 2850-2860). Using real-time PCR, it was determined that challenging nrpb2-3 seedlings by heat stress (called HS here) lead to a mild increase in ONSEN ecDNA compared to the wild type (Fig. 1 a). This result was supported by the observed increase in ONSEN ecDNA after pharmacological inactivation of Pol II with 5 pg/ml a-amanitin (called A here), a potent Pol II inhibitor that does not affect Pol IV or Pol V (Haag, J.R. et al., Mol Cell,

2012, 48: 81 1 -818) (Fig 1 a,b). Transcription by RNA Polymerase II (Pol II) is inhibited by a- amanitin, derivatives thereof or other Pol II inhibitors. Global inhibition of DNA methylation is achieved by treatments with zebularine or 5-aza-2'-deoxycytidine (and derivatives thereof). In order to test the interaction between Pol ll-mediated repression of TE activation and DNA methylation the inventors grew wild-type and nrpb2-3 plants on media supplemented with moderate amounts of zebularine (called Z here, 40 μΜ for wild-type plants, 10 μΜ for nrpb2- 3 plants to ensure the viability of nrpb2-3 seedlings), an inhibitor of DNA methyltransferases active in plants (Baubec, T. et al., Plant J, 2009, 57: 542-554) and submitted them to HS. The presence of Z in the medium during HS generally enhanced the production of ONSEN ecDNA. Notably, this induced increase in ecDNA accumulation was more distinct in the nrpi)2-3-backg round (Fig. 1 a). This indicated that both, DNA methylation and Pol II transcriptional activity contribute to the repression of ONSEN ecDNA production. Because both DNA methylation and Pol II can be specifically inhibited by the addition of different drugs the inventors tested if treating wild-type plants with both A and Z at the same time could strongly activate and even mobilize ONSEN after a heat stress treatment. The inventors grew WT seedlings on MS medium supplemented with each drug individually and both combined. In conformity with the strong activation of ONSEN in heat stressed and Z-treated nrpi)2-3-seedlings, the combined treatment (A+Z) of the WT gave rise to a very high (Fig. 1 b) and HS-dependent (Fig. 2) accumulation of ONSEN ecDNA comparable to the nrpdl mutant (Fig. 3c).

Example 2

In order to better understand the effect the drugs had at the DNA level underlying the increased activation of ONSEN after HS, the inventors assessed how they influenced DNA methylation at the long terminal repeat (LTR) of a selected ONSEN endogenous locus (AT1 TE12295) and at an unrelated well characterized RdDM target (soloLTR). Treating plants with A or Z individually already resulted in reduced CHH methylation levels at the ONSEN LTR after CS (Fig. 3a). Combining the two drugs lead to a loss of DNA methylation comparable to the nrpdl mutant. DNA methylation at the soloLTR showed a different response to the drug treatments as a reduction in DNA methylation levels was only observed in plants submitted to a combined A and Z treatment. The inventors then checked by Northern Blot whether the degree of reduction in CHH methylation would coincide with increased ONSEN-transcript-levels directly after HS. The inventors found that treatment with Z alone already resulted in the highest ΟΛ/SE/v-transcript levels after HS (Fig. 3b). From this observation, the inventors concluded that these additional Z-induced transcripts were not suitable templates for the production of ONSEN ecDNA (compare Fig. 1 and Fig. 3b).

In Drosophila, it has been shown that Pol ll-mediated antisense transcription results in the production of TE-derived siRNAs in a Dicer-2 dependent manner (Russo, J. et al. Genetics, 2016, 202:107-21 ). Supporting this notion for Arabidopsis, a recent publication pointed out the importance of DCL3 in regulating ONSEN in the ddml background (Panda, K. et al. Genome Biol, 2016, 17:1 -19). To elucidate whether the effect of Pol II inhibition was also dicer-dependent, the inventors grew both the rdr6- and the dcl2/3/4-triple mutant (defective in three of the four plant dicer-like enzymes, DCLs) on A, applied HS and measured ONSEN ecDNA. The inventors found that A was still enhancing ecDNA accumulation in rdr6 whereas inhibition of Pol II had no effect in the dcl2/3/4 triple-mutant (Fig. 3c). This finding supports the notion that Pol II acts upstream of the processing step catalyzed by the DCLs.

