DAMEfinder: a method to detect differential allele-specific methylation.
ABSTRACT: BACKGROUND:DNA methylation is a highly studied epigenetic signature that is associated with regulation of gene expression, whereby genes with high levels of promoter methylation are generally repressed. Genomic imprinting occurs when one of the parental alleles is methylated, i.e., when there is inherited allele-specific methylation (ASM). A special case of imprinting occurs during X chromosome inactivation in females, where one of the two X chromosomes is silenced, to achieve dosage compensation between the sexes. Another more widespread form of ASM is sequence dependent (SD-ASM), where ASM is linked to a nearby heterozygous single nucleotide polymorphism (SNP). RESULTS:We developed a method to screen for genomic regions that exhibit loss or gain of ASM in samples from two conditions (treatments, diseases, etc.). The method relies on the availability of bisulfite sequencing data from multiple samples of the two conditions. We leverage other established computational methods to screen for these regions within a new R package called DAMEfinder. It calculates an ASM score for all CpG sites or pairs in the genome of each sample, and then quantifies the change in ASM between conditions. It then clusters nearby CpG sites with consistent change into regions. In the absence of SNP information, our method relies only on reads to quantify ASM. This novel ASM score compares favorably to current methods that also screen for ASM. Not only does it easily discern between imprinted and non-imprinted regions, but also females from males based on X chromosome inactivation. We also applied DAMEfinder to a colorectal cancer dataset and observed that colorectal cancer subtypes are distinguishable according to their ASM signature. We also re-discover known cases of loss of imprinting. CONCLUSION:We have designed DAMEfinder to detect regions of differential ASM (DAMEs), which is a more refined definition of differential methylation, and can therefore help in breaking down the complexity of DNA methylation and its influence in development and disease.
Project description:It is now widely accepted that allele-specific DNA methylation (ASM) commonly occurs at non-imprinted loci. Most of the non-imprinted ASM regions observed both within and outside of the CpG island show a strong correlation with DNA polymorphisms. However, what polymorphic cis-acting elements mediate non-imprinted ASM of the CpG island remains unclear. In this study, we investigated the impact of polymorphic GT microsatellites within the gene promoter on non-imprinted ASM of the local CpG island in goldfish. We generated various goldfish heterozygotes, in which the length of GT microsatellites or some non-repetitive sequences in the promoter of no tail alleles was different. By examining the methylation status of the downstream CpG island in these heterozygotes, we found that polymorphisms of a long GT microsatellite can lead to the ASM of the downstream CpG island during oogenesis and embryogenesis, polymorphisms of short GT microsatellites and non-repetitive sequences in the promoter exhibited no significant effect on the methylation of the CpG island. We also observed that the ASM of the CpG island was associated with allele-specific expression in heterozygous embryos. These results suggest that a long polymorphic GT microsatellite within a gene promoter mediates non-imprinted ASM of the local CpG island in a goldfish inter-strain hybrid.
Project description:Genomic imprinting arises from allele-specific epigenetic modifications that are established during gametogenesis and that are maintained throughout somatic development. These parental-specific modifications include DNA methylation and post-translational modifications to histones, which create allele-specific active and repressive domains at imprinted regions. Through the use of a high-density genomic tiling array, we generated DNA and histone methylation profiles at 11 imprinted gene clusters in the mouse from DNA and from chromatin immunoprecipitated from sperm, heart, and cerebellum. Our analysis revealed that despite high levels of differential DNA methylation at non-CpG islands within these regions, imprinting control regions (ICRs) and secondary differentially methylated regions (DMRs) were identified by an overlapping pattern of H3K4 trimethylation (active chromatin) and H3K9 trimethylation (repressive chromatin) modifications in somatic tissue, and a sperm differentially methylated region (sDMR; sperm not equal somatic tissue). Using these features as a common signature of DMRs, we identified 11 unique regions that mapped to known imprinted genes, to uncharacterized genes, and to intergenic regions flanking known imprinted genes. A common feature among these regions was the presence of a CpG island and an array of tandem repeats. Collectively, this study provides a comprehensive analysis of DNA methylation and histone H3K4me3 and H3K9me3 modifications at imprinted gene clusters, and identifies common epigenetic and genetic features of regions regulating genomic imprinting.
