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1.  How a Retrotransposon Exploits the Plant's Heat Stress Response for Its Activation 
PLoS Genetics  2014;10(1):e1004115.
Retrotransposons are major components of plant and animal genomes. They amplify by reverse transcription and reintegration into the host genome but their activity is usually epigenetically silenced. In plants, genomic copies of retrotransposons are typically associated with repressive chromatin modifications installed and maintained by RNA-directed DNA methylation. To escape this tight control, retrotransposons employ various strategies to avoid epigenetic silencing. Here we describe the mechanism developed by ONSEN, an LTR-copia type retrotransposon in Arabidopsis thaliana. ONSEN has acquired a heat-responsive element recognized by plant-derived heat stress defense factors, resulting in transcription and production of full length extrachromosomal DNA under elevated temperatures. Further, the ONSEN promoter is free of CG and CHG sites, and the reduction of DNA methylation at the CHH sites is not sufficient to activate the element. Since dividing cells have a more pronounced heat response, the extrachromosomal ONSEN DNA, capable of reintegrating into the genome, accumulates preferentially in the meristematic tissue of the shoot. The recruitment of a major plant heat shock transcription factor in periods of heat stress exploits the plant's heat stress response to achieve the transposon's activation, making it impossible for the host to respond appropriately to stress without losing control over the invader.
Author Summary
Transposons are programmed to amplify within their host genomes. In defense, hosts have evolved mechanisms to impede transposon activation, often by epigenetic transcriptional silencing. A constant and likely unending arms race between host and invader has brought about different strategies to mutually counteract the tricks of the other. Several such strategies are combined in one transposon in the Arabidopsis genome. Its promoter is devoid of symmetric sites necessary for stable maintenance of repressive DNA methylation, and a reduction of methylation at the remaining cytosines does not activate the element. More sophisticated still: its promoter shares a sequence motif with heat stress-responsive plant genes and is recognized by a heat-induced plant transcription factor. Whenever the plants must activate their heat stress defense under high temperatures, the transposon is able to generate new extrachromosomal DNA copies that can potentially integrate into new sites of the genome. In addition, the heat response is especially strong in tissue with dividing cells, which form consequently the largest amount of extrachromosomal transposon copies. We see this as an example of a “wolf in sheep's clothing” strategy, whereby the transposon becomes visible as such only under specific stress conditions of its host.
PMCID: PMC3907296  PMID: 24497839
2.  Deposition of Histone Variant H2A.Z within Gene Bodies Regulates Responsive Genes 
PLoS Genetics  2012;8(10):e1002988.
The regulation of eukaryotic chromatin relies on interactions between many epigenetic factors, including histone modifications, DNA methylation, and the incorporation of histone variants. H2A.Z, one of the most conserved but enigmatic histone variants that is enriched at the transcriptional start sites of genes, has been implicated in a variety of chromosomal processes. Recently, we reported a genome-wide anticorrelation between H2A.Z and DNA methylation, an epigenetic hallmark of heterochromatin that has also been found in the bodies of active genes in plants and animals. Here, we investigate the basis of this anticorrelation using a novel h2a.z loss-of-function line in Arabidopsis thaliana. Through genome-wide bisulfite sequencing, we demonstrate that loss of H2A.Z in Arabidopsis has only a minor effect on the level or profile of DNA methylation in genes, and we propose that the global anticorrelation between DNA methylation and H2A.Z is primarily caused by the exclusion of H2A.Z from methylated DNA. RNA sequencing and genomic mapping of H2A.Z show that H2A.Z enrichment across gene bodies, rather than at the TSS, is correlated with lower transcription levels and higher measures of gene responsiveness. Loss of H2A.Z causes misregulation of many genes that are disproportionately associated with response to environmental and developmental stimuli. We propose that H2A.Z deposition in gene bodies promotes variability in levels and patterns of gene expression, and that a major function of genic DNA methylation is to exclude H2A.Z from constitutively expressed genes.
Author Summary
Eukaryotes package their DNA to fit within the nucleus using well-conserved proteins, called histones, that form the building blocks of nucleosomes, the fundamental units of chromatin. Histone variants are specialized versions of these proteins that change the chromatin landscape by altering the biochemical properties and interacting partners of the nucleosome. H2A.Z, a conserved eukaryotic histone variant, is preferentially enriched at the beginnings of genes, though the significance of this enrichment remains unknown. We and others have shown that H2A.Z is conspicuously absent from methylated DNA across the genome in plants and animals. Typically considered a mark of epigenetic silencing, DNA methylation has more recently been discovered in the bodies of many genes. Here, we present evidence that the genome-wide anticorrelation between DNA methylation and H2A.Z enrichment in Arabidopsis is the result of DNA methylation acting to prevent H2A.Z incorporation. We demonstrate that the presence of H2A.Z within gene bodies is correlated with lower transcription levels and higher variability in expression patterns across tissue types and environmental conditions, and we propose that a major function of gene-body DNA methylation is to exclude H2A.Z from the bodies of highly and constitutively expressed genes.
PMCID: PMC3469445  PMID: 23071449
3.  Reprogramming of H3K27me3 Is Critical for Acquisition of Pluripotency from Cultured Arabidopsis Tissues 
PLoS Genetics  2012;8(8):e1002911.
In plants, multiple detached tissues are capable of forming a pluripotent cell mass, termed callus, when cultured on media containing appropriate plant hormones. Recent studies demonstrated that callus resembles the root-tip meristem, even if it is derived from aerial organs. This finding improves our understanding of the regeneration process of plant cells; however, the molecular mechanism that guides cells of different tissue types to form a callus still remains elusive. Here, we show that genome-wide reprogramming of histone H3 lysine 27 trimethylation (H3K27me3) is a critical step in the leaf-to-callus transition. The Polycomb Repressive Complex 2 (PRC2) is known to function in establishing H3K27me3. By analyzing callus formation of mutants corresponding to different histone modification pathways, we found that leaf blades and/or cotyledons of the PRC2 mutants curly leaf swinger (clf swn) and embryonic flower2 (emf2) were defective in callus formation. We identified the H3K27me3-covered loci in leaves and calli by a ChIP–chip assay, and we found that in the callus H3K27me3 levels decreased first at certain auxin-pathway genes. The levels were then increased at specific leaf genes but decreased at a number of root-regulatory genes. Changes in H3K27me3 levels were negatively correlated with expression levels of the corresponding genes. One possible role of PRC2-mediated H3K27me3 in the leaf-to-callus transition might relate to elimination of leaf features by silencing leaf-regulatory genes, as most leaf-preferentially expressed regulatory genes could not be silenced in the leaf explants of clf swn. In contrast to the leaf explants, the root explants of both clf swn and emf2 formed calli normally, possibly because the root-to-callus transition bypasses the leaf gene silencing process. Furthermore, our data show that PRC2-mediated H3K27me3 and H3K27 demethylation act in parallel in the reprogramming of H3K27me3 during the leaf-to-callus transition, suggesting a general mechanism for cell fate transition in plants.
