- Open Access
Epigenetic signatures associated with imprinted paternally expressed genes in the Arabidopsis endosperm
© The Author(s). 2019
- Received: 30 October 2018
- Accepted: 12 February 2019
- Published: 21 February 2019
Imprinted genes are epigenetically modified during gametogenesis and maintain the established epigenetic signatures after fertilization, causing parental-specific gene expression.
In this study, we show that imprinted paternally expressed genes (PEGs) in the Arabidopsis endosperm are marked by an epigenetic signature of Polycomb Repressive Complex2 (PRC2)-mediated H3K27me3 together with heterochromatic H3K9me2 and CHG methylation, which specifically mark the silenced maternal alleles of PEGs. The co-occurrence of H3K27me3 and H3K9me2 on defined loci in the endosperm drastically differs from the strict separation of both pathways in vegetative tissues, revealing tissue-specific employment of repressive epigenetic pathways in plants. Based on the presence of this epigenetic signature on maternal alleles, we are able to predict known PEGs at high accuracy and identify several new PEGs that we confirm using INTACT-based transcriptomes generated in this study.
The presence of the three repressive epigenetic marks, H3K27me3, H3K9me2, and CHG methylation on the maternal alleles in the endosperm serves as a specific epigenetic signature that allows prediction of genes with parental-specific gene expression. Our study reveals that there are substantially more PEGs than previously identified, indicating that paternal-specific gene expression is of higher functional relevance than currently estimated. The combined activity of PRC2-mediated H3K27me3 together with the heterochromatic H3K9me3 has also been reported to silence the maternal Xist locus in mammalian preimplantation embryos, suggesting convergent employment of both pathways during the evolution of genomic imprinting.
- CHG methylation
- Genomic imprinting
- Paternally expressed genes
Genomic imprinting is an epigenetic phenomenon causing maternal and paternal alleles to be differentially expressed after fertilization. In plants, genomic imprinting is mainly confined to the endosperm, an ephemeral nutritive tissue supporting embryo growth, similar to the placenta in mammals . The endosperm is the product of a double fertilization event, where one of the haploid sperm cells fertilizes the haploid egg cell giving rise to the diploid embryo, while the second sperm cell fertilizes the diploid central cell to give rise to the triploid endosperm . Imprinted genes are epigenetically modified during gamete formation, and the established epigenetic asymmetry is maintained after fertilization. Differential DNA methylation is established by the DNA glycosylase DEMETER (DME) that removes methylated cytosine residues in the central cell of the female gametophyte . DME is not active in sperm, leading to differential DNA methylation between male and female genomes in the endosperm. DME acts on small transposable elements (TEs) in the vicinity of genes  and its activity has been connected to the expression of maternally expressed imprinted genes (MEGs). Hypomethylation can furthermore cause repression [5, 6], possibly by exposing binding sites for the Fertilization-Independent Seed (FIS)-Polycomb Repressive Complex 2 (PRC2) [7, 8], an evolutionary conserved chromatin modifying complex that applies a trimethylation mark on histone H3 at lysine 27 (H3K27me3) . The Arabidopsis FIS-PRC2 consists of the subunits MEDEA (MEA), FIS2, FERTILIZATION-INDEPENDENT ENDOSPERM (FIE), and MULTICOPY SUPPRESSOR OF IRA1 (MSI1)  and is specifically active in the central cell of the female gametophyte and in the endosperm . Repression of the maternal alleles of PEGs is mediated by the activity of the FIS-PRC2 , consistent with maternal PEG alleles being marked by H3K27me3 . In this manuscript, we addressed the mechanism of maternal allele repression in PEGs. We surprisingly found that PEGs are regulated by two otherwise largely exclusive epigenetic repressive pathways, the FIS2-PRC2 and the pathway establishing the heterochromatin-localized H3K9me2 modification . We demonstrate that both modifications, H3K27me3 and H3K9me2, overlap on the maternal alleles of the majority of PEGs. Our data suggest that most likely FIS-PRC2 acts first and is required to establish H3K9me2. Furthermore, we find maternal alleles of PEGs to be marked by CHG methylation in the central cell, indicating that repressive pathways establishing H3K27me3, H3K9me2, and CHG methylation act in the central cell of the female gametophyte. Finally, we use the presence of the three modifications to predict novel PEGs and propose that the number of PEGs predicted based on expression data strongly underestimates the real number of PEGs.
