Influence of the Prader-Willi syndrome imprinting center on the DNA methylation landscape in the mouse brain

Reduced representation bisulfite sequencing (RRBS) was used to analyze DNA methylation patterns across the mouse brain genome in mice carrying a deletion of the Prader-Willi syndrome imprinting center (PWS-IC) on either the maternally- or paternally-inherited chromosome. Within the ∼3.7 Mb imprinted Angelman/Prader-Willi syndrome (AS/PWS) domain, 254 CpG sites were interrogated for changes in methylation due to PWS-IC deletion. Paternally-inherited deletion of the PWS-IC increased methylation levels ∼2-fold at each CpG site (compared to wild-type controls) at differentially methylated regions (DMRs) associated with 5′ CpG island promoters of paternally-expressed genes; these methylation changes extended, to a variable degree, into the adjacent CpG island shores. Maternal PWS-IC deletion yielded little or no changes in methylation at these DMRs, and methylation of CpG sites outside of promoter DMRs also was unchanged upon maternal or paternal PWS-IC deletion. Using stringent ascertainment criteria, ∼750,000 additional CpG sites were also interrogated across the entire mouse genome. This analysis identified 26 loci outside of the imprinted AS/PWS domain showing altered DNA methylation levels of ≥25% upon PWS-IC deletion. Curiously, altered methylation at 9 of these loci was a consequence of maternal PWS-IC deletion (maternal PWS-IC deletion by itself is not known to be associated with a phenotype in either humans or mice), and 10 of these loci exhibited the same changes in methylation irrespective of the parental origin of the PWS-IC deletion. These results suggest that the PWS-IC may affect DNA methylation at these loci by directly interacting with them, or may affect methylation at these loci through indirect downstream effects due to PWS-IC deletion. They further suggest the PWS-IC may have a previously uncharacterized function outside of the imprinted AS/PWS domain.     

Genome‐wide DNA methylation analysis: no evidence for stable hemimethylation in the sheep muscle genome

The importance of maintaining DNA methylation patterns and faithful transmission of these patterns during cell division to ensure appropriate gene expression has been known for many decades now. It has largely been assumed that the symmetrical nature of CpG motifs, the most common site for DNA methylation in mammals, together with the presence of maintenance methylases able to methylate newly synthesised DNA, ensures that there is concordance of methylation on both strands. However, although this assumption is compelling in theory, little experimental evidence exists that either supports or refutes this assumption. Here, we have undertaken a genome‐wide single‐nucleotide resolution analysis to determine the frequency with which hemimethylated CpG sites exist in sheep muscle tissue. Analysis of multiple independent samples provides strong evidence that stably maintained hemimethylation is a very rare occurrence, at least in this tissue. Given the rarity of stably maintained hemimethylation, next‐generation sequencing data from both DNA strands may be carefully combined to increase the accuracy with which DNA methylation can be measured at single‐nucleotide resolution.     

Heritability of DNA methylation in threespine stickleback (Gasterosteus aculeatus)

Epigenetic mechanisms underlying phenotypic change are hypothesized to contribute to population persistence and adaptation in the face of environmental change. To date, few studies have explored the heritability of intergenerationally stable methylation levels in natural populations, and little is known about the relative contribution of cis- and trans-regulatory changes to methylation variation. Here, we explore the heritability of DNA methylation, and conduct methylation quantitative trait loci (meQTLs) analysis to investigate the genetic architecture underlying methylation variation between marine and freshwater ecotypes of threespine stickleback (Gasterosteus aculeatus). We quantitatively measured genome-wide DNA methylation in fin tissue using reduced representation bisulfite sequencing of F1 and F2 crosses, and their marine and freshwater source populations. We identified cytosines (CpG sites) that exhibited stable methylation levels across generations. We found that additive genetic variance explained an average of 24–35% of the methylation variance, with a number of CpG sites possibly autonomous from genetic control. We also detected both cis- and trans-meQTLs, with only trans-meQTLs overlapping with previously identified genomic regions of high differentiation between marine and freshwater ecotypes. Finally, we identified the genetic architecture underlying two key CpG sites that were differentially methylated between ecotypes. These findings demonstrate a potential role for DNA methylation in facilitating adaptation to divergent environments and improve our understanding of the heritable basis of population epigenomic variation.     

