Imprinting in Plants and Its Underlying Mechanisms

Genomic imprinting (or imprinting) refers to an epigenetic phenomenon by which the allelic expression of a gene depends on the parent of origin. It has evolved independently in placental mammals and flowering plants. In plants, imprinting is mainly found in endosperm. Recent genome-wide surveys in Arabidopsis, rice, and maize identified hundreds of imprinted genes in endosperm. Since these genes are of diverse functions, endosperm development is regulated at different regulatory levels. The imprinted expression of only a few genes is conserved between Arabidopsis and monocots, suggesting that imprinting evolved quickly during speciation. In Arabidopsis, DEMETER (DME) mediates hypomethylation in the maternal genome at numerous loci (mainly transposons and repeats) in the central cell and results in many differentially methylated regions between parental genomes in the endosperm, and subsequent imprinted expression of some genes. In addition, histone modification mediated by Polycomb group (PcG) proteins is also involved in regulating imprinting. DME-induced hypomethylated alleles in the central cell are considered to produce small interfering RNAs (siRNAs) which are imported to the egg to reinforce DNA methylation. In parallel, the activity of DME in the vegetative cell of the male gametophyte demethylates many regions which overlap with the demethylated regions in the central cell. siRNAs from the demethylated regions are hypothesized to be also transferred into sperm to reinforce DNA methylation. Imprinting is partly the result of genome-wide epigenetic reprogramming in the central cell and vegetative cell and evolved under different selective pressures.     

Genomic imprinting in plants: observations and evolutionary implications

The epigenetic phenomenon of genomic imprinting occurs among both plants and animals. In species where imprinting is observed, there are parent-of-origin effects on the expression of imprinted genes in offspring. This review focuses on imprinting in plants with examples from maize, where gene imprinting was first described, and Arabidopsis. Our current understanding of imprinting in plants is presented in the context of cytosine methylation and imprinting in mammals, where developmentally essential genes are imprinted. Important considerations include the structure and organization of imprinted genes and the role of regional, differential methylation. Imprinting in plants may be related to other epigenetic phenomena including paramutation and transgene silencing. Finally, we discuss the role of gene structure and evolutionary implications of imprinting in plants.     

Reproductive barrier and genomic imprinting in the endosperm of flowering plants

In flowering plants, success or failure of seed development is determined by various genetic mechanisms. During sexual reproduction, double fertilization produces the embryo and endosperm, which both contain maternally and paternally derived genomes. In endosperm, a reproductive barrier is often observed in inter-specific crosses. Endosperm is a tissue that provides nourishment for the embryo within the seed, in a similar fashion to the placenta of mammals, and for the young seedling after germination. This review considers the relationship between the reproductive barrier in endosperm and genomic imprinting. Genomic imprinting is an epigenetic mechanism that results in mono-allelic gene expression that is parent-of-origin dependent. In Arabidopsis, recent studies of several imprinted gene loci have identified the epigenetic mechanisms that determine genomic imprinting. A crucial feature of genomic imprinting is that the maternally and paternally derived imprinted genes must carry some form of differential mark, usually DNA methylation and/or histone modification. Although the epigenetic marks should be complementary on maternally and paternally imprinted genes within a single species, it is possible that neither the patterns of epigenetic marks nor expression of imprinted genes are the same in different species. Moreover, in hybrid endosperm, the regulation of expression of imprinted genes can be affected by upstream regulatory mechanisms in the male and female gametophytes. Species-specific variations in epigenetic marks, the copy number of imprinted genes, and the epigenetic regulation of imprinted genes in hybrids might all play a role in the reproductive barriers observed in the endosperm of interspecific and interploidy crosses. These predicted molecular mechanisms might be related to earlier models such as the "endosperm balance number" (EBN) and "polar nuclei activation" (PNA) hypotheses.     