Author Summary
Callus formation is a necessary step in regenerating a new plant from detached plant tissues, and the nature of the callus is similar to that of the root meristem. In this study, we intended to address the molecular basis that directs different plant tissues to form the root-meristem-like callus. We found that leaves and cotyledons, but not roots, of PRC2 mutants curly leaf-50 swinger-1 and embryonic flower2 lost the ability to form a callus. Using ChIP–chip analysis, we identified genes that were changed markedly in the histone H3 lysine 27 trimethylation (H3K27me3) levels during callus formation from leaf blades. Among these genes, a number of leaf-regulatory genes were repressed through PRC2-mediated H3K27me3. Conversely, certain auxin pathway genes and many root-regulatory genes were derepressed through H3K27 demethylation. Our data indicate that genome-wide H3K27me3 reprogramming, through the PRC2-mediated H3K27me3 and the H3K27 demethylation pathways, is critical in directing cell fate transition.
PMCID: PMC3426549  PMID: 22927830
4.  DNA Methyltransferases Are Required to Induce Heterochromatic Re-Replication in Arabidopsis 
PLoS Genetics  2012;8(7):e1002808.
The relationship between epigenetic marks on chromatin and the regulation of DNA replication is poorly understood. Mutations of the H3K27 methyltransferase genes, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, result in re-replication (repeated origin firing within the same cell cycle). Here we show that mutations that reduce DNA methylation act to suppress the re-replication phenotype of atxr5 atxr6 mutants. This suggests that DNA methylation, a mark enriched at the same heterochromatic regions that re-replicate in atxr5/6 mutants, is required for aberrant re-replication. In contrast, RNA sequencing analyses suggest that ATXR5/6 and DNA methylation cooperatively transcriptionally silence transposable elements (TEs). Hence our results suggest a complex relationship between ATXR5/6 and DNA methylation in the regulation of DNA replication and transcription of TEs.
Author Summary
Before cell division the genome is required to replicate once to ensure that each daughter cell inherits a full copy of genomic DNA. Eukaryotic DNA is wrapped around histones to form nucleosomes. Chemical modifications of DNA and histones are known to regulate gene expression. There is growing evidence that these modifications also regulate DNA replication, however very little is understood. Two histone methyltransferases, ARABIDOPSIS TRITHORAX-RELATED PROTEIN5 (ATXR5) and ATXR6, are required to prevent over-replication of normally silent regions of the genome called heterochromatin. Heterochromatin is enriched with transposable elements (TEs) that are silenced by modifications such as DNA methylation. We find that losses of DNA methylation suppress the over-replication defect in an atxr5 atxr6 mutant background. This suggests that DNA methylation positively regulates DNA replication in the absence of ATXR5/6. We further study the relationship between ATXR5/6 and DNA methylation in regulating the expression of TEs and find that they cooperatively silence TEs. Together these findings reveal relationships between DNA and histone modifications in regulating basic biological processes such as DNA replication and gene expression.
PMCID: PMC3390372  PMID: 22792077
5.  Dynamic Deposition of Histone Variant H3.3 Accompanies Developmental Remodeling of the Arabidopsis Transcriptome 
PLoS Genetics  2012;8(5):e1002658.
In animals, replication-coupled histone H3.1 can be distinguished from replication-independent histone H3.3. H3.3 variants are enriched at active genes and their promoters. Furthermore, H3.3 is specifically incorporated upon gene activation. Histone H3 variants evolved independently in plants and animals, and it is unclear whether different replication-independent H3.3 variants developed similar properties in both phyla. We studied Arabidopsis H3 variants in order to find core properties of this class of histones. Here we present genome-wide maps of H3.3 and H3.1 enrichment and the dynamic changes of their profiles upon cell division arrest. We find H3.3 enrichment to positively correlate with gene expression and to be biased towards the transcription termination site. In contrast with H3.1, heterochromatic regions are mostly depleted of H3.3. We report that, in planta, dynamic changes in H3.3 profiles are associated with the extensive remodeling of the transcriptome that occurs during cell differentiation. We propose that H3.3 dynamics are linked to transcription and are involved in resetting covalent histone marks at a genomic scale during plant development. Our study suggests that H3 variants properties likely result from functionally convergent evolution.
Author Summary
Histone proteins are assembled into nucleosomes to build the skeleton of chromosomes. Beyond their role as DNA scaffold, histones participate in the regulation of gene activity. Studies in animals have shown that the deposition of two different histone H3 variants, H3.1 and H3.3, requires distinct pathways and results in distinct profiles throughout the genome. H3 variants evolved independently in plants and animals. Hence, H3 variants' properties shared by plants and animals would reflect core functions that have been selected during evolution. Our study indicates that these core properties include the high enrichment of H3.3 at active genes and a relative low deposition of H3.3 over regions deprived of genes or with inactive genes. In contrast with H3.1, H3.3 incorporation is dynamic and accompanies global changes of gene activity at major developmental transitions. We anticipate that the dynamic link between H3.3 variants and transcription enables remodeling of histone modifications that contribute to developmental transitions.
PMCID: PMC3342937  PMID: 22570629
6.  Gene Expression and Stress Response Mediated by the Epigenetic Regulation of a Transposable Element Small RNA 
PLoS Genetics  2012;8(2):e1002474.