Quantitative DNA methylation analysis improves epigenotype-phenotype correlations in Beckwith-Wiedemann syndrome

Beckwith-Wiedemann syndrome (BWS) is a rare disorder characterized by overgrowth and predisposition to embryonal tumors. BWS is caused by various epigenetic and/or genetic alterations that dysregulate the imprinted genes on chromosome region 11p15.5. Molecular analysis is required to reinforce the clinical diagnosis of BWS and to identify BWS patients with cancer susceptibility. This is particularly crucial prenatally because most signs of BWS cannot be recognized in utero. We established a reliable molecular assay by pyrosequencing to quantitatively evaluate the methylation profiles of ICR1 and ICR2. We explored epigenotype-phenotype correlations in 19 patients that fulfilled the clinical diagnostic criteria for BWS, 22 patients with suspected BWS, and three fetuses with omphalocele. Abnormal methylation was observed in one prenatal case and 19 postnatal cases, including seven suspected BWS. Seven cases showed ICR1 hypermethylation, five cases showed ICR2 hypomethylation, and eight cases showed abnormal methylation of ICR1 and ICR2 indicating paternal uniparental disomy (UPD). More cases of ICR1 alterations and UPD were found than expected. This is likely due to the sensitivity of this approach, which can detect slight deviations in methylation from normal levels. There was a significant correlation (p < 0.001) between the percentage of ICR1 methylation and BWS features: severe hypermethylation (range: 75–86%) was associated with macroglossia, macrosomia, and visceromegaly, whereas mild hypermethylation (range: 55–59%) was associated with umbilical hernia and diastasis recti. Evaluation of ICR1 and ICR2 methylation by pyrosequencing in BWS can improve epigenotype-phenotype correlations, detection of methylation alterations in suspected cases, and identification of UPD.     

Erasure of methylation imprinting of Igf2r during mouse primordial germ-cell development

During germ cell differentiation in mice, the genome undergoes specific epigenetic modifications. These include demethylation of imprinted genes and subsequent establishment of parental allele-specific methylation. The mouse Igf2r gene is an imprinted gene that shows maternal-specific expression. Maternal-specific methylation of differentially methylated region 2 (DMR2) of this gene may be necessary for its maternal-specific expression. Before the allele-specific methylation is established, DMR2 is demethylated in both male and female primordial germ cells (PGCs) by 13.5 days post coitum (dpc), indicating that the demethylation of this region occurs earlier in PGC development. The timing of the demethylation has been, however, unknown. In this study, we attempted to determine the timing of methylation erasure of Igf2r DMR2 in developing PGCs, using transgenic mice expressing green fluorescent protein specifically in the germ line. We purified migrating PGCs from the transgenic mice and examined the methylation status of DMR2. The results show that some CpG sites within DMR2 start demethylation at 9.5 dpc in some migrating PGCs, before the cells colonize genital ridges, and the progression of demethylation is rapid after colonization of the genital ridges. To examine whether the gonadal environment is involved in demethylation, we analyzed the methylation of DMR2 after culturing migrating PGCs in the absence of a gonadal environment. These culture experiments support the idea that a gonadal environment is not required for demethylation of the region in at least a fraction of PGCs.     

Genomic imprinting and human hereditary disorders

Modern data are reviewed that concern hereditary disorders caused by abnormal expression of imprinted genes rather than mutations and structural aberrations. As an example, the molecular organization of the critical chromosomal region 15(q11.2–q13) and the possible pathogenetic mechanisms are described in detail for Prader-Willi and Angelman syndromes.     

