Demeter Research Articles

HMG Domain Containing SSRP1 Is Required for DNA Demethylation and Genomic Imprinting in Arabidopsis

In Arabidopsis, DEMETER (DME) DNA demethylase contributes to reprogramming of the epigenetic state of the genome in the central cell. However, other aspects of the active DNA demethylation processes remain elusive. Here we show that Arabidopsis SSRP1, known as an HMG domain-containing component of FACT histone chaperone, is required for DNA demethylation and for activation and repression of many parentally imprinted genes in the central cell. Although loss of DNA methylation releases silencing of the imprinted FWA-GFP, double ssrp1-3;met1-3 mutants surprisingly showed limited activation of maternal FWA-GFP in the central cell, and only became fully active after several nuclear divisions in the endosperm. This behavior was in contrast to the dme-1;met1 double mutant in which hypomethylation of FWA-GFP by met1 suppressed the DNA demethylation defect of dme-1. We propose that active DNA demethylation by DME requires SSRP1 function through a distinctly different process from direct DNA methylation control.

Function of the DEMETER DNA glycosylase in the Arabidopsis thaliana male gametophyte

In double fertilization, the vegetative cell of the male gametophyte (pollen) germinates and forms a pollen tube that brings to the female gametophyte two sperm cells that fertilize the egg and central cell to form the embryo and endosperm, respectively. The 5-methylcytosine DNA glycosylase DEMETER (DME), expressed in the central cell, is required for maternal allele demethylation and gene imprinting in the endosperm. By contrast, little is known about the function of DME in the male gametophyte. Here we show that reduced transmission of the paternal mutant dme allele in certain ecotypes reflects, at least in part, defective pollen germination. DME RNA is detected in pollen, but not in isolated sperm cells, suggesting that DME is expressed in the vegetative cell. Bisulfite sequencing experiments show that imprinted genes (MEA and FWA) and a repetitive element (Mu1a) are hypomethylated in the vegetative cell genome compared with the sperm genome, which is a process that requires DME. Moreover, we show that MEA and FWA RNA are detectable in pollen, but not in isolated sperm cells, suggesting that their expression occurs primarily in the vegetative cell. These results suggest that DME is active and demethylates similar genes and transposons in the genomes of the vegetative and central cells in the male and female gametophytes, respectively. Although the genome of the vegetative cell does not participate in double fertilization, its DME-mediated demethylation is important for male fertility and may contribute to the reconfiguration of the methylation landscape that occurs in the vegetative cell genome.

Genome-Wide Transcript Profiling of Endosperm without Paternal Contribution Identifies Parent-of-Origin–Dependent Regulation of AGAMOUS-LIKE36

Seed development in angiosperms is dependent on the interplay among different transcriptional programs operating in the embryo, the endosperm, and the maternally-derived seed coat. In angiosperms, the embryo and the endosperm are products of double fertilization during which the two pollen sperm cells fuse with the egg cell and the central cell of the female gametophyte. In Arabidopsis, analyses of mutants in the cell-cycle regulator CYCLIN DEPENDENT KINASE A;1 (CKDA;1) have revealed the importance of a paternal genome for the effective development of the endosperm and ultimately the seed. Here we have exploited cdka;1 fertilization as a novel tool for the identification of seed regulators and factors involved in parent-of-origin–specific regulation during seed development. We have generated genome-wide transcription profiles of cdka;1 fertilized seeds and identified approximately 600 genes that are downregulated in the absence of a paternal genome. Among those, AGAMOUS-LIKE (AGL) genes encoding Type-I MADS-box transcription factors were significantly overrepresented. Here, AGL36 was chosen for an in-depth study and shown to be imprinted. We demonstrate that AGL36 parent-of-origin–dependent expression is controlled by the activity of METHYLTRANSFERASE1 (MET1) maintenance DNA methyltransferase and DEMETER (DME) DNA glycosylase. Interestingly, our data also show that the active maternal allele of AGL36 is regulated throughout endosperm development by components of the FIS Polycomb Repressive Complex 2 (PRC2), revealing a new type of dual epigenetic regulation in seeds.