Regulation and flexibility of genomic imprinting during seed development

Genomic imprinting results in monoallelic gene expression in a parent-of-origin-dependent manner. It is achieved by the differential epigenetic marking of parental alleles. Over the past decade, studies in the model systems Arabidopsis thaliana and maize (Zea mays) have shown a strong correlation between silent or active states with epigenetic marks, such as DNA methylation and histone modifications, but the nature of the primary imprint has not been clearly established for all imprinted genes. Phenotypes and expression patterns of imprinted genes have fueled the perception that genomic imprinting is specific to the endosperm, a seed tissue that does not contribute to the next generation. However, several lines of evidence suggest a potential role for imprinting in the embryo, raising questions as to how imprints are erased and reset from one generation to the next. Imprinting regulation in flowering plants shows striking similarities, but also some important differences, compared with the mechanisms of imprinting described in mammals. For example, some imprinted genes are involved in seed growth and viability in plants, which is similar in mammals, where imprinted gene regulation is essential for embryonic development. However, it seems to be more flexible in plants, as imprinting requirements can be bypassed to allow the development of clonal offspring in apomicts.     

High-resolution analysis of parent-of-origin allelic expression in the Arabidopsis Endosperm

Genomic imprinting is an epigenetic phenomenon leading to parent-of-origin specific differential expression of maternally and paternally inherited alleles. In plants, genomic imprinting has mainly been observed in the endosperm, an ephemeral triploid tissue derived after fertilization of the diploid central cell with a haploid sperm cell. In an effort to identify novel imprinted genes in Arabidopsis thaliana, we generated deep sequencing RNA profiles of F1 hybrid seeds derived after reciprocal crosses of Arabidopsis Col-0 and Bur-0 accessions. Using polymorphic sites to quantify allele-specific expression levels, we could identify more than 60 genes with potential parent-of-origin specific expression. By analyzing the distribution of DNA methylation and epigenetic marks established by Polycomb group (PcG) proteins using publicly available datasets, we suggest that for maternally expressed genes (MEGs) repression of the paternally inherited alleles largely depends on DNA methylation or PcG-mediated repression, whereas repression of the maternal alleles of paternally expressed genes (PEGs) predominantly depends on PcG proteins. While maternal alleles of MEGs are also targeted by PcG proteins, such targeting does not cause complete repression. Candidate MEGs and PEGs are enriched for cis-proximal transposons, suggesting that transposons might be a driving force for the evolution of imprinted genes in Arabidopsis. In addition, we find that MEGs and PEGs are significantly faster evolving when compared to other genes in the genome. In contrast to the predominant location of mammalian imprinted genes in clusters, cluster formation was only detected for few MEGs and PEGs, suggesting that clustering is not a major requirement for imprinted gene regulation in Arabidopsis.     

H19 and Igf2--enhancing the confusion?

Genomic imprinting, whereby certain genes are expressed dependent on whether they are maternally or paternally inherited, is restricted to mammals and angiosperm plants. This unusual mode of gene regulation results from the complex interplay between cis-regulatory elements, leading to parent-of-origin-dependent epigenetic modifications and tissue-specific patterns of imprinted gene expression. Many studies of imprinting and imprinted genes have focused on epigenetic effects, such as DNA methylation and chromatin structure. However, it is equally important to explore the interconnected role of regulatory elements at imprinted domains by genetic experiments, including the use of transgenes and deletions.     

Evolution, function, and regulation of genomic imprinting in plant seed development

Genomic imprinting is an epigenetic phenomenon whereby genetically identical alleles are differentially expressed dependent on their parent-of-origin. Genomic imprinting has independently evolved in flowering plants and mammals. In both organism classes, imprinting occurs in embryo-nourishing tissues, the placenta and the endosperm, respectively, and it has been proposed that imprinted genes regulate the transfer of nutrients to the developing progeny. Many imprinted genes are located in the vicinity of DNA-methylated transposon or repeat sequences, implying that transposon insertions are associated with the evolution of imprinted loci. The antagonistic action of DNA methylation and Polycomb group-mediated histone methylation seems important for the regulation of many imprinted plant genes, whereby the position of such epigenetic modifications can determine whether a gene will be mainly expressed from either the maternally or paternally inherited alleles. Furthermore, long non-coding RNAs seem to play an as yet underappreciated role for the regulation of imprinted plant genes. Imprinted expression of a number of genes is conserved between monocots and dicots, suggesting that long-term selection can maintain imprinted expression at some loci.     