The epigenetic activity of transposable elements (TEs) can influence the regulation of genes; though, this regulation is confined to the genes, promoters, and enhancers that neighbor the TE. This local cis regulation of genes therefore limits the influence of the TE's epigenetic regulation on the genome. TE activity is suppressed by small RNAs, which also inhibit viruses and regulate the expression of genes. The production of TE heterochromatin-associated endogenous small interfering RNAs (siRNAs) in the reference plant Arabidopsis thaliana is mechanistically distinct from gene-regulating small RNAs, such as microRNAs or trans-acting siRNAs (tasiRNAs). Previous research identified a TE small RNA that potentially regulates the UBP1b mRNA, which encodes an RNA–binding protein involved in stress granule formation. We demonstrate that this siRNA, siRNA854, is under the same trans-generational epigenetic control as the Athila family LTR retrotransposons from which it is produced. The epigenetic activation of Athila elements results in a shift in small RNA processing pathways, and new 21–22 nucleotide versions of Athila siRNAs are produced by protein components normally not responsible for processing TE siRNAs. This processing results in siRNA854's incorporation into ARGONAUTE1 protein complexes in a similar fashion to gene-regulating tasiRNAs. We have used reporter transgenes to demonstrate that the UPB1b 3′ untranslated region directly responds to the epigenetic status of Athila TEs and the accumulation of siRNA854. The regulation of the UPB1b 3′ untranslated region occurs both on the post-transcriptional and translational levels when Athila TEs are epigenetically activated, and this regulation results in the phenocopy of the ubp1b mutant stress-sensitive phenotype. This demonstrates that a TE's epigenetic activity can modulate the host organism's stress response. In addition, the ability of this TE siRNA to regulate a gene's expression in trans blurs the lines between TE and gene-regulating small RNAs.
Author Summary
The portion of the genome that does not encode for genes is often overlooked as a source of cellular regulatory information. Here, we demonstrate that regulatory information controlling expression and protein production from a gene called UBP1b is coming from a distant non-gene transposable element (TE). TEs are fragments of DNA that, unlike genes, are capable of duplicating themselves from one location in the genome to another, and occupy nearly half of the human genome. TEs are often referred to as “junk DNA,” as the study of cellular regulation and function is focused on genes. The regulation of TEs is distinct from genes, as a process termed epigenetic silencing heritably represses TE expression and activity. We have demonstrated that the epigenetic status (active versus silenced) of the Athila TE family regulates the UBP1b gene through the activity of a TE small RNA. The function of the UPB1b gene is to respond to and regulate cellular stress, and the epigenetic regulatory status of the Athila TE therefore modulates this stress response. This demonstrates that the epigenetic regulation of TEs can be a source of gene regulatory information, influencing a basic cellular function such as the stress response.
PMCID: PMC3276544  PMID: 22346759
7.  Genetic Rearrangements Can Modify Chromatin Features at Epialleles 
PLoS Genetics  2011;7(10):e1002331.
Analogous to genetically distinct alleles, epialleles represent heritable states of different gene expression from sequence-identical genes. Alleles and epialleles both contribute to phenotypic heterogeneity. While alleles originate from mutation and recombination, the source of epialleles is less well understood. We analyze active and inactive epialleles that were found at a transgenic insert with a selectable marker gene in Arabidopsis. Both converse expression states are stably transmitted to progeny. The silent epiallele was previously shown to change its state upon loss-of-function of trans-acting regulators and drug treatments. We analyzed the composition of the epialleles, their chromatin features, their nuclear localization, transcripts, and homologous small RNA. After mutagenesis by T-DNA transformation of plants carrying the silent epiallele, we found new active alleles. These switches were associated with different, larger or smaller, and non-overlapping deletions or rearrangements in the 3′ regions of the epiallele. These cis-mutations caused different degrees of gene expression stability depending on the nature of the sequence alteration, the consequences for transcription and transcripts, and the resulting chromatin organization upstream. This illustrates a tight dependence of epigenetic regulation on local structures and indicates that sequence alterations can cause epigenetic changes at some distance in regions not directly affected by the mutation. Similar effects may also be involved in gene expression and chromatin changes in the vicinity of transposon insertions or excisions, recombination events, or DNA repair processes and could contribute to the origin of new epialleles.
Author Summary
In contrast to alleles, epialleles have identical DNA sequence and differ only in gene expression and chromatin features. Epialleles are heritable and can also contribute to phenotypes. How this variation originates is unclear. In this study, we analyzed two epialleles found in Arabidopsis for the difference between their chromatin features and their potential to change state. We mutagenized plants with the inactive epiallele and recovered mutants with restored gene expression. In several cases, this was connected with different rearrangements downstream of the epiallele that caused a switch of the epigenetic configuration further upstream. Therefore, sequence alterations, for example by transposon activity or recombination events, may trigger similar heritable changes of chromatin and gene expression in their proximity and could create new epialleles.
PMCID: PMC3197671  PMID: 22028669
8.  Coexistence of Trichome Variation in a Natural Plant Population: A Combined Study Using Ecological and Candidate Gene Approaches 
PLoS ONE  2011;6(7):e22184.
The coexistence of distinct phenotypes within populations has long been investigated in evolutionary ecology. Recent studies have identified the genetic basis of distinct phenotypes, but it is poorly understood how the variation in candidate loci is maintained in natural environments. In this study, we examined fitness consequences and genetic basis of variation in trichome production in a natural population of Arabidopsis halleri subsp. gemmifera. Half of the individuals in the study population produced trichomes while the other half were glabrous, and the leaf beetle Phaedon brassicae imposed intensive damage to both phenotypes. The fitness of hairy and glabrous plants showed no significant differences in the field during two years. A similar result was obtained when sibling hairy and glabrous plants were transplanted at the same field site, whereas a fitness cost of trichome production was detected under a weak herbivory condition. Thus, equivalent fitness of hairy and glabrous plants under natural herbivory allows their coexistence in the contemporary population. The pattern of polymorphism of the candidate trichome gene GLABROUS1 (GL1) showed no evidence of long-term maintenance of trichome variation within the population. Although balancing selection under fluctuating biotic environments is often proposed to explain the maintenance of defense variation, the lack of clear evidence of balancing selection in the study population suggests that other factors such as gene flow and neutral process may have played relatively large roles in shaping trichome variation at least for the single population level.
