Abstract
The Arabidopsis DEMETER (DME) DNA glycosylase demethylates the maternal genome in the central cell prior to fertilization, and is essential for seed viability. DME preferentially targets small transposons that flank coding genes, influencing their expression and initiating plant gene imprinting. DME also targets intergenic and heterochromatic regions, and how it is recruited to these differing chromatin landscapes is unknown. The C-terminal DME catalytic core consists of three conserved regions required for catalysis in vitro. We show that the catalytic core of DME guides active demethylation at endogenous targets, rescuing the developmental and genomic hypermethylation phenotypes of DME mutants. However, without the N-terminus, heterochromatin demethylation is significantly impeded, and abundant CG-methylated genic sequences are ectopically demethylated. We used comparative analysis to reveal that the conserved DME N-terminal domains are only present in the flowering plants, whereas the domain architecture of DME-like proteins in non-vascular plants mainly resembles the catalytic core, suggesting that it might represent the ancestral form of the 5mC DNA glycosylase found in all plant lineages. We propose a bipartite model for DME protein action and suggest that the DME N-terminus was acquired late during land plant evolution to improve specificity and facilitate demethylation at heterochromatin targets.
Introduction
DNA methylation is a covalent modification that influences the transcription of nearby genes and regulates important processes in eukaryotic genomes, including cell differentiation, transposable element (TE) silencing, and genomic imprinting (1, 2). Plant DNA methylation occurs in CG, CHG, and CHH sequence contexts (H = A, C, or T) and is primarily targeted to TEs. Flowering plants and mammals can also exhibit gene body methylation (gbM) in the CG context, generally in constitutively expressed genes, but the function of gbM is not fully understood (3, 4).
Maintaining DNA methylation homeostasis is essential for genome stability, notably in maintaining TE silencing, and for the stable and long-term inheritance of epigenetic information (5, 6). In plants, this is achieved by maintenance and de novo DNA methylation, and by active DNA demethylation (1, 7–10), Active DNA demethylation is catalyzed by a family of novel DNA glycosylases, REPRESSOR OF SILENCING 1 (ROS1), DEMETER (DME), DEMETER-LIKE 2 (DML2) and DML3 through a base excision repair pathway (11, 12). However, epigenetic profiles are dynamic in response to biotic and abiotic stress, and during reproduction and development. Similar to mammals, complete epigenetic reprogramming is required during flowering plant gamete formation, characterized by extensive DNA demethylation, which in Arabidopsis is performed by DME (13, 14).
DME encodes a bifunctional 5mC DNA glycosylase/lyase that is essential for reproduction (11). Paralogs ROS1, DML2, and DML3 function primarily in the sporophyte to counteract the spread of DNA methylation mediated by RNA-dependent DNA methylation (RdDM) (15, 16). The A, Glycosylase, and B regions of the C-terminal half of DME are conserved among the DME/ROS1 DNA glycosylase clade, and are absolutely required for DME 5mC excision activity in vitro, comprising the minimal catalytic core for direct excision of 5mC from DNA (11, 17, 18). DME acts specifically in the central cell of the female gametophyte and the vegetative nucleus of pollen (17, 19, 20). The pollen grain contains the vegetative nucleus and two sperm cells, and the vegetative nucleus contributes to the germination and growth of the pollen tube, which delivers the to sperm cells to the female gametophyte. Following double fertilization, the egg and central cell respectively develop into the embryo and nutritive endosperm, which accumulates starch, lipids, and storage proteins to nourish the developing embryo (21, 22). The endosperm is also the site of plant genomic imprinting, i.e. parent-of-origin specific expression, resulting from allelic inheritance of differential epigenetic states. DNMT1 homolog MET1-mediated DNA methylation and DME-mediated demethylation are primary players in the epigenetic regulation of endosperm imprinting (11, 23–26). For example, DME-mediated DNA demethylation is required for the activation of MEA, FIS2, and FWA expression in the central cell, which persists in the endosperm, while MET1 is responsible for the silencing of FIS2 and FWA paternal alleles (23, 25). Imprinting is essential for reproduction in Arabidopsis, and seeds that inherit a maternal dme allele abort due to failure to activate MEA and FIS2, essential components of the endosperm Polycomb Repressive Complex 2 (PRC2) required for seed viability (17, 23).