Domain structure of the DEMETER 5-methylcytosine DNA glycosylase

DNA glycosylases initiate the base excision repair (BER) pathway by excising damaged, mismatched, or otherwise modified bases. Animals and plants independently evolved active BER-dependent DNA demethylation mechanisms important for epigenetic reprogramming. One such DNA demethylation mechanism is uniquely initiated in plants by DEMETER (DME)-class DNA glycosylases. Arabidopsis DME family glycosylases contain a conserved helix-hairpin-helix domain present in both prokaryotic and eukaryotic DNA glycosylases as well as two domains A and B of unknown function that are unique to this family. Here, we employed a mutagenesis approach to screen for DME residues critical for DNA glycosylase activity. This analysis revealed that amino acids clustered in all three domains, but not in the intervening variable regions, are required for in vitro 5-methylcytosine excision activity. Amino acids in domain A were found to be required for nonspecific DNA binding, a prerequisite for 5-methylcytosine excision. In addition, mutational analysis confirmed the importance of the iron-sulfur cluster motif to base excision activity. Thus, the DME DNA glycosylase has a unique structure composed of three essential domains that all function in 5-methylcytosine excision.

Imprinting genes and it’s expression in <I>Arabidopsis</I>

Genomic imprinting refers to the phenomenon that the expression of a gene copy depends on its parent of origin. The Arabidopsis imprinted FIS (Fertilisation-independent seed) genes, mea, fis2, and fie, play essential roles in the repression of central cell and the regulation of early endosperm development. fis mutants display two phenotypes: autonomous diploid endosperm development when fertilization is absent and un-cellularised endosperm formation when fertilization occurs. The FIS Polycomb protein complex including the above three FIS proteins catalyzes histone H3 K27 tri-methylation on target loci. DME (DEMETER), a DNA glycosylase, and AtMET1 (Methyltransferase1), a DNA methyltransferase, are involved in the regulation of imprinted expression of both mea and fis2. This review summarizes the studies on the Arabidopsis imprinted FIS genes and other related genes. Recent works have shown that the insertion of transposons may affect nearby gene expression, which may be the main driving force behind the evolution of genomic imprinting. This summary covers the achievements on Arabidopsis imprinted genes will provide important information for studies on genomic imprinting in the important crops such as rice and maize.

Methylation-independent DNA Binding Modulates Specificity of Repressor of Silencing 1 (ROS1) and Facilitates Demethylation in Long Substrates

DNA cytosine methylation is an epigenetic mark that promotes gene silencing and performs critical roles during reproduction and development in both plants and animals. The genomic distribution of DNA methylation is the dynamic outcome of opposing methylation and demethylation processes. In plants, active demethylation occurs through a base excision repair pathway initiated by 5-methycytosine (5-meC) DNA glycosylases of the REPRESSOR OF SILENCING 1 (ROS1)/DEMETER (DME) family. To gain insight into the mechanism by which Arabidopsis ROS1 recognizes and excises 5-meC, we have identified those protein regions that are required for efficient DNA binding and catalysis. We have found that a short N-terminal lysine-rich domain conserved in members of the ROS1/DME family mediates strong methylation-independent binding of ROS1 to DNA and is required for efficient activity on 5-meC.G, but not for T.G processing. Removal of this domain does not significantly affect 5-meC excision from short molecules, but strongly decreases ROS1 activity on long DNA substrates. This region is not required for product binding and is not involved in the distributive behavior of the enzyme on substrates containing multiple 5-meC residues. Altogether, our results suggest that methylation-independent DNA binding allows ROS1 to perform a highly redundant search for efficient excision of a nondamaged, correctly paired base such as 5-meC in long stretches of DNA. These findings may have implications for understanding the evolution of structure and target specificity in DNA glycosylases.