Example 3

Mobilization of endogenous TEs in plants has so far been very inefficient, thus limiting their use in basic research and plant breeding. We have previously not observed ONSEN transposition in HS treated wild-type plants (Ito, H. et al. Nature, 201 1 , 472:1 15-1 19). Because the A&Z drug treatment resulted in an increased ONSEN ecDNA accumulation to a similar degree like in nrpdl, the inventors tested if the combined drug treatment could lead to an efficient ONSEN mobilization in wild-type plants. First, the inventors assessed by real- time PCR if, and at which frequencies, new ONSEN copies could be detected in the progeny of A&Z-treated and heat stressed plants. The inventors found new ONSEN insertions in 29.4% of the tested F1 pools (n=51 ) with mean copy numbers of the pools reaching up to 52 (Fig. 5). The inventors then confirmed stable novel ONSEN insertions in a subset of independent individual high copy plants by transposon display (Fig. 4a), real-time PCR (Fig. 4b) and sequencing of some insertions in a selected high-copy line (#3) (Fig. 6). The combination of HS, A and Z resulted in a similar extrachromosomal ONSEN copy number as has been previously observed in RdDM deficient plants. The inventors detected novel ONSEN insertions in the progeny of 27 % of the treated plants. According to qPCR measurements, up to 90 +/-6 inserted copies were detected in individual plants in the F2 and successive generations of A, Z and HS treated plants (Figure 4a). These insertions were further confirmed by transposon display. The inventors did not observe further increases in ONSEN copy numbers over three generations indicating that the new insertions were stable and that ONSEN was not transposing anymore (Fig. 4b).

TE insertions can interrupt genes or alter their expression by either recruiting epigenetic marks or by stress-dependent readout transcription from the 3'LTR into flanking regions (Lisch, D., Nat Rev Genet, 2013, 14: 49-61 ). To test this, the inventors grew the F2 generation of the aforementioned selected high copy lines under long and short day conditions. The inventors observed that many lines showed clear and homogenous phenotypes in response to the different growth conditions (plant size, chlorophyll content and flowering time, Fig. 4c and d).

Example 4

The inventors tested if Pol II plays a more general role in repressing TEs in plants. Due to its significantly different epigenetic landscape compared to Arabidopsis the inventors chose the genetically well characterized monocotyledonous rice crop O. sativa (Kawahara, Y. et al., 2013, Rice, 6: 4-10). In order to capture drug-induced mobilized TEs, the inventors characterized the active mobilome in O. sativa seedlings that were grown on MS medium supplemented either with no drugs, A only, Z only or the combination of A and Z, using a method that allows to specifically sequence extrachromosomal circular DNA (eccDNA). eccDNA is a byproduct of the LTR retrotransposon life cycle. Using this approach, the inventors identified Houba, a copia like retrotransposon (Panaud, O. et al., Mol. Genet. Genomics, 2002, 268:1 13-121 ), as highly activated only when plants were treated with both A and Z (Fig. 7a). The sequencing data were confirmed by an eccDNA-specific PCR on the Houba LTRs (Fig. 7b and c).

Example 5

Because the treatment with A alone reduced DNA methylation (Fig. 3a) in Arabidopsis, the inventors wanted to test the robustness and generality of this treatment. In order to confirm the robustness, plants were treated with A (20pg/ml), Z (10 pg/ml) and A&Z. A alone already strongly reduced DNA methylation at this higher concentration (Fig. 10a), this result was then further supported by the assessment of DNA methylation in the CHH context by bisulfite sequencing (average of 10 sequenced clones for each sample). Because A inhibits the highly conserved RNA Pol II enzyme and that A is also active in human cells, the inventors tested the effect of A on DNA methylation in the A549 human cancer cell line. Global DNA methylation content in the cells was assessed and compared to untreated or Z-treated cells. Supplementation of the growth medium with A (0.5 pg/ml) resulted in a 40% reduction of DNA methylation. This reduction was comparable to a treatment with the DNA demethylating agent Z (350 μΜ) (Fig. 10c). The authors then also assessed the DNA methylation levels at the long interspersed element 1 (LINE-1) retrotransposon. At LINE-1 A had an even more pronounced effect on the reduction of DNA methylation than Z (40% versus 20% reduction, respectively). These results demonstrate that an inhibitor of transcription can be used as a potent DNA demethylating agent in eukaryotic cells.