Although DME preferentially targets small, AT-rich, and nucleosome-poor euchromatic transposons, it also demethylates intergenic and heterochromatin targets (13). How DME is recruited to distinct target sites with various chromatin structures is largely unknown, although the FAcilitates Chromatin Transactions (FACT) histone chaperone is required at heterochromatic targets and some imprinted loci (27, 28). Other than the glycosylase domain, the catalytic core region of the DME/ROS1-like DNA glycosylases contains multiple conserved globular domains of unknown function.
Here, we show that expressing a nuclear-localized DME C-terminal catalytic region controlled by a native DME promoter complements dme seed abortion and pollen germination defects, and partially rescues the DNA hypermethylation phenotype in endosperm. DNA methylation analysis revealed that the majority of canonical DME target sites are demethylated by the catalytic core, indicating that this region is sufficient to direct DME targeting and localization. However, without the N-terminal region, the degree of demethylation is reduced across all target sites and demethylation of heterochromatin targets is particularly impeded. In addition, we observed prevalent ectopic demethylation specifically at genic sequences by the catalytic core. Thus, the N-terminal region of DME is needed for the full breadth and depth of demethylation and to prevent gene body demethylation. We provide evidence that the N-terminal conserved domains are specific to the angiosperm lineage, acquired late during land plant evolution, potentially to ensure robust demethylation in nucleosome-rich heterochromatin targets.
Results
Nuclear-Localized Catalytic Core of DME Rescues dme Associated Developmental Defects
DME encodes at least two alternatively spliced variants, capable of producing two hypothetical polypeptides of 1,729 (DME.1) and 1,987 (DME.2) amino-acids in length (17, 29). The amino acid positions denoted in this study correspond to DME.2, the predominant isoform expressed in floral tissues (19). First, we detailed the domain structure and predicted characteristics of the catalytic and non-catalytic regions of the DME protein. The N-terminal half of DME consists of a large portion of unstructured, low complexity sequences (aa 364-947, N-terminal variable region, Fig. 1A), a stretch of basic amino acid-rich repeats (aa 291-363, Basic stretch) previously shown to direct nuclear localization (17), and an 120 amino-acid N-terminal domain (aa 1-120, DemeN) of unknown function present only among the angiosperm DME/ROS1-like proteins. Within the DemeN domain is a sWIPxTPxKs motif that is highly conserved (Fig. S1) but is absent in the shorter DME.1 isoform. This motif has a hydrophobic core and may mediate protein-protein interactions and/or contribute to DME regulation by post-translational modification i.e. phosphorylation or methylation at the lysine residue. The basic stretch region is highly conserved among angiosperm DME-like proteins and is reminiscent of the AT-hook motifs that can bind DNA in a non-sequence specific manner (30), suggesting the basic stretch might also bind DNA, in addition to directing DME to the nucleus.
