Antisilencing role of the RNA-directed DNA methylation pathway and a histone acetyltransferase in Arabidopsis

REPRESSOR OF SILENCING 1 (ROS1) is a DNA demethylation enzyme that was previously identified during a genetic screen for the silencing of both RD29A-LUC and 35S-NPTII transgenes on a T-DNA construct. Here we performed a genetic screen to identify additional mutants in which the 35S-NPTII transgene is silenced. We identified several alleles of ros1 and of the following components of the RNA-directed DNA methylation (RdDM) pathway: NRPD1 (the largest subunit of polymerase IV), RDR2, NRPE1 (the largest subunit of polymerase V), NRPD2, AGO4, and DMS3. Our results show that the silencing of 35S-NPTII in the RdDM pathway mutants is due to the reduced expression of ROS1 in the mutants. We also identified a putative histone acetyltransferase (ROS4) from the genetic screen. The acetyltransferase contains a PHD-finger domain that binds to unmethylated histone H3K4. The mutation in ROS4 led to reduction of H3K18 and H3K23 acetylation levels. We show that the silencing of 35S-NPTII and some transposable element genes was released by the ddm1 mutation but that this also required ROS4. Our study identifies a unique antisilencing factor, and reveals that the RdDM pathway has an antisilencing function due to its role in maintaining ROS1 expression.     

Dnmt2-dependent methylomes lack defined DNA methylation patterns

Several organisms have retained methyltransferase 2 (Dnmt2) as their only candidate DNA methyltransferase gene. However, information about Dnmt2-dependent methylation patterns has been limited to a few isolated loci and the results have been discussed controversially. In addition, recent studies have shown that Dnmt2 functions as a tRNA methyltransferase, which raised the possibility that Dnmt2-only genomes might be unmethylated. We have now used whole-genome bisulfite sequencing to analyze the methylomes of Dnmt2-only organisms at single-base resolution. Our results show that the genomes of Schistosoma mansoni and Drosophila melanogaster lack detectable DNA methylation patterns. Residual unconverted cytosine residues shared many attributes with bisulfite deamination artifacts and were observed at comparable levels in Dnmt2-deficient flies. Furthermore, genetically modified Dnmt2-only mouse embryonic stem cells lost the DNA methylation patterns found in wild-type cells. Our results thus uncover fundamental differences among animal methylomes and suggest that DNA methylation is dispensable for a considerable number of eukaryotic organisms.     

Deficiency in DNA methylation increases meiotic crossover rates in euchromatic but not in heterochromatic regions in Arabidopsis

Meiotic recombination is tightly regulated by cis- and trans-acting factors. Although DNA methylation and chromatin remodeling affect chromosome structure, their impact on meiotic recombination is not well understood. To study the effect of DNA methylation on the landscape of chromosomal recombination, we analyzed meiotic recombination in the decreased DNA methylation 1 (ddm1) mutant. DDM1 is a SWI2/SNF2-like chromatin-remodeling protein necessary for DNA methylation and heterochromatin maintenance in Arabidopsis thaliana. The rate of meiotic recombination between markers located in euchromatic regions was significantly higher in both heterozygous (DDM1/ddm1) and homozygous (ddm1/ddm1) backgrounds than in WT plants. The effect on recombination was similar for both male and female meiocytes. Contrary to expectations, ddm1 had no effect on the number of crossovers between markers in heterochromatic pericentric regions that underwent demethylation. These results are surprising, because the pericentromeric regions are hypermethylated and were expected to be the regions most affected by demethylation. Thus, DDM1 loss of function may trigger changes that enhance meiotic recombination in euchromatin regions but are not sufficient to induce the same events in heterochromatic segments. This work uncovers the repressive role of methylation on meiotic recombination in euchromatic regions and suggests that additional factors may have a role in controlling the suppression of recombination in heterochromatin.     

Clinical implications of DNA methylation in hepatocellular carcinoma

Background

Epigenetics is a rapidly evolving field of genetic study applicable to nearly every aspect of genome-related research. The importance of epigenetics has been recognised in human hepatocellular carcinoma (HCC). Changes in DNA methylation patterns, including global hypomethylation and promoter hypermethylation, are thought to be early events in hepatocarcinogenesis.

