Single-cell,locus-specific bisulfite sequencing (SLBS) for direct detection of epimutations in DNA methylation patterns

Stochastic epigenetic changes drive biological processes, such as development, aging and disease. Yet, epigenetic information is typically collected from millions of cells, thereby precluding a more precise understanding of cell-to-cell variability and the pathogenic history of epimutations. Here we present a novel procedure for directly detecting epimutations in DNA methylation patterns using single-cell, locus-specific bisulfite sequencing (SLBS). We show that within gene promoter regions of mouse hepatocytes the epimutation rate is two orders of magnitude higher than the mutation rate.     

The epigenetic footprint of poleward range‐expanding plants in apomictic dandelions

Epigenetic modifications, such as DNA methylation variation, can generate heritable phenotypic variation independent of the underlying genetic code. However, epigenetic variation in natural plant populations is poorly documented and little understood. Here, we test whether northward range expansion of obligate apomicts of the common dandelion (Taraxacum officinale) is associated with DNA methylation variation. We characterized and compared patterns of genetic and DNA methylation variation in greenhouse‐reared offspring of T. officinale that were collected along a latitudinal transect of northward range expansion in Europe. Genetic AFLP and epigenetic MS‐AFLP markers revealed high levels of local diversity and modest but significant heritable differentiation between sampling locations and between the southern, central and northern regions of the transect. Patterns of genetic and epigenetic variation were significantly correlated, reflecting the genetic control over epigenetic variation and/or the accumulation of lineage‐specific spontaneous epimutations, which may be selectively neutral. In addition, we identified a small component of DNA methylation differentiation along the transect that is independent of genetic variation. This epigenetic differentiation might reflect environment‐specific induction or, in case the DNA methylation variation affects relevant traits and fitness, selection of heritable DNA methylation variants. Such generated epigenetic variants might contribute to the adaptive capacity of individual asexual lineages under changing environments. Our results highlight the potential of heritable DNA methylation variation to contribute to population differentiation along ecological gradients. Further studies are needed using higher resolution methods to understand the functional significance of such natural occurring epigenetic differentiation.     

Genome-Wide Locations of Potential Epimutations Associated with Environmentally Induced Epigenetic Transgenerational Inheritance of Disease Using a Sequential Machine Learning Prediction Approach

Environmentally induced epigenetic transgenerational inheritance of disease and phenotypic variation involves germline transmitted epimutations. The primary epimutations identified involve altered differential DNA methylation regions (DMRs). Different environmental toxicants have been shown to promote exposure (i.e., toxicant) specific signatures of germline epimutations. Analysis of genomic features associated with these epimutations identified low-density CpG regions (<3 CpG / 100bp) termed CpG deserts and a number of unique DNA sequence motifs. The rat genome was annotated for these and additional relevant features. The objective of the current study was to use a machine learning computational approach to predict all potential epimutations in the genome. A number of previously identified sperm epimutations were used as training sets. A novel machine learning approach using a sequential combination of Active Learning and Imbalance Class Learner analysis was developed. The transgenerational sperm epimutation analysis identified approximately 50K individual sites with a 1 kb mean size and 3,233 regions that had a minimum of three adjacent sites with a mean size of 3.5 kb. A select number of the most relevant genomic features were identified with the low density CpG deserts being a critical genomic feature of the features selected. A similar independent analysis with transgenerational somatic cell epimutation training sets identified a smaller number of 1,503 regions of genome-wide predicted sites and differences in genomic feature contributions. The predicted genome-wide germline (sperm) epimutations were found to be distinct from the predicted somatic cell epimutations. Validation of the genome-wide germline predicted sites used two recently identified transgenerational sperm epimutation signature sets from the pesticides dichlorodiphenyltrichloroethane (DDT) and methoxychlor (MXC) exposure lineage F3 generation. Analysis of this positive validation data set showed a 100% prediction accuracy for all the DDT-MXC sperm epimutations. Observations further elucidate the genomic features associated with transgenerational germline epimutations and identify a genome-wide set of potential epimutations that can be used to facilitate identification of epigenetic diagnostics for ancestral environmental exposures and disease susceptibility.     

