Heritable Methylation Research Articles

Research progress of regulatory mechanism of DNA methylation in complex traits using monozygotic twins.

Coming from a single zygote, monozygotic (MZ) twins share nearly all of their genetic materials and are almost strikingly similar in appearance. Yet they are often discordant for important phenotypes including complex diseases. The study of discordant MZ twins allows us to understand the role of epigenetics in complex traits by controlling many potential confounders, such as genetic factors, maternal effects, age and sex. As we know, DNA methylation is the most stable epigenetic modification. In human, many causal genes, regulating disease status by DNA methylation modification in imprinting disorders, psychological disorders, autoimmune diseases and cancers, have been discovered, which provides a foundation for studying epigenetic regulation of diseases and application of epigenetic drugs. This article reviews the latest findings on DNA methylation status, estimates of DNA methylation heritability as well as studies of DNA methylation regulation in complex traits using MZ twins, in order to provide reference for research on epigenetic modification in complex traits.

Familial resemblances in blood leukocyte DNA methylation levels

ABSTRACTEpigenetic factors such as DNA methylation are DNA alterations affecting gene expression that can convey environmental information through generations. Only a few studies have demonstrated epigenetic inheritance in humans. Our objective is to quantify genetic and common environmental determinants of familial resemblances in DNA methylation levels, using a family based sample. DNA methylation was measured in 48 French Canadians from 16 families as part of the GENERATION Study. We used the Illumina HumanMethylation450 BeadChip array to measure DNA methylation levels in blood leukocytes on 485,577 CpG sites. Heritability was assessed using the variance components method implemented in the QTDT software, which partitions the variance into polygenic (G), common environmental (C), and non-shared environmental (E) effects. We computed maximal heritability, genetic heritability, and common environmental effect for all probes (12.7%, 8.2%, and 4.5%, respectively) and for statistically significant probes (81.8%, 26.9%, and 54.9%, respectively). Higher maximal heritability was observed in the Major Histocompatibility Complex region on chromosome 6. In conclusion, familial resemblances in DNA methylation levels are mainly attributable to genetic factors when considering the average across the genome, but common environmental effect plays an important role when considering statistically significant probes. Further epigenome-wide studies on larger samples combined with genome-wide genotyping studies are needed to better understand the underlying mechanisms of DNA methylation heritability.

Human DNMT1 transition state structure.

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys(1226) to cytosine (Cyt) C6, methyl transfer from S-adenosyl-l-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys(1226) attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.

Structure and extent of DNA methylation-based epigenetic variation in wild emmer wheat (T. turgidum ssp. dicoccoides) populations.

BackgroundThe genetic structure and differentiation of wild emmer wheat suggests that genetic diversity is eco-geographically structured. However, very little is known about the structure and extent of the heritable epigenetic variation and its influence on local adaptation in natural populations.ResultsThe structure and extent of the heritable methylation-based epigenetic variation were assessed within and among natural populations of Triticum turgidum ssp. dicoccoides. We used methylation sensitive amplified polymorphism (MSAP) and transposon methylation display (TMD) techniques, to assess the methylation status of random genomic CCGG sites and CCGG sites flanking transposable elements (TEs), respectively. Both techniques were applied to the DNA of 50 emmer accessions which were collected from five different geographically isolated regions. In order to ensure the assessment of heritable epigenetic variation, all accessions were grown under common garden conditions for two generations. In all accessions, the difference in methylation levels of CCGG sites, including CCGG sites that flanked TEs, were not statistically significant and relatively high, ranging between 46 and 76 %. The pattern of methylation was significantly different among accessions, such that clear and statistically significant population-specific methylation patterns were observed.ConclusionIn this study, we have observed population-unique heritable methylation patterns in emmer wheat accessions originating from five geographically isolated regions. Our data indicate that methylation-based epigenetic diversity might be eco-geographically structured and might be partly determined by climatic and edaphic factors.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-015-0544-z) contains supplementary material, which is available to authorized users.

Transient DNMT1 suppression reveals hidden heritable marks in the genome.

