RNA-directed DNA methylation prevents rapid and heritable reversal of transposon silencing under heat stress in Zea mays

Abstract

In large complex plant genomes, RNA-directed DNA methylation (RdDM) ensures that epigenetic silencing is maintained at the boundary between genes and flanking transposable elements. In maize, RdDM is dependent on Mediator of Paramutation1 (Mop1), a gene encoding a putative RNA dependent RNA polymerase. Here we show that although RdDM is essential for the maintenance of DNA methylation of a silenced MuDR transposon in maize, a loss of that methylation does not result in a restoration of activity. Instead, heritable maintenance of silencing is maintained by histone modifications. At one terminal inverted repeat (TIR) of this element, heritable silencing is mediated via histone H3 lysine 9 dimethylation (H3K9me2), and histone H3 lysine 27 dimethylation (H3K27me2), even in the absence of DNA methylation. At the second TIR, heritable silencing is mediated by histone H3 lysine 27 trimethylation (H3K27me3), a mark normally associated with somatically inherited gene silencing. We find that a brief exposure of high temperature in a mop1 mutant rapidly reverses both of these modifications in conjunction with a loss of transcriptional silencing. These reversals are heritable, even in mop1 wild-type progeny in which methylation is restored at both TIRs. These observations suggest that DNA methylation is neither necessary to maintain silencing, nor is it sufficient to initiate silencing once has been reversed. However, given that heritable reactivation only occurs in a mop1 mutant background, these observations suggest that DNA methylation is required to buffer the effects of environmental stress on transposable elements.

Fig 1. DNA methylation patterns at TIRA and TIRB of stably silenced F₂ plants.

(A) qPCR analysis of mudrA and mudrB expression from MuDR*/-; mop1/+ and MuDR*/-;mop1/mop1 plants. MuDR: active element. MuDR*: inactive element. Tub2 is used as an internal control gene. Six biological replicates are used for each experiment; two of six biological replicates are pooled together for each amplification. Error bars indicate mean ± standard deviation (SD) of three individuals. (B) DNA methylation patterns at TIRA and TIRB. Ten individual clones were sequenced from amplification of bisulfite-treated samples of the indicated genotypes. The cytosines in different sequence contexts are represented by different colors (red, CG; blue, CHG; green, CHH, where H = A, C, or T). For each genotype, DNA from six biological replicates were pooled.

Fig 2. ChIP-qPCR analysis of enrichment of histone marks H3K9me2 and H3K27me3 at TIRA and TIRB in mop1 mutants.

ChIP-qPCR analysis of enrichment of histone marks, H3K9me2 and H3K27me3 at TIRA and TIRB. (A) Relative enrichment of H3K9me2 and H3K27me3 in leaf 3 of plants of the indicated genotypes. MuDR: active element. MuDR*: inactive element. (B) Relative enrichment of H3K9me2 and H3K27me3 in leaf 3 of plants of the indicated genotypes. qPCR signal was normalized to Copia and then to the value of input sample. An unpaired t-test was performed. Error bars indicate mean ± standard deviation (SD) of the three biological replicates. *P<0.05; **P < 0.01

Fig 3. Expression of mudrA and mudrB in plants under heat stress.

(A) Schematic diagram of the heat-reactivation experiment. (B) qRT-PCR of mudrA and mudrB in leaf 3 and leaf 7 in plants of the indicated genotypes. Twelve biological replicates are used for each experiment. Tub2 is an internal control gene. Additional controls for each experiment include MuDR/-, pooled samples from twelve heated MuDR*/-; mop1/+ plants and twelve unheated plants. Red text is used to indicate samples that were subjected to heat stress.

Fig 4. ChIP-qPCR analysis of histone marks TIRA and TIRB under heat stress.

Relative enrichment of H3K9me2 and H3K27me3 at TIRA (A) and TIRB (B) in leaf 3 of plants of the indicated genotypes. (Relative enrichment of H3K4me3 at TIRA (C) and TIRB (D) in leaf 3 of plants of the indicated genotypes. qPCR signals were normalized to Copia and then to the value of input samples. MuDR* refers to a silenced MuDR element. MuDR^~ refers to a reactivated element. Red text indicates a sample that has been heat-treated. Error bars indicate mean ± standard deviation (SD) of the three biological replicates. **P < 0.01; ***P < 0.001

Fig 5. Expression of mudrA and mudrB in new emerging tissues following heat stress.

(A) Diagram of the experiment. (B) qPCR was performed to measure transcript levels of mudrA and mudrB using expression of Tub2 as an internal control. Expression levels were normalized to that of an active MuDR element. Error bars indicate mean ± standard deviation (SD) of the ten biological replicates.

Fig 6. Testing transgenerational inheritance.

(A) A schematic diagram showing the crosses used to determine transgenerational inheritance. (B) Ears derived from heat-treated and control individuals. (C) Crosses done in the generations following heat stress. (D) Ratios of spotted kernels in subsequent generations following the heat stress (H₁) generation. Red text indicates a sample that has been heat-reactivated.

Fig 7. DNA methylation patterns at TIRA and TIRB in H₂, H₄ and H₅ progeny of heat-treated plants.

(A) DNA methylation patterns at TIRA. (B) DNA methylation patterns at TIRB. Ten individual clones were sequenced from each amplification of bisulfite-treated sample. The cytosines in different sequence contexts are represented by different colors (red, CG; blue, CHG; green, CHH, where H = A, C, or T). Red text indicates plants derived from heat-treated plants. MuDR* refers to a silenced MuDR element. MuDR^~ refers to a reactivated element. For each assay, six independent samples were pooled together.

Fig 8. ChIP-qPCR analysis of enrichment of histone marks, H3K9me2, H3K27me3 and H3K4me3 at TIRA and TIRB.

Relative enrichment of H3K9me2, H3K27me3 and H3K4me3 at TIRA and TIRB in leaf 3 of plants of the indicated genotypes. qPCR signals were normalized to Copia and then to the value of input samples. Red text indicates plants derived from heat-treated plants. MuDR* refers to a silenced MuDR element. MuDR^~ refers to a reactivated element. An unpaired t-test was performed. Error bars indicate mean ± standard deviation (SD) of the three biological replicates. *P<0.05; **P < 0.01; ***P<0.001.