Project description:DNA methylation mediates imprinted gene expression by passing an epigenomic state across generations and differentially marking specific regulatory regions on maternal and paternal alleles. Imprinting has been tied to the evolution of the placenta in mammals and defects of imprinting have been associated with human diseases. Although recent advances in genome sequencing have revolutionized the study of DNA methylation, existing methylome data remain largely untapped in the study of imprinting. We present a statistical model to describe allele-specific methylation (ASM) in data from high-throughput short-read bisulfite sequencing. Simulation results indicate technical specifications of existing methylome data, such as read length and coverage, are sufficient for full-genome ASM profiling based on our model. We used our model to analyze methylomes for a diverse set of human cell types, including cultured and uncultured differentiated cells, embryonic stem cells and induced pluripotent stem cells. Regions of ASM identified most consistently across methylomes are tightly connected with known imprinted genes and precisely delineate the boundaries of several known imprinting control regions. Predicted regions of ASM common to multiple cell types frequently mark noncoding RNA promoters and represent promising starting points for targeted validation. More generally, our model provides the analytical complement to cutting-edge experimental technologies for surveying ASM in specific cell types and across species.
Project description:Differential methylation of the two parental genomes in placental mammals is essential for genomic imprinting and embryogenesis. To systematically study this epigenetic process, we have generated a base-resolution, allele-specific DNA methylation (ASM) map in the mouse genome. We find parent-of-origin dependent (imprinted) ASM at 1,952 CG dinucleotides. These imprinted CGs form 55 discrete clusters including virtually all known germline differentially methylated regions (DMRs) and 23 previously unknown DMRs, with some occurring at microRNA genes. We also identify sequence-dependent ASM at 131,765 CGs. Interestingly, methylation at these sites exhibits a strong dependence on the immediate adjacent bases, allowing us to define a conserved sequence preference for the mammalian DNA methylation machinery. Finally, we report a surprising presence of non-CG methylation in the adult mouse brain, with some showing evidence of imprinting. Our results provide a resource for understanding the mechanisms of imprinting and allele-specific gene expression in mammalian cells.
Project description:In diploid mammalian genomes, parental alleles can exhibit different methylation patterns (allele-specific DNA methylation, ASM), which have been documented in a small number of cases except for the imprinted regions and X chromosomes in females. We carried out a chromosome-wide survey of ASM across 16 human pluripotent and adult cell lines using Illumina bisulfite sequencing. We applied the principle of linkage disequilibrium (LD) analysis to characterize the correlation of methylation between adjacent CpG sites on single DNA molecules, and also investigated the correlation between CpG methylation and single nucleotide polymorphisms (SNPs). We observed ASM on 23% approximately 37% heterozygous SNPs in any given cell line. ASM is often cell-type-specific. Furthermore, we found that a significant fraction (38%-88%) of ASM regions is dependent on the presence of heterozygous SNPs in CpG dinucleotides that disrupt their methylation potential. This study identified distinct types of ASM across many cell types and suggests a potential role for CpG-SNP in connecting genetic variation with the epigenome.
Project description:Differential DNA methylation plays a critical role in the regulation of imprinted genes. The differentially methylated state of the imprinting control region is inherited via the gametes at fertilization, and is stably maintained in somatic cells throughout development, influencing the expression of genes across the imprinting cluster. In contrast, DNA methylation patterns are more labile at secondary differentially methylated regions which are established at imprinted loci during post-implantation development. To investigate the nature of these more variably methylated secondary differentially methylated regions, we adopted a hairpin linker bisulfite mutagenesis approach to examine CpG dyad methylation at differentially methylated regions associated with the murine Dlk1/Gtl2 imprinting cluster on both complementary strands.We observed homomethylation at greater than 90% of the methylated CpG dyads at the IG-DMR, which serves as the imprinting control element. In contrast, homomethylation was only observed at 67-78% of the methylated CpG dyads at the secondary differentially methylated regions; the remaining 22-33% of methylated CpG dyads exhibited hemimethylation.We propose that this high degree of hemimethylation could explain the variability in DNA methylation patterns at secondary differentially methylated regions associated with imprinted loci. We further suggest that the presence of 5-hydroxymethylation at secondary differentially methylated regions may result in hemimethylation and methylation variability as a result of passive and/or active demethylation mechanisms.
Project description:To test whether regions undergoing genomic imprinting have unique genomic characteristics, imprinted and nonimprinted human loci were compared for nucleotide and retroelement composition. Maternally and paternally expressed subgroups of imprinted genes were found to differ in terms of guanine and cytosine, CpG, and retroelement content, indicating a segregation into distinct genomic compartments. Imprinted regions have been normally permissive to L1 long interspersed transposable element retroposition during mammalian evolution but universally and significantly lack short interspersed transposable elements (SINEs). The primate-specific Alu SINEs, as well as the more ancient mammalian-wide interspersed repeat SINEs, are found at significantly low densities in imprinted regions. The latter paleogenomic signature indicates that the sequence characteristics of currently imprinted regions existed before the mammalian radiation. Transitions from imprinted to nonimprinted genomic regions in cis are characterized by a sharp inflection in SINE content, demonstrating that this genomic characteristic can help predict the presence and extent of regions undergoing imprinting. During primate evolution, SINE accumulation in imprinted regions occurred at a decreased rate compared with control loci. The constraint on SINE accumulation in imprinted regions may be mediated by an active selection process. This selection could be because of SINEs attracting and spreading methylation, as has been found at other loci. Methylation-induced silencing could lead to deleterious consequences at imprinted loci, where inactivation of one allele is already established, and expression is often essential for embryonic growth and survival.
