Inheritance of an epigenetic mark: the CpG DNA methyltransferase 1 is required for de novo establishment of a complex pattern of non-CpG methylation

Site-specific methylation of cytosines is a key epigenetic mark of vertebrate DNA. While a majority of the methylated residues are in the symmetrical (meC)pG:Gp(meC) configuration, a smaller, but significant fraction is found in the CpA, CpT and CpC asymmetric (non-CpG) dinucleotides. CpG methylation is reproducibly maintained by the activity of the DNA methyltransferase 1 (Dnmt1) on the newly replicated hemimethylated substrates (meC)pG:GpC. On the other hand, establishment and hereditary maintenance of non-CpG methylation patterns have not been analyzed in detail. We previously reported the occurrence of site- and allele-specific methylation at both CpG and non-CpG sites. Here we characterize a hereditary complex of non-CpG methylation, with the transgenerational maintenance of three distinct profiles in a constant ratio, associated with extensive CpG methylation. These observations raised the question of the signal leading to the maintenance of the pattern of asymmetric methylation. The complete non-CpG pattern was reinstated at each generation in spite of the fact that the majority of the sperm genomes contained either none or only one methylated non-CpG site. This observation led us to the hypothesis that the stable CpG patterns might act as blueprints for the maintenance of non-CpG DNA methylation. As predicted, non-CpG DNA methylation profiles were abrogated in a mutant lacking Dnmt1, the enzymes responsible for CpG methylation, but not in mutants defective for either Dnmt3a or Dnmt2.     

DNMT1, DNMT3A and DNMT3B proteins are differently expressed in mouse oocytes and early embryos

DNA methylation is one of the epigenetic mechanisms and plays important roles during oogenesis and early embryo development in mammals. DNA methylation is basically known as adding a methyl group to the fifth carbon atom of cytosine residues within cytosine–phosphate–guanine (CpG) and non-CpG dinucleotide sites. This mechanism is composed of two main processes: de novo methylation and maintenance methylation, both of which are catalyzed by specific DNA methyltransferase (DNMT) enzymes. To date, six different DNMTs have been characterized in mammals defined as DNMT1, DNMT2, DNMT3A, DNMT3B, DNMT3C, and DNMT3L. While DNMT1 primarily functions in maintenance methylation, both DNMT3A and DNMT3B are essentially responsible for de novo methylation. As is known, either maintenance or de novo methylation processes appears during oocyte and early embryo development terms. The aim of the present study is to investigate spatial and temporal expression levels and subcellular localizations of the DNMT1, DNMT3A, and DNMT3B proteins in the mouse germinal vesicle (GV) and metaphase II (MII) oocytes, and early embryos from 1-cell to blastocyst stages. We found that there are remarkable differences in the expressional levels and subcellular localizations of the DNMT1, DNMT3A and DNMT3B proteins in the GV and MII oocytes, and 1-cell, 2-cell, 4-cell, 8-cell, morula, and blastocyst stage embryos. The fluctuations in the expression of DNMT proteins in the analyzed oocytes and early embryos are largely compatible with DNA methylation changes and genomic imprintestablishment appearing during oogenesis and early embryo development. To understand precisemolecular biological meaning of differently expressing DNMTs in the early developmental periods, further studies are required.     

Alu element mutation spectra: molecular clocks and the effect of DNA methylation

In primate genomes more than 40% of CpG islands are found within repetitive elements. With more than one million copies in the human genome, the Alu family of retrotransposons represents the most successful short interspersed element (SINE) in primates and CpG dinucleotides make up about 20% of Alu sequences. It is generally thought that CpG dinucleotides mutate approximately ten times faster than other dinucleotides due to cytosine methylation and the subsequent deamination and conversion of C-->T. However, the disparity of Alu subfamily age estimations based upon CpG or non-CpG substitution density indicates a more complex relationship between CpG and non-CpG substitutions within the Alu elements. Here we report an analysis of the mutation patterns for 5296 Alu elements comprising 20 subfamilies. Our results indicate a relatively constant CpG versus non-CpG substitution ratio of approximately 6 for the young (AluY) and intermediate (AluS) Alu subfamilies. However, a more complex non-linear relationship between CpG and non-CpG substitutions was observed when old (AluJ) subfamilies were included in the analysis. These patterns may be the result of the slowdown of the neutral mutation rate during primate evolution and/or an increase in the CpG mutation rate as the consequence of increased DNA methylation in response to a burst of retrotransposition activity approximately 35 million years ago.     