PMCID: PMC3139618  PMID: 21811571
9.  A Genome-Wide Survey of Imprinted Genes in Rice Seeds Reveals Imprinting Primarily Occurs in the Endosperm 
PLoS Genetics  2011;7(6):e1002125.
Genomic imprinting causes the expression of an allele depending on its parental origin. In plants, most imprinted genes have been identified in Arabidopsis endosperm, a transient structure consumed by the embryo during seed formation. We identified imprinted genes in rice seed where both the endosperm and embryo are present at seed maturity. RNA was extracted from embryos and endosperm of seeds obtained from reciprocal crosses between two subspecies Nipponbare (Japonica rice) and 93-11 (Indica rice). Sequenced reads from cDNA libraries were aligned to their respective parental genomes using single-nucleotide polymorphisms (SNPs). Reads across SNPs enabled derivation of parental expression bias ratios. A continuum of parental expression bias states was observed. Statistical analyses indicated 262 candidate imprinted loci in the endosperm and three in the embryo (168 genic and 97 non-genic). Fifty-six of the 67 loci investigated were confirmed to be imprinted in the seed. Imprinted loci are not clustered in the rice genome as found in mammals. All of these imprinted loci were expressed in the endosperm, and one of these was also imprinted in the embryo, confirming that in both rice and Arabidopsis imprinted expression is primarily confined to the endosperm. Some rice imprinted genes were also expressed in vegetative tissues, indicating that they have additional roles in plant growth. Comparison of candidate imprinted genes found in rice with imprinted candidate loci obtained from genome-wide surveys of imprinted genes in Arabidopsis to date shows a low degree of conservation, suggesting that imprinting has evolved independently in eudicots and monocots.
Author Summary
The expression of maternal or paternal alleles in either a preferentially or exclusively uniparental manner, termed imprinting, is prevalent in the transient endosperm of seeds in the model plant Arabidopsis. Cereals form seeds where both the embryo and endosperm are present at seed maturity. They are an important world food source. To date, very few imprinted genes have been identified in cereal seeds. How parental gene expression biases contribute to rice seed development has not yet been studied in detail. The deep resolution of transcript sequencing platforms was used to identify loci expressed in a parentally biased manner in the embryo and endosperm of Indica and Japonica rice at a genome-wide level. We identified 262 candidate imprinted loci expressed in the endosperm, experimentally verified 56 of these, and found novel features pertaining to their expression. Only one gene was found to be imprinted in the rice embryo. Imprinting in Arabidopsis and rice seeds is confined primarily to the endosperm, but the identified loci do not share extensive sequence conservation. Imprinting thus appears to have evolved independently in these plant species.
PMCID: PMC3121744  PMID: 21731498
10.  High-Resolution Analysis of Parent-of-Origin Allelic Expression in the Arabidopsis Endosperm 
PLoS Genetics  2011;7(6):e1002126.
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.
Author Summary
Genomic imprinting poses a violation to the Mendelian rules of inheritance, which state functional equality of maternally and paternally inherited alleles. Imprinted genes are expressed dependent on their parent-of-origin, implicating an epigenetic asymmetry of maternal and paternal alleles. Genomic imprinting occurs in mammals and flowering plants. In both groups of organisms, nourishing of the progeny depends on ephemeral tissues, the placenta and the endosperm, respectively. In plants, genomic imprinting predominantly occurs in the endosperm, which is derived after fertilization of the diploid central cell with a haploid sperm cell. In this study we identify more than 60 potentially imprinted genes and show that there are different epigenetic mechanisms causing maternal and paternal-specific gene expression. We show that maternally expressed genes are regulated by DNA methylation or Polycomb group (PcG)-mediated repression, while paternally expressed genes are predominantly regulated by PcG proteins. From an evolutionary perspective, we also show that imprinted genes are associated with transposons and are more rapidly evolving than other genes in the genome. Many MEGs and PEGs encode for transcriptional regulators, implicating important functional roles of imprinted genes for endosperm and seed development.
PMCID: PMC3116908  PMID: 21698132
11.  Arabidopsis HDA6 Regulates Locus-Directed Heterochromatin Silencing in Cooperation with MET1 
PLoS Genetics  2011;7(4):e1002055.
Heterochromatin silencing is pivotal for genome stability in eukaryotes. In Arabidopsis, a plant-specific mechanism called RNA–directed DNA methylation (RdDM) is involved in heterochromatin silencing. Histone deacetylase HDA6 has been identified as a component of such machineries; however, its endogenous targets and the silencing mechanisms have not been analyzed globally. In this study, we investigated the silencing mechanism mediated by HDA6. Genome-wide transcript profiling revealed that the loci silenced by HDA6 carried sequences corresponding to the RDR2-dependent 24-nt siRNAs, however their transcript levels were mostly unaffected in the rdr2 mutant. Strikingly, we observed significant overlap of genes silenced by HDA6 to those by the CG DNA methyltransferase MET1. Furthermore, regardless of dependence on RdDM pathway, HDA6 deficiency resulted in loss of heterochromatic epigenetic marks and aberrant enrichment for euchromatic marks at HDA6 direct targets, along with ectopic expression of these loci. Acetylation levels increased significantly in the hda6 mutant at all of the lysine residues in the H3 and H4 N-tails, except H4K16. Interestingly, we observed two different CG methylation statuses in the hda6 mutant. CG methylation was sustained in the hda6 mutant at some HDA6 target loci that were surrounded by flanking DNA–methylated regions. In contrast, complete loss of CG methylation occurred in the hda6 mutant at the HDA6 target loci that were isolated from flanking DNA methylation. Regardless of CG methylation status, CHG and CHH methylation were lost and transcriptional derepression occurred in the hda6 mutant. Furthermore, we show that HDA6 binds only to its target loci, not the flanking methylated DNA, indicating the profound target specificity of HDA6. We propose that HDA6 regulates locus-directed heterochromatin silencing in cooperation with MET1, possibly recruiting MET1 to specific loci, thus forming the foundation of silent chromatin structure for subsequent non-CG methylation.