Objectives

This review aimed to summarise the role of epigenetics in HCC, to describe the mechanisms of epigenetic changes in HCC and to examine the clinical relevance of epigenetics in HCC.

Methods

This review examines the role of CpG-rich regions and DNA methylation, and describes an epigenetic model of cancer, tumour type-specific methylation, the relationships among methylation, cirrhosis and hepatocarcinogenesis, and the role of DNA methylation in HCC. The clinical implications of epigenetics in HCC are discussed.

Results

A multivariate predictor model based on traditional clinical factors and DNA methylation profile may have important applications in the early detection of neoplastic transformation in populations at high risk for HCC. CpG methylation may be valuable in HCC prognostics. DNA methylation profiles may enable clinical prediction in pre-therapy patient biopsies, paraffin-embedded samples or plasma DNA.

Conclusions

Epigenetic changes and profiles may correlate to the biological behaviour of tumours and clinical outcome of HCC patients. The use of DNA methylation profiles as a surrogate biomarker remains an active area of clinical cancer research.     

PolIVb influences RNA-directed DNA methylation independently of its role in siRNA biogenesis

DNA-dependent RNA polymerase (Pol)IV in Arabidopsis exists in two isoforms (PolIVa and PolIVb), with NRPD1a and NRPD1b as their respective largest subunits. Both isoforms are implicated in production and activity of siRNAs and in RNA-directed DNA methylation (RdDM). Deep sequence analysis of siRNAs in WT Arabidopsis flowers and in nrpd1a and nrpd1b mutants identified >4,200 loci producing siRNAs in a PolIV-dependent manner, with PolIVb reinforcing siRNA production by PolIVa. Transposable element identity and pericentromeric localization are both features that predispose a locus for siRNA production via PolIV proteins and determine the extent to which siRNA production relies on PolIVb. Detailed analysis of DNA methylation at PolIV-dependent loci revealed unexpected deviations from the previously noted association of PolIVb-dependent siRNA production and RdDM. Notably, PolIVb functions independently in DNA methylation and siRNA generation. Additionally, we have uncovered siRNA-directed loss of DNA methylation, a process requiring both PolIV isoforms. From these findings, we infer that the role of PolIVb in siRNA production is secondary to a role in chromatin modification and is influenced by chromatin context.     

Total kinetic analysis reveals how combinatorial methylation patterns are established on lysines 27 and 36 of histone H3

We have developed a targeted method to quantify all combinations of methylation on an H3 peptide containing lysines 27 and 36 (H3K27-K36). By using stable isotopes that separately label the histone backbone and its methylations, we tracked the rates of methylation and demethylation in myeloma cells expressing high vs. low levels of the methyltransferase MMSET/WHSC1/NSD2. Following quantification of 99 labeled H3K27-K36 methylation states across time, a kinetic model converged to yield 44 effective rate constants qualifying each methylation and demethylation step as a function of the methylation state on the neighboring lysine. We call this approach MS-based measurement and modeling of histone methylation kinetics (M4K). M4K revealed that, when dimethylation states are reached on H3K27 or H3K36, rates of further methylation on the other site are reduced as much as 100-fold. Overall, cells with high MMSET have as much as 33-fold increases in the effective rate constants for formation of H3K36 mono- and dimethylation. At H3K27, cells with high MMSET have elevated formation of K27me1, but even higher increases in the effective rate constants for its reversal by demethylation. These quantitative studies lay bare a bidirectional antagonism between H3K27 and H3K36 that controls the writing and erasing of these methylation marks. Additionally, the integrated kinetic model was used to correctly predict observed abundances of H3K27-K36 methylation states within 5% of that actually established in perturbed cells. Such predictive power for how histone methylations are established should have major value as this family of methyltransferases matures as drug targets.     