It was previously determined that the C-terminal half of DME (aa 936-1987, hereafter the DMECTD) constitutes the catalytic core for 5mC excision in vitro (11, 31). This core contains the catalytic glycosylase domain of the HhH (helix-hairpin-helix) motif followed by the [4Fe-4S] cluster loop (FCL) motif (32). The conserved B region contains a domain predicted to adopt an RNA Recognition Motif-like fold (RRMF) and a divergent, circularly permuted version of a singlecopy methylated CpG-discriminating CXXC domain (32). The presence of a permuted CXXC and RRMF raises the possibility that the enzymatic core might contain intrinsic targeting information (33, 34). To test this possibility, we investigated whether DMECTD can rescue dme seed abortion phenotype when expressed by an endogenous DME promoter. A classical SV40 NLS (PKKKPKV) was included to ensure robust nuclear localization (Fig. 1A). The resulting transgene (nDMECTD) was transformed directly into DME/dme-2 heterozygous Col-g/ plants via the floral dipping procedure (35). Self-pollinated non-transformed DME/dme-2 plants produce 50% viable (inherited DME maternal allele) and 50% aborted (inherited dme-2 maternal allele) F1 seeds. In contrast, transgenic lines that were DME/dme-2 and hemizygous for a single locus nDMECTD transgene, produced a 3:1 ratio of viable to aborted F1 seeds when self-pollinated (1292:439, 3:1, χ2 = 0.12, P > 0.7, Table S1), indicating that nDMECTD fully complemented the dme-mediated seed abortion phenotype. When the nDMECTD transgene was transformed into dme-2/dme-2 homozygous plants (Materials and Methods), the resulting T1 transgenic lines (dme-2/dme-2;nDMECTD/~) displayed 50% to 75% viable seed rates when self-pollinated (Table S1), compared to selfed non-transformed dme-2/dme-2 plants which bear < 0.05% viable seeds, again indicating full seed abortion complementation by nDMECTD (Fig. 1B, C, Table S1). These results show that nDMECTD can rescue the dme-2 seed abortion phenotype. Since dme mutant seed abortion is partially due to defects in activating imprinted Polycomb Repressor Complex 2 (PRC2) genes (36), we used qRT-PCR to measure expression of PRC2 subunit FIS2, and FWA, whose maternal expression is enabled by DME-mediated DNA demethylation, and found that nDMECTD restored the expression of these genes (Fig. F1D).
DME is also expressed in the vegetative cell of pollen, and mutations in DME reduce pollen germination, resulting in lower transmission of the paternal mutant dme allele in certain ecotypes (20). Under our growth conditions, when DME/dme-2 heterozygous Col-gl plants were self-pollinated, about 20-30% (compared to the expected 50% if dme-2 allele is normally transmitted) of the F1 progeny were dme-2 heterozygotes (Table S2). To test whether nDMECTD can rescue the dme pollen phenotype, we pollinated wild-type Col-0 with pollen derived from transgenic lines that are homozygous for the dme-2 allele and carry a single locus of the nDMECTD transgene (dme-2/dme-2; nDMECTD/~ lines with ~50% seed abortion rates, Table S1). If nDMECTD does not complement dme-2 pollen germination defects, we expect roughly half of the F1 progeny will carry the nDMECTD transgene (hygromycin resistant) because mutant pollen with or without the transgene would germinate with equal frequency. Instead, we observed 65% - 90% of the F1 progeny are hygromycin resistant (resistant: sensitive = 190:52, 1:1, χ2 = 79.69, P = 7.3E−19; Table S3), indicating that nDMECTD rescues dme-2 pollen defects. These results show that nDMECTD can rescue the dme developmental phenotype, which indicates that DME targeting and recruitment information is contained within the catalytic core region.
Canonical DME target loci are demethylated by nDMECTD
The molecular cause of DME mutant phenotypes are a loss of DME-mediated genome-wide DNA demethylation. To test the extent of nDMECTD complementation, we compared the methylome of the nDMECTD-complemented endosperm to those of wild-type and dme-2 endosperm (13). nDMECTD-complemented endosperm methylomes from three independent lines (dme-2/dme-2, nDMECTD/nDMECTD, F2 and F3 generation of dme-2 homozygotes) were generated and the reads combined for downstream analyses (Pearson correlation coefficients between independent lines showed they were highly concordant >0.93 (Table S4)). We compared the differentially methylated regions (DMRs) between dme-2 and wild-type endosperm (DME targets), and the DMRs between dme-2 and nDMECTD-complemented endosperm (nDMECTD targets) (Materials and Methods) (13). Looking genome-wide, but excluding all DME and nDMECTD targets, the Pearson correlation coefficients between our combined independent lines and the previously published wild-type and dme-2 endosperm datasets ranged between 0.92 and 0.94 (Table S5), indicating a high level of concordance between the methylomes used in this study.