The maternal alleles of PEGs are marked by H3K27me3, H3K9me2, and CHG methylation
Maternal-specific CHG methylation is established in the central cell and depends on FIS-PRC2
Previous work revealed that loss of FIS-PRC2 function caused activation of maternal PEG alleles . We addressed the question whether loss of CMT3 and the redundantly acting SUVH4,5,6  similarly affect silencing of maternal PEG alleles. Based on published expression studies, CMT3, SUVH4, and SUVH6 are expressed in the central cell of the female gametophyte and in the endosperm [21, 22]. The maternal alleles of seven tested PEGs remained silenced in reciprocal crosses of wild type with cmt3 and suvh4,5,6 triple mutants (Additional file 1: Figure S4A-B), indicating that stable silencing of the maternal alleles of PEGs does not depend on CMT3 and SUVH4,5,6 activity before fertilization. We tested the requirement of CMT3 and SUVH4,5,6 for PEG regulation after fertilization by monitoring PEG expression in homozygous mutant cmt3 and suvh4,5,6 seeds. None of the seven tested genes was significantly upregulated in seeds of cmt3 or suvh4,5,6 mutants (Additional file 1: Figure S4C), indicating that CMT3 and SUVH4,5,6 are not required for repression of maternal PEG alleles after fertilization. In the suvh4,5,6 triple mutant, H3K9me2 is genome-wide eliminated in vegetative tissues ; however, whether H3K9me2 is similarly eliminated in the central cell and endosperm remains to be shown. Therefore, deciphering the role of H3K9me2 in repressing the maternal alleles of PEGs remains to be subject of future investigations.
Paternally biased expression coincides with the combination of H3K27me3, H3K9me2, and CHG methylation
Maternal seed coat contamination restricts the identification of PEGs
In this study, we identified the concomitant presence of maternal-specific CHG methylation, H3K27me3, and H3K9me2 as an epigenetic signature for paternally biased expression in the endosperm. We furthermore predict that there are substantially more PEGs than previously reported in Arabidopsis [12, 14, 26], suggesting that in Arabidopsis the number of PEGs exceeds the number of MEGs. Recent re-evaluation of published imprintome data of Arabidopsis revealed that a large number of previously predicted MEGs were seed coat expressed genes, while the number of PEGs was underestimated . Our study supports and extends this notion by showing that there is a large number of paternally biased genes that likely failed to be identified in previous studies because of maternal seed coat contamination or early stage-specific expression.
A previous study reported that the maternal alleles of PEGs in A. lyrata are marked by CHG methylation and implicated that closely related species use different mechanisms to regulate imprinted gene expression . Our study reveals that similar to A. lyrata the maternal alleles of many PEGs are also marked by CHG methylation in A. thaliana, highlighting that epigenetic mechanisms employed to maintain monoallelic expression are rather conserved between related species. Interestingly, while the maternal alleles of PEGs in maize are marked by H3K27me3, they are not marked by CHG methylation , indicating that diverged species may use a different mechanism in maintaining maternal allele repression.
How H3K9me2 and CHG methylation are established at PRC2 target genes remains to be studied; however, the strong activation of maternal PEG alleles upon loss of FIS-PRC2 function  suggests that H3K9me2 and CHG methylation require FIS-PRC2 function. The PRC2 is generally targeting genes with specific roles during development , while complexes establishing H3K9me2 are mainly targeting TEs localized in heterochromatic regions of the genome . This functional division of PRC2 and machineries establishing heterochromatic marks is conserved in plants as well as in mammals ; however, there are notable exceptions to this rule in both groups of organisms. In rice seedlings, about one third of H3K27me3 marked genes are also marked by CHG and CHH methylation . Similar to the findings reported in our study, higher levels of H3K27me3 correlate with higher CHG and CHH methylation in gene bodies of rice . The rice H3K27me3 methyltransferase SDG711 physically interacts with the CHH methyltransferase OsDMR2 and the SRA-domain containing SUVH protein SDG703, uncovering a mechanistic connection between PRC2 and non-CG methylation.
Which methyltransferases establish H3K9me2 in the central cell of the female gametophyte and in the endosperm of Arabidopsis remains to be investigated. SUVH4,5,6 are the main H3K9me2 methyltransferases in sporophytic tissues of Arabidopsis  and SUVH4 and SUVH6 are expressed in the central cell and in the endosperm [21, 22]. However, the imprinted genes SUVH7 and SUVH8  encode for two potential H3K9me2 methyltransferases that despite lack of in vitro activity  may be active in the endosperm. Increased expression of SUVH7 is detrimental in triploid seeds , indicating that SUVH7 is functionally active in the endosperm. Similarly, CMT3 is expressed in the central cell and in the endosperm [21, 22]; however, functional redundancies with other CMT genes cannot be ruled out based on available data. Identifying the H3K9me2 and CHG methyltransferases acting in the endosperm will be a major step to address the functional role of H3K9me2 and CHG methylation in the stable repression of maternal PEG alleles.