RNA-directed DNA methylation enforces boundaries between heterochromatin and euchromatin in the maize genome

The maize genome is relatively large (∼2.3 Gb) and has a complex organization of interspersed genes and transposable elements, which necessitates frequent boundaries between different types of chromatin. The examination of maize genes and conserved noncoding sequences revealed that many of these are flanked by regions of elevated asymmetric CHH (where H is A, C, or T) methylation (termed mCHH islands). These mCHH islands are quite short (∼100 bp), are enriched near active genes, and often occur at the edge of the transposon that is located nearest to genes. The analysis of DNA methylation in other sequence contexts and several chromatin modifications revealed that mCHH islands mark the transition from heterochromatin-associated modifications to euchromatin-associated modifications. The presence of an mCHH island is fairly consistent in several distinct tissues that were surveyed but shows some variation among different haplotypes. The presence of insertion/deletions in promoters often influences the presence and position of an mCHH island. The mCHH islands are dependent upon RNA-directed DNA methylation activities and are lost in mop1 and mop3 mutants, but the nearby genes rarely exhibit altered expression levels. Instead, loss of an mCHH island is often accompanied by additional loss of DNA methylation in CG and CHG contexts associated with heterochromatin in nearby transposons. This suggests that mCHH islands and RNA-directed DNA methylation near maize genes may act to preserve the silencing of transposons from activity of nearby genes.The cytosine bases in a genome can be modified to 5-methylcytosine by adding a methyl group at the 5′ position. This process, called DNA methylation, is conserved from algae to animals and plants (1, 2). DNA methylation can be separated into different types based on the local sequence context. In plants DNA methylation is found at the symmetric CG or CHG (where H = A, C, or T) sites or at nonsymmetric CHH sites. CG and CHG methylation are maintained at high fidelity following DNA replication due to activity of maintenance methyltransferases such as MET1 or chromomethylase (CMT) 3 (3, 4), whereas CHH methylation (mCHH) requires targeting by either domains rearranged methylase 2 (DRM2) or CMT2 (3–6). The DRM2 targeting occurs via RNA-directed DNA methylation (RdDM) and requires the activity of polymerase IV (PolIV) and polymerase V (PolV) complexes (3, 4). There is evidence that recruitment of PolIV and PolV may require the presence of dimethylation of lysine 9 of histone H3 (H3K9me2) or DNA methylation at the targeted genomic regions (7, 8). The specific mechanisms that recruit CMT2 are not well characterized but may require specific histone modifications (5, 6).Much of our knowledge of DNA methylation in plants is derived from studies of the model plant Arabidopsis thaliana, which has a relatively small genome and relatively few examples of genes with nearby transposons (36.3%; ref. 9). The maize genome is much more complex, with the majority (85.5%) of genes positioned within 1 kb of transposons. In both species, transposons tend to have quite high levels of CG and CHG methylation whereas genes have much lower levels (10). mCHH is often thought to provide an important component for silencing transposons, yet the maize genome has relatively low levels of mCHH despite the high transposon context (11). This is partially attributed to the lack of a CMT2 ortholog in maize (5), which may explain the reduced levels of mCHH in the middle of larger transposons. Although mCHH is low in maize, there are still genomic regions with elevated mCHH (12). Genomic profiles of mCHH in maize revealed that this modification is often found near genes (termed mCHH islands) and is dependent upon RdDM activity (12–14). This elevation of mCHH in regions surrounding genes is much less prevalent in Arabidopsis (10). A recent study showed that high mCHH can also be induced near genes that are up-regulated in plants subjected to phosphate starvation (15).In this study we further probed the basis and function of these mCHH islands. We found that mCHH islands are short regions of elevated mCHH that flank nearly half of the genes in maize and many conserved noncoding sequences (CNSs). These mCHH islands mark a transition for CG and CHG DNA methylation, several histone modifications, and chromatin accessibility. The mCHH islands are relatively stable across different tissues but show some variation among haplotypes that are often associated with sequence insertions/deletions (InDels). The loss of mCHH islands does not strongly affect gene expression, but instead leads to an additional loss of CG and CHG methylation in some transposons flanking maize genes.     

