Reprogramming of DNA methylation in pollen guides epigenetic inheritance via small RNA

Abstract

Epigenetic inheritance is more widespread in plants than in mammals, in part because mammals erase epigenetic information by germline reprogramming. We sequenced the methylome of three haploid cell types from developing pollen: the sperm cell, the vegetative cell, and their precursor, the postmeiotic microspore, and found that unlike in mammals the plant germline retains CG and CHG DNA methylation. However, CHH methylation is lost from retrotransposons in microspores and sperm cells and restored by de novo DNA methyltransferase guided by 24 nt small interfering RNA, both in the vegetative nucleus and in the embryo after fertilization. In the vegetative nucleus, CG methylation is lost from targets of DEMETER (DME), REPRESSOR OF SILENCING 1 (ROS1), and their homologs, which include imprinted loci and recurrent epialleles that accumulate corresponding small RNA and are premethylated in sperm. Thus genome reprogramming in pollen contributes to epigenetic inheritance, transposon silencing, and imprinting, guided by small RNA.

Figure 1. DNA methylation and small RNA accumulation during pollen development

(A) Pollen development: the uninucleate microspore divides asymmetrically giving rise to bicellular pollen, which consists of a larger vegetative cell embedding a smaller generative cell. A second mitotic division of the generative cell originates two sperm cells. The three cell types analyzed in this study were stained with DAPI to highlight heterochromatin, which is lost in the vegetative nucleus (VN) but not in the sperm cell nuclei (SC) (bar = 10μm). (B) Heat map representation of DNA methylation. Bisulfite sequencing of genomic DNA from each cell type was performed as described. Methylation density is represented in 10kb blocks, separated by context and cell type. CG (CG methylation), CHG (CHG methylation), CHH (CHH methylation), INF (Inflorescence), MS (Microspore), VN (Vegetative nucleus), SC (Sperm Cell), EMB (Embryo). The maximum value of the heat map is calibrated to the VN. The outer annotation track highlights the position of transposons (TEs).

Figure 2. Differentially Methylated Regions (DMRs) during pollen development

(A) DMRs were detected in a pairwise manner by comparing the bisulfite-sequence profiles from each of the three pollen cell types (vegetative nucleus-VN, sperm cell-SC, and microspore-MS) in each methylation context (CG, CHG, CHH). Annotated features (Genic, TE and Intergenic) overlapping one or more DMR in each cell type and methylation context were identified using TAIR10 annotation. Bars represent the number of DMRs overlapping each feature class. Where a DMR overlaps two or more features each feature is counted once. (B) Scaled distribution of transposon classes overlapping DMRs in the VN. TEs that matched each DMR were identified. Where a DMR overlaps two or more TE superfamilies each overlap is counted once. DMRs that lost CG methylation in the VN were enriched for class II DNA transposons, while DMRs that lost CHH methylation in sperm cells were enriched for class I LTR/gypsy transposons. There were very few CHG DMRs (data not shown) and these did not overlap transposons. (C) CG DMRs (red, upper left) and CHH DMRs (green, upper right) were similar in pairwise comparisons between the VN and the microspore, and the VN and the SC. CG DMRs in the VN (blue, bottom left) overlap with DMRs detected between WT endosperm and dme endosperm (green, bottom left), which are targets of DEMETER (Hsieh et al., 2009), and with DMRs between inflorescence and ros1/dml2/dml3 inflorescence (Lister et al., 2008) which are targets of ROS1 and its homologs (orange, bottom left). In the VN, CG DMRs (pink, bottom right) and CHH DMRs (green, bottom right) do not overlap.

Figure 3. DRM2 expression during pollen development

GFP expression (green) was visualized in pollen from a pDRM2-DRM2::GFP transgenic plant, counterstained with DAPI (blue). Microspores and pollen at the bicellular, tricellular and mature stages are shown. DRM2 was expressed at a low level in the microspore and sperm cells, and at a much higher level in the vegetative nucleus at the bicellular and tricellular stage.

Figure 4. Small RNA from Differentially Methylated Regions (DMRs)

Small RNA in sperm cells (Slotkin et al., 2009) and seeds (Lu et al., 2012) were mapped to DMRs and plotted according to size. CHH DMRs were hypomethylated in sperm cells, while CG DMRs were hypermethylated. CG DMRs flanking Maternally and Paternally Expressed imprinted Genes (MEGs and PEGs) were also analyzed separately. Relative abundance of size classes is shown as proportions.

Figure 5. DNA methylation and small RNA abundance at imprinted genes in pollen

(A) Genome browser view of the Maternally Expressed Gene (MEG) SDC and the Paternally Expressed Gene (PEG) PHE1. Tracks display CG (red) and CHH (green) methylation as well as 24nt siRNAs (blue) from pollen, seeds and purified sperm cells. Methylation is represented on a scale of 0–100% and siRNAs for total normalized reads from 0–20 RPM (reads per million). MS (microspore), SC (sperm cell), VN (vegetative nucleus), INF (Inflorescence). (B) Box-plot representation of DNA methylation percentages at MEGs and PEGs. TEs neighboring both MEGs and PEGs are demethylated in the CG context specifically in the vegetative nucleus. Higher CHH methylation levels were detected at MEGs in comparison with PEGs. (C) Box plot representation of 24nt siRNA corresponding to TEs surrounding PEGs and MEGs in total pollen, sperm cells, and seeds. Boxes represent lower and upper quartiles surrounding the median (line). Triangles represent the mean.

Figure 6. DNA methylation at hypervariable recurrent epialleles

100 hypervariable epialleles gain DNA methylation recurrently in plants propagated by single seed descent (Becker et al., 2011; Schmitz et al., 2011). Many are targets of ROS1 and its homologs DML1 and DML2 (RDD). An example is shown (ATCOPIA51, At4g09455), along with a neighboring MuDR element for comparison. Tracks represent the RDD target region, and methylation levels in CG and CHH contexts in microspores (MS), vegetative nucleus (VN), and sperm cells (SC), along with inflorescence (INF) and embryo. CG methylation at the RDD target site is found in rdd triple mutant inflorescence (rdd INF) (Lister et al., 2008) and in pollen, but not in inflorescence or embryo. Small RNA from sperm cells (Slotkin et al., 2009) and seed (Lu et al., 2012) are also shown.

Figure 7. Genome reprogramming during pollen development

Differential expression of DRM2, MET1, ROS1, DME and DDM1 is depicted in bicellular pollen and persists in tricellular and mature pollen after the vegetative nucleus (VN, blue) and sperm cells (SC, red) differentiate (not shown). This results in reprogramming of transposons, imprinted genes and epialleles, as shown. Transposon reprogramming. DRM2 is down regulated in the microspore and sperm cells, so that CHH methylation is lost from retrotransposons, and is only restored after fertilization in the embryo (green), guided in part by maternal 24nt siRNA. DRM2 restores CHH methylation in the VN, guided by pollen 24nt siRNAs. In the vegetative cell, the chromatin remodeler DDM1 is lost, and retrotransposon activation generates 21nt siRNA that accumulate in sperm cells (arrow). Epigenetic inheritance. In the VN the DNA glycosylases DME and ROS1 target specific transposons for demethylation, including those that flank imprinted genes. In SC, CG methylation is maintained, and 24nt siRNA accumulate specifically from transposons that flank Maternally Expressed imprinted Genes (MEGs). These 24nt siRNAs may arise in the VN, resembling 21nt retrotransposon siRNA in this respect. A similar mechanism targets recurrent epialleles in pollen, contributing to their sporadic occurrence and to their subsequent inheritance in the embryo.