In mammalian cells, H3K9 methyltransferases colocalize with PRC2 [35–37], revealing crosstalk between these two major epigenetic silencing pathways that likely is required for stable gene silencing. The imprinted maternal Xist locus encoding an X-linked long-noncoding RNA is covered by H3K27me3 and H3K9me3 in preimplantation embryos [38, 39]. Importantly, loss of maternal H3K27me3 induces Xist activation, indicating that maternal H3K27me3 is the major imprinting mark of Xist . This is strikingly similar to findings made in this study revealing H3K27me3 as the major repressive mark for PEGs. Recent work revealed that maternal H3K27me3 controls DNA methylation-independent imprinting in mammalian preimplantation embryos . While imprinted expression of most genes is lost in the embryonic cell lineage, few genes maintain their imprinted expression in the extra-embryonic cell lineage . The Xist locus that is marked by H3K27me3 and H3K9me3 is among those loci that remain imprinted in extra-embryonic tissues . Whether the presence of both marks distinguishes those genes that maintain their imprinted expression from those that become biallelically expressed remains to be tested, but we consider this a very attractive hypothesis. We speculate that the presence of both marks in certain tissue types of mammals and flowering plants is a conserved epigenetic signature marking stably repressed genes.
We discovered the co-occurrence of the PRC2-mediated H3K27me3 and heterochromatic H3K9me2 and CHG methylation as an epigenetic signature marking the silenced maternal alleles of PEGs. This signature can be used to predict PEGs at high accuracy, and based on this prediction, we estimate that the number of PEGs is substantially larger than previously estimated. We hypothesize that the common use of PRC2 and H3K9 methylation to silence target loci during reproduction has convergently evolved in flowering plants and mammals to ensure stable silencing during this sensitive life stage.
Plant material and growth conditions
All seeds were surface sterilized (5% sodium hypochlorite and 0.01% Triton X-100), stratified for 2 days at 4 °C, and germinated on half-strength Murashige and Skoog medium containing 1% sucrose under long-day conditions (16 h light/8 h darkness, 21 °C). Plants were transferred to soil after 10 to 12 days and grown under long-day conditions. The cmt3-11 (SALK_148381; ) and suvh456 mutants (kindly provided by Judith Bender) used in this study are in the Col-0 background.
Imprinting assays and expression analysis
To generate siliques of indicated crosses, three to five flowers were emasculated, hand-pollinated, and harvested at 4 DAP. RNA extraction was performed using the MagJET Plant RNA Purification Kit (Thermo Scientific) following the manufacturer’s instructions. Residual DNA was removed using Invitrogen DNase I (Amplification Grade), and cDNA was synthesized using the Fermentas first-strand cDNA synthesis kit according to the manufacturer’s instructions. Quantitative PCR was performed using a MyiQ5 real-time PCR detection system (Bio-Rad) and Solis BioDyne-5x Hot FIREPol EvaGreen qPCR Mix Plus (ROX, Solis BioDyne). For the imprinting-by-sequencing assay, the PCR products were purified and analyzed by Sanger sequencing. For the imprinting-by-restriction enzyme digestion assay, the PCR products were purified and digested. Restriction enzymes and primers used are listed in the Additional file 1: Table S6.
We made use of endosperm-specific ChIP-seq data that have been previously generated in our group . Data correspond to pooled biological triplicates with ChIP signals being normalized with H3 ChIP data by calculating the log2 ratio in 150-bp bins across the genome. Data were standardized and normalized with a z-score transformation . Metagene plots over genes were constructed between − 2 and + 2 kb by calculating mean levels of methylation signals in 100-bp bins in the flanks of the genes and in 40 equally long bins between the transcriptional start and stop. Gene z-scores were calculated as an average of z-scores over the gene body. DNA methylation data of fie and dme and their corresponding wild types are from . DNA methylation data of the central cell, sperm, and vegetative cells are from [3, 17]. Endosperm expression data are from  and seed coat expression data from .