Author Summary
Eukaryotes are defended from potentially harmful DNA elements, such as transposons, by forming inactive genomic structure. Chromatin, which consists of DNA and histone proteins, is densely packed in the silent structure, and chromatin chemical modifications such as DNA methylation and histone modifications are known to be essential for this packing. In plants, small RNA molecules have been thought to trigger DNA methylation and resulting silent chromatin formation. We revealed that elimination of specific histone modifications concomitant with DNA methylation is pivotal for the silent chromatin. Furthermore, the histone deacetylase was shown to have more profound target specificity than the DNA methyltransferase and is required for locus-directed DNA methylation, implying the involvement of the histone deacetylase for targeting the DNA methyltransferase to specific places on the genome. These proteins and their functions for gene silencing are evolutionarily conserved in higher eukaryotes, and several proteins involved in small RNA production are plant-specific. Thus, we present a hypothesis that the plant genome may build the protecting foundation by the conserved genome surveillance in eukaryotes, and the reinforcing machinery involving small RNAs could be evolutionarily added to the plant heterochromatin silencing system.
PMCID: PMC3084210  PMID: 21552333
12.  Dynamic Regulation of H3K27 Trimethylation during Arabidopsis Differentiation 
PLoS Genetics  2011;7(4):e1002040.
During growth of multicellular organisms, identities of stem cells and differentiated cells need to be maintained. Cell fate is epigenetically controlled by the conserved Polycomb-group (Pc-G) proteins that repress their target genes by catalyzing histone H3 lysine 27 trimethylation (H3K27me3). Although H3K27me3 is associated with mitotically stable gene repression, a large fraction of H3K27me3 target genes are tissue-specifically activated during differentiation processes. However, in plants it is currently unclear whether H3K27me3 is already present in undifferentiated cells and dynamically regulated to permit tissue-specific gene repression or activation. We used whole-genome tiling arrays to identify the H3K27me3 target genes in undifferentiated cells of the shoot apical meristem and in differentiated leaf cells. Hundreds of genes gain or lose H3K27me3 upon differentiation, demonstrating dynamic regulation of an epigenetic modification in plants. H3K27me3 is correlated with gene repression, and its release preferentially results in tissue-specific gene activation, both during differentiation and in Pc-G mutants. We further reveal meristem- and leaf-specific targeting of individual gene families including known but also likely novel regulators of differentiation and stem cell regulation. Interestingly, H3K27me3 directly represses only specific transcription factor families, but indirectly activates others through H3K27me3-mediated silencing of microRNA genes. Furthermore, H3K27me3 targeting of genes involved in biosynthesis, transport, perception, and signal transduction of the phytohormone auxin demonstrates control of an entire signaling pathway. Based on these and previous analyses, we propose that H3K27me3 is one of the major determinants of tissue-specific expression patterns in plants, which restricts expression of its direct targets and promotes gene expression indirectly by repressing miRNA genes.
Author Summary
All organs and differentiated tissues in multicellular organisms are derived from undifferentiated pluripotent stem cells. The evolutionarily conserved Polycomb-group (Pc-G) proteins control stem cell identity and maintenance, likely by repressing genes involved in differentiation processes. Pc-G proteins are epigenetic regulators, thus they maintain stable expression states of their target genes through cell divisions that are not accompanied by changes in their DNA sequence. In this study, we asked whether Pc-G–mediated gene regulation is also dynamically regulated in plant development to confer stable, but flexible gene expression states that may switch in response to developmental or environmental cues. We therefore generated genome-wide maps of Pc-G activity of undifferentiated stem cell and differentiated leaf cell tissues which revealed dynamic regulation of Pc-G activity in plants. Pc-G activity is correlated with gene repression and its tissue-specific release results in local gene activation. Pc-G proteins target specific gene families in the two analyzed tissues, indicating a role for Pc-G proteins in balancing pluripotency and differentiation in plants. Based on our analyses, we propose that Pc-G activity not only permits long-term gene regulation but also has a more basic gene regulatory function in fine-tuning expression patterns of specific gene families during differentiation.
PMCID: PMC3072373  PMID: 21490956
13.  H3K27me3 Profiling of the Endosperm Implies Exclusion of Polycomb Group Protein Targeting by DNA Methylation 
PLoS Genetics  2010;6(10):e1001152.
Polycomb group (PcG) proteins act as evolutionary conserved epigenetic mediators of cell identity because they repress transcriptional programs that are not required at particular developmental stages. Each tissue is likely to have a specific epigenetic profile, which acts as a blueprint for its developmental fate. A hallmark for Polycomb Repressive Complex 2 (PRC2) activity is trimethylated lysine 27 on histone H3 (H3K27me3). In plants, there are distinct PRC2 complexes for vegetative and reproductive development, and it was unknown so far whether these complexes have target gene specificity. The FERTILIZATION INDEPENDENT SEED (FIS) PRC2 complex is specifically expressed in the endosperm and is required for its development; loss of FIS function causes endosperm hyperproliferation and seed abortion. The endosperm nourishes the embryo, similar to the physiological function of the placenta in mammals. We established the endosperm H3K27me3 profile and identified specific target genes of the FIS complex with functional roles in endosperm cellularization and chromatin architecture, implicating that distinct PRC2 complexes have a subset of specific target genes. Importantly, our study revealed that selected transposable elements and protein coding genes are specifically targeted by the FIS PcG complex in the endosperm, whereas these elements and genes are densely marked by DNA methylation in vegetative tissues, suggesting that DNA methylation prevents targeting by PcG proteins in vegetative tissues.
Author Summary
Cell identity is established by the evolutionary conserved Polycomb group (PcG) proteins that repress transcriptional programs which are not required at particular developmental stages. The plant FERTILIZATION INDEPENDENT SEED (FIS) PcG complex is specifically expressed in the endosperm where it is essential for normal development. The endosperm nourishes the embryo, similar to the physiological function of the placenta in mammals. In this study, we established the cell type–specific epigenome profile of PcG activity in the endosperm. The endosperm has reduced levels of DNA methylation, and based on our data we propose that PcG proteins are specifically targeted to hypomethylated sequences in the endosperm. Among these endosperm-specific PcG targets are genes with functional roles in endosperm cellularization and chromatin architecture, implicating a fundamental role of PcG proteins in regulating endosperm development. Importantly, we identified transposable elements and genes among the specific PcG targets in the endosperm that are densely marked by DNA methylation in vegetative tissues, suggesting an antagonistic placement of DNA methylation and H3K27me3 at defined sequences.