Genome-wide demethylation and remethylation of DNA during early embryogenesis is essential for development. Imprinted germline differentially methylated domains (gDMDs) established by sex-specific methylation in either male or female germ cells, must escape these dynamic changes and sustain precise inheritance of both methylated and unmethylated parental alleles. To identify other, gDMD-like sequences with the same epigenetic inheritance properties, we used a modified embryonic stem (ES) cell line that emulates the early embryonic demethylation and remethylation waves. Transient DNMT1 suppression revealed gDMD-like sequences requiring continuous DNMT1 activity to sustain a highly methylated state. Remethylation of these sequences was also compromised in vivo in a mouse model of transient DNMT1 loss in the preimplantation embryo. These novel regions, possessing heritable epigenetic features similar to imprinted-gDMDs are required for normal physiological and developmental processes and when disrupted are associated with disorders such as cancer and autism spectrum disorders. This study presents new perspectives on DNA methylation heritability during early embryo development that extend beyond conventional imprinted-gDMDs.

Genetic and environmental exposures constrain epigenetic drift over the human life course

Epigenetic mechanisms such as DNA methylation (DNAm) are essential for regulation of gene expression. DNAm is dynamic, influenced by both environmental and genetic factors. Epigenetic drift is the divergence of the epigenome as a function of age due to stochastic changes in methylation. Here we show that epigenetic drift may be constrained at many CpGs across the human genome by DNA sequence variation and by lifetime environmental exposures. We estimate repeatability of DNAm at 234,811 autosomal CpGs in whole blood using longitudinal data (2–3 repeated measurements) on 478 older people from two Scottish birth cohorts—the Lothian Birth Cohorts of 1921 and 1936. Median age was 79 yr and 70 yr, and the follow-up period was ∼10 yr and ∼6 yr, respectively. We compare this to methylation heritability estimated in the Brisbane Systems Genomics Study, a cross-sectional study of 117 families (offspring median age 13 yr; parent median age 46 yr). CpG repeatability in older people was highly correlated (0.68) with heritability estimated in younger people. Highly heritable sites had strong underlying cis-genetic effects. Thirty-seven and 1687 autosomal CpGs were associated with smoking and sex, respectively. Both sets were strongly enriched for high repeatability. Sex-associated CpGs were also strongly enriched for high heritability. Our results show that a large number of CpGs across the genome, as a result of environmental and/or genetic constraints, have stable DNAm variation over the human lifetime. Moreover, at a number of CpGs, most variation in the population is due to genetic factors, despite some sites being highly modifiable by the environment.

Characterization of genome-methylome interactions in 22 nuclear pedigrees.

Genetic polymorphisms can shape the global landscape of DNA methylation, by either changing substrates for DNA methyltransferases or altering the DNA binding affinity of cis-regulatory proteins. The interactions between CpG methylation and genetic polymorphisms have been previously investigated by methylation quantitative trait loci (mQTL) and allele-specific methylation (ASM) analysis. However, it remains unclear whether these approaches can effectively and comprehensively identify all genetic variants that contribute to the inter-individual variation of DNA methylation levels. Here we used three independent approaches to systematically investigate the influence of genetic polymorphisms on variability in DNA methylation by characterizing the methylation state of 96 whole blood samples in 52 parent-child trios from 22 nuclear pedigrees. We performed targeted bisulfite sequencing with padlock probes to quantify the absolute DNA methylation levels at a set of 411,800 CpG sites in the human genome. With mid-parent offspring analysis (MPO), we identified 10,593 CpG sites that exhibited heritable methylation patterns, among which 70.1% were SNPs directly present in methylated CpG dinucleotides. We determined the mQTL analysis identified 49.9% of heritable CpG sites for which regulation occurred in a distal cis-regulatory manner, and that ASM analysis was only able to identify 5%. Finally, we identified hundreds of clusters in the human genome for which the degree of variation of CpG methylation, as opposed to whether or not CpG sites were methylated, was associated with genetic polymorphisms, supporting a recent hypothesis on the genetic influence of phenotypic plasticity. These results show that cis-regulatory SNPs identified by mQTL do not comprise the full extent of heritable CpG methylation, and that ASM appears overall unreliable. Overall, the extent of genome-methylome interactions is well beyond what is detectible with the commonly used mQTL and ASM approaches, and is likely to include effects on plasticity.