Environmental and genealogical effects on DNA methylation in a widespread apomictic dandelion lineage

DNA methylation in plant genomes occurs in different sequences and genomic contexts that have very different properties. DNA methylation that occurs in CG (mCG) sequence context shows transgenerational stability and high epimutation rate, and can thus provide genealogical information at short time scales. However, due to meta-stability and because mCG variants may arise due to other factors than epimutation, such as environmental stress exposure, it is not clear how well mCG captures genealogical information at micro-evolutionary time scales. Here, we analysed DNA methylation variation between accessions from a geographically widespread, apomictic common dandelion (Taraxacum officinale) lineage when grown experimentally under different light conditions. Using a reduced-representation bisulphite sequencing approach, we show that the light treatment induced differentially methylated cytosines (DMCs) in all sequence contexts, with a bias towards transposable elements. Accession differences were associated mainly with DMCs in CG context. Hierarchical clustering of samples based on total mCG profiles revealed a perfect clustering of samples by accession identity, irrespective of light conditions. Using microsatellite information as a benchmark of genetic divergence within the clonal lineage, we show that genetic divergence between accessions correlates strongly with overall mCG profiles. However, our results suggest that environmental effects that do occur in CG context may produce a heritable signal that partly dilutes the genealogical signal. Our study shows that methylation information in plants can be used to reconstruct micro-evolutionary genealogy, providing a useful tool in systems that lack genetic variation such as clonal and vegetatively propagated plants.     

Epigenetic variability in plants: Heritability,adaptability, evolutionary significance

DNA methylation is the most stable epigenetic modification with a well studied maintenance mechanism in the mitotically dividing cell generations. The plant DNA is methylated at sites of three types, CG, CHG and CHH. The methylation mechanisms of these sites are different and involve functional activity of various DNA methyltransferases and their accessory factors, that largely define the genome locus specificity of methylation. The genome methylation pattern, DNA methylome, in plants is inheritable not only in the dividing cell generations but also to a considerable extent in generations of the whole plants. A great number of spontaneous epimutations, both natural and experimental ones, are known, that have discernible phenotypic manifestations and are stably inheritable in the plant generations as Mendelian traits. A fundamental distinction of such epimutations from classical mutations is their reversibility. The higher plants epigenome is much more flexible compared with their genome. The single-nucleotide epimutation frequency is hundredfolds higher than the mutation frequency. This variability is probably a main source of the plant phenotypic plasticity, that enables them to adapt to changing environment on the time scales too short for adaptive mutations to occur. A dramatic increase in the plant population epigenetic variability on a practically unchanged genetic context is observed when the essential environmental factors are rapidly changing. Being flexible enough for such adaptive changes, on the other hand, epigenome is stable enough for these adaptive variations to be inheritable between the plant generations. Obviously, the epigenetic variations, that enable plants to adapt to the fast changing environmental factors, serve as material for natural selection and other evolutionary processes on the respective time scales. A still another aspect of evolutionary significance is a capability of epigenetic mechanisms to induce transient bursts of genetic variability by transposon mobilization.     

Extensive Natural Epigenetic Variation at a De Novo Originated Gene

Epigenetic variation, such as heritable changes of DNA methylation, can affect gene expression and thus phenotypes, but examples of natural epimutations are few and little is known about their stability and frequency in nature. Here, we report that the gene Qua-Quine Starch (QQS) of Arabidopsis thaliana, which is involved in starch metabolism and that originated de novo recently, is subject to frequent epigenetic variation in nature. Specifically, we show that expression of this gene varies considerably among natural accessions as well as within populations directly sampled from the wild, and we demonstrate that this variation correlates negatively with the DNA methylation level of repeated sequences located within the 5′end of the gene. Furthermore, we provide extensive evidence that DNA methylation and expression variants can be inherited for several generations and are not linked to DNA sequence changes. Taken together, these observations provide a first indication that de novo originated genes might be particularly prone to epigenetic variation in their initial stages of formation.     