Plants and growth conditions

After stratification for two days at 4°C, Arabidopsis thaliana plants (accession Col-0) were grown on sterile ½ MS medium with 1 % sucrose and a pH of 5.8 (control medium) under long day conditions (16 h light) at 24°C (day) and 22°C (night), respectively. Oryza sativa plants were grown on sterile ½ MS medium with 1 % sucrose and a pH of 5.8 (control medium) 16 h at 28 °C (day) and 27 °C (night), respectively.

In order to analyze successive generations, seedlings were transferred to soil and grown under long day conditions (16 h light) at 24 °C (day) and 22 °C (night) (A. thaliana) in a Sanyo MLR-350 growth chamber until seed maturity.

For phenotyping, A. thaliana plants were grown under long day conditions (16 h light) at 24°C (day) and 22 °C (night) and short day conditions (10 h light) at 21 °C (day) and 18 °C (night).

The induction of epigenetic changes and the activation and stable integration of transposable elements in Arabidopsis seedlings was enhanced by germinating and growing them on ½

MS-medium that contained zebularine (final concentration: 10-40 μΜ), α-amanitin (final concentration: 0.005-20 pg/ml) or a combination of both chemicals (inductive media). In order to trigger the transposition of the heat-responsive retrotransposon ONSEN, seven days old seedlings were exposed to a cold shock for 24h at 6°C followed by a heat-stress for 24h at 37°C (heat stress, HS) under controlled conditions in a growth chamber (Sanyo). Control plants were transferred back to longday-conditions 24°C (day) and 22°C (night) after the cold treatment at 6°C for 24h (CS, control stress, according to Ito et al., 201 1 ).

In order to trigger a biotic-stress response, nine days old -Arab/c ops/s-seedlings were grown for nine days on 5 ug/ul alpha-amanitin and 40 uM zebularine and sprayed with flg22 (10 μΜ). After 5h of incubation, total DNA from the aerial part of seedlings was extracted and TE copy number assessed by qPCR. qPCRs on total DNA to measure ONSEN and COPIA 17 copy numbers

Total DNA from seedlings and adult plants was isolated using a DNeasy Plant Mini Kit (QIAGEN).

In preparation to the measurement of extrachromosomal DNA copies of ONSEN in CS/HS and untreated/treated seedlings, roots were dissected directly after the heat stress and plants were immediately frozen in liquid nitrogen until DNA extraction.

To track ONSEN copy numbers in the F1-F3 generations of control and high copy lines, DNA from true leaves was extracted.

For the estimation of the ONSEN transposition frequency, total DNA of pools consisting of at least eight seedlings of the progeny of HS+A&Z-treated plants was isolated. The DNA concentration was measured with a Qubit Fluorometer (Thermo Fisher Scientific).

The copy numbers of ONSEN were determined with qPCRs on total DNA using a TaqMan master mix (Life Technologies) in a final volume of 10 μΙ in the Light-Cycler 480 (Roche). ACTOPIA17 copy number was measured by quantitative PCR (qPCR) in a Light-Cycler 480 (Roche), using XYBR 421 Green I Master Mix. Actin2 (At3g18780) served as a standard gene for normalization. The sequences of the primers and probes for the qPCRs are listed in table 2.

For the mobilome-seq analysis DNA from the aerial parts of three O. sativa seedlings was extracted as previously reported (Mette, M. et al., EMBOJ, 1999, 18: 241 -248).