Epigenetic Resetting of a Gene Imprinted in Plant Embryos

Genomic imprinting resulting in the differential expression of maternal and paternal alleles in the fertilization products has evolved independently in placental mammals and flowering plants. In most cases, silenced alleles carry DNA methylation [1]. Whereas these methylation marks of imprinted genes are generally erased and reestablished in each generation in mammals [2], imprinting marks persist in endosperms [3], the sole tissue of reported imprinted gene expression in plants. Here we show that the maternally expressed in embryo 1 (mee1) gene of maize is imprinted in both the embryo and endosperm and that parent-of-origin-specific expression correlates with differential allelic methylation. This epigenetic asymmetry is maintained in the endosperm, whereas the embryonic maternal allele is demethylated on fertilization and remethylated later in embryogenesis. This report of imprinting in the plant embryo confirms that, as in mammals, epigenetic mechanisms operate to regulate allelic gene expression in both embryonic and extraembryonic structures. The embryonic methylation profile demonstrates that plants evolved a mechanism for resetting parent-specific imprinting marks, a necessary prerequisite for parent-of-origin-dependent gene expression in consecutive generations. The striking difference between the regulation of imprinting in the embryo and endosperm suggests that imprinting mechanisms might have evolved independently in both fertilization products of flowering plants.     

Parent-of-origin effects on gene expression and DNA methylation in the maize endosperm

Imprinting describes the differential expression of alleles based on their parent of origin. Deep sequencing of RNAs from maize (Zea mays) endosperm and embryo tissue 14 d after pollination was used to identify imprinted genes among a set of ~12,000 genes that were expressed and contained sequence polymorphisms between the B73 and Mo17 genotypes. The analysis of parent-of-origin patterns of expression resulted in the identification of 100 putative imprinted genes in maize endosperm, including 54 maternally expressed genes (MEGs) and 46 paternally expressed genes (PEGs). Three of these genes have been previously identified as imprinted, while the remaining 97 genes represent novel imprinted maize genes. A genome-wide analysis of DNA methylation identified regions with reduced endosperm DNA methylation in, or near, 19 of the 100 imprinted genes. The reduced levels of DNA methylation in endosperm are caused by hypomethylation of the maternal allele for both MEGs and PEGs in all cases tested. Many of the imprinted genes with reduced DNA methylation levels also show endosperm-specific expression patterns. The imprinted maize genes were compared with imprinted genes identified in genome-wide screens of rice (Oryza sativa) and Arabidopsis thaliana, and at least 10 examples of conserved imprinting between maize and each of the other species were identified.     

An atypical epigenetic mechanism affects uniparental expression of Pol IV-dependent siRNAs

Background

Small RNAs generated by RNA polymerase IV (Pol IV) are the most abundant class of small RNAs in flowering plants. In Arabidopsis thaliana Pol IV-dependent short interfering (p4-si)RNAs are imprinted and accumulate specifically from maternal chromosomes in the developing seeds. Imprinted expression of protein-coding genes is controlled by differential DNA or histone methylation placed in gametes. To identify epigenetic factors required for maternal-specific expression of p4-siRNAs we analyzed the effect of a series of candidate mutations, including those required for genomic imprinting of protein-coding genes, on uniparental expression of a representative p4-siRNA locus.

Results

Paternal alleles of imprinted genes are marked by DNA or histone methylation placed by DNA METHYLTRANSFERASE 1 or the Polycomb Repressive Complex 2. Here we demonstrate that repression of paternal p4-siRNA expression at locus 08002 is not controlled by either of these mechanisms. Similarly, loss of several chromatin modification enzymes, including a histone acetyltransferase, a histone methyltransferase, and two nucleosome remodeling proteins, does not affect maternal expression of locus 08002. Maternal alleles of imprinted genes are hypomethylated by DEMETER DNA glycosylase, yet expression of p4-siRNAs occurs irrespective of demethylation by DEMETER or related glycosylases.

