[Show abstract][Hide abstract] ABSTRACT: Centromeres mediate chromosome segregation and are defined by the centromere-specific histone H3 variant (CenH3)/centromere pro-tein A (CENP-A). Removal of CenH3 from centromeres is a general property of terminally differentiated cells, and the persistence of CenH3 increases the risk of diseases such as cancer. However, active mechanisms of centromere disassembly are unknown. Nondividing Arabidopsis pollen vegetative cells, which transport engulfed sperm by extended tip growth, undergo loss of CenH3; centromeric het-erochromatin decondensation; and bulk activation of silent rRNA genes, accompanied by their translocation into the nucleolus. Here, we show that these processes are blocked by mutations in the evo-lutionarily conserved AAA-ATPase molecular chaperone, CDC48A, homologous to yeast Cdc48 and human p97 proteins, both of which are implicated in ubiquitin/small ubiquitin-like modifier (SUMO)-tar-geted protein degradation. We demonstrate that CDC48A physically associates with its heterodimeric cofactor UFD1-NPL4, known to bind ubiquitin and SUMO, as well as with SUMO1-modified CenH3 and mutations in NPL4 phenocopy cdc48a mutations. In WT vegeta-tive cell nuclei, genetically unlinked ribosomal DNA (rDNA) loci are uniquely clustered together within the nucleolus and all major rRNA gene variants, including those rDNA variants silenced in leaves, are transcribed. In cdc48a mutant vegetative cell nuclei, however, these rDNA loci frequently colocalized with condensed centromeric het-erochromatin at the external periphery of the nucleolus. Our results indicate that the CDC48A NPL4 complex actively removes sumoylated CenH3 from centromeres and disrupts centromeric het-erochromatin to release bulk rRNA genes into the nucleolus for ri-bosome production, which fuels single nucleus-driven pollen tube growth and is essential for plant reproduction. centromere disassembly | heterochromatin decondensation | chromosome dynamics | rDNA activation | pollen tip growth C entromeres are required for chromosomes to attach to spindle fibers and segregate during mitosis and meiosis. Of central importance to centromeres are nucleosomes with the cen-tromere-specific histone H3 variant (CenH3)/centromere protein A (CENP-A) (1). Centromeres are intimately associated with heterochromatin, which is frequently formed adjacent to an array of CenH3 nucleosomes, upon which the kinetochore is assembled, and heterochromatin is required to promote CenH3 assembly at centromeres (2). In both mammals and plants, constitutively het-erochromatic megabase-sized centromeric regions are rich in highly repetitive tandem arrays of satellite DNA and moderately repetitive sequences, such as transposons, and show a low level of transcription (3). Centromeric heterochromatin remains condensed after mitosis and is associated with repressive chromatin marks, including DNA cytosine methylation (4) and histone H3 Lys9 methylation (5). Chromosomes from terminally differentiated cells that have exited the cell cycle often have little CenH3 (6), presumably to prevent them from undergoing defective mitoses and oncogenesis (7). In contrast to the relatively well-known pro-cess of de novo CenH3 assembly at centromeres, little is known about how CenH3 is actively removed from centromeres, leading to centromere disassembly. Unlike animals, whose gametes represent the direct products of meiosis, flowering plants form multicellular haploid gametophytes by means of postmeiotic mitoses (8). In stamen, each haploid pollen microspore undergoes an asymmetrical mitosis to produce a large, terminally differentiated vegetative cell and a smaller generative cell, which have different fates. The generative cell, engulfed in the cytoplasm of the vegetative cell, undergoes a second mitosis to form two sperm cells. The primary role of the vegetative cell in plant reproduction is to germinate and form a rapid tip-growing pollen tube, sometimes centimeters in length, which transports two sperm cells to the female gametophyte for double fertilization. We reported previously that the Arabidopsis thaliana pollen vegetative cell invariably undergoes CenH3 removal from centromeres, coincident with loss of the demethylated histone H3 Lys9 (H3K9me2) heterochromatin mark and centromeric heterochromatin decondensation (9), although the mechanism and biological significance of these processes remain unclear.
[Show abstract][Hide abstract] ABSTRACT: Dnmt1 epigenetically propagates symmetrical CG methylation in many eukaryotes. Their genomes are typically depleted of CG dinucleotides because of imperfect repair of deaminated methylcytosines. Here, we extensively survey diverse species lacking Dnmt1 and show that, surprisingly, symmetrical CG methylation is nonetheless frequently present and catalyzed by a different DNA methyltransferase family, Dnmt5. Numerous Dnmt5-containing organisms that diverged more than a billion years ago exhibit clustered methylation, specifically in nucleosome linkers. Clustered methylation occurs at unprecedented densities and directly disfavors nucleosomes, contributing to nucleosome positioning between clusters. Dense methylation is enabled by a regime of genomic sequence evolution that enriches CG dinucleotides and drives the highest CG frequencies known. Species with linker methylation have small, transcriptionally active nuclei that approach the physical limits of chromatin compaction. These features constitute a previously unappreciated genome architecture, in which dense methylation influences nucleosome positions, likely facilitating nuclear processes under extreme spatial constraints.
[Show abstract][Hide abstract] ABSTRACT: Transposons are selfish genetic sequences that can increase their copy number and inflict substantial damage on their hosts. To combat these genomic parasites, plants have evolved multiple pathways to identify and silence transposons by methylating their DNA. Plants have also evolved mechanisms to limit the collateral damage from the antitransposon machinery. In this review, we examine recent developments that have elucidated many of the molecular workings of these pathways. We also highlight the evidence that the methylation and demethylation pathways interact, indicating that plants have a highly sophisticated, integrated system of transposon defense that has an important role in the regulation of gene expression.
Trends in Plant Science 03/2014; · 11.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Arabidopsis thaliana endosperm, a transient tissue that nourishes the embryo, exhibits extensive localized DNA demethylation on maternally inherited chromosomes. Demethylation mediates parent-of-origin-specific (imprinted) gene expression but is apparently unnecessary for the extensive accumulation of maternally biased small RNA (sRNA) molecules detected in seeds. Endosperm DNA in the distantly related monocots rice and maize is likewise locally hypomethylated, but whether this hypomethylation is generally parent-of-origin specific is unknown. Imprinted expression of sRNA also remains uninvestigated in monocot seeds. Here, we report high-coverage sequencing of the Kitaake rice cultivar that enabled us to show that localized hypomethylation in rice endosperm occurs solely on the maternal genome, preferring regions of high DNA accessibility. Maternally expressed imprinted genes are enriched for hypomethylation at putative promoter regions and transcriptional termini and paternally expressed genes at promoters and gene bodies, mirroring our recent results in A. thaliana. However, unlike in A. thaliana, rice endosperm sRNA populations are dominated by specific strong sRNA-producing loci, and imprinted 24-nt sRNAs are expressed from both parental genomes and correlate with hypomethylation. Overlaps between imprinted sRNA loci and imprinted genes expressed from opposite alleles suggest that sRNAs may regulate genomic imprinting. Whereas sRNAs in seedling tissues primarily originate from small class II (cut-and-paste) transposable elements, those in endosperm are more uniformly derived, including sequences from other transposon classes, as well as genic and intergenic regions. Our data indicate that the endosperm exhibits a unique pattern of sRNA expression and suggest that localized hypomethylation of maternal endosperm DNA is conserved in flowering plants.
Proceedings of the National Academy of Sciences 04/2013; · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Nucleosome remodelers of the DDM1/Lsh family are required for DNA methylation of transposable elements, but the reason for this is unknown. How DDM1 interacts with other methylation pathways, such as small-RNA-directed DNA methylation (RdDM), which is thought to mediate plant asymmetric methylation through DRM enzymes, is also unclear. Here, we show that most asymmetric methylation is facilitated by DDM1 and mediated by the methyltransferase CMT2 separately from RdDM. We find that heterochromatic sequences preferentially require DDM1 for DNA methylation and that this preference depends on linker histone H1. RdDM is instead inhibited by heterochromatin and absolutely requires the nucleosome remodeler DRD1. Together, DDM1 and RdDM mediate nearly all transposon methylation and collaborate to repress transposition and regulate the methylation and expression of genes. Our results indicate that DDM1 provides DNA methyltransferases access to H1-containing heterochromatin to allow stable silencing of transposable elements in cooperation with the RdDM pathway.
[Show abstract][Hide abstract] ABSTRACT: Plants undergo alternation of generation in which reproductive cells develop in the plant body ("sporophytic generation") and then differentiate into a multicellular gamete-forming "gametophytic generation." Different populations of helper cells assist in this transgenerational journey, with somatic tissues supporting early development and single nurse cells supporting gametogenesis. New data reveal a two-way relationship between early reproductive cells and their helpers involving complex epigenetic and signaling networks determining cell number and fate. Later, the egg cell plays a central role in specifying accessory cells, whereas in both gametophytes, companion cells contribute non-cell-autonomously to the epigenetic landscape of the gamete genomes.
[Show abstract][Hide abstract] ABSTRACT: The most well-studied function of DNA methylation in eukaryotic cells is the transcriptional silencing of genes and transposons. More recent results showed that many eukaryotes methylate the bodies of genes as well and that this methylation correlates with transcriptional activity rather than repression. The purpose of gene body methylation remains mysterious, but is potentially related to the histone variant H2A.Z. Studies in plants and animals have shown that the genome-wide distributions of H2A.Z and DNA methylation are strikingly anticorrelated. Furthermore, we and other investigators have shown that this relationship is likely to be the result of an ancient but unknown mechanism by which DNA methylation prevents the incorporation of H2A.Z. Recently, we discovered strong correlations between the presence of H2A.Z within gene bodies, the degree to which a gene's expression varies across tissue types or environmental conditions, and transcriptional misregulation in an h2a.z mutant. We propose that one basal function of gene body methylation is the establishment of constitutive expression patterns within housekeeping genes by excluding H2A.Z from their bodies.
Cold Spring Harbor Symposia on Quantitative Biology 12/2012;
[Show abstract][Hide abstract] ABSTRACT: 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.
[Show abstract][Hide abstract] ABSTRACT: The Arabidopsis thaliana central cell, the companion cell of the egg, undergoes DNA demethylation before fertilization, but the targeting preferences, mechanism, and biological significance of this process remain unclear. Here, we show that active DNA demethylation mediated by the DEMETER DNA glycosylase accounts for all of the demethylation in the central cell and preferentially targets small, AT-rich, and nucleosome-depleted euchromatic transposable elements. The vegetative cell, the companion cell of sperm, also undergoes DEMETER-dependent demethylation of similar sequences, and lack of DEMETER in vegetative cells causes reduced small RNA-directed DNA methylation of transposons in sperm. Our results demonstrate that demethylation in companion cells reinforces transposon methylation in plant gametes and likely contributes to stable silencing of transposable elements across generations.
[Show abstract][Hide abstract] ABSTRACT: EMBRYONIC FLOWER1 (EMF1) is a plant-specific gene crucial to Arabidopsis vegetative development. Loss of function mutants in the EMF1 gene mimic the phenotype caused by mutations in Polycomb Group protein (PcG) genes, which encode epigenetic repressors that regulate many aspects of eukaryotic development. In Arabidopsis, Polycomb Repressor Complex 2 (PRC2), made of PcG proteins, catalyzes trimethylation of lysine 27 on histone H3 (H3K27me3) and PRC1-like proteins catalyze H2AK119 ubiquitination. Despite functional similarity to PcG proteins, EMF1 lacks sequence homology with known PcG proteins; thus, its role in the PcG mechanism is unclear. To study the EMF1 functions and its mechanism of action, we performed genome-wide mapping of EMF1 binding and H3K27me3 modification sites in Arabidopsis seedlings. The EMF1 binding pattern is similar to that of H3K27me3 modification on the chromosomal and genic level. ChIPOTLe peak finding and clustering analyses both show that the highly trimethylated genes also have high enrichment levels of EMF1 binding, termed EMF1_K27 genes. EMF1 interacts with regulatory genes, which are silenced to allow vegetative growth, and with genes specifying cell fates during growth and differentiation. H3K27me3 marks not only these genes but also some genes that are involved in endosperm development and maternal effects. Transcriptome analysis, coupled with the H3K27me3 pattern, of EMF1_K27 genes in emf1 and PRC2 mutants showed that EMF1 represses gene activities via diverse mechanisms and plays a novel role in the PcG mechanism.
[Show abstract][Hide abstract] ABSTRACT: Accumulating evidence points toward diverse functions for plant chromatin. Remarkable progress has been made over the last few years in elucidating the mechanisms for a number of these functions. Activity of the histone demethylase IBM1 accurately targets DNA methylation to silent repeats and transposable elements, not to genes. A genetic screen uncovered the surprising role of H2A.Z-containing nucleosomes in sensing precise differences in ambient temperature and consequent gene regulation. Precise maintenance of chromosome number is assured by a histone modification that suppresses inappropriate DNA replication and by centromeric histone H3 regulation of chromosome segregation. Histones and noncoding RNAs regulate FLOWERING LOCUS C, the expression of which quantitatively measures the duration of cold exposure, functioning as memory of winter. These findings are a testament to the power of using plants to research chromatin organization, and demonstrate examples of how chromatin functions to achieve biological accuracy, precision, and memory.
Current opinion in genetics & development 02/2012; 22(2):132-8. · 8.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Little is known about chromatin remodeling events immediately after fertilization. A recent report by Autran et al. (2011) in Cell now shows that chromatin regulatory pathways that silence transposable elements are responsible for global delayed activation of gene expression in the early Arabidopsis embryo.
[Show abstract][Hide abstract] ABSTRACT: Imprinted genes are expressed primarily or exclusively from either the maternal or paternal allele, a phenomenon that occurs in flowering plants and mammals. Flowering plant imprinted gene expression has been described primarily in endosperm, a terminal nutritive tissue consumed by the embryo during seed development or after germination. Imprinted expression in Arabidopsis thaliana endosperm is orchestrated by differences in cytosine DNA methylation between the paternal and maternal genomes as well as by Polycomb group proteins. Currently, only 11 imprinted A. thaliana genes are known. Here, we use extensive sequencing of cDNA libraries to identify 9 paternally expressed and 34 maternally expressed imprinted genes in A. thaliana endosperm that are regulated by the DNA-demethylating glycosylase DEMETER, the DNA methyltransferase MET1, and/or the core Polycomb group protein FIE. These genes encode transcription factors, proteins involved in hormone signaling, components of the ubiquitin protein degradation pathway, regulators of histone and DNA methylation, and small RNA pathway proteins. We also identify maternally expressed genes that may be regulated by unknown mechanisms or deposited from maternal tissues. We did not detect any imprinted genes in the embryo. Our results show that imprinted gene expression is an extensive mechanistically complex phenomenon that likely affects multiple aspects of seed development.
Proceedings of the National Academy of Sciences 02/2011; 108(5):1755-62. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytosine methylation silences transposable elements in plants, vertebrates, and fungi but also regulates gene expression. Plant methylation is catalyzed by three families of enzymes, each with a preferred sequence context: CG, CHG (H = A, C, or T), and CHH, with CHH methylation targeted by the RNAi pathway. Arabidopsis thaliana endosperm, a placenta-like tissue that nourishes the embryo, is globally hypomethylated in the CG context while retaining high non-CG methylation. Global methylation dynamics in seeds of cereal crops that provide the bulk of human nutrition remain unknown. Here, we show that rice endosperm DNA is hypomethylated in all sequence contexts. Non-CG methylation is reduced evenly across the genome, whereas CG hypomethylation is localized. CHH methylation of small transposable elements is increased in embryos, suggesting that endosperm demethylation enhances transposon silencing. Genes preferentially expressed in endosperm, including those coding for major storage proteins and starch synthesizing enzymes, are frequently hypomethylated in endosperm, indicating that DNA methylation is a crucial regulator of rice endosperm biogenesis. Our data show that genome-wide reshaping of seed DNA methylation is conserved among angiosperms and has a profound effect on gene expression in cereal crops.
Proceedings of the National Academy of Sciences 10/2010; 107(43):18729-34. · 9.81 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Cytosine methylation is an ancient process with conserved enzymology but diverse biological functions that include defense against transposable elements and regulation of gene expression. Here we will discuss the evolution and biological significance of eukaryotic DNA methylation, the likely drivers of that evolution, and major remaining mysteries.
Current biology: CB 09/2010; 20(17):R780-5. · 10.99 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic cytosine methylation represses transcription but also occurs in the bodies of active genes, and the extent of methylation biology conservation is unclear. We quantified DNA methylation in 17 eukaryotic genomes and found that gene body methylation is conserved between plants and animals, whereas selective methylation of transposons is not. We show that methylation of plant transposons in the CHG context extends to green algae and that exclusion of histone H2A.Z from methylated DNA is conserved between plants and animals, and we present evidence for RNA-directed DNA methylation of fungal genes. Our data demonstrate that extant DNA methylation systems are mosaics of conserved and derived features, and indicate that gene body methylation is an ancient property of eukaryotic genomes.
[Show abstract][Hide abstract] ABSTRACT: Parent-of-origin-specific (imprinted) gene expression is regulated in Arabidopsis thaliana endosperm by cytosine demethylation of the maternal genome mediated by the DNA glycosylase DEMETER, but the extent of the methylation changes is not known. Here, we show that virtually the entire endosperm genome is demethylated, coupled with extensive local non-CG hypermethylation of small interfering RNA-targeted sequences. Mutation of DEMETER partially restores endosperm CG methylation to levels found in other tissues, indicating that CG demethylation is specific to maternal sequences. Endosperm demethylation is accompanied by CHH hypermethylation of embryo transposable elements. Our findings demonstrate extensive reconfiguration of the endosperm methylation landscape that likely reinforces transposon silencing in the embryo.
[Show abstract][Hide abstract] ABSTRACT: DNA methylation is an ancient process found in all domains of life. Although the enzymes that mediate methylation have remained highly conserved, DNA methylation has been adapted for a variety of uses throughout evolution, including defense against transposable elements and control of gene expression. Defects in DNA methylation are linked to human diseases, including cancer. Methylation has been lost several times in the course of animal and fungal evolution, thus limiting the opportunity for study in common model organisms. In the past decade, plants have emerged as a premier model system for genetic dissection of DNA methylation. A recent combination of plant genetics with powerful genomic approaches has led to a number of exciting discoveries and promises many more.
Current Opinion in Plant Biology 10/2008; 11(5):554-9. · 8.46 Impact Factor
[Show abstract][Hide abstract] ABSTRACT: Eukaryotic chromatin is separated into functional domains differentiated by post-translational histone modifications, histone variants and DNA methylation. Methylation is associated with repression of transcriptional initiation in plants and animals, and is frequently found in transposable elements. Proper methylation patterns are crucial for eukaryotic development, and aberrant methylation-induced silencing of tumour suppressor genes is a common feature of human cancer. In contrast to methylation, the histone variant H2A.Z is preferentially deposited by the Swr1 ATPase complex near 5' ends of genes where it promotes transcriptional competence. How DNA methylation and H2A.Z influence transcription remains largely unknown. Here we show that in the plant Arabidopsis thaliana regions of DNA methylation are quantitatively deficient in H2A.Z. Exclusion of H2A.Z is seen at sites of DNA methylation in the bodies of actively transcribed genes and in methylated transposons. Mutation of the MET1 DNA methyltransferase, which causes both losses and gains of DNA methylation, engenders opposite changes (gains and losses) in H2A.Z deposition, whereas mutation of the PIE1 subunit of the Swr1 complex that deposits H2A.Z leads to genome-wide hypermethylation. Our findings indicate that DNA methylation can influence chromatin structure and effect gene silencing by excluding H2A.Z, and that H2A.Z protects genes from DNA methylation.