The evidence for functional non-CpG methylation in mammalian cells

In mammalian genomes, the methylation of cytosine residues within CpG dinucleotides is crucial to normal development and cell differentiation. However, methylation of cytosines in the contexts of CpA, CpT, and CpC (non-CpG methylation) has been reported for decades, yet remains poorly understood. In recent years, whole genome bisulphite sequencing (WGBS) has confirmed significant levels of non-CpG methylation in specific tissues and cell types. Non-CpG methylation has several properties that distinguish it from CpG methylation. Here we review the literature describing non-CpG methylation in mammalian cells, describe the important characteristics that distinguish it from CpG methylation, and discuss its functional importance.     

The evidence for functional non-CpG methylation in mammalian cells

In mammalian genomes, the methylation of cytosine residues within CpG dinucleotides is crucial to normal development and cell differentiation. However, methylation of cytosines in the contexts of CpA, CpT, and CpC (non-CpG methylation) has been reported for decades, yet remains poorly understood. In recent years, whole genome bisulphite sequencing (WGBS) has confirmed significant levels of non-CpG methylation in specific tissues and cell types. Non-CpG methylation has several properties that distinguish it from CpG methylation. Here we review the literature describing non-CpG methylation in mammalian cells, describe the important characteristics that distinguish it from CpG methylation, and discuss its functional importance.     

Methylation of DNA repeats of decreasing sizes in Ascobolus immersus.

In Ascobolus immersus, DNA duplications are subject to the process of methylation induced premeiotically (MIP), which methylates the cytosine residues within the repeats and results in reversible gene silencing. The triggering of MIP requires pairing of the repeats, and its detection requires maintenance of the resulting methylation. MIP of kilobase-size duplications occurs frequently and leads to the methylation of all C residues in the repeats, including those belonging to non-CpG sequences. Using duplications of decreasing sizes, we observed that tandem repeats never escaped MIP when larger than 630 bp and showed a sudden and drastic drop in MIP frequencies when their sizes decreased from 630 to 317 bp. This contrasted with the progressive decrease of MIP frequencies observed with ectopic repeats, in which apparently the search for homology influences the MIP triggering efficiency. The minimal size actually required for a repeat to undergo detectable MIP was found to be close to 300 bp. Genomic sequencing and Southern hybridization analyses using restriction enzymes sensitive to C methylation showed a loss of methylation at non-CpG sites in short DNA segments, methylation being restricted to a limited number of CpG dinucleotides. Our data suggest the existence of two distinct mechanisms underlying methylation maintenance, one responsible for methylation at CpG sites and the other responsible for methylation at non-CpG sites.     

Methylation status of differentially methylated regions at Igf2/H19 locus in porcine gametes and preimplantation embryos

The aim of this study was to demonstrate how differential methylation imprints are established during porcine preimplantation embryo development. For the methylation analysis, the primers for the three Igf2/H19 DMRs were designed and based upon previously published sequences. The methylation marks of Igf2/H19 DMRs were analysed in sperm and MII oocytes with our results showing that these regions are fully methylated in sperm but remain unmethylated in MII oocytes. In order to identify the methylation pattern at the pronuclear stage, we indirectly compared the methylation profile of Igf2/H19 DMR3 in each zygote derived by in vitro fertilization, parthenogenesis, and androgenesis. Interestingly, this region was found to be differently methylated according to parental origins; DMR3 was hemimethylated in in vitro fertilized zygotes, fully methylated in parthenogenetic zygotes, and demethylated in androgenetic zygotes. These results indicate that the methylation mark of the paternal allele is erased by active demethylation, and that of the maternal one is de novo methylated. We further examined the methylation imprints of Igf2/H19 DMR3 during early embryonic development. The hemimethylated pattern as seen in zygotes fertilized in vitro was observed up to the 4-cell embryo stage. However, this mark was exclusively demethylated at the 8-cell stage and then restored at the morula stage. These results suggest that methylation imprints are established via dynamic changes during early embryonic development in porcine embryos.     