Conclusions

Differential DNA methylation and other chromatin modifications associated with epigenetic silencing are not required for maternal-specific expression of p4-siRNAs at locus 08002. These data indicate that there is an as yet unknown epigenetic mechanism causing maternal-specific p4-siRNA expression that is distinct from the well-characterized mechanisms associated with DNA methylation or the Polycomb Repressive Complex 2.     

Deeper into the maize: new insights into genomic imprinting in plants

Current models for regulation of parent-specific gene expression in plants have been based on a small number of imprinted genes in Arabidopsis. These present repression as the default state, with expression requiring targeted activation. In general, repression is associated with maintenance methylation of cytosines, while no role has been found in Arabidopsis imprinting for de novo methylation--unlike the case in mammals. A recent paper both reinforces and challenges the model drawn from Arabidopsis. Methylation patterns of two imprinted loci in maize were tracked from gametes to offspring, enabling an exploration of the timing of imprinting. For one gene, fie1, the results were as expected: parent-specific methylation patterns were inherited from the three types of gamete: egg, central cell and sperm. The behaviour of fie2, however, was a surprise: no alleles were methylated in the gametes, although paternally contributed fie2 is methylated and silent in the endosperm, indicating that, in some cases, plant imprinting requires de novo DNA methylation. This work significantly broadens our understanding of plant imprinting and points to a greater diversity in imprinting mechanisms than has previously been appreciated.     

基因组印记与疾病研究进展

基因组印记是一种特别的非孟德尔遗传现象,即来自双亲的等位基因在子代中的差异性表达,是遗传后的基因调控方式,主要与基因组甲基化模式有关,包括去甲基化、重新甲基化及甲基化维持三个过程。印记基因主要通过对启动子、边界元件及非编码RNA的作用来调控基因表达。基因组印记异常与一些先天性疾病相关,也与肿瘤发生和易感性有关,     

Evolution and control of imprinted FWA genes in the genus Arabidopsis

A central question in genomic imprinting is how a specific sequence is recognized as the target for epigenetic marking. In both mammals and plants, imprinted genes are often associated with tandem repeats and transposon-related sequences, but the role of these elements in epigenetic gene silencing remains elusive. FWA is an imprinted gene in Arabidopsis thaliana expressed specifically in the female gametophyte and endosperm. Tissue-specific and imprinted expression of FWA depends on DNA methylation in the FWA promoter, which is comprised of two direct repeats containing a sequence related to a SINE retroelement. Methylation of this element causes epigenetic silencing, but it is not known whether the methylation is targeted to the SINE-related sequence itself or the direct repeat structure is also necessary. Here we show that the repeat structure in the FWA promoter is highly diverse in species within the genus Arabidopsis. Four independent tandem repeat formation events were found in three closely related species. Another related species, A. halleri, did not have a tandem repeat in the FWA promoter. Unexpectedly, even in this species, FWA expression was imprinted and the FWA promoter was methylated. In addition, our expression analysis of FWA gene in vegetative tissues revealed high frequency of intra-specific variation in the expression level. In conclusion, we show that the tandem repeat structure is dispensable for the epigenetic silencing of the FWA gene. Rather, SINE-related sequence is sufficient for imprinting, vegetative silencing, and targeting of DNA methylation. Frequent independent tandem repeat formation events in the FWA promoter led us to propose that they may be a consequence, rather than cause, of the epigenetic control. The possible significance of epigenetic variation in reproductive strategies during evolution is also discussed.     