We found several DME-regulated imprinting control regions of maternally and paternally expressed genes (MEGs and PEGs, respectively) to be hypomethylated in the nDMECTD-complemented endosperm compared to dme-2 endosperm (Fig. S2), suggesting that nDMECTD is active at these loci. CG methylation does not return to wild-type levels, however, indicating that the genome is demethylated to a lesser degree by nDMECTD than by wild-type DME (Fig. 2A and Fig. S3C). The DME and nDMECTD DMRs largely overlap (Fig. 2B), and for the DMRs that appear unique to DME, the same regions are also demethylated by nDMECTD (Fig 2C, black solid-line trace), but to a reduced degree (the solid black peak is on the left of the dotted peak) that falls below our DMR cutoff (fractional CG methylation difference ≥ 0.3, Fisher’s exact test p-value < 10−10). The shared DMRs are also slightly less demethylated in nDMECTD-complemented endosperm compared to wild-type endosperm (Fig. 2D, red trace is to the left of the black trace). Together, these data show that nDMECTD rescues the dme-associated endosperm hypermethylation phenotype, but only partially. This reduced demethylation was not a result of low transgene expression as far as we could ascertain, since qRT-PCR analyses of endosperm tissue showed that the nDMECTD is abundantly expressed (Fig. S2D).
To investigate whether chromatin features influence nDMECTD recruitment and demethylation, we assessed the specific histone marks and genomic characteristics (37) in target sites that are nDMECTD-unique, DME-unique, or shared between the two. Compared to nDMECTD-DME shared DMRs, DME-unique target sites are highly enriched for heterochromatin states 8 and 9 (Fig. 2E). nDMECTD DMRs (unique and shared) are enriched for chromatin states 1, 6, and 7 that correspond to open chromatin states, but show a significant reduction in heterochromatic chromatin states 8 and 9 when compared to DME DMRs (Fig. 2E)(37). Thus, nDMECTD demethylates poorly at heterochromatic loci and preferentially targets euchromatin sites.
The DME N-Terminal Region is Required for Effective Demethylation at Long Heterochromatic Target Sites
Longer DME DMRs almost exclusively reside in heterochromatin (86.3% of 1-1.5 kb and 95.5% of ≥ 1.5 kb, Fig. 3A). We postulate that this is due to the dense methylation associated with heterochromatin, which may result in longer stretches of DNA demethylation during DME occupancy at these sites. Interestingly, the number of long DMRs is dramatically reduced in nDMECTD-complemented endosperm (Fig. 3A, B). This reduction in the number of longer DMRs was not due to a lack of nDMECTD targeting to these sites, since the partial demethylation characteristic of nDMECTD activity occurred in all targets regardless of their length (Fig. S3E). However, when we analyzed the length of the nDMECTD demethylated regions, we found that it produced much shorter DMRs (Fig. S5). For example, there are 250 DME DMRs that are longer than 1.5 kb (median = 1.9kb). Among them, 165 are also DMRs of nDMECTD, but are much shorter in length (median = 400bp) (Fig. 3C-D). Thus, removal of the DME N-terminal region significantly reduced the extent of demethylation in these long targets.
