Epigenetic control of CACTA transposon mobility in Arabidopsis thaliana

Epigenetic mutation, heritable developmental variation not based on a change in nucleotide sequence, is widely reported in plants. However, the developmental and evolutionary significance of such mutations remains enigmatic. On the basis of our studies of the endogenous Arabidopsis transposon CACTA, we propose that the inheritance of epigenetic gene silencing over generations can function as a transgenerational genome defense mechanism against deleterious movement of transposons. We previously reported that silent CACTA1 is mobilized by the DNA hypomethylation mutation ddm1 (decrease in DNA methylation). In this study, we report that CACTA activated by the ddm1 mutation remains mobile in the presence of the wild-type DDM1 gene, suggesting that de novo silencing is not efficient for the defense of the genome against CACTA movement. The defense depends on maintenance of transposon silencing over generations. In addition, we show that the activated CACTA1 element transposes throughout the genome in DDM1 plants, as reported previously for ddm1 backgrounds. Furthermore, the CACTA1 element integrated into both the ddm1-derived and the DDM1-derived chromosomal regions in the DDM1 wild-type plants, demonstrating that this class of transposons does not exhibit targeted integration into heterochromatin, despite its accumulation in the pericentromeric regions in natural populations. The possible contribution of natural selection as a mechanism for the accumulation of transposons and evolution of heterochromatin is discussed.     

RNAi, DRD1, and histone methylation actively target developmentally important non-CG DNA methylation in arabidopsis

Cytosine DNA methylation protects eukaryotic genomes by silencing transposons and harmful DNAs, but also regulates gene expression during normal development. Loss of CG methylation in the Arabidopsis thaliana met1 and ddm1 mutants causes varied and stochastic developmental defects that are often inherited independently of the original met1 or ddm1 mutation. Loss of non-CG methylation in plants with combined mutations in the DRM and CMT3 genes also causes a suite of developmental defects. We show here that the pleiotropic developmental defects of drm1 drm2 cmt3 triple mutant plants are fully recessive, and unlike phenotypes caused by met1 and ddm1, are not inherited independently of the drm and cmt3 mutations. Developmental phenotypes are also reversed when drm1 drm2 cmt3 plants are transformed with DRM2 or CMT3, implying that non-CG DNA methylation is efficiently re-established by sequence-specific signals. We provide evidence that these signals include RNA silencing though the 24-nucleotide short interfering RNA (siRNA) pathway as well as histone H3K9 methylation, both of which converge on the putative chromatin-remodeling protein DRD1. These signals act in at least three partially intersecting pathways that control the locus-specific patterning of non-CG methylation by the DRM2 and CMT3 methyltransferases. Our results suggest that non-CG DNA methylation that is inherited via a network of persistent targeting signals has been co-opted to regulate developmentally important genes.     

Analysis of target sequences of DDM1s in Brassica rapa by MSAP

DNA methylation is an important epigenetic modification regulating gene expression and transposon silencing. Although epigenetic regulation is involved in some agricultural traits, there has been relatively little research on epigenetic modifications of genes in Brassica rapa, which includes many important vegetables. In B. rapa, orthologs of DDM1, a chromatin remodeling factor required for maintenance of DNA methylation, have been characterized and DNA hypomethylated knock-down plants by RNAi (ddm1-RNAi plants) have been generated. In this study, we investigated differences of DNA methylation status at the genome-wide level between a wild-type (WT) plant and a ddm1-RNAi plant by methylation-sensitive amplification polymorphism (MSAP) analysis. MSAP analysis detected changes of DNA methylation of many repetitive sequences in the ddm1-RNAi plant. Search for body methylated regions in the WT plant revealed no difference in gene body methylation levels between the WT plant and the ddm1-RNAi plant. These results indicate that repetitive sequences are preferentially methylated by DDM1 genes in B. rapa.     