De novo DNA methylation independent establishment of maternal imprint on X chromosome in mouse oocytes

In female mouse embryos, the paternal X chromosome (Xp) is preferentially inactivated during preimplantation development and trophoblast differentiation. This imprinted X-chromosome inactivation (XCI) is partly due to an activating imprint on the maternal X chromosome (Xm), which is set during oocyte growth. However, the nature of this imprint is unknown. DNA methylation is one candidate, and therefore we examined whether disruptions of the two de novo DNA methyltransferases in growing oocytes affect imprinted XCI. We found that accumulation of histone H3 lysine-27 trimethylation, a hallmark of XCI, occurs normally on the Xp, and not on the Xm, in female blastocysts developed from the mutant oocytes. Furthermore, the allelic expression patterns of X-linked genes including Xist and Tsix were unchanged in preimplantation embryos and also in the trophoblast. These results show that a maternal disruption of the DNA methyltransferases has no effect on imprinted XCI and argue that de novo DNA methylation is dispensable for Xm imprinting. This underscores the difference between imprinted XCI and autosomal imprinting.     

DNA methylation and imprinting in plants: machinery and mechanisms

Imprinting is an epigenetic phenomenon in which genes are expressed selectively from either the maternal or paternal alleles. In plants, imprinted gene expression is found in a tissue called the endosperm. Imprinting is often set by a unique epigenomic configuration in which the maternal chromosomes are less DNA methylated than their paternal counterparts. In this review, we synthesize studies that paint a detailed molecular portrait of the distinctive endosperm methylome. We will also discuss the molecular machinery that shapes and modifies this methylome, and the role of DNA methylation in imprinting.     

Sequence-specific methylation of the mouse H19 gene in embryonic cells deficient in the Dnmt-1 gene

We have used Dnmt^c/c ES cells that are homozygous for disruption of the DNA methyltransferase gene to address how de novo methylation is propagated and whether it is directed to specific sites in the early embryo. We examined the imprinted H19 gene and the specific-sequence region implicated as an “imprinting mark” to determine whether de novo methylation was occurring at a restricted set of sites. Since the “imprinting mark” was found to be methylated differentially at all stages of development, we reasoned that the sequence may still be a target for the de novo methylation activity found in the Dnmt^c/c cells, even though the loss of maintenance methylase activity renders the H19 promoter active. We used bisulfite genomic sequencing to determine the methylation state of the imprinted region of the H19 gene and found a low level of DNA methylation at specific single CpG sites in the upstream region of the imprinted H19 sequence in the Dnmt^c/c mutant ES cells. Moreover, these CpG sites appeared to be favoured targets for further de novo methylation of neighbouring CpG sites in rescued ES cells, which possess apparently normal maintenance activity. Our data provide further evidence for a separate methylating activity in ES cells and indicate that this activity displays sequence specificity. Dev. Genet. 22:111–121, 1998. © 1998 Wiley-Liss, Inc.     

An ecologist's guide for studying DNA methylation variation in wild vertebrates

The field of molecular biology is advancing fast with new powerful technologies, sequencing methods and analysis software being developed constantly. Commonly used tools originally developed for research on humans and model species are now regularly used in ecological and evolutionary research. There is also a growing interest in the causes and consequences of epigenetic variation in natural populations. Studying ecological epigenetics is currently challenging, especially for vertebrate systems, because of the required technical expertise, complications with analyses and interpretation, and limitations in acquiring sufficiently high sample sizes. Importantly, neglecting the limitations of the experimental setup, technology and analyses may affect the reliability and reproducibility, and the extent to which unbiased conclusions can be drawn from these studies. Here, we provide a practical guide for researchers aiming to study DNA methylation variation in wild vertebrates. We review the technical aspects of epigenetic research, concentrating on DNA methylation using bisulfite sequencing, discuss the limitations and possible pitfalls, and how to overcome them through rigid and reproducible data analysis. This review provides a solid foundation for the proper design of epigenetic studies, a clear roadmap on the best practices for correct data analysis and a realistic view on the limitations for studying ecological epigenetics in vertebrates. This review will help researchers studying the ecological and evolutionary implications of epigenetic variation in wild populations.     