5pg of genomic DNA for each sample were purified using a Geneclean kit (MPBio, USA) according to the manufacturer's instructions. ecDNA was isolated from the GeneClean product using the PlasmidSafe DNase (Epicentre, USA) according to the manufacturer's instructions, except that the 37°C incubation was performed for 17h. DNA samples were precipitated by adding 0.1 volume of 3M sodium acetate (pH 5.2), 2.5 volumes of ethanol and 1 μΙ of glycogen (Fisher, USA) and incubating overnight at -20°C. The precipitated circular DNA was amplified by random rolling circle amplification using the lllustra TempliPhi kit (GE Healthcare, USA) according to the manufacturer's instructions except that the incubation was performed for 65h at 28°C. The DNA concentration was determined using the DNA PicoGreen kit (Invitrogen, USA) using a LightCycler480 (Roche, USA). One nanogram of amplified ecDNA from each sample was used to prepare the libraries using the Nextera XT library kit (lllumina, USA) according to the manufacturer's instructions. DNA quality and concentration were determined using a high sensitivity DNA Bioanalyzer chip (Agilent Technologies, USA). Samples were pooled and loaded onto a MiSeq platform (lllumina, USA) and 2x250 nucleotides paired-end sequencing was performed. Quality control of FASTQ files was evaluated using the FastQC tool (version 0.10.1 ). To remove any read originating from organelle circular genomes, reads were mapped 198 against the mitochondria and chloroplast genomes using the program Bowtie2 version 2.2.2 71 with— sensitive local mapping. Unmapped reads were mapped against the reference genome IRGSP1.0 (http://rgp.dna.affrc.go.jp/E/IRGSP/ Build5.html) using the following parameters: - sensitive local, -k 1. DNA from both mitochondria and chloroplast genomes integrated in nuclear genomes was masked (1 ,697,400 bp), The TE containing regions cover 194,224,800 bp in O. sativa. Finally, for each library, a .bam alignment file corresponding to enriched genomic regions was considered for statistical analysis and visualized with the Integrative Genomics Viewer (IGV) software (https://www.broadinstitute.org/igv/home)

Table 2: Sequences of primers and probes that were used for the qPCRs (TaqMan, Life Technologies) to measure total number of extrachromosomal ONSEN DNA-copies. Actin 2 served as a control gene for normalization.

Primer Sequence 5 ' -> 3 '

SEQ ID No 001 CCACAAGAGGAACCAACGAA

( ONSEN RT fw)

SEQ ID No 002 TTCGATCATGGAAGACCGG

( ONSEN RT rev)

SEQ ID No 003 (FAM) AAGTCGGCAATAGCTTTGGCGAAGA (BHQ1)

( ONSEN probe)

SEQ ID No 004 TGCCAATCTACGAGGGTTTC

(Actin2 RT fw)

SEQ ID No 005 TTACAATTTCCCGCTCTGCT

(Actin2 RT rev)

SEQ ID No 006 (JOE) TCCGTCTTGACCTTGCTGGACG (BHQ- 1)

(Actin2 probe)

SEQ ID No 007 GTAATACGACTCACTATAGGGCACGCGTGGTCGACGGCCCGGGCTG

(GenWalkAdaptatorl ) GT

GenWalkAdaptor2 (PHOS) ACCAGCCC (AMINO)

SEQ ID No 008 GTAATACGACTCACTATAGGGC

(API)

SEQ ID No 009 AACACTTAAACACTTTCTCCA

(Copia78 3' LTR)

SEQ ID NO 010 TTAGTATAAGGCTGAGCTGGAAACTG 706_ATCOPIA17 QT R

SEQ ID NO Oil CAAGCCTAACCCTCAGCTACATG

705 ATCOPIA17 QT F

Transposon display to confirm insertion of new ONSEN copies

The stable integration of additional copies of the ONSEN TE into the genome of heat stressed and treated plants was ascertained by a simplified transposon display based on the GenomeWalker Universal kit (Clontech Laboratories), according to Ito et al. 201 1.

300 ng of total DNA from adult plants from the F2 generation of HS +/- A & Z was extracted with a DNeasy Plant Mini Kit (QIAGEN) and digested with a blunt cutter restriction enzyme (Dra I). After purification with a High Pure PCR Product Purification Kit (Roche) digested DNA was ligated to the annealed GenWalkAdapters 1 & 2. For the PCR, the adaptor specific Primer AP1 and the ΟΛ/SE/v-specific Primer Copia 78 3 ' LTR was used. The PCR products were separated on a 2 % agarose gel that was stained with a Midori Green Nucleic Acid Staining Solution. For sequence information, see tables 2 and 3.

Cloning, sequencing and genotyping of new insertions

In order to identify the genomic region of new ONSEN insertions, the PCR product of the transposon display was purified using a High Pure PCR Product Purification Kit (Roche), ligated into a pGEM-T Vector (Promega) and transformed into E. coli. After a blue white selection, positive clones were used for the insert amplification and sequencing (StarSEQ). The obtained sequences were analyzed with Geneious 8.2.1 and blasted against the Arabidopsis thaliana reference genome. The standard genotyping-PCRs to prove novel ONSEN insertions were performed with combinations of the ΟΛ/SE/v-specific primer "Copia 78 TE display S ' LTR" and primers listed in tables 2 and 3.