Project description:<h4>Background</h4>Allele-specific methylation (ASM) occurs when DNA methylation patterns exhibit asymmetry among alleles. ASM occurs at imprinted loci, but its presence elsewhere across the human genome is indicative of wider importance in terms of gene regulation and disease risk. Here, we studied ASM by focusing on blood-based DNA collected from 24 subjects comprising a 3-generation pedigree from the Norfolk Island genetic isolate. We applied a genome-wide bisulphite sequencing approach with a genotype-independent ASM calling method to map ASM across the genome. Regions of ASM were then tested for enrichment at gene regulatory regions using Genomic Association Test (GAT) tool.<h4>Results</h4>In total, we identified 1.12 M CpGs of which 147,170 (13%) exhibited ASM (P???0.05). When including contiguous ASM signal spanning???2 CpGs, this condensed to 12,761 ASM regions (AMRs). These AMRs tagged 79% of known imprinting regions and most (98.1%) co-localised with known single nucleotide variants. Notably, miRNA and lncRNA showed a 3.3- and 1.8-fold enrichment of AMRs, respectively (P?<?0.005). Also, the 5' UTR and start codons each showed a 3.5-fold enrichment of AMRs (P?<?0.005). There was also enrichment of AMRs observed at subtelomeric regions of many chromosomes. Five out of 11 large AMRs localised to the protocadherin cluster on chromosome 5.<h4>Conclusions</h4>This study shows ASM extends far beyond genomic imprinting in humans and that gene regulatory regions are hotspots for ASM. Future studies of ASM in pedigrees should help to clarify transgenerational inheritance patterns in relation to genotype and disease phenotypes.
Project description:While DNA methylation is usually thought to be symmetrical across both alleles, there are some notable exceptions. Genomic imprinting and X chromosome inactivation are two well-studied sources of allele-specific methylation (ASM), but recent research has indicated a more complex pattern in which genotypic variation can be associated with allelically-skewed DNA methylation in cis. Given the known heterogeneity of DNA methylation across tissues and cell types we explored inter- and intra-individual variation in ASM across several regions of the human brain and whole blood from multiple individuals. Consistent with previous studies, we find widespread ASM with > 4% of the ?220,000 loci interrogated showing evidence of allelically-skewed DNA methylation. We identify ASM flanking known imprinted regions, and show that ASM sites are enriched in DNase I hypersensitivity sites and often located in an extended genomic context of intermediate DNA methylation. We also detect examples of genotype-driven ASM, some of which are tissue-specific. These findings contribute to our understanding of the nature of differential DNA methylation across tissues and have important implications for genetic studies of complex disease. As a resource to the community, ASM patterns across each of the tissues studied are available in a searchable online database: http://epigenetics.essex.ac.uk/ASMBrainBlood.
Project description:BACKGROUND:In mammals, the regulation of imprinted genes is controlled by differential methylation at imprinting control regions which acquire parent of origin-specific methylation patterns during gametogenesis and retain differences in allelic methylation status throughout fertilization and subsequent somatic cell divisions. In addition, many imprinted genes acquire differential methylation during post-implantation development; these secondary differentially methylated regions appear necessary to maintain the imprinted expression state of individual genes. Despite the requirement for both types of differentially methylated sequence elements to achieve proper expression across imprinting clusters, methylation patterns are more labile at secondary differentially methylated regions. To understand the nature of this variability, we analyzed CpG dyad methylation patterns at both paternally and maternally methylated imprinted loci within multiple imprinting clusters. RESULTS:We determined that both paternally and maternally methylated secondary differentially methylated regions associated with imprinted genes display high levels of hemimethylation, 29-49%, in comparison to imprinting control regions which exhibited 8-12% hemimethylation. To explore how hemimethylation could arise, we assessed the differentially methylated regions for the presence of 5-hydroxymethylcytosine which could cause methylation to be lost via either passive and/or active demethylation mechanisms. We found enrichment of 5-hydroxymethylcytosine at paternally methylated secondary differentially methylated regions, but not at the maternally methylated sites we analyzed in this study. CONCLUSIONS:We found high levels of hemimethylation to be a generalizable characteristic of secondary differentially methylated regions associated with imprinted genes. We propose that 5-hydroxymethylcytosine enrichment may be responsible for the variability in methylation status at paternally methylated secondary differentially methylated regions associated with imprinted genes. We further suggest that the high incidence of hemimethylation at secondary differentially methylated regions must be counteracted by continuous methylation acquisition at these loci.