Epigenetic reprogramming that prevents transgenerational inheritance of the vernalized state

Abstract

The reprogramming of epigenetic states in gametes and embryos is essential for correct development in plants and mammals. 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 next. 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 embryos.

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

a, We started with a population of EMS-mutagenized Arabidopsis Landsberg erecta (Ler) plants carrying an FLC::luciferase (FLC::LUC) translational fusion and an active FRIGIDA transgene. The mutants we screened for were early flowering (from low FLC expression) in the generation following vernalization, but which did not flower early (and whose FLC expression was near normal) without vernalization. b, To discriminate early flowering from resetting mutants early flowering M2 plants were backcrossed to the parental line and the F2 phenotype evaluated without vernalization. Those showing no early flowering segregants were considered to be resetting mutants. In the figure, we use superscript characters to note if 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 the generation following vernalization and the parental wild-type line. Flowering time was assayed as total leaf number under non-vernalized long day conditions. Means + s.e.m., n = 8. b, The earlier flowering time of the mutant the generation following vernalization was stable at least three generations without vernalization. Means + s.e.m., n = 10.

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

Histogram showing the relationship between FLC::LUC levels in 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, elf6-3 shows reduced FLC expression compared to Col wild-type. b, the null elf6-3 allele suppresses the high FLC expression induced by FRI before vernalization. This pre-vernalization phenotype of the null allele precludes observing the role of ELF6 during FLC resetting after vernalization. All graphs show 10 day-old non-vernalized seedlings. Means + s.e.m., n = 3.

Extended Data Figure 5. siRNA production in elf6-5

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

Extended Data Figure 6. ELF6 has no H3K4/K9/K36me demethylase activity in a N. benthamiana transient assay

Overexpression of a YFP-ELF6 fusion protein, using wild-type ELF6 sequence has no effect on H3K4me3/K9me2/K36me3 methylation. Histone methylation was visualized by immunostaining with rabbit polyclonal modification-specific antibodies followed by Alexa fluor 555-conjugated goat anti-rabbit (red; right panels). Transfected nuclei were visualized by the YFP signal (green; middle panels). Nuclei were stained with DAPI (blue; left panels). Arrows indicate transfected nuclei.

Extended Data Figure 7. H3K27me3 accumulation at the FLC locus

a, Schematic representation of the FLC locus and regions analysed in the chromatin immunoprecipitation. b, H3K27me3 levels at FLC in Ler-FRI seedlings grown without vernalization (Seedling NV), 7 days after vernalization (Seedling VER) and siliques from vernalized seedlings (Siliques VER). Means + s.d., n = 2.

Figure 1. Isolation and characterization of the resetting mutant

a, Logic of the genetic screen. Parental wild-type is Ler-FRI FLC::luciferase. b-d, The resetting mutant is early flowering (b) with fewer total leaves and maintains low FLC expression shown by FLC luciferase imaging of 8 day-old seedlings (c) or Q-RT-PCR analysis normalised to UBC (d). NV is non-vernalized, VER is vernalized, NV(V) is non-vernalized following vernalization in the previous generation. Means + s.e.m., n = 20 (b) and n = 3 (d), ***P < 0.001.

Figure 2. Mapping of the resetting mutant

a, b ELF6 genomic construct complements the resetting mutant. FLC luciferase imaging (a) and FLC Q-RT-PCR data (b) of mature siliques from vernalized WT, elf6-5 and representative T2 elf6-5 [pELF6::ELF6] lines. c-f, ELF6::GUS expression profile in 7 day-old seedling (c), ovules (d), globular embryo (e) and mature embryo (f). g, The ELF6 residue mutated in elf6-5 is conserved (red A). Sequence alignment of the JmJC domain of Arabidopsis ELF6 and REF6, and human JMJD3 and UTX proteins. Highly conserved residues are shadowed in grey. Numbering refers to ELF6 amino acid position.

Figure 3. Characterization of the elf6-5 resetting mutant

a, FLC luciferase imaging (b) and FLC Q-RT-PCR data of tissues from WT and elf6-5 the generation following vernalization. Means +/− s.e.m., n = 6. c, Q-RT-PCR data of vernalized WT and elf6-5 siliques: just after fertilization with petals still attached (SQ16); small without petals (SQ17a); first (SQ17b1) and last (SQ17b2) mature green siliques; yellow siliques (SQ18). Means + s.e.m., n = 4, *P < 0.05, **P < 0.01, ***P < 0.001. d, FLC::GUS expression in vernalized WT and elf6-5 early globular embryos. e, Q-RT-PCR shows COOLAIR levels are not affected in elf6-5 siliques. Means + s.e.m., n = 5. f, Q-RT-PCR showing that MAF2-5 genes are misregulated in elf6-5 seedlings a generation following vernalization. Means + s.e.m., n = 3. g, FLM has reduced expression in elf6-5 vernalized siliques. Means + s.e.m., n = 3.

Figure 4. ELF6 shows H3K27 histone demethylase activity

a, Overexpression of a YFP::ELF6 fusion protein reduces H3K27me3, H3K27me2 but not H3K27me1. b, Overexpression of YFP::ELF6^A424V has no effect on H3K27 methylation. Histone methylation was visualized by immunostaining (red; right panels). Transfected nuclei (arrowed) were visualized by YFP signal (green; middle panels), and stained with DAPI (blue; left panels). Graphs quantify methylation levels of transfected (red) versus non-transfected (blue) nuclei. Means ± s.d. c, FLC regions analysed in ChIP. d, H3K27me3 levels in elf6-5 and WT siliques (stage SQ16-SQ17a) from vernalized plants. Means + s.e.m., n = 3. H3K27me3 in WT (V) plants is significantly lower than elf6-5 (V) in FLC region +4088 at *P < 0.05. e, H3K27me3 levels in progeny derived from parents that had (V) or had not (NV) been vernalized. Means + s.e.m., n = 3. H3K27me3 in WT (V) plants is significantly lower than elf6-5 (V) in FLC region +459 at *P < 0.05.