Genome-wide survey of imprinted genes

The developmental failure of mammalian parthenogenote has been a mystery for a long time and posed a question as to why bi-parental reproduction is necessary for development to term. In the 1980s, it was proven that this failure was not due to the genetic information itself, but to epigenetic modification of genomic DNA. In the following decade, several studies successfully identified imprinted genes which were differentially expressed in a parent-of-origin-specific manner, and it was shown that the differential expression depended on the pattern of DNA methylation. These facts prompted development of genome-wide systematic screening methods based on DNA methylation and differential gene expression to identify imprinted genes. Recently computational approaches and microarray technology have been introduced to identify imprinted genes/loci, contributing to the expansion of our knowledge. However, it has been shown that the gene silencing derived from genomic imprinting is accomplished by several mechanisms in addition to direct DNA methylation, indicating that novel approaches are further required for comprehensive understanding of genomic imprinting. To unveil the mechanism of developmental failure in mammalian parthenogenote, systematic screenings for imprinted genes/loci have been developed. In this review, we describe genomic imprinting focusing on the history of genome-wide screening.     

Maintenance of DNA methylation during the Arabidopsis life cycle is essential for parental imprinting

Imprinted genes are expressed predominantly from either their paternal or their maternal allele. To date, all imprinted genes identified in plants are expressed in the endosperm. In Arabidopsis thaliana, maternal imprinting has been clearly demonstrated for the Polycomb group gene MEDEA (MEA) and for FWA. Direct repeats upstream of FWA are subject to DNA methylation. However, it is still not clear to what extent similar cis-acting elements may be part of a conserved molecular mechanism controlling maternally imprinted genes. In this work, we show that the Polycomb group gene FERTILIZATION-INDEPENDENT SEED2 (FIS2) is imprinted. Maintenance of FIS2 imprinting depends on DNA methylation, whereas loss of DNA methylation does not affect MEA imprinting. DNA methylation targets a small region upstream of FIS2 distinct from the target of DNA methylation associated with FWA. We show that FWA and FIS2 imprinting requires the maintenance of DNA methylation throughout the plant life cycle, including male gametogenesis and endosperm development. Our data thus demonstrate that parental genomic imprinting in plants depends on diverse cis-elements and mechanisms dependent or independent of DNA methylation. We propose that imprinting has evolved under constraints linked to the evolution of plant reproduction and not by the selection of a specific molecular mechanism.     

Imprinting and epigenetic changes in the early embryo

Imprinted genes are epigenetically regulated so that only one allele is expressed in a parent-of-origin-dependent manner. Although they represent a small subset of the mammalian genome, imprinted genes are essential for normal development. The regulatory mechanisms underlying imprinting are complex and have been the subject of extensive investigation. DNA methylation is the best-established epigenetic mark that is critical for the allele-specific expression of imprinted genes. This mark must be correctly established in the germline, maintained throughout life, and erased and reestablished in the germline the next generation. These events coincide with the genome-wide epigenetic reprogramming that occurs during gametogenesis and early embryogenesis; therefore, the establishment and maintenance of DNA methylation must be tightly regulated. Studies on enzymes that participate in both de novo methylation and its maintenance (i.e., the DNMT family) have provided information on how methylation influences imprinting. However, many aspects of the regulation of DNA methylation are unknown, including how methylation complexes are targeted and the molecular mechanisms underlying DNA demethylation. In this review we focus on the epigenetic changes that occur in the germline and early embryo, with an emphasis on imprinting. We summarize recent findings on factors influencing DNA methylation establishment, maintenance, and erasure that have further elucidated the mechanisms of imprinting, while highlighting topics that require further investigation.     

Chromatin regulators of genomic imprinting

Genomic imprinting is an epigenetic phenomenon in which genes are expressed monoallelically in a parent-of-origin-specific manner. Each chromosome is imprinted with its parental identity. Here we will discuss the nature of this imprinting mark. DNA methylation has a well-established central role in imprinting, and the details of DNA methylation dynamics and the mechanisms that target it to imprinted loci are areas of active investigation. However, there is increasing evidence that DNA methylation is not solely responsible for imprinted expression. At the same time, there is growing appreciation for the contributions of post-translational histone modifications to the regulation of imprinting. The integration of our understanding of these two mechanisms is an important goal for the future of the imprinting field. This article is part of a Special Issue entitled: Chromatin and epigenetic regulation of animal development.