Genomic distribution and inter-sample variation of non-CpG methylation across human cell types

DNA methylation plays an important role in development and disease. The primary sites of DNA methylation in vertebrates are cytosines in the CpG dinucleotide context, which account for roughly three quarters of the total DNA methylation content in human and mouse cells. While the genomic distribution, inter-individual stability, and functional role of CpG methylation are reasonably well understood, little is known about DNA methylation targeting CpA, CpT, and CpC (non-CpG) dinucleotides. Here we report a comprehensive analysis of non-CpG methylation in 76 genome-scale DNA methylation maps across pluripotent and differentiated human cell types. We confirm non-CpG methylation to be predominantly present in pluripotent cell types and observe a decrease upon differentiation and near complete absence in various somatic cell types. Although no function has been assigned to it in pluripotency, our data highlight that non-CpG methylation patterns reappear upon iPS cell reprogramming. Intriguingly, the patterns are highly variable and show little conservation between different pluripotent cell lines. We find a strong correlation of non-CpG methylation and DNMT3 expression levels while showing statistical independence of non-CpG methylation from pluripotency associated gene expression. In line with these findings, we show that knockdown of DNMTA and DNMT3B in hESCs results in a global reduction of non-CpG methylation. Finally, non-CpG methylation appears to be spatially correlated with CpG methylation. In summary these results contribute further to our understanding of cytosine methylation patterns in human cells using a large representative sample set.     

Characterizing the strand-specific distribution of non-CpG methylation in human pluripotent cells

DNA methylation is an important defense and regulatory mechanism. In mammals, most DNA methylation occurs at CpG sites, and asymmetric non-CpG methylation has only been detected at appreciable levels in a few cell types. We are the first to systematically study the strand-specific distribution of non-CpG methylation. With the divide-and-compare strategy, we show that CHG and CHH methylation are not intrinsically different in human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). We also find that non-CpG methylation is skewed between the two strands in introns, especially at intron boundaries and in highly expressed genes. Controlling for the proximal sequences of non-CpG sites, we show that the skew of non-CpG methylation in introns is mainly guided by sequence skew. By studying subgroups of transposable elements, we also found that non-CpG methylation is distributed in a strand-specific manner in both short interspersed nuclear elements (SINE) and long interspersed nuclear elements (LINE), but not in long terminal repeats (LTR). Finally, we show that on the antisense strand of Alus, a non-CpG site just downstream of the A-box is highly methylated. Together, the divide-and-compare strategy leads us to identify regions with strand-specific distributions of non-CpG methylation in humans.     

Heterogeneous genomic molecular clocks in primates

Using data from primates, we show that molecular clocks in sites that have been part of a CpG dinucleotide in recent past (CpG sites) and non-CpG sites are of markedly different nature, reflecting differences in their molecular origins. Notably, single nucleotide substitutions at non-CpG sites show clear generation-time dependency, indicating that most of these substitutions occur by errors during DNA replication. On the other hand, substitutions at CpG sites occur relatively constantly over time, as expected from their primary origin due to methylation. Therefore, molecular clocks are heterogeneous even within a genome. Furthermore, we propose that varying frequencies of CpG dinucleotides in different genomic regions may have contributed significantly to conflicting earlier results on rate constancy of mammalian molecular clock. Our conclusion that different regions of genomes follow different molecular clocks should be considered when inferring divergence times using molecular data and in phylogenetic analysis.     

Accumulation and loss of asymmetric non-CpG methylation during male germ-cell development

DNA methylation is a well-characterized epigenetic modification involved in gene regulation and transposon silencing in mammals. It mainly occurs on cytosines at CpG sites but methylation at non-CpG sites is frequently observed in embryonic stem cells, induced pluriotent stem cells, oocytes and the brain. The biological significance of non-CpG methylation is unknown. Here, we show that non-CpG methylation is also present in male germ cells, within and around B1 retrotransposon sequences interspersed in the mouse genome. It accumulates in mitotically arrested fetal prospermatogonia and reaches the highest level by birth in a Dnmt3l-dependent manner. The preferential site of non-CpG methylation is CpA, especially CpApG and CpApC. Although CpApG (and CpTpG) sites contain cytosines at symmetrical positions, hairpin-bisulfite sequencing reveals that they are hemimethylated, suggesting the absence of a template-dependent copying mechanism. Indeed, the level of non-CpG methylation decreases after the resumption of mitosis in the neonatal period, whereas that of CpG methylation does not. The cells eventually lose non-CpG methylation by the time they become spermatogonia. Our results show that non-CpG methylation accumulates in non-replicating, arrested cells but is not maintained in mitotically dividing cells during male germ-cell development.     