Distinct mechanisms determine transposon inheritance and methylation via small interfering RNA and histone modification

Heritable, but reversible, changes in transposable element activity were first observed in maize by Barbara McClintock in the 1950s. More recently, transposon silencing has been associated with DNA methylation, histone H3 lysine-9 methylation (H3mK9), and RNA interference (RNAi). Using a genetic approach, we have investigated the role of these modifications in the epigenetic regulation and inheritance of six Arabidopsis transposons. Silencing of most of the transposons is relieved in DNA methyltransferase (met1), chromatin remodeling ATPase (ddm1), and histone modification (sil1) mutants. In contrast, only a small subset of the transposons require the H3mK9 methyltransferase KRYPTONITE, the RNAi gene ARGONAUTE1, and the CXG methyltransferase CHROMOMETHYLASE3. In crosses to wild-type plants, epigenetic inheritance of active transposons varied from mutant to mutant, indicating these genes differ in their ability to silence transposons. According to their pattern of transposon regulation, the mutants can be divided into two groups, which suggests that there are distinct, but interacting, complexes or pathways involved in transposon silencing. Furthermore, different transposons tend to be susceptible to different forms of epigenetic regulation.     

ddm1 plants are sensitive to methyl methane sulfonate and NaCl stresses and are deficient in DNA repair

Plant response to stress includes changes in gene expression and chromatin structure. Our previous work showed that Arabidopsis thaliana Dicer-like (DCL) mutants were impaired in transgenerational response to stress that included an increase in recombination frequency, cytosine methylation and stress tolerance. It can be hypothesized that changes in chromatin structure are important for an efficient stress response. To test this hypothesis, we analyzed the stress response of ddm1, a mutant impaired in DDM1, a member of the SWI/SNF family of adenosine triphosphate-dependent chromatin remodeling genes. We exposed Arabidopsis thaliana ddm1 mutants to methyl methane sulfonate (MMS) and NaCl and found that these plants were more sensitive. At the same time, ddm1 plants were similar to wild-type plants in sensitivity to temperature and bleomycin stresses. Direct comparison to met1 plants, deficient in maintenance methyltransferase MET1, showed higher sensitivity of ddm1 plants to NaCl. The level of DNA strand breaks upon exposure to MMS increased in wild-type plants but decreased in ddm1 plants. DNA methylation analysis showed that heterozygous ddm1/DDM1 plants had lower methylation as compared to fourth generation of homozygous ddm1/ddm1 plants. Exposure to MMS resulted in a decrease in methylation in wild-type plants and an increase in ddm1 plants. Finally, in vitro DNA excision repair assay showed lower capacity for ddm1 mutant. Our results provided a new example of a link between genetic genome stability and epigenetic genome stability. Key message We demonstrate that heterozygous ddm1/DDM1 plants are more sensitive to stress and have more severe changes in methylation than homozygous ddm1/ddm1 plants.     

The CpG island methylator phenotype correlates with long-range epigenetic silencing in colorectal cancer

The CpG island methylator phenotype (CIMP), characterized by an exceptionally high frequency of methylation of discrete CpG islands, is observed in 18% to 25% of sporadic colorectal cancers. Another hypermethylation pattern found in colorectal cancers, termed long-range epigenetic silencing, is associated with DNA/histone methylation in three distinct gene clusters at chromosome 2q14.2, showing that DNA hypermethylation can span larger chromosomal domains and lead to the silencing of flanking, unmethylated genes. We investigated whether these two phenotypes are interrelated in colorectal cancers. The CIMP status of 148 sporadic colorectal cancers was determined by methylation-specific PCR. We determined the BRAF V600E mutation by mutant allele-specific PCR amplification. The methylation status of the MLH1 gene and of three CpG islands (EN1, SCTR, and INHBB), corresponding to three distinct clusters along 2q14.2, was determined by methylation-specific PCR. The average number of sites showing methylation in CIMP+ tumors was 2.21, compared with 1.22 for CIMP- individuals, and this difference was highly significant (P = 3.6 x 10(-8), Mann-Whitney test). Moreover, all CIMP+ tumors showed hypermethylation of at least one of these loci, in contrast to CIMP- tumors, where 18 (16%) samples remained unmethylated. The mean number of simultaneously hypermethylated CpG islands at 2q14.2 differs significantly between CIMP- and CIMP+ tumors, suggesting varying effects of domain silencing in this region. Given that the number of hypermethylated loci at 2q14.2 likely affects the range of silenced flanking genes, high frequency of simultaneous hypermethylation of three CpG islands (EN1, SCTR, and INHBB) may have potential influence on specific characteristics of CIMP+ colorectal cancers.