PMCID: PMC2951372  PMID: 20949070
14.  Dosage-Sensitive Function of RETINOBLASTOMA RELATED and Convergent Epigenetic Control Are Required during the Arabidopsis Life Cycle 
PLoS Genetics  2010;6(6):e1000988.
The plant life cycle alternates between two distinct multi-cellular generations, the reduced gametophytes and the dominant sporophyte. Little is known about how generation-specific cell fate, differentiation, and development are controlled by the core regulators of the cell cycle. In Arabidopsis, RETINOBLASTOMA RELATED (RBR), an evolutionarily ancient cell cycle regulator, controls cell proliferation, differentiation, and regulation of a subset of Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the male and female gametophytes, as well as cell fate establishment in the male gametophyte. Here we demonstrate that RBR is also essential for cell fate determination in the female gametophyte, as revealed by loss of cell-specific marker expression in all the gametophytic cells that lack RBR. Maintenance of genome integrity also requires RBR, because diploid plants heterozygous for rbr (rbr/RBR) produce an abnormal portion of triploid offspring, likely due to gametic genome duplication. While the sporophyte of the diploid mutant plants phenocopied wild type due to the haplosufficiency of RBR, genetic analysis of tetraploid plants triplex for rbr (rbr/rbr/rbr/RBR) revealed that RBR has a dosage-dependent pleiotropic effect on sporophytic development, trichome differentiation, and regulation of PRC2 subunit genes CURLY LEAF (CLF) and VERNALIZATION 2 (VRN2), and MET1 in leaves. There were, however, no obvious cell cycle and cell proliferation defects in these plant tissues, suggesting that a single functional RBR copy in tetraploids is capable of maintaining normal cell division but is not sufficient for distinct differentiation and developmental processes. Conversely, in leaves of mutants in sporophytic PRC2 subunits, trichome differentiation was also affected and expression of RBR and MET1 was reduced, providing evidence for a RBR-PRC2-MET1 regulatory feedback loop involved in sporophyte development. Together, dosage-sensitive RBR function and its genetic interaction with PRC2 genes and MET1 must have been recruited during plant evolution to control distinct generation-specific cell fate, differentiation, and development.
Author Summary
Understanding the convergent developmental mechanisms of core cell cycle genes is highly instructive in biology. When these genes are essential in development, lethality precludes mutation analysis throughout the life cycle of an organism. We subjected a homozygous lethal mutation in RETINOBLASTOMA RELATED (RBR) of Arabidopsis for tetraploid genetic analysis to study the function of RBR during the plant life cycle. In diploids, while RBR–deficient female gametophytes with features of aberrant cell fate and differentiation were analogous to what was previously reported for male gametophytes, we provide evidence that RBR controls gametic genome duplication, thus genome integrity in the gametophyte-derived progeny. Quantitative reduction of RBR in tetraploids led to identification of rbr heterozygous plants that displayed novel RBR dosage-dependent phenotypes in differentiation and development of the sporophyte albeit the absence of cell cycle defects. These phenotypes coincided with deregulation of conserved epigenetic factors such as Polycomb Repressive Complex 2 (PRC2) genes and METHYLTRANSFERASE 1 (MET1) in the sporophyte, as shown for the gametophytes as well. However, unlike the repression by the PRC2 in gametophytes, RBR is activated by the sporophytic PRC2 subunits, suggesting that distinct modules of the conserved RBR-PRC2-MET1 loop control gametophyte and sporophyte generations in plants.
PMCID: PMC2887464  PMID: 20585548
15.  siRNA–Mediated Methylation of Arabidopsis Telomeres 
PLoS Genetics  2010;6(6):e1000986.
Chromosome termini form a specialized type of heterochromatin that is important for chromosome stability. The recent discovery of telomeric RNA transcripts in yeast and vertebrates raised the question of whether RNA–based mechanisms are involved in the formation of telomeric heterochromatin. In this study, we performed detailed analysis of chromatin structure and RNA transcription at chromosome termini in Arabidopsis. Arabidopsis telomeres display features of intermediate heterochromatin that does not extensively spread to subtelomeric regions which encode transcriptionally active genes. We also found telomeric repeat–containing transcripts arising from telomeres and centromeric loci, a portion of which are processed into small interfering RNAs. These telomeric siRNAs contribute to the maintenance of telomeric chromatin through promoting methylation of asymmetric cytosines in telomeric (CCCTAAA)n repeats. The formation of telomeric siRNAs and methylation of telomeres relies on the RNA–dependent DNA methylation pathway. The loss of telomeric DNA methylation in rdr2 mutants is accompanied by only a modest effect on histone heterochromatic marks, indicating that maintenance of telomeric heterochromatin in Arabidopsis is reinforced by several independent mechanisms. In conclusion, this study provides evidence for an siRNA–directed mechanism of chromatin maintenance at telomeres in Arabidopsis.
Author Summary
Telomeres are protein–DNA structures that protect the ends of eukaryotic chromosomes. A failure in this protective structure can lead to chromosomal instabilities and contribute to cancer and aging. The protective nature of telomeres relies on complex interactions between repetitive telomeric DNA and associated proteins. One major question is how telomeric proteins, including telomere-associated nucleosomes, are modified in order to achieve this protection. In this study, we have discovered that Arabidopsis telomeric nucleosomes contain a unique mixture of both active and inactive chromatin marks. Additionally, the telomeric DNA itself is modified by methylation of cytosines within the telomeric repeat. Regulation of DNA methylation is achieved by telomeric repeat–containing small RNAs, which are derived from the processing of telomeric transcripts by the RNA–dependent DNA methylation pathway. From these data, we infer that the formation of a proper telomere structure is partly regulated by non-coding telomeric RNAs.
PMCID: PMC2883606  PMID: 20548962
16.  Redundant and Specific Roles of the ARGONAUTE Proteins AGO1 and ZLL in Development and Small RNA-Directed Gene Silencing 
PLoS Genetics  2009;5(9):e1000646.