Loss of genomic imprinting in mouse embryos with fast rates of preimplantation development in culture

Currently, the stage of embryo development has been proposed as one of many criteria for identifying healthy embryos in infertility clinics with the fastest embryos being highlighted as the healthiest. However the validity of this as an accurate criterion with respect to genomic imprinting is unknown. Given that embryo development in culture generally requires an extra day compared to in vivo development, we hypothesized that loss of imprinting correlates with slower rates of embryonic development. To evaluate this, embryos were recovered at the 2-cell stage, separated into four groups based on morphological stage at two predetermined time points, and cultured to blastocysts. We examined cell number, embryo volume, embryo sex, imprinted Snrpn and H19 methylation, imprinted Snrpn, H19, and Cdkn1c expression, and expression of genes involved in embryo metabolism-Atp1a1, Slc2a1, and Mapk14-all within the same individual embryo. Contrary to our hypothesis, we observed that faster developing embryos exhibited greater cell numbers and embryo volumes as well as greater perturbations in genomic imprinting and metabolic marker expression. Embryos with slower rates of preimplantation development were most similar to in vivo derived embryos, displaying similar cell numbers, embryo volumes, Snrpn and H19 imprinted methylation, H19 imprinted expression, and Atp1a1 and Slc2a1 expression. We conclude that faster development rates in vitro are correlated with loss of genomic imprinting and aberrant metabolic marker expression. Importantly, we identified a subset of in vitro cultured embryos that, according to the parameters evaluated, are very similar to in vivo derived embryos and thus are likely most suitable for embryo transfer.     

DNA methylation is critical for Arabidopsis embryogenesis and seed viability

DNA methylation (5-methylcytosine) in mammalian genomes predominantly occurs at CpG dinucleotides, is maintained by DNA methyltransferase1 (Dnmt1), and is essential for embryo viability. The plant genome also has 5-methylcytosine at CpG dinucleotides, which is maintained by METHYLTRANSFERASE1 (MET1), a homolog of Dnmt1. In addition, plants have DNA methylation at CpNpG and CpNpN sites, maintained, in part, by the CHROMOMETHYLASE3 (CMT3) DNA methyltransferase. Here, we show that Arabidopsis thaliana embryos with loss-of-function mutations in MET1 and CMT3 develop improperly, display altered planes and numbers of cell division, and have reduced viability. Genes that specify embryo cell identity are misexpressed, and auxin hormone gradients are not properly formed in abnormal met1 embryos. Thus, DNA methylation is critical for the regulation of plant embryogenesis and for seed viability.     

Patterns of DNA methylation in development, division of labor and hybridization in an ant with genetic caste determination

Background

DNA methylation is a common regulator of gene expression, including acting as a regulator of developmental events and behavioral changes in adults. Using the unique system of genetic caste determination in Pogonomyrmex barbatus, we were able to document changes in DNA methylation during development, and also across both ancient and contemporary hybridization events.

Methodology/Principal Findings

Sodium bisulfite sequencing demonstrated in vivo methylation of symmetric CG dinucleotides in P. barbatus. We also found methylation of non-CpG sequences. This validated two bioinformatics methods for predicting gene methylation, the bias in observed to expected ratio of CpG dinucleotides and the density of CpG/TpG single nucleotide polymorphisms (SNP). Frequencies of genomic DNA methylation were determined for different developmental stages and castes using ms-AFLP assays. The genetic caste determination system (GCD) is probably the product of an ancestral hybridization event between P. barbatus and P. rugosus. Two lineages obligately co-occur within a GCD population, and queens are derived from intra-lineage matings whereas workers are produced from inter-lineage matings. Relative DNA methylation levels of queens and workers from GCD lineages (contemporary hybrids) were not significantly different until adulthood. Virgin queens had significantly higher relative levels of DNA methylation compared to workers. Worker DNA methylation did not vary among developmental stages within each lineage, but was significantly different between the currently hybridizing lineages. Finally, workers of the two genetic caste determination lineages had half as many methylated cytosines as workers from the putative parental species, which have environmental caste determination.

Conclusions/Significance

These results suggest that DNA methylation may be a conserved regulatory mechanism moderating division of labor in both bees and ants. Current and historic hybridization appear to have altered genomic methylation levels suggesting a possible link between changes in overall DNA methylation and the origin and regulation of genetic caste determination in P. barbatus.