The histone chaperone FACT complex is required for demethylation of about half of DME targets in Arabidopsis, particularly those in longer TEs in heterochromatin regions (27). DME co-localizes with SPT16 (the larger of the two FACT subunits) in an in vivo BiFC assay, suggesting that DME might recruit the FACT complex to these heterochromatic target loci (27). Of the 250 long DME DMRs, 87% of them require FACT activity, raising the possibility that nDMECTD might be defective in recruiting FACT. To test this hypothesis, we examined how nDMECTD demethylates FACT-dependent vs independent loci. In wild-type endosperm, both target groups are demethylated to a similar degree (Fig. 3E, blue and green traces have similar shape and peak). In the nDMECTD-complemented endosperm, FACT-independent loci are only slightly less demethylated compared to wild-type endosperm (Fig. 3E, orange trace moderately shifted to the left of blue and green traces). In contrast, demethylation at the FACT-dependent loci is more severely impeded (Fig. 3E, red trace). These results support a model whereby DME recruits FACT via the N-terminal region to make heterochromatic regions accessible.
Prevalent Ectopic Gene Body Demethylation by nDMECTD
Our analyses revealed a set of new DMRs that were unique to nDMECTD, which we term as ‘ectopic’ targets (Fig 2B). The CG methylation difference between dme-2 and wild-type endosperm for these DMRs is minimal but not absent (Fig. 2C, although the red trace peak is close to zero, there is a positive right-hand shoulder). As shown in Fig. 4A, we plotted the methylation status of nDMECTD-unique loci in wild-type endosperm to assess how they are demethylated by DME. This resulted in positive peaks for shorter (red trace) or longer (blue trace) transposable elements (TEs), and intergenic sequences (green trace), showing that these nDMECTD-unique loci are also demethylated by DME but not enough to reach the DMR cutoff (fractional CG methylation difference ≥ 0.3, Fisher’s exact test p-value < 10−10). In contrast, most nDMECTD-unique loci within coding sequences are not demethylated by DME (Fig. 4A, orange trace). This indicates that genic sequences are the primary ectopic targets of nDMECTD. This is also reflected by the increased nDMECTD DMR frequency (Fig. 4B, red and orange traces), the decreased average CG methylation (Fig. S4) within coding genes. These results show that nDMECTD preferentially targets methylated coding genes.
Gene body CG methylation, or gbM, is an evolutionarily conserved feature in mammals and angiosperms, but the origin of gbM remains unknown and its function is highly debated (3, 4). Around 15% (~5,000) of Arabidopsis genes contain gbM (38). In our endosperm methylomes, we found 2,202 and 3,213 genes are associated with DME or nDMECTD DMRs, respectively, which were largely mutually exclusive and thus constituted almost all of the genes with gbM (Fig. 4C). Among them, 2,260 genes were ectopically targeted by nDMECTD (Fig. 4C, Dataset S1). These nDMECTD-unique genes have higher CG methylation and higher expression levels compared to DME-targeted coding genes (Fig. 4D). They include genes across most actively used cellular processes (Table S6), consistent with the current theory that moderate gene body methylation positively correlates with constitutively transcribed genes (3). These observations suggest the presence of an active mechanism that prevents DME from targeting genes undergoing active transcription in wild-type plants, but which is impaired by nDMECTD.
The Evolutionary History of the DME/ROS1 Glycosylase Family
Our data show that the C-terminal catalytic region of DME is able to efficiently demethylate DNA in vivo, and also harbors targeting ability. We also demonstrate that the N-terminal domain is important for fine-tuning DME targeting to heterochromatin and restricting it from gene bodies. To provide clues as to the evolutionary origin of these bipartite domains, we carried out comparative analyses across plant lineages. Using various DME homologs as query sequences, we revealed a diversity of N-terminal domains associated with the DME catalytic core across various clades (Fig. 5). These indicated that a shorter protein, comprising only the C-terminal of Arabidopsis DME, may represent the ancestral form of the 5mC glycosylase that is found in all plant lineages. Variation of the N-terminal domains included land plants and charophytes (streptophyta), which possess a divergent circularly permuted CXXC domain between the FCL and RRMF domains. In contrast, one or more copies of the classical CXXC can be found in chlorophyte and stramenopile algae at distinct positions. Chlorophyte and stramenopile homologs also possess additional chromatin readers such as Tudor and PHD, DNA-binding domains such as the AT-hook domain, and the chaperone Hsp70-interacting DnaJ domains (32, 39). These accessory domains suggest a mode of regulating DNA glycosylase activity according to methylation status (via CXXC) or chromatin states (via PHD, Tudor). The DemeN and basic-stretch of the DME N-terminal region are restricted to the angiosperm lineage and appear to be a late acquisition during land plant evolution (Fig. 5). The acquisition of this region thus coincides with the origins of double fertilization in plants and emergence of plant gene imprinting.