DNA cytosine methylation in plant development

Cytosine bases of the nuclear genome in higher plants are often extensively methylated. Cytosine methylation has been implicated in the silencing of both transposable elements (TEs) and endogenous genes, and loss of methylation may have severe functional consequences. The recent methylation profiling of the entire Arabidopsis genome has provided novel insights into the extent and pattern of cytosine methylation and its relationships with gene activity. In addition, the fresh studies also revealed the more dynamic nature of this epigenetic modification across plant development than previously believed. Cytosine methylation of gene promoter regions usually inhibits transcription, but methylation in coding regions (gene-body methylation) does not generally affect gene expression. Active demethylation (though probably act synergistically with passive loss of methylation) of promoters by the 5-methyl cytosine DNA glycosylase or DEMETER (DME) is required for the uni-parental expression of imprinting genes in endosperm, which is essential for seed viability. The opinion that cytosine methylation is indispensible for normal plant development has been reinforced by using single or combinations of diverse loss-of-function mutants for DNA methyltransferases, DNA glycosylases, components involved in siRNA biogenesis and chromatin remodeling factors. Patterns of cytosine methylation in plants are usually faithfully maintained across organismal generations by the concerted action of epigenetic inheritance and progressive correction of strayed patterns. However, some variant methylation patterns may escape from being corrected and hence produce novel epialleles in the affected somatic cells. This, coupled with the unique property of plants to produce germline cells late during development, may enable the newly acquired epialleles to be inherited to future generations, which if visible to selection may contribute to adaptation and evolution.

DNA LIGASE I exerts a maternal effect on seed development inArabidopsis thaliana

Maternal effects are defined by mutations that affect the next generation when they are maternally inherited. To date, most indepth studies of maternal effects in plants have attributed their origin to genomic imprinting that restricts expression to the maternal allele. The DNA glycosylase DEMETER (DME) removes methylated cytosine residues, causing transcriptional activation of the maternal allele of imprinted genes. In this study, we show that loss-of-function of the major DNA LIGASE I (AtLIG1) in Arabidopsis thaliana causes maternal effects in the endosperm, which is the seed tissue that nurtures embryo development. AtLIG1 expression is not imprinted and has a limited impact on imprinted gene expression. Genetic interaction analyses further indicate that AtLIG1 acts downstream of DME. The removal of methylated cytosine residues by DME involves the creation of DNA single-strand breaks and our results suggest that AtLIG1 repairs these breaks.

Tissue culture-induced variation at simple sequence repeats in sorghum (Sorghum bicolor L.) is genotype-dependent and associated with down-regulated expression of a mismatch repair gene, MLH3

Somaclonal variation is a common phenomenon associated with plant tissue culture. Microsatellites or simple sequence repeats (SSRs) are ubiquitous components of eukaryotic genomes, and are intrinsically unstable under various stress conditions including tissue culture. Here, we assessed genetic stability of a set of 29 mapped SSR loci in calli and regenerated plants derived from a pair of reciprocal sorghum inter-strain F1 hybrids and their pure line parents. We further measured the steady-state transcripts of a set of nine mismatch repair (MMR)-encoding genes and a DEMETER (DME), a DNA glycosylase domain protein-encoding gene in these lines, and tested for a possible relationship between altered expression of a given MMR or DME gene and the SSR variations. We found that SSR variations occurred in calli and regenerated plants of both the studied pure lines though at sharply different frequencies (20.7 vs. 6.9%), but no variation was detected in calli and regenerated plants of the pair of F1 hybrids. Compared with the donor seed plants, markedly altered expression of all nine studied MMR genes and the DME gene was observed in calli, and more conspicuously, in the regenerated plants. However, only one gene, i.e., MLH3, showed an altered expression pattern that is genotype specific and significantly correlated with the occurrence of SSR instability.

Small RNAs: How Seeds Remember To Obey Their Mother

The endosperm is one of two products from the double fertilization event that occurs during sexual reproduction in flowering plants. A series of recent reports highlights the unusual genetic regulatory mechanisms that occur in endosperm and suggests a role for transposon regulation in imprinting.