Endosperm nuclei isolation and RNA sequencing
We performed Col × Ler reciprocal crosses using Arabidopsis lines expressing PHE1::NTF and PHE1::BirA (lines referred as INT hereafter) . To facilitate the crosses, we used the male sterile mutants pistillata (pi-1, in Ler accession) and dde2 (in Col accession containing INT) as female parents and pollinated them with the INT line (Col accession) and Ler wild type, respectively. A total of 500 mg of siliques for the first replicates and 250 mg of siliques for the second and third replicates were collected at 4 DAP. Tissue homogenization, nuclei purification, RNA extraction, and library preparation were performed from three biological replicates as previously described [43, 44]. Samples were sequenced at the National Genomic Infrastructure (NGI) from SciLife Laboratory (Uppsala, Sweden) on an Illumina HiSeq2500 in paired-end 125 bp read length. Mapping and discrimination of parental reads was done as previously described . We calculated contamination levels based on the deviation of the read counts from the expected 2:1 maternal/paternal genome ratio in the endosperm  (Additional file 1: Table S6). Based on this analysis, the first replicates of both cross directions Col × Ler and Ler × Col were not included in the downstream analyses. To increase the statistical power to detect parentally biased genes, we merged libraries from two replicates of Col × Ler and three replicates of Ler × Col for downstream analysis.
Allele-specific expression analysis
We followed our previously published analysis pipeline to define imprinted genes . Briefly, we defined a minimum threshold of 20 informative reads for Col × Ler (2 replicates) and Ler × Col (2 replicates) crosses, respectively. Statistical differences between maternal and paternal read counts for each gene were calculated using a chi-square test, considering genes with a false discovery rate adjusted P value of less than 0.01. Additionally, MEGs required to have at least 85% maternal informative reads in both directions of the reciprocal cross and PEGs to have at least 50% paternal informative reads in both directions of the reciprocal cross, following previously defined conditions . Quality of sequencing samples is shown in Additional file 1: Table S7.
Generation of reporter constructs and transgenic lines
For the generation of reporter constructs, we used the ClonExpress® MultiS One Step Cloning kit. Promoters (≃ 2 kb) and genic sequences of AT2G33620, AT1G43580, AT1G47530, AT1G64660, AT2G30590, AT4G15390, and AT5G53160 were amplified from WT Col-0 genomic DNA using primers specified in Additional file 1: Table S6 and cloned into vector pB7FWG.0. Constructs were transformed into Agrobacterium tumefaciens strain GV3101, and Arabidopsis plants were transformed using the floral dip method . Ten transgenic lines per construct were generated and analyzed.
For reciprocal crosses, designated female partners were emasculated at 1–2 days prior to anthesis. Two days after emasculation, pistils were hand-pollinated with respective pollen donors. Seeds were dissected from the siliques and mounted on a microscope slide for imaging and counting at 2 and 4 DAP. For fluorescence analyses, seeds were stained with 0.1 mg/mL propidium iodide (PI) solution in 7% glucose. Seeds of reciprocal crosses of reporter lines were analyzed under confocal microscopy on a Zeiss 800 Inverted Axio Observer with a supersensitive GaASp detector. Images were acquired, analyzed, and exported using Zeiss ZEN software. For each reporter construct, 50–60 seeds per line were analyzed.
Sequencing was performed by the SNP&SEQ Technology Platform, Science for Life Laboratory at Uppsala University, a national infrastructure supported by the Swedish Research Council (VRRFI) and the Knut and Alice Wallenberg Foundation.
This research was supported by a European Research Council Starting Independent Researcher grant (to C.K.), a grant from the Swedish Science Foundation (to C.K.), a grant from the Knut and Alice Wallenberg Foundation (to C.K.), the Göran Gustafsson Foundation for Research in Natural Sciences and Medicine (to C.K.), and an EMBO fellowship (to G.D.T.D.L).
Availability of data and materials
The RNA-seq data generated in this study are available through GEO (GSE119915) publicly available at https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE119915 . We furthermore used endosperm RNA expression data from  (GSE52814), seed coat expression data from  (GSE12404), parental-specific histone and DNA methylation data from  (GSE66585), central cell DNA methylation profiles from  (GSE89789), and DNA methylation data from sperm cell, vegetative cell and endosperm of fie and dme mutants from  (GSE38935).
JMR, VKY, and GDTDL executed the experimental procedures. JMR, JSG, GDTDL, VKY, and CK analyzed the data. JMR and CK wrote the manuscript. All authors discussed the results and commented on the manuscript. All authors read and approved the final manuscript.
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