Loss of DNA methylation affects the recombination landscape in Arabidopsis

During sexual reproduction, one-half of the genetic material is deposited in gametes, and a complete set of chromosomes is restored upon fertilization. Reduction of the genetic information before gametogenesis occurs in meiosis, when cross-overs (COs) between homologous chromosomes secure an exchange of their genetic information. COs are not evenly distributed along chromosomes and are suppressed in chromosomal regions encompassing compact, hypermethylated centromeric and pericentromeric DNA. Therefore, it was postulated that DNA hypermethylation is inhibitory to COs. Here, when analyzing meiotic recombination in mutant plants with hypomethylated DNA, we observed unexpected and counterintuitive effects of DNA methylation losses on CO distribution. Recombination was further promoted in the hypomethylated chromosome arms while it was inhibited in heterochromatic regions encompassing pericentromeric DNA. Importantly, the total number of COs was not affected, implying that loss of DNA methylation led to a global redistribution of COs along chromosomes. To determine by which mechanisms altered levels of DNA methylation influence recombination--whether directly in cis or indirectly in trans by changing expression of genes encoding recombination components--we analyzed CO distribution in wild-type lines with randomly scattered and well-mapped hypomethylated chromosomal segments. The results of these experiments, supported by expression profiling data, suggest that DNA methylation affects meiotic recombination in cis. Because DNA methylation exhibits significant variation even within a single species, our results imply that it may influence the evolution of plant genomes through the control of meiotic recombination.     

DNA methylation is widespread and associated with differential gene expression in castes of the honeybee,Apis mellifera

The recent, unexpected discovery of a functional DNA methylation system in the genome of the social bee Apis mellifera underscores the potential importance of DNA methylation in invertebrates. The extent of genomic DNA methylation and its role in A. mellifera remain unknown, however. Here we show that genes in A. mellifera can be divided into 2 distinct classes, one with low-CpG dinucleotide content and the other with high-CpG dinucleotide content. This dichotomy is explained by the gradual depletion of CpG dinucleotides, a well-known consequence of DNA methylation. The loss of CpG dinucleotides associated with DNA methylation also may explain the unusual mutational patterns seen in A. mellifera that lead to AT-rich regions of the genome. A detailed investigation of this dichotomy implicates DNA methylation in A. mellifera development. High-CpG genes, which are predicted to be hypomethylated in germlines, are enriched with functions associated with developmental processes, whereas low-CpG genes, predicted to be hypermethylated in germlines, are enriched with functions associated with basic biological processes. Furthermore, genes more highly expressed in one caste than another are overrepresented among high-CpG genes. Our results highlight the potential significance of epigenetic modifications, such as DNA methylation, in developmental processes in social insects. In particular, the pervasiveness of DNA methylation in the genome of A. mellifera provides fertile ground for future studies of phenotypic plasticity and genomic imprinting.     

The role of microRNA in myelodysplastic syndromes: beyond DNA methylation and histone modification

Myelodysplastic syndromes (MDS) are heterogeneous group of hematologic disorders of mostly elderly and based on distinct clinical phenotypes. Current paradigm of their pathogenesis relies on somatic gene mutations combined with the predisposing defective osteohematopoietic niche, but due to the breakout in epigenetic research scientific focus has steered toward two most common epigenetic modifications: methylation mechanisms and histone modification. At the same time, relatively few studies have been undertaken regarding the third epigenetic pathway – microRNAs – in MDS. The main aim of this review is to provide the basics of microRNA biology and function in oncogenesis, showing the complexity of mechanisms behind this single‐stranded 22 nucleotides long RNA molecule, with further focus on its implication in MDS pathology and clinical context. By extensive literature search, we have shown enough evidence for their deregulation in MDS. However, few studies have addressed the issue on pathogenic events in MDS and its association with specific microRNAs. Preliminary research in clinical setting has shown the possible utility of microRNAs in terms of prognosis and therapy, although we are only beginning to understand various implications of microRNAs in MDS and further extensive research is warranted to answer multiple questions arising from interconnection of this epigenetic mechanism in MDS.