The Arabidopsis ARGONAUTE1 (AGO1) and ZWILLE/PINHEAD/AGO10 (ZLL) proteins act in the miRNA and siRNA pathways and are essential for multiple processes in development. Here, we analyze what determines common and specific function of both proteins. Analysis of ago1 mutants with partially compromised AGO1 activity revealed that loss of ZLL function re-establishes both siRNA and miRNA pathways for a subset of AGO1 target genes. Loss of ZLL function in ago1 mutants led to increased AGO1 protein levels, whereas AGO1 mRNA levels were unchanged, implicating ZLL as a negative regulator of AGO1 at the protein level. Since ZLL, unlike AGO1, is not subjected to small RNA-mediated repression itself, this cross regulation has the potential to adjust RNA silencing activity independent of feedback dynamics. Although AGO1 is expressed in a broader pattern than ZLL, expression of AGO1 from the ZLL promoter restored transgene PTGS and most developmental defects of ago1, whereas ZLL rescued only a few AGO1 functions when expressed from the AGO1 promoter, suggesting that the specific functions of AGO1 and ZLL are mainly determined by their protein sequence. Protein domain swapping experiments revealed that the PAZ domain, which in AGO1 is involved in binding small RNAs, is interchangeable between both proteins, suggesting that this common small RNA-binding domain contributes to redundant functions. By contrast, the conserved MID and PIWI domains, which are involved in 5′-end small RNA selectivity and mRNA cleavage, and the non-conserved N-terminal domain, to which no function has been assigned, provide specificity to AGO1 and ZLL protein function.
Author Summary
In eukaryotes, short RNAs (21–24 nucleotides long) have broad effects on gene expression through the action of ARGONAUTE (AGO) proteins. The model flowering plant Arabidopsis thaliana contains ten AGO proteins, among which AGO1 and ZLL/PNH/AGO10 play a major role in regulating gene expression through small RNA-directed RNA cleavage and translational repression. Here, we address the common and specific effects of zll and ago1 loss of function in Arabidopsis. We show that zll mutations lead to increased AGO1 protein levels and suppress a subset of small RNA-directed gene regulatory defects of weak ago1 mutations. Although AGO1 and ZLL proteins are highly similar in sequence, we show that only the PAZ domain, which in AGO1 is involved in binding small RNAs, can be exchanged between the two proteins. By contrast, the PIWI domain, that is responsible for the RNA cleaving activity of AGO1, the MID domain, which is involved in 5′ nucleotide selection of small RNAs, and the functionally uncharacterized N-terminal domain contribute to their individual functions during small RNA-directed gene regulation and development.
PMCID: PMC2730571  PMID: 19763164
17.  An Arabidopsis jmjC domain protein protects transcribed genes from DNA methylation at CHG sites 
The EMBO Journal  2009;28(8):1078-1086.
Differential cytosine methylation of genes and transposons is important for maintaining integrity of plant genomes. In Arabidopsis, transposons are heavily methylated at both CG and non-CG sites, whereas the non-CG methylation is rarely found in active genes. Our previous genetic analysis suggested that a jmjC domain-containing protein IBM1 (increase in BONSAI methylation 1) prevents ectopic deposition of non-CG methylation, and this process is necessary for normal Arabidopsis development. Here, we directly determined the genomic targets of IBM1 through high-resolution genome-wide analysis of DNA methylation. The ibm1 mutation induced extensive hyper-methylation in thousands of genes. Transposons were unaffected. Notably, long transcribed genes were most severely affected. Methylation of genes is limited to CG sites in wild type, but CHG sites were also methylated in the ibm1 mutant. The ibm1-induced hyper-methylation did not depend on previously characterized components of the RNAi-based DNA methylation machinery. Our results suggest novel transcription-coupled mechanisms to direct genic methylation not only at CG but also at CHG sites. IBM1 prevents the CHG methylation in genes, but not in transposons.
PMCID: PMC2653724  PMID: 19262562
chromatin; IBM1; transposon
18.  SRA-Domain Proteins Required for DRM2-Mediated De Novo DNA Methylation 
PLoS Genetics  2008;4(11):e1000280.
De novo DNA methylation and the maintenance of DNA methylation in asymmetrical sequence contexts is catalyzed by homologous proteins in plants (DRM2) and animals (DNMT3a/b). In plants, targeting of DRM2 depends on small interfering RNAs (siRNAs), although the molecular details are still unclear. Here, we show that two SRA-domain proteins (SUVH9 and SUVH2) are also essential for DRM2-mediated de novo and maintenance DNA methylation in Arabidopsis thaliana. At some loci, SUVH9 and SUVH2 act redundantly, while at other loci only SUVH2 is required, and this locus specificity correlates with the differing DNA-binding affinity of the SRA domains within SUVH9 and SUVH2. Specifically, SUVH9 preferentially binds methylated asymmetric sites, while SUVH2 preferentially binds methylated CG sites. The suvh9 and suvh2 mutations do not eliminate siRNAs, suggesting a role for SUVH9 and SUVH2 late in the RNA-directed DNA methylation pathway. With these new results, it is clear that SRA-domain proteins are involved in each of the three pathways leading to DNA methylation in Arabidopsis.
Author Summary
Our genetic heritage plays an important role in determining who we are and the characteristics we possess. However, in the past decade it has become increasingly clear that in addition to the genes we inherit, a second level of information is critical for expression of these genes. This information takes the form of modifications to either the DNA (DNA methylation) or the proteins that package the DNA (histones). These modifications can determine whether a gene is expressed or silenced. In this paper, we identify two new genes that are part of a DNA methylation–targeting pathway in the model plant A. thaliana. Disruption of these two closely related genes prevents DNA methylation by one of the cellular DNA methyltransferases. However, these genes are not simply redundant. They are both capable of binding methylated DNA, but differ in their preference for specific sequences in the genome. This ability to bind to methylated DNA suggests that these proteins help target or retain the modification apparatus at particular regions of the genome. These results are important in that they identify two new players in this vital cellular process and bring us closer to understanding how epigenetic modifications can be targeted to specific genes.
PMCID: PMC2582956  PMID: 19043555
19.  Divergent Evolution of CHD3 Proteins Resulted in MOM1 Refining Epigenetic Control in Vascular Plants 
PLoS Genetics  2008;4(8):e1000165.