DNA demethylation: a lesson from the garden

Gene silencing by DNA methylation is well documented and known to be essential for various biological phenomena in many organisms. In contrast, the processes that convert the silent state of a gene whose DNA is methylated and predicted to form facultative heterochromatin to the actively transcribed state remain elusive. In Arabidopsis, recent studies have shown that the DNA glycosylases DEMETER (DME) and REPRESSOR OF SILENCING1 (ROS1) participate in DNA demethylation. DME is necessary for genomic imprinting in the endosperm, while ROS1 is involved in pruning DNA methylation patterns in transposons and genic regions of vegetative tissues. These findings provide us with molecular clues for understanding the underlying mechanisms of DNA demethylation and gene activation. In this review, we will consider and discuss the processes of controlling gene activation through DNA demethylation, which are predicted to include the recognition of target sequences, DNA demethylation, the transformation of the chromatin to the active state, and transcription. Many of these processes remain poorly understood at this stage.

MATERNALLY EXPRESSED PAB C-TERMINAL, a Novel Imprinted Gene inArabidopsis, Encodes the Conserved C-Terminal Domain of Polyadenylate Binding Proteins

Parental imprinting is important for seed development, but few imprinted genes have been identified in plants. The four known imprinted genes in Arabidopsis thaliana encode transcriptional regulators. Here, we describe a novel imprinted gene, MATERNALLY EXPRESSED PAB C-TERMINAL (MPC), which encodes the C-terminal domain of poly(A) binding proteins (PABPs). PABPs play roles in mRNA stability and translation. MPC interacts with proteins that also interact with the C-terminal domain of typical PABPs, suggesting that MPC may regulate translation by modulating PABP activity. In the endosperm, MPC is expressed only from the maternal allele. Reduction of MPC expression affects seed development. In dna methyltransferase1 (met1) mutants, MPC is ectopically expressed, and the paternal allele is active in the endosperm. CGs in the 5' flanking region and gene body of MPC lose methylation in a met1 background. Both regions are required to confer imprinted reporter expression, suggesting that the gene body contains imprinting control region elements. In Arabidopsis, DEMETER (DME) activates expression of maternal alleles. MPC expression is reduced in flowers and seeds in a dme-4 mutant but only after fertilization in dme-1. We conclude that other factors along with DME promote MPC expression and that DME has indirect effects on imprinted gene expression in endosperm.

Retinoblastoma and Its Binding Partner MSI1 Control Imprinting in Arabidopsis

Parental genomic imprinting causes preferential expression of one of the two parental alleles. In mammals, differential sex-dependent deposition of silencing DNA methylation marks during gametogenesis initiates a new cycle of imprinting. Parental genomic imprinting has been detected in plants and relies on DNA methylation by the methyltransferase MET1. However, in contrast to mammals, plant imprints are created by differential removal of silencing marks during gametogenesis. In Arabidopsis, DNA demethylation is mediated by the DNA glycosylase DEMETER (DME) causing activation of imprinted genes at the end of female gametogenesis. On the basis of genetic interactions, we show that in addition to DME, the plant homologs of the human Retinoblastoma (Rb) and its binding partner RbAp48 are required for the activation of the imprinted genes FIS2 and FWA. This Rb-dependent activation is mediated by direct transcriptional repression of MET1 during female gametogenesis. We have thus identified a new mechanism required for imprinting establishment, outlining a new role for the Retinoblastoma pathway, which may be conserved in mammals.

Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks

Cytosine DNA methylation is a stable epigenetic mark for maintenance of gene silencing across cellular divisions, but it is a reversible modification. Genetic and biochemical studies have revealed that the Arabidopsis DNA glycosylase domain-containing proteins ROS1 (REPRESSOR OF SILENCING 1) and DME (DEMETER) initiate erasure of 5-methylcytosine through a base excision repair process. The Arabidopsis genome encodes two paralogs of ROS1 and DME, referred to as DEMETER-LIKE proteins DML2 and DML3. We have found that DML2 and DML3 are 5-methylcytosine DNA glycosylases that are expressed in a wide range of plant organs. We analyzed the distribution of methylation marks at two methylated loci in wild-type and dml mutant plants. Mutations in DML2 and/or DML3 lead to hypermethylation of cytosine residues that are unmethylated or weakly methylated in wild-type plants. In contrast, sites that are heavily methylated in wild-type plants are hypomethylated in mutants. These results suggest that DML2 and DML3 are required not only for removing DNA methylation marks from improperly-methylated cytosines, but also for maintenance of high methylation levels in properly targeted sites.

Novel Festuca arundinacea Shreb. and Dactylis glomerata L. germplasm to improve adaptation for marginal environments

Tall fescue (Festuca arundinacea Schreb., Lolium arundinceum Schreb., S.J. Darbyshire) and cocksfoot (Dactylis glomerata L.) were identified for improvement for low to medium rainfall (400–700 mm) environments where persistence of common cultivars has been unreliable. Over 200 accessions and experimental varieties of tall fescue and cocksfoot sourced from the Mediterranean basin were screened over 2 years at sites on the North-West Slopes of NSW and on the Central Highlands of Victoria, respectively. These were compared with some locally naturalised plants and most of the cultivars available in Australasia as well as cultivars developed for warm temperate and Mediterranean climates in Italy, France, Uruguay and the USA. To date, the screened accessions and experimental varieties have exhibited varying degrees of summer activity and other attributes of commercial value. Six tall fescue accessions were selected for development of synthetic varieties. These included three Sardinian accessions that after 2 years had superior persistence to cv. Demeter and recorded the highest yield scores, the mean of which exceeded that of the best performing cultivars by 34% and that of Demeter by 64%. A further three select North African accessions of tall fescue had similar yield ratings to Demeter and that of the best performing winter-active, summer-dormant cultivars. After 2 years, four Mediterranean accessions of cocksfoot were selected. These had recorded the highest yield scores, the mean of which was 34% greater than that recorded for the highest yielding cultivars and 40% greater than cv. Currie, compared with which these accessions were densely tillered and fine-leafed. The four select Mediterranean cocksfoot accessions exhibited 100% persistence; the persistence of the cultivars ranged from 31–97%. Select plants of the best performing accessions were subsequently removed from the field sites and transferred to pollen-proof glasshouse chambers for synthesis of experimental varieties. The endophyte-free tall fescue synthetics were based on Sardinian accessions selected for year round production and persistence or North African accessions that had similar yield to Demeter but with improved winter production and some summer activity. The cocksfoot synthetics were based on select plants of accessions from North Africa and included both D. glomerata and D. glomerata ssp. glomerata × spp. hispanica hybrids exhibiting persistence, dense tillering and seasonal productivity.

Identification of putative Arabidopsis DEMETER target genes by GeneChip analysis

The Arabidopsis DEMETER (DME) DNA glycosylase is required for the maternal allele expression of imprinted Polycomb group ( MEDEA and FIS2) and transcription factor ( FWA) genes in the endosperm. Expression of DME in the central cell, not in pollen or stamen, establishes gene imprinting by hypomethylating maternal alleles. However, little is known about other genes regulated by DME. To identify putative DME target genes, we generated CaMV:DME plants which ectopically express DME in pollen and stamens. Comparison of mRNA profiles revealed 94 genes induced by ectopic DME expression in both stamen and pollen. Gene ontology analysis identified three molecular functions enriched in the DME-inducible RNA list: DNA or RNA binding, kinase activity, and transcription factor activity. Semi-quantitative RT-PCR verified the candidate genes identified by GeneChip analysis. The putative target genes identified in this study will provide insights into the regulatory mechanism of DME DNA glycosylase and the functions of DNA demethylation.

