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Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state

Nature volume 515, pages 587–590 (2014)Cite this article

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Abstract

The reprogramming of epigenetic states in gametes and embryos is essential for correct development in plants and mammals1. In plants, the germ line arises from somatic tissues of the flower, necessitating the erasure of chromatin modifications that have accumulated at specific loci during development or in response to external stimuli. If this process occurs inefficiently, it can lead to epigenetic states being inherited from one generation to the next2,3,4. However, in most cases, accumulated epigenetic modifications are efficiently erased before the next generation. An important example of epigenetic reprogramming in plants is the resetting of the expression of the floral repressor locus FLC in Arabidopsis thaliana. FLC is epigenetically silenced by prolonged cold in a process called vernalization. However, the locus is reactivated before the completion of seed development, ensuring the requirement for vernalization in every generation. In contrast to our detailed understanding of the polycomb-mediated epigenetic silencing induced by vernalization, little is known about the mechanism involved in the reactivation of FLC. Here we show that a hypomorphic mutation in the jumonji-domain-containing protein ELF6 impaired the reactivation of FLC in reproductive tissues, leading to the inheritance of a partially vernalized state. ELF6 has H3K27me3 demethylase activity, and the mutation reduced this enzymatic activity in planta. Consistent with this, in the next generation of mutant plants, H3K27me3 levels at the FLC locus stayed higher, and FLC expression remained lower, than in the wild type. Our data reveal an ancient role for H3K27 demethylation in the reprogramming of epigenetic states in plant and mammalian embryos5,6,7.

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Figure 1: Isolation and characterization of the resetting mutant.
Figure 2: Mapping of the resetting mutant.
Figure 3: Characterization of the elf6-5 resetting mutant.
Figure 4: ELF6 shows H3K27 histone demethylase activity.

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Accession codes

Primary accessions

European Nucleotide Archive

Data deposits

Genomic DNA deep sequencing data for the parental Ler-derived plant and the resetting mutant line have been deposited in the European Nucleotide Archive database under accession number PRJEB6498.

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Acknowledgements

We thank Dean laboratory members and A. Surani for discussions. The Dean laboratory is supported by the UK Biotechnology and Biological Sciences Research Council grants BB/G009562/1 and BB/C517633/1 and by a European Research Council Advanced Investigator grant (233039 ENVGENE). The Cao laboratory is supported by National Basic Research Program of China grants 2013CB967300 and 2011CB915400 and by the National Natural Science Foundation of China grant 31271363.

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Author notes

  1. Pedro Crevillén & Christiaan Greeff

    Present address: Present addresses: Centro de Biotecnología y Genómica de Plantas, UPM-INIA, 28223 Madrid, Spain (P.C.); Department of Biology, Copenhagen University, DK-2200 Copenhagen, Denmark (C.G.).,

Authors and Affiliations

  1. Department of Cell & Developmental Biology, John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK,

    Pedro Crevillén, Hongchun Yang, Christiaan Greeff, Martin Trick & Caroline Dean

  2. State Key Laboratory of Plant Genomics and National Center for Plant Gene Research, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences. Beijing 100101, China

    Xia Cui, Qi Qiu & Xiaofeng Cao

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Contributions

P.C. and C.D. designed the research. P.C., H.Y., X. Cui, C.G. and Q.Q. performed experiments. M.T. conducted deep sequencing data analysis. X. Cao contributed new reagents and analytical tools. P.C., H.Y. and C.D. analysed the data and wrote the paper. All authors discussed the results and commented on the manuscript.

Corresponding author

Correspondence to Caroline Dean.

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C.D. holds stock in Mendel Biotechnology.

Extended data figures and tables

Extended Data Figure 1 Screening for mutants with impaired epigenetic reprogramming of FLC.

a, We started with a population of ethylmethane sulphonate (EMS)-mutagenized A. thaliana Ler plants carrying an FLC::LUC translational fusion and an active FRI transgene. We screened for mutants that were early flowering (as a result of low FLC expression) in the generation following vernalization but that did not flower early (and whose FLC expression was almost normal) without vernalization. b, To discriminate between early flowering and resetting mutants, early flowering M2 plants were backcrossed to the parental line, and the F2 phenotype was evaluated without vernalization. Those plants showing no early flowering segregants were considered to be resetting mutants. Superscript characters denote whether the plant was vernalized in the previous generation.

Extended Data Figure 2 Characterization of the first resetting mutant.

a, The first resetting mutation was found to be recessive. F1 plants were generated from a cross between the mutant in the generation following vernalization and the parental wild-type line. Flowering time was assayed as total leaf number under non-vernalized long-day conditions. The data are presented as the mean + s.e.m., n = 8. b, The earlier flowering time of the mutant in the generation following vernalization was stable for at least three generations without vernalization. The data are presented as the mean + s.e.m., n = 10.

Extended Data Figure 3 The elf6-5 SNP is linked to the resetting of FLC expression.

Histogram showing the relationship between FLC::LUC levels in the reproductive organs of vernalized plants and the elf6-5 SNP (n = 154).

Extended Data Figure 4 FLC expression levels in the null elf6-3 T-DNA insertion allele.

a, The elf6-3 mutant expresses less FLC than the Col wild type. b, The null elf6-3 allele suppresses the high FLC expression induced by FRI before vernalization. This pre-vernalization phenotype of plants carrying the null allele precludes observation of the role of ELF6 during FLC resetting after vernalization. All graphs show 10-day-old non-vernalized seedlings. The data are presented as the mean + s.e.m., n = 3.

Extended Data Figure 5 siRNA production in elf6-5 mutants.

The production of specific siRNAs associated with the epigenetic reactivation of transposable elements is not affected in elf6-5 mutants. Total RNA was extracted from vernalized mature siliques, and the detection of siRNAs was performed as described in Methods.

Extended Data Figure 6 ELF6 has no H3K4me, H3K9me or H3K36me demethylase activity in an N. benthamiana transient assay.

Overexpression of a yellow fluorescent protein (YFP)–ELF6 fusion protein, using the wild-type ELF6 sequence had no effect on H3K4me3, H3K9me2 or H3K36me3 methylation. Histone methylation was visualized by immunostaining with polyclonal rabbit modification-specific antibodies followed by Alexa-Fluor-555-conjugated goat anti-rabbit antibody (red; right). The nuclei of transfected cells were visualized by the YFP signal (green; centre). Nuclei were stained with DAPI (4′,6-diamidino-2-phenylindole) (blue; left). Arrows indicate the nuclei of transfected cells.

Extended Data Figure 7 H3K27me3 accumulation at the FLC locus.

a, Schematic representation of the FLC locus and the regions analysed in the ChIP assays. b, H3K27me3 levels at FLC in Ler (FRI) seedlings grown without vernalization (seedling NV), 7 days after vernalization (seedling VER) and in siliques from vernalized seedlings (siliques VER). The data are presented as the mean + s.d., n = 2.

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Crevillén, P., Yang, H., Cui, X. et al. Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state. Nature 515, 587–590 (2014). https://doi.org/10.1038/nature13722

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