Increased transgenerational epigenetic variation,but not predictable epigenetic variants,after environmental exposure in two apomictic dandelion lineages

DNA methylation is one of the mechanisms underlying epigenetic modifications. DNA methylations can be environmentally induced and such induced modifications can at times be transmitted to successive generations. However, it remains speculative how common such environmentally induced transgenerational DNA methylation changes are and if they persist for more than one offspring generation. We exposed multiple accessions of two different apomictic dandelion lineages of the Taraxacum officinale group (Taraxacum alatum and T. hemicyclum) to drought and salicylic acid (SA) treatment. Using methylation‐sensitive amplified fragment length polymorphism markers (MS‐AFLPs) we screened anonymous methylation changes at CCGG restriction sites throughout the genome after stress treatments and assessed the heritability of induced changes for two subsequent unexposed offspring generations. Irrespective of the initial stress treatment, a clear buildup of heritable DNA methylation variation was observed across three generations, indicating a considerable background rate of heritable epimutations. Less evidence was detected for environmental effects. Drought stress showed some evidence for accession‐specific methylation changes, but only in the exposed generation and not in their offspring. By contrast, SA treatment caused an increased rate of methylation change in offspring of treated plants. These changes were seemingly undirected resulting in increased transgenerational epigenetic variation between offspring individuals, but not in predictable epigenetic variants. While the functional consequences of these MS‐AFLP‐detected DNA methylation changes remain to be demonstrated, our study shows that (1) stress‐induced transgenerational DNA methylation modification in dandelions is genotype and context‐specific; and (2) inherited environmental DNA methylation effects are mostly undirected and not targeted to specific loci.     

Mutations and epimutations in mammalian cells

Early studies on heritable variation in cultured mammalian cells suggested that both mutation and epigenetic events might be involved. The importance of mutations has subsequently been fully documented, but only recently has an alternative form of inheritance been uncovered. This is based on the post-synthetic methylation of cytosine in regulatory regions of genes. The pattern of methylation is heritable, and in almost all cases studied, methylation of a region is associated with lack of gene expression. Such silent genes can be reactivated by the powerful demethylating agent 5-azacytidine (5-aza-CR). Changes in heritable DNA methylation which alter phenotype are referred to as epimutations. It now seems very likely that the well known ‘functional hemizygosity’ in CHO cells and other near diploid cell lines is due to the existence of one active and one silent gene at many autosomal loci. It is clear that permanent cell lines inactive genes by de novo methylation, whereas normal diploid cells do not have this activity. This has important implications for our understanding of cellular transformation, tumor progression, and the increase in chromosome number frequently associated with these cellular changes. It is likely that both mutations and epimutations are important in the emergence of fully transformed tumorigenic cells. Agents which increase or reduce DNA methylation in cells can be regarded as epimutagens, although in many cases the mechanisms of inducing hypo- or hyper-methylation are not understood. Two exceptions are 5-aza-CR which inhibits the normal DNA maintenance methylase activity, and 5-methyldeoxycytidine triphosphate which is incorporated into cellular DNA following electroporation and has been shown to silence genes.     

Role of CpG deserts in the epigenetic transgenerational inheritance of differential DNA methylation regions

Background

Previously a variety of environmental toxicants were found to promote the epigenetic transgenerational inheritance of disease through differential DNA methylation regions (DMRs), termed epimutations, present in sperm. The transgenerational epimutations in sperm and somatic cells identified in a number of previous studies were further investigated.

Results

The epimutations from six different environmental exposures were found to be predominantly exposure specific with negligible overlap. The current report describes a major genomic feature of all the unique epimutations identified (535) as a very low (<10 CpG/100 bp) CpG density in sperm and somatic cells associated with transgenerational disease. The genomic locations of these epimutations were found to contain DMRs with small clusters of CpG within a general region of very low density CpG. The potential role of these epimutations on gene expression is suggested to be important.