Contribution of genetic variation to transgenerational inheritance of DNA methylation.

BackgroundDespite the important role DNA methylation plays in transcriptional regulation, the transgenerational inheritance of DNA methylation is not well understood. The genetic heritability of DNA methylation has been estimated using twin pairs, although concern has been expressed whether the underlying assumption of equal common environmental effects are applicable due to intrauterine differences between monozygotic and dizygotic twins. We estimate the heritability of DNA methylation on peripheral blood leukocytes using Illumina HumanMethylation450 array using a family based sample of 614 people from 117 families, allowing comparison both within and across generations.ResultsThe correlations from the various available relative pairs indicate that on average the similarity in DNA methylation between relatives is predominantly due to genetic effects with any common environmental or zygotic effects being limited. The average heritability of DNA methylation measured at probes with no known SNPs is estimated as 0.187. The ten most heritable methylation probes were investigated with a genome-wide association study, all showing highly statistically significant cis mQTLs. Further investigation of one of these cis mQTL, found in the MHC region of chromosome 6, showed the most significantly associated SNP was also associated with over 200 other DNA methylation probes in this region and the gene expression level of 9 genes.ConclusionsThe majority of transgenerational similarity in DNA methylation is attributable to genetic effects, and approximately 20% of individual differences in DNA methylation in the population are caused by DNA sequence variation that is not located within CpG sites.

Patterns of methylation heritability in a genome-wide analysis of four brain regions

DNA methylation has been implicated in a number of diseases and other phenotypes. It is, therefore, of interest to identify and understand the genetic determinants of methylation and epigenomic variation. We investigated the extent to which genetic variation in cis-DNA sequence explains variation in CpG dinucleotide methylation in publicly available data for four brain regions from unrelated individuals, finding that 3–4% of CpG loci assayed were heritable, with a mean estimated narrow-sense heritability of 30% over the heritable loci. Over all loci, the mean estimated heritability was 3%, as compared with a recent twin-based study reporting 18%. Heritable loci were enriched for open chromatin regions and binding sites of CTCF, an influential regulator of transcription and chromatin architecture. Additionally, heritable loci were proximal to genes enriched in several known pathways, suggesting a possible functional role for these loci. Our estimates of heritability are conservative, and we suspect that the number of identified heritable loci will increase as the methylome is assayed across a broader range of cell types and the density of the tested loci is increased. Finally, we show that the number of heritable loci depends on the window size parameter commonly used to identify candidate cis-acting single-nucleotide polymorphism variants.

The Influence of cis-Regulatory Elements on DNA Methylation Fidelity

It is now established that, as compared to normal cells, the cancer cell genome has an overall inverse distribution of DNA methylation (“methylome”), i.e., predominant hypomethylation and localized hypermethylation, within “CpG islands” (CGIs). Moreover, although cancer cells have reduced methylation “fidelity” and genomic instability, accurate maintenance of aberrant methylomes that underlie malignant phenotypes remains necessary. However, the mechanism(s) of cancer methylome maintenance remains largely unknown. Here, we assessed CGI methylation patterns propagated over 1, 3, and 5 divisions of A2780 ovarian cancer cells, concurrent with exposure to the DNA cross-linking chemotherapeutic cisplatin, and observed cell generation-successive increases in total hyper- and hypo-methylated CGIs. Empirical Bayesian modeling revealed five distinct modes of methylation propagation: (1) heritable (i.e., unchanged) high- methylation (1186 probe loci in CGI microarray); (2) heritable (i.e., unchanged) low-methylation (286 loci); (3) stochastic hypermethylation (i.e., progressively increased, 243 loci); (4) stochastic hypomethylation (i.e., progressively decreased, 247 loci); and (5) considerable “random” methylation (582 loci). These results support a “stochastic model” of DNA methylation equilibrium deriving from the efficiency of two distinct processes, methylation maintenance and de novo methylation. A role for cis-regulatory elements in methylation fidelity was also demonstrated by highly significant (p<2.2×10−5) enrichment of transcription factor binding sites in CGI probe loci showing heritably high (118 elements) and low (47 elements) methylation, and also in loci demonstrating stochastic hyper-(30 elements) and hypo-(31 elements) methylation. Notably, loci having “random” methylation heritability displayed nearly no enrichment. These results demonstrate an influence of cis-regulatory elements on the nonrandom propagation of both strictly heritable and stochastically heritable CGIs.