DNA demethylation in the Arabidopsis genome

Cytosine DNA methylation is considered to be a stable epigenetic mark, but active demethylation has been observed in both plants and animals. In Arabidopsis thaliana, DNA glycosylases of the DEMETER (DME) family remove methylcytosines from DNA. Demethylation by DME is necessary for genomic imprinting, and demethylation by a related protein, REPRESSOR OF SILENCING1, prevents gene silencing in a transgenic background. However, the extent and function of demethylation by DEMETER-LIKE (DML) proteins in WT plants is not known. Using genome-tiling microarrays, we mapped DNA methylation in mutant and WT plants and identified 179 loci actively demethylated by DML enzymes. Mutations in DML genes lead to locus-specific DNA hypermethylation. Reintroducing WT DML genes restores most loci to the normal pattern of methylation, although at some loci, hypermethylated epialleles persist. Of loci demethylated by DML enzymes, >80% are near or overlap genes. Genic demethylation by DML enzymes primarily occurs at the 5' and 3' ends, a pattern opposite to the overall distribution of WT DNA methylation. Our results show that demethylation by DML DNA glycosylases edits the patterns of DNA methylation within the Arabidopsis genome to protect genes from potentially deleterious methylation.

Arabidopsis DEMETER DNA glycosylase requires non‐catalytic domains for 5‐methylcytosine excision

Arabidopsis DEMETER (DME) encodes a 5-methylcytosine DNA glycosylase and its maternal expression is necessary for establishment of gene imprinting, transcription activation and endosperm development. DME is a bifunctional DNA glycosylase/AP-lyase which specifically removes 5-methylcytosine in the genome regardless of the sequence context. DME is an exceptionally large glycosylase (1,729 aa) with the conserved aspartic acid (1304) and lysine (1286) catalytic residues. DME was shown to require an iron-sulfur cluster following the helix-hairpin-helix motif for 5-methylcytosine excision. A series of terminal deletions revealed two additional conserved domains necessary for the DME activity, which is unique to the DME family genes in Arabidopsis. Expression of active DME in E. coli showed cytotoxicity and this property was exploited to identify critical residues important for DME function. The entire DME region was subjected to random mutagenesis and the resulting mutant library was screened for loss of DME activity as a consequence of amino acid substitutions. We found that the conserved domains were highly susceptible to amino acid substitutions, which implies the functional/structural importance of these uncharacterized regions for 5-methylcytosine glycosylase activity of DME. This study was supported by the NIH (GM069415).

DEMETER and REPRESSOR OF SILENCING 1 encode 5-methylcytosine DNA glycosylases.

Cytosine methylation is an epigenetic mark that promotes gene silencing and plays important roles in development and genome defense against transposons. Methylation patterns are established and maintained by DNA methyltransferases that catalyze transfer of a methyl group from S-adenosyl-L-methionine to cytosine bases in DNA. Erasure of cytosine methylation occurs during development, but the enzymatic basis of active demethylation remains controversial. In Arabidopsis thaliana, DEMETER (DME) activates the maternal expression of two imprinted genes silenced by methylation, and REPRESSOR OF SILENCING 1 (ROS1) is required for release of transcriptional silencing of a hypermethylated transgene. DME and ROS1 encode two closely related DNA glycosylase domain proteins, but it is unknown whether they participate directly in a DNA demethylation process or counteract silencing through an indirect effect on chromatin structure. Here we show that DME and ROS1 catalyze the release of 5-methylcytosine (5-meC) from DNA by a glycosylase/lyase mechanism. Both enzymes also remove thymine, but not uracil, mismatched to guanine. DME and ROS1 show a preference for 5-meC over thymine in the symmetric dinucleotide CpG context, where most plant DNA methylation occurs. Nevertheless, they also have significant activity on both substrates at CpApG and asymmetric sequences, which are additional methylation targets in plant genomes. These findings suggest that a function of ROS1 and DME is to initiate erasure of 5-meC through a base excision repair process and provide strong biochemical evidence for the existence of an active DNA demethylation pathway in plants.

Diotima and Demeter as Mystagogues in Plato'sSymposium

Diotima and Demeter as Mystagogues in Plato's<i>Symposium</i>