Discussion
DME is an essential epigenetic modifier that regulates gene imprinting and influences trans-generational epigenetic inheritance in Arabidopsis (40). DME demethylates the central and vegetative gamete companion cell genomes at thousands of loci, but the mechanism of DME targeting remains elusive. This is due to its restricted expression to these ephemeral nuclei, embedded within Arabidopsis gametophytes and largely prohibiting biochemical interrogation, at least with current tools. In contrast, genetic analysis, coupled with endosperm transcriptome and methylome profiling, has been instrumental in revealing DME’s molecular function (11, 13, 14, 27, 41). Here, we used genetic complementation and endosperm DNA methylation profiling assays to show that the catalytic core of the DME protein, containing the DNA glycosylase, FCL, permuted CXXC and divergent RRMF domains, is sufficient to rescue the dme seed abortion phenotype and complement the pollen germination defects caused by the dme mutation (Fig. 1). We also present evidence that nDMECTD can be recruited to, and demethylate, most canonical DME target sites, and reason that the DME C-terminal catalytic region must contain sufficient information for targeting and recruitment. We go on to suggest the DME protein has a bipartite structure (Fig. S7) and demonstrate a requirement for the N-terminal domain in targeting DME to heterochromatin, possibly via FACT.
Although our nDMECTD complemented dme-associated developmental defects (Fig 1B, Table S1, S3), it did not fully rescue the dme endosperm DNA hypermethylation phenotype. Instead, we observed a reduced degree of demethylation by nDMECTD in all of the endogenous DME target loci, regardless of their length (Fig. 2A, S2E). From this, we deduce that the DME N-terminal region is likely required for full and robust demethylation activity. To achieve this, the N-terminal region may assist the glycosylase by tightly binding to DNA templates (via the AT-hook) to promote complete and thorough demethylation. Previous studies of ROS1 support such a model: the basic stretch/AT-hook region of ROS1 was shown to bind strongly to DNA templates in vitro in a non-sequence-specific manner, and removal of the ROS1 basic-rich region impaired the sliding capacity of ROS1 on the DNA template (42), significantly reducing ROS1 5mC excision activity (30).
DME preferentially targets smaller euchromatic transposons that flank coding genes, it also targets gene-poor heterochromatin regions for demethylation (13). Since heterochromatin regions are compacted, demethylation in these regions requires substantial chromatin remodeling, including the temporary eviction of nucleosomes in order for DME to gain access to DNA. This is reminiscent of the requirement for the ATP-dependent chromatin remodeler DDM1 at linker histone H1-associated heterochromatin for methyltransferases access (43). The nucleosome remodeler FACT was previously shown to be required for DME-mediated demethylation, primarily at heterochromatin targets, including those associated with H1 (27), and we additionally noted that these sites have increased nucleosome occupancy (Fig. S6). It is tempting to speculate that the DME N-terminal region is required to recruit factors such as FACT, and indeed we found the overwhelming majority of the 250 longer DME DMRs (87%) that were not demethylated in the absence of the N-terminal region, also require FACT activity for demethylation (Dataset S2). SPT16 was shown to co-localize with DME in vivo (27), suggesting that a direct or indirect interaction between DME and FACT does occur.
nDMECTD also displayed a reduced capacity in demethylating FACT-independent loci. Thus, it is possible that the N-terminal region is needed to recruit other chromatin remodelers at FACT-independent targets, i.e. if nucleosomes are a natural barrier for DME demethylation in euchromatin as well as heterochromatin (Fig. S6). We envision a working model (Fig. S7) where the catalytic region is sufficient for directing DME to target sites, whilst the N-terminal region is required to interact with the local chromatin environment, stabilizing binding to the chromosomal template, and/or assisting demethylation of flanking sequences by remodeling nucleosomes.