A candidate CpG SNP approach identifies a breast cancer associated ESR1‐SNP

Altered DNA methylation is often seen in malignant cells, potentially contributing to carcinogenesis by suppressing gene expression. We hypothesized that heritable methylation potential might be a risk factor for breast cancer and evaluated possible association with breast cancer for single nucleotide polymorphisms (SNPs) either involving CpG sequences in extended 5'-regulatory regions of candidate genes (ESR1, ESR2, PGR, and SHBG) or CpG and missense coding SNPs in genes involved in methylation (MBD1, MECP2, DNMT1, MGMT, MTHFR, MTR, MTRR, MTHFD1, MTHFD2, BHMT, DCTD, and SLC19A1). Genome-wide searches for genetic risk factors for breast cancers have in general not investigated these SNPs, because of low minor allele frequency or weak haplotype associations. Genotyping was performed using Mass spectrometry-Maldi-Tof in a screening panel of 538 cases and 1,067 controls. Potential association to breast cancer was identified for 15 SNPs and one of these SNPs (rs7766585 in ESR1) was found to associate strongly with breast cancer, OR 1.30 (95% CI 1.17-1.45; p-value 2.1 × 10(-6)), when tested in a verification panel consisting of 3,211 unique breast cancer cases and 4,223 unique controls from five European biobank cohorts. In conclusion, a candidate gene search strategy focusing on methylation-related SNPs did identify a SNP that associated with breast cancer at high significance.

Genetic Control of Individual Differences in Gene-Specific Methylation in Human Brain

We have observed extensive interindividual differences in DNA methylation of 8590 CpG sites of 6229 genes in 153 human adult cerebellum samples, enriched in CpG island "shores" and at further distances from CpG islands. To search for genetic factors that regulate this variation, we performed a genome-wide association study (GWAS) mapping of methylation quantitative trait loci (mQTLs) for the 8590 testable CpG sites. cis association refers to correlation of methylation with SNPs within 1 Mb of a CpG site. 736 CpG sites showed phenotype-wide significant cis association with 2878 SNPs (after permutation correction for all tested markers and methylation phenotypes). In trans analysis of methylation, which tests for distant regulation effects, associations of 12 CpG sites and 38 SNPs remained significant after phenotype-wide correction. To examine the functional effects of mQTLs, we analyzed 85 genes that were with genetically regulated methylation we observed and for which we had quality gene expression data. Ten genes showed SNP-methylation-expression three-way associations-the same SNP simultaneously showed significant association with both DNA methylation and gene expression, while DNA methylation was significantly correlated with gene expression. Thus, we demonstrated that DNA methylation is frequently a heritable continuous quantitatively variable trait in human brain. Unlike allele-specific methylation, genetic polymorphisms mark both cis- and trans-regulatory genetic sites at measurable distances from their CpG sites. Some of the genetically regulated DNA methylation is directly connected with genetically regulated gene expression variation.