Arabidopsis MOM1 is required for the heritable maintenance of transcriptional gene silencing (TGS). Unlike many other silencing factors, depletion of MOM1 evokes transcription at selected loci without major changes in DNA methylation or histone modification. These loci retain unusual, bivalent chromatin properties, intermediate to both euchromatin and heterochromatin. The structure of MOM1 previously suggested an integral nuclear membrane protein with chromatin-remodeling and actin-binding activities. Unexpected results presented here challenge these presumed MOM1 activities and demonstrate that less than 13% of MOM1 sequence is necessary and sufficient for TGS maintenance. This active sequence encompasses a novel Conserved MOM1 Motif 2 (CMM2). The high conservation suggests that CMM2 has been the subject of strong evolutionary pressure. The replacement of Arabidopsis CMM2 by a poplar motif reveals its functional conservation. Interspecies comparison suggests that MOM1 proteins emerged at the origin of vascular plants through neo-functionalization of the ubiquitous eukaryotic CHD3 chromatin remodeling factors. Interestingly, despite the divergent evolution of CHD3 and MOM1, we observed functional cooperation in epigenetic control involving unrelated protein motifs and thus probably diverse mechanisms.
Author Summary
Epigenetic regulation of transcription usually involves changes in histone modifications, as well as DNA methylation changes in plants and mammals. Previously, we found an exceptional epigenetic regulator in Arabidopsis, MOM1, acting independently of these epigenetic marks. Interestingly, MOM1 controls loci associated with bivalent chromatin marks, intermediate to active euchromatin and silent heterochromatin. Such bivalent marks are often associated with newly inserted and/or potentially active transposons, silent transgenes, and certain chromosomal loci. Notably, bivalent chromatin seems to be characteristic for embryonic stem cells, where such loci change their activity and determination of epigenetic marks during cell differentiation. Here, we provide evidence that in vascular plants, the MOM1-like proteins evolved from the ubiquitous eukaryotic chromatin remodeling factor CHD3. The domains necessary for CHD3 function degenerated in MOM1, became dispensable for its gene silencing activity, and were replaced by a novel, unrelated domain providing silencing function. Therefore, MOM1-like proteins use a different silencing mechanism compared to the ancestral CHD3s. In spite of this divergent evolution, CHD3 and MOM1 seem to retain a functional cooperation in control of transcriptionally silent loci. Our results provide an unprecedented example of an evolutionary path for epigenetic components resulting in increased complexity of an epigenetic regulatory network characteristic for multicellular eukaryotes.
PMCID: PMC2507757  PMID: 18725928
20.  Three SRA-Domain Methylcytosine-Binding Proteins Cooperate to Maintain Global CpG Methylation and Epigenetic Silencing in Arabidopsis 
PLoS Genetics  2008;4(8):e1000156.
Methylcytosine-binding proteins decipher the epigenetic information encoded by DNA methylation and provide a link between DNA methylation, modification of chromatin structure, and gene silencing. VARIANT IN METHYLATION 1 (VIM1) encodes an SRA (SET- and RING-associated) domain methylcytosine-binding protein in Arabidopsis thaliana, and loss of VIM1 function causes centromere DNA hypomethylation and centromeric heterochromatin decondensation in interphase. In the Arabidopsis genome, there are five VIM genes that share very high sequence similarity and encode proteins containing a PHD domain, two RING domains, and an SRA domain. To gain further insight into the function and potential redundancy among the VIM proteins, we investigated strains combining different vim mutations and transgenic vim knock-down lines that down-regulate multiple VIM family genes. The vim1 vim3 double mutant and the transgenic vim knock-down lines showed decreased DNA methylation primarily at CpG sites in genic regions, as well as repeated sequences in heterochromatic regions. In addition, transcriptional silencing was released in these plants at most heterochromatin regions examined. Interestingly, the vim1 vim3 mutant and vim knock-down lines gained ectopic CpHpH methylation in the 5S rRNA genes against a background of CpG hypomethylation. The vim1 vim2 vim3 triple mutant displayed abnormal morphological phenotypes including late flowering, which is associated with DNA hypomethylation of the 5′ region of FWA and release of FWA gene silencing. Our findings demonstrate that VIM1, VIM2, and VIM3 have overlapping functions in maintenance of global CpG methylation and epigenetic transcriptional silencing.
Author Summary
Methylation of cytosine bases provides one layer of epigenetic information that is superimposed on the nucleotide sequence of a genome. Proteins that bind methylated cytosines and also help maintain that DNA modification are important linchpins in a self-propagating system mediating memory of epigenetic states. We previously demonstrated that the VIM1 (VARIANT IN METHYLATION 1) protein from the flowering plant Arabidopsis thaliana binds DNA that contains methylated cytosine and is required for complete methylation and compaction of centromeric DNA. In this study, we show that VIM1 works in concert with two related proteins, VIM2 and VIM3, to maintain cytosine methylation not only at centromeres but throughout the genome. VIM proteins act specifically in the DNA methylation pathway that targets CpG dinucleotides, which plants share with animals, rather than the plant-specific non-CpG methylation pathways. Loss of VIM1, VIM2, and VIM3 function also causes a reduction in transcriptional gene silencing at a variety of sequences, and leads to abnormal developmental phenotypes, including late flowering associated with loss of FWA gene silencing. Our results demonstrate that these three related VIM family proteins have overlapping functions in the MET1-mediated CpG methylation pathway.
PMCID: PMC2491724  PMID: 18704160
21.  Evolution and Control of Imprinted FWA Genes in the Genus Arabidopsis 
PLoS Genetics  2008;4(4):e1000048.
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.
Author Summary
Genomic imprinting, mono-allelic gene expression depending on the parent-of-origin, is an epigenetic process known in mammals and flowering plants. 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. The FWA promoter 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 necessary. Here we show that the direct repeat structure is highly diverse in species within the genus Arabidopsis. Unexpectedly, we found that the direct repeat structure is dispensable for the epigenetic silencing and methylation of the FWA promoter. Rather, the SINE-related promoter sequence is sufficient for these features. Frequent independent formation of the tandem repeats suggests that they may be a consequence of the epigenetically controlled system.
PMCID: PMC2270340  PMID: 18389059

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