Conclusions

Observations suggest a potential regulatory role for lower density CpG regions termed “CpG deserts”. The potential evolutionary origins of these regions is also discussed.     

Inheritance Patterns and Stability of DNA Methylation Variation in Maize Near-Isogenic Lines

DNA methylation is a chromatin modification that contributes to epigenetic regulation of gene expression. The inheritance patterns and trans-generational stability of 962 differentially methylated regions (DMRs) were assessed in a panel of 71 near-isogenic lines (NILs) derived from maize (Zea mays) inbred lines B73 and Mo17. The majority of DMRs exhibit inheritance patterns that would be expected for local (cis) inheritance of DNA methylation variation such that DNA methylation level was coupled to local genotype. There are few examples of DNA methylation that exhibit trans-acting control or paramutation-like patterns. The cis-inherited DMRs provide an opportunity to study the stability of inheritance for DNA methylation variation. There was very little evidence for alterations of DNA methylation levels at these DMRs during the generations of the NIL population development. DNA methylation level was associated with local genotypes in nearly all of the >30,000 potential cases of inheritance. The majority of the DMRs were not associated with small RNAs. Together, our results suggest that a significant portion of DNA methylation variation in maize exhibits locally (cis) inherited patterns, is highly stable, and does not require active programming by small RNAs for maintenance.DNA methylation may contribute to heritable epigenetic information in many eukaryotic genomes. In this study, we have documented the inheritance patterns and trans-generational stability for nearly 1000 DNA methylation variants in a segregating maize population. At most loci studied, the DNA methylation differences are locally inherited and are not influenced by the other allele or other genomic regions. The inheritance of DNA methylation levels across generations is quite robust with almost no examples of unstable inheritance, suggesting that DNA methylation differences can be quite stably inherited, even in segregating populations.     

Epigenetic status of imprinted genes in placenta during recurrent pregnancy loss

An analysis of differential methylation of 47 imprinted genes in placenta tissues of spontaneous abortions at the first trimester of pregnancy from women with recurrent pregnancy loss or with one sporadic abortion was performed using the DNA-microarray approach. We showed that epimutations of the imprinted genes were registered significantly more often in abortions from women with recurrent miscarriage in contrast to the embryos from women with sporadic pregnancy loss with frequency of 6.2 and 3.7% per locus, respectively (p < 0.01). The predominant type of epimutation appeared to be a postzygotic hypomethylation of the imprinted genes on chromosomes of maternal origin, which was observed in the examined samples in 5.1 and 2.89% of cases, respectively. Replicative study of the methylation status of seven imprinted genes (DLK1, PEG10, PLAGL1, KCNQ1OT1, PEG3, GRB10, and PEG1/MEST) in the enlarged embryo samples supported the results of microarray analysis in respect to both epimutation frequency and predominance of somatic hypomethylation of maternal alleles. It was also demonstrated that pregnancy loss was associated with multilocus methylation defects of imprinted genes, the frequency of which was also significantly increased in the placental tissues of spontaneous abortions in women with recurrent miscarriage.     

DNA methylation, epimutations and cancer predisposition

Hereditary cancer syndromes caused by germline mutations give rise to distinct spectra of cancers with characteristic clinico-pathological features. Many of these hereditary cancer genes are silenced by methylation in a similar spectrum of sporadic cancers. It is likely that the initiating event in some of those cases of sporadic cancer is the somatic epigenetic inactivation (epimutation) of the same hereditary cancer gene. Recently, it has been shown that epimutations of certain hereditary cancer genes can be constitutional i.e. present throughout the soma. These epimutations may be inherited or arise very early in the germline. The heritability of these epimutations is very low as in most cases they are erased by passage through the germline. In other cases, predisposition to epimutations rather than the epimutations themselves can be inherited. These cases are characterised by Mendelian inheritance and are likely to be associated with sequence variants. Other sequence variants and environmental influences may also affect methylation propensity at a global level.