Common methods for cytosine methylation analysis in DNA

The review considers the methods most commonly used to detect DNA methylation, their advantages, potential limitations, and selection for various purposes. A detailed protocol is described for bisulfite treatment, which is used as a preliminary step in the majority of DNA methylation assays.     

Strategies for DNA methylation analysis in developmental studies

Developmental processes in eukaryotes are highly dependent on DNA methylation. 5-methylcytosine (m(5) C) is the most prevalent and best understood DNA modification implicated in maintenance of genomic integrity and function across species. Although m(5) C occurs almost exclusively in symmetrical CpG context in vertebrates, additional asymmetrical distribution in CpHpG and CpHpH sites has been observed in plants and embryonic stem cells. To this end, accurate and reproducible methodology for full analysis of the DNA methylome is highly demanded. Fortunately, a variety of methods enable quantitative DNA methylation mapping at a single-base resolution and in a large scale. Here, we provide a critical overview of methods applied primarily to m(5) C detection with particular emphasis on technical improvements of the classical bisulfite-conversion protocol. We further describe strategies in combination with emerging technologies that allow acquisition of highly reliable data for developmental studies.     

Histone H1 affects gene imprinting and DNA methylation in Arabidopsis

Imprinting, i.e. parent-of-origin expression of alleles, plays an important role in regulating development in mammals and plants. DNA methylation catalyzed by DNA methyltransferases plays a pivotal role in regulating imprinting by silencing parental alleles. DEMETER (DME), a DNA glycosylase functioning in the base-excision DNA repair pathway, can excise 5-methylcytosine from DNA and regulate genomic imprinting in Arabidopsis. DME demethylates the maternal MEDEA (MEA) promoter in endosperm, resulting in expression of the maternal MEA allele. However, it is not known whether DME interacts with other proteins in regulating gene imprinting. Here we report the identification of histone H1.2 as a DME-interacting protein in a yeast two-hybrid screen, and confirmation of their interaction by the in vitro pull-down assay. Genetic analysis of the loss-of-function histone h1 mutant showed that the maternal histone H1 allele is required for DME regulation of MEA, FWA and FIS2 imprinting in Arabidopsis endosperm but the paternal allele is dispensable. Furthermore, we show that mutations in histone H1 result in an increase of DNA methylation in the maternal MEA and FWA promoter in endosperm. Our results suggest that histone H1 is involved in DME-mediated DNA methylation and gene regulation at imprinted loci.     

生殖细胞及早期胚胎基因组印记的表观调控

基因组印记是一种区别父母等位基因的表观遗传过程,可导致父源和母源基因特异性表达。印记是在配子发生过程中全基因组表观重编程时获得的,且在早期胚胎发育过程中得以维持。因此,在全基因组重编程过程中,对印记的识别和维持十分重要。本文概述了原始生殖细胞的印记清除、双亲原始生殖细胞的印记获得以及早期胚胎发育过程中印记维持的相关过程,并对在印记区域内保护印记基因免受全基因组DNA去甲基化的表观遗传因子的相关作用机制进行了讨论。     

Paradoxical methylation of the tyrosine hydroxylase gene in mouse preimplantation embryos

The mouse tyrosine hydroxylase (Th) gene is located in an evolutionarily conserved imprinted gene cluster on distal chromosome 7. It is associated with a CpG island that spans the promoter of the gene. Using a bisulfite sequencing method we show that the Th promoter is fully methylated in both male and female mouse germ cells and in human spermatozoa, suggesting that it belongs to the newly identified category of CpG islands, the similarly methylated regions (SMRs). Contrary to other tissue-specific gene sequences, the mouse Th promoter escapes the initial wave of genome demethylation during the first few cell cycles, but becomes demethylated between the morula and the blastocyst stages. This unusual methylation ontogeny may be a characteristic of the SMRs and/or related to the localization of the Th gene in an imprinted gene cluster.