Table 3: Names, purpose and sequences of primers

836 Chop PCR ACT2 R TTGGCACAGTGTGAGACACACCA

SEQ ID 19

houba_F2 SEQ ID 20 ATCCTGGGAAGAACAAACCATTAA PCR on houba_R2 SEQ ID 21 GAGTTCGAGTACCTTAGCCATGGT circular rice TE

Chloroplast eye F ACAACCACTG ATG AAG G ATT and

SEQ ID 22 chloroplast

Chloroplast eye R AGAAAGAAAAGCAACGACTG control

SEQ ID 23

Prove TED 2_20 R ACCTAGCTCTGAGTGATGAA Genotyping of SEQ ID 24 novel ONSEN

Prove TED4_27 F TGGATATACACATTGGTTGCA insertions SEQ ID 25

Prove TED 2_19 F GGAGAAAGCTGAAAACTTGG

SEQ ID 26

Prove TED4_30_rev CTAGGTTGGTGACTGATGAG

SEQ ID 27

Prove TED 2_17 F AAGAATGGGAGCAGCATTAA

SEQ ID 28

Prove3_2R SEQ ID 29 GCAGTACTATAACCGGGACT

prove TED3_1 Fw GAACTTTCCGTTGTTACCGG

SEQ ID 30

Prove TED3 F SEQ ID 31 ATG AG ACAG G GAG CTTATCT

Prove TED1 R SEQ ID 32 GGTGTGAACCGAACCTAAAT

Prove TED 4_25 F AAACACCAGAAATCTTTCGC

SEQ ID 33

PCRs on extrachromosomal Houba DNA

The presence of circular Houba-copies was proven by an inverse PCR on 7 ng of the rolling- circle amplified template that was also used for sequencing. A PCR specific to a chloroplast DNA served as a loading control. PCR products were separated on a 1 % agarose gel that was stained with a Midori Green Nucleic Acid Staining Solution (Nippon Genetics Europe). Primer sequences are given in supplementary Table 4. Table 4: Sequences of primers and probes that were used for the PCRs to measure total number of extrachromosomal Houba DNA.

Primer /SEQ ID NO Sequence 5 -> 3 '

286 OnsenLTRchopF SEQ ID 34 GGTTGAAGGGTYAAAGAGTAAAT

287 OnsenLTRchopR SEQ ID 35 CCTCCAAACTACAAAATATCTAAAA

Houba F2 SEQ ID 36 ATCCTGGGAAGAACAAACCATTAA

Houba R2 SEQ ID 37 GAGTTCGAGTACCTTAGCCATGGT

RNA analysis and Northern Blot

Total RNA from the aerial part of Arabidopsis seedlings was isolated using the TRI Reagent (Sigma) according to manufacturer's recommendations. RNA concentration was measured (Qubit RNA HS Assay Kit, Thermo Fisher), 15 pg of RNA was separated on a denaturing 1.5% Agarose gel, blotted on a Hybond-N+ (GE Healthcare) membrane and hybridized with 25 ng of a gel-purified and P32 labelled probe (Megaprime DNA Labelling System, GE Healthcare) specific to the full length ONSEN transcript (See table 3 for primer sequences). DNA methylation analysis

20 ng of total genomic DNA isolated from Arabidopsis seedlings was digested with the methylation sensitive restriction enzyme, Dde1 (NEB) at 37 °C over night. Following heat inactivation at 60°C for 20 min, the digested DNA was used as a template for the chopPCR. Actin2 served as a control for the digest. Undigested DNA was used as a loading control. PCR products were separated on a 1 % agarose gel and stained with Midori Green.

For the A549 human cancer cell line cells were grown in medium without treatment or supplemented with either Z (350 μΜ) or A (0.5 pg/ml), DNA was extracted by using the QiaAmp DNA mini Kit (Qiagen, France). Next, global DNA methylation was estimated by quantifying the presence of 5-methylcytosine 5-mC DNA ELISA Kit (Zymo Research) according to the manufacturers's instructions. DNA methylation at the LINE-1 transposons were assessed with the Global DNA Methylation Assay-LINE-1 kit (Active Motif).