Further evidence for heritability of an epimutation in one of 12 cases with MLH1 promoter methylation in blood cells clinically displaying HNPCC

Germline mutations in mismatch repair (MMR) genes, tumours with high microsatellite instability (MSI-H) and loss of MMR protein expression are the hallmarks of HNPCC (Lynch syndrome). While somatic MLH1 promoter hypermethylation is generally accepted in the tumorigenesis of sporadic tumours, abnormal MLH1 promoter methylation in normal body cells is controversially discussed as a mechanism predisposing patients to HNPCC. In all 94 patients suspected of HNPCC-syndrome with a mean age of onset of 45.5 years, MLH1-deficiency in their tumours but no germline mutation, underwent methylation-specific PCR-screening for MLH1 promoter methylation. In peripheral blood cells of 12 patients an MLH1 promoter methylation, in seven informative cases allele-specific, was found. Normal colonic tissue, buccal mucosa, and tumour tissue available from three patients also presented abnormal methylation in the MLH1 promoter. The heredity of aberrant methylation is questionable. Pro: MLH1 promoter methylation was found in a patient and his mother giving evidence for a familial predisposition for an epimutation in MLH1. Contra: a de novo set-up of methylation in one patient, a mosaic or incomplete methylation pattern in six patients, and no evidence for inheritance of MLH1 promoter methylation in the remaining families. Our findings provide strong evidence that MLH1 promoter methylation in normal body cells mimics HNPCC and constitutes a pathogenic pre-lesion in MLH1. The identification of hypermethylation as an epigenetic defect has important implications for surveillance recommendations, as these patients should be treated like Lynch syndrome patients, whereas the heritability of methylation is still under investigation.

Epigenetic Decisions in Mammalian Germ Cells

Specific sequences are designated for de novo DNA methylation at CpG dinucleotides in mammalian germ cells. The result is the long-term transcriptional silencing of the methylated sequences, most of which are retrotransposons and CpG-rich sequences associated with imprinted genes. There is profound sexual dimorphism in both the nature of the sequences that undergo de novo methylation in germ cells and in the mechanism by which de novo methylation is regulated. The restriction of future gene expression by the imposition of heritable methylation patterns in germ cell genomes is characteristic of mammals but is rare in other taxa.

Heritable rather than age-related environmental and stochastic factors dominate variation in DNA methylation of the human IGF2/H19 locus

Epigenetic variation may significantly contribute to the risk of common disease. Currently, little is known about the extent and causes of epigenetic variation. Here, we investigated the contribution of heritable influences and the combined effect of environmental and stochastic factors to variation in DNA methylation of the IGF2/H19 locus. Moreover, we tested whether this locus was subject to age-related degeneration of epigenetic patterns as was previously suggested for global methylation. We measured methylation of the H19 and IGF2 differentially methylated regions (DMRs) in 196 adolescent and 176 middle-aged twins using a recently developed mass spectrometry-based method. We observed substantial variation in DNA methylation across individuals, underscoring that DNA methylation is a quantitative trait. Analysis of data in monozygotic and dizygotic twins revealed that a significant part of this variation could be attributed to heritable factors. The heritability of methylation of individual CpG sites varied between 20 and 74% for the H19 DMR and was even higher, between 57 and 97%, for the IGF2 DMR. Remarkably, the combined influence of environmental and stochastic factors on DNA methylation was not greater in middle-age than in adolescence, suggesting a limited role for age-related degeneration of methylation patterns at this locus. Single nucleotide polymorphisms in the IGF2/H19 locus were significantly associated with DNA methylation of the IGF2 DMR (P = 0.004). A preliminary analysis suggested an association between H19 DMR methylation and body size (P < 0.05). Our study shows that variation in DNA methylation of the IGF2/H19 locus is mainly determined by heritable factors and single nucleotide polymorphisms (SNPs) in cis, rather than the cumulative effect of environmental and stochastic factors occurring with age.

Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes

The mechanism by which radiation-induced genomic instability is initiated, propagated and effected is currently under intense scrutiny. We have investigated the potential role of altered genomic methylation patterns in the cellular response to irradiation and have found evidence for widespread dysregulation of CpG methylation persisting up to 20 population doublings post-irradiation. Similar effects are seen with cells treated with medium from irradiated cells (the ‘bystander effect’) rather than subjected to direct irradiation. Using an arbitrarily primed methylation sensitive PCR screening method we have demonstrated that irradiation causes reproducible alterations in the methylation profile of a human keratinocyte cell line, HPV-G, and have further characterised one of these sequences as being a member of a retrotransposon element derived sequence family on chromosome 7; MLT1A. Multiple changes were also detected in the screen, which indicate that although the response of cells is predominantly hypermethylation, specific hypomethylation occurs as well. Sequence specific changes are also reported in the methylation of the pericentromeric SAT2 satellite sequence. This is the first demonstration that irradiation results in the induction of heritable methylation changes in mammalian cells, and provides a link between the various non-radiological instigators of genomic instability, the perpetuation of the unstable state and several of its manifestations.

DNA methylation and epigenetic inheritance in plants and filamentous fungi.

Plants and filamentous fungi share with mammals enzymes responsible for DNA methylation. In these organisms, DNA methylation is associated with gene silencing and transposon control. However, plants and fungi differ from mammals in the genomic distribution, sequence specificity, and heritability of methylation. We consider the role that transposons play in establishing methylation patterns and the epigenetic consequences of their perturbation.

Evolutionary developmental genetics of floral symmetry: The revealing power of Linnaeus' monstrous flower

Actinomorphic flowers have several planes of reflectional symmetry while zygomorphic flowers have just one. In a number of independent cases, actinomorphic flowers have arisen from zygomorphic ones during evolution. A famous example, studied by Linnaeus, is an actinomorphic variety of the common toadflax Linaria vulgaris. It has been shown now that this mutant carries a defect in LCYC, a homolog of the CYC gene, which controls zygomorphy in Antirrhinum majus.(1) Interestingly, the mutant phenotype is not due to changes in the LCYC nucleotide sequence but rather to an extensive, heritable methylation of the gene.(1) A second gene controlling zygomorphy in snapdragon, DICH, has recently also been shown to be a CYC homolog and both genes share significant sequence similarity with TB1, one of the key genes of maize domestication. The respective family of genes, probably encoding transcription factors, might thus become both a useful instrument and a target of future plant evolutionary developmental genetics. BioEssays 22:209–213, 2000. © 2000 John Wiley & Sons, Inc.

Evolutionary developmental genetics of floral symmetry: the revealing power of Linnaeus' monstrous flower.

Actinomorphic flowers have several planes of reflectional symmetry while zygomorphic flowers have just one. In a number of independent cases, actinomorphic flowers have arisen from zygomorphic ones during evolution. A famous example, studied by Linnaeus, is an actinomorphic variety of the common toadflax Linaria vulgaris. It has been shown now that this mutant carries a defect in LCYC, a homolog of the CYC gene, which controls zygomorphy in Antirrhinum majus.((1)) Interestingly, the mutant phenotype is not due to changes in the LCYC nucleotide sequence but rather to an extensive, heritable methylation of the gene.((1)) A second gene controlling zygomorphy in snapdragon, DICH, has recently also been shown to be a CYC homolog and both genes share significant sequence similarity with TB1, one of the key genes of maize domestication. The respective family of genes, probably encoding transcription factors, might thus become both a useful instrument and a target of future plant evolutionary developmental genetics.

Epigenetic silencing of plant transgenes as a consequence of diverse cellular defence responses.

Linked and unlinked copies of transgenes and related endogenous genes in plants can be epigenetically silenced by homology-based mechanisms that operate at either the transcriptional or post-transcriptional level. Transcriptional inactivation is associated with promoter homology and meiotically heritable methylation. Post-transcriptional silencing requires homology in protein-coding regions and is fully reversed during meiosis. Recently, the notion that both of these processes reflect the action of different host defence systems has been strengthened: (i) Obvious parallels have emerged between promoter homology-dependent silencing/methylation of transgenes and paramutation of endogenous genes that contain transposable elements in their promoters: (ii) remarkable similarities have been observed between post-transcriptional silencing involving transgenes and natural forms of virus resistance in nontransgenic plants. These results and others implicate two distinct cellular defence responses in transgene silencing. One is active in the nucleus and is manifested by transgene methylation, a reaction that might have originated as a means to oppose the spread of transposable elements. A second line of defence resides in the cytoplasm and operates through enhanced RNA turnover, a process that might help plants overcome viral infection.