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.

Genome-wide quantitative analysis of DNA methylation from bisulfite sequencing data.

UNLABELLED: Here we present the open-source R/Bioconductor software package BEAT (BS-Seq Epimutation Analysis Toolkit). It implements all bioinformatics steps required for the quantitative high-resolution analysis of DNA methylation patterns from bisulfite sequencing data, including the detection of regional epimutation events, i.e. loss or gain of DNA methylation at CG positions relative to a reference. Using a binomial mixture model, the BEAT package aggregates methylation counts per genomic position, thereby compensