Hidetoshi Saze

Maintenance of CpG methylation is essential for epigenetic inheritance during plant gametogenesis

In mammals, the DNA methyltransferase 1 (Dnmt1) faithfully copies the pattern of cytosine methylation at CpG sites to the newly synthesized strand, and this is essential for epigenetic inheritance. In Arabidopsis thaliana, several DNA methyltransferases or chromatin modifiers coupled to methylation changes have been characterized, and mutations that cause loss of their function are recessive. This is surprising because plant gametogenesis includes postmeiotic DNA replication in haploid nuclei before fertilization. Therefore, the recessive character of the mutations excludes the affected components from a regulatory role in postmeiotic maintenance or modification of epigenetic states. Here we show, however, that depletion of A. thaliana MET1, a homolog of mammalian Dnmt1 (ref. 8), results in immense epigenetic diversification of gametes. This diversity seems to be a consequence of passive postmeiotic demethylation, leading to gametes with fully demethylated and hemidemethylated DNA, followed by remethylation of hemimethylated templates once MET1 is again supplied in a zygote.

Erasure of CpG methylation in Arabidopsis alters patterns of histone H3 methylation in heterochromatin

In mammals and plants, formation of heterochromatin is associated with hypermethylation of DNA at CpG sites and histone H3 methylation at lysine 9. Previous studies have revealed that maintenance of DNA methylation in Neurospora and Arabidopsis requires histone H3 methylation. A feedback loop from DNA methylation to histone methylation, however, is less understood. Its recent examination in Arabidopsis with a partial loss of function in DNA methyltransferase 1 (responsible for maintenance of CpG methylation) yielded conflicting results. Here we report that complete removal of CpG methylation in an Arabidopsis mutant null for DNA maintenance methyltransferase results in a clear loss of histone H3 methylation at lysine 9 in heterochromatin and also at heterochromatic loci that remain transcriptionally silent. Surprisingly, these dramatic alterations are not reflected in heterochromatin relaxation.

Control of genic DNA methylation by a jmjC domain-containing protein in Arabidopsis thaliana.

Differential cytosine methylation of repeats and genes is important for coordination of genome stability and proper gene expression. Through genetic screen of mutants showing ectopic cytosine methylation in a genic region, we identified a jmjC-domain gene, IBM1 (increase in bonsai methylation 1), in Arabidopsis thaliana. In addition to the ectopic cytosine methylation, the ibm1 mutations induced a variety of developmental phenotypes, which depend on methylation of histone H3 at lysine 9. Paradoxically, the developmental phenotypes of the ibm1 were enhanced by the mutation in the chromatin-remodeling gene DDM1 (decrease in DNA methylation 1), which is necessary for keeping methylation and silencing of repeated heterochromatin loci. Our results demonstrate the importance of chromatin remodeling and histone modifications in the differential epigenetic control of repeats and genes.

Heritable epigenetic mutation of a transposon-flanked Arabidopsis gene due to lack of the chromatin-remodeling factor DDM1

Epigenetically silent transposons and repeats constitute a substantial proportion of eukaryotic genomes, but their impact on cellular gene function remains largely unexplored. In Arabidopsis, transposons are silenced by DNA methylation, and this methylation is often abolished by mutations in a chromatin‐remodeling gene DDM1 (DECREASE IN DNA METHYLATION 1). The ddm1 mutation induces various types of developmental abnormalities through de‐repression of transposons and repeats. Here, we report a novel mechanism for a ddm1‐induced syndrome, called bonsai (bns). We identified the gene responsible for the bns phenotypes by genetic linkage analysis and subsequent transcriptional analysis. The bns phenotypes are due to silencing of a putative Anaphase‐Promoting Complex (APC) 13 gene. The BNS gene silencing was associated with DNA hypermethylation, which is in contrast to the ddm1‐induced hypomethylation in the other genomic regions. This paradoxical BNS hypermethylation was reproducibly induced during self‐pollination of the ddm1 mutant, and it was mediated by a long interspersed nuclear element (LINE) retrotransposon flanking the BNS gene. We discuss possible molecular mechanisms and the evolutionary implications of transposon‐mediated epigenetic changes in the BNS locus.

An Arabidopsis jmjC domain protein protects transcribed genes from DNA methylation at CHG sites

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.

DNA Methylation in Plants: Relationship to Small RNAs and Histone Modifications, and Functions in Transposon Inactivation

DNA methylation is a type of epigenetic marking that strongly influences chromatin structure and gene expression in plants and mammals. Over the past decade, DNA methylation has been intensively investigated in order to elucidate its control mechanisms. These studies have shown that small RNAs are involved in the induction of DNA methylation, that there is a relationship between DNA methylation and histone methylation, and that the base excision repair pathway has an important role in DNA demethylation. Some aspects of DNA methylation have also been shown to be shared with mammals, suggesting that the regulatory pathways are, in part at least, evolutionarily conserved. Considerable progress has been made in elucidating the mechanisms that control DNA methylation; however, many aspects of the mechanisms that read the information encoded by DNA methylation and mediate this into downstream regulation remain uncertain, although some candidate proteins have been identified. DNA methylation has a vital role in the inactivation of transposons, suggesting that DNA methylation is a key factor in the evolution and adaptation of plants.

Differentiation of epigenetic modifications between transposons and genes.

Transposable elements (TEs) and repeats are methylated and silenced epigenetically in a variety of organisms including plants. Recent results in Arabidopsis suggest that the TE silencing can be reprogrammed by small RNA during gametogenesis. On the other hand, TE-specific DNA methylation independent of small RNA can be induced by H3K9 methylation through mechanisms conserved between plants and fungi. Methylation of CG sites is found not only in TEs but also in the body of constitutively transcribed genes. Although the function of gene-body methylation is still elusive, the distribution and control of this type of DNA methylation are very similar between plants and animals. Possible interactions of these multiple layers of epigenetic marks and their evolution are discussed.

Epigenetic memory transmission through mitosis and meiosis in plants.

Gene activities can be regulated by epigenetic modifications of nucleotides and chromatin that are stably propagated through somatic cell divisions and, in some cases, across generations. The mechanisms that control epigenetic marks have recently been uncovered using model organisms, such as the flowering plant Arabidopsis thaliana. In Arabidopsis, perturbation of epigenetic gene activity often results in heritable developmental phenotypes. Stable, but potentially reversible, changes in epigenetic status can also be sources for phenotypic variations in natural plant populations.

Autocatalytic differentiation of epigenetic modifications within the Arabidopsis genome

In diverse eukaryotes, constitutively silent sequences, such as transposons and repeats, are marked by methylation at histone H3 lysine 9 (H3K9me). Although selective H3K9me is critical for maintaining genome integrity, mechanisms to exclude H3K9me from active genes remain largely unexplored. Here, we show in Arabidopsis that the exclusion depends on a histone demethylase gene, IBM1 (increase in BONSAI methylation). Loss‐of‐function ibm1 mutation results in ectopic H3K9me and non‐CG methylation in thousands of genes. The ibm1‐induced genic H3K9me depends on both histone methylase KYP/SUVH4 and DNA methylase CMT3, suggesting interdependence of two epigenetic marks—H3K9me and non‐CG methylation. Notably, IBM1 enhances loss of H3K9me in transcriptionally de‐repressed sequences. Furthermore, disruption of transcription in genes induces ectopic non‐CG methylation, which mimics the loss of IBM1 function. We propose that active chromatin is stabilized by an autocatalytic loop of transcription and H3K9 demethylation. This process counteracts a similarly autocatalytic accumulation of silent epigenetic marks, H3K9me and non‐CG methylation.

Epigenetic regulation of intragenic transposable elements impacts gene transcription in Arabidopsis thaliana

Genomes of higher eukaryotes, including plants, contain numerous transposable elements (TEs), that are often silenced by epigenetic mechanisms, such as histone modifications and DNA methylation. Although TE silencing adversely affects expression of nearby genes, recent studies reveal the presence of intragenic TEs marked by repressive heterochromatic epigenetic marks within transcribed genes. However, even for the well-studied plant model Arabidopsis thaliana, the abundance of intragenic TEs, how they are epigenetically regulated, and their potential impacts on host gene expression, remain unexplored. In this study, we comprehensively analyzed genome-wide distribution and epigenetic regulation of intragenic TEs in A. thaliana. Our analysis revealed that about 3% of TEs are located within gene bodies, dominantly at intronic regions. Most of them are shorter and less methylated than intergenic TEs, but they are still targeted by RNA-directed DNA methylation-dependent and independent pathways. Surprisingly, the heterochromatic epigenetic marks at TEs are maintained within actively transcribed genes. Moreover, the heterochromatic state of intronic TEs is critical for proper transcription of associated genes. Our study provides the first insight into how intragenic TEs affect the transcriptional landscape of the A. thaliana genome, and suggests the importance of epigenetic mechanisms for regulation of TEs within transcriptional gene units.