Our observation that nDMECTD demethylated a large number of ectopic sites in genic sequences was unexpected. Gene body methylation is evolutionarily conserved and around 20% of all Arabidopsis genes contain gbM (38, 44–47). Genes that are CG-methylated are preferentially constitutively expressed housekeeping genes (48), raising the possibility that these genic methylated genes reside in open chromatin regions, easily accessible by chromatin or DNA modifying enzymes, so that nDMECTD was able to act in those regions, whereas the more tightly-regulated endogenous DME is not. It is also possible that DME is actively repelled by certain open chromatin histone marks such as H3K4me1, and the region responsible for repulsion is missing in nDMECTD. This scenario would be analogous to the mammalian de novo DNA methyltransferase DNMT3, where its binding to an allosteric activator, unmethylated histone H3, is strongly inhibited by H3K4 methylation (49, 50).
Tracing the evolutionary history of the DME-like genes (Fig. 5), we found that a bacterial version of the HhH-FCL pair underwent a horizontal gene transfer to the ancestor of plants, followed by a gene duplication. One copy was fused to an RRMF domain and further acquired an insert in the glycosylase domain, giving the ancestral form of DME in plants. This was likely then transferred to the stramenopiles from a secondary chlorophyte endosymbiont of this lineage. Finally, at the base of the streptophyte radiation, DME acquired a permuted CXXC, and later the DemeN domain and associated charged repeats were acquired in angiosperms, possibly to facilitate and ensure robust and thorough DNA demethylation. Thus, the adoption of a DME-based demethylation system for DNA base modification appears to have occurred early in the plant lineage. The presence of several accessory domains in addition to the conserved core suggests variation in the chromatin environments in specific lineages. For example, the presence of the DemeN and basic stretch/AT-hook motifs in angiosperms and the permuted CXXC domain in the streptophyta lineage likely reflect adjustment to the unique methylation and chromatin environment of the larger Streptophyta and land plant genomes.
In summary, our data show that the catalytic core of DME was present in ancient plant ancestors, and is alone capable of targeting and demethylation. We provide evidence that the N-terminal domain, confined to flowering plant DME proteins, evolved to facilitate access to diverse chromatin states, in turn mediating gene imprinting and the transgenerational silencing of transposons (13).
Materials and Methods
Complete details concerning plant materials, experimental methods, and data analyses are provided in the Supplemental Information.
Author contributions
C.Z., Y.-H.H., R.L.F. and T.-F.H. designed research; C.Z., Y.-H.H., X.-Q.Z., and F.L. performed research; C.Z., Y.-H.H., X.-Q.Z., D.Z., J.M.F., L.M.I., L.A., and T.-F.H. analyzed data; J.M.F, R.L.F. and T.-F.H. wrote the paper, and W.X., L.M.I., L.A., J.H.H. provided insightful comments on the research and the manuscript.
Data availability
All data generated or analyzed in this study are included in this article and Supplementary Information files. BS-seq sequencing data are available in NCBI (GEO XXXXXX).
Acknowledgements
We thank J.-Y. Lin (UCLA), P.-H. Hsieh (JGI), D.B. Lyons (UC Berkeley), and Y.H. Choi (Seoul National University) for suggestions and critical reading of the manuscript. This work is supported by the Hatch Project (No. 02413) of National Institute of Food and Agriculture, USDA to T.-F.H., the National Science Foundation Grant MCB-1715115 to T.-F.H. and W.Y.X. LMI and LA are supported by the IRP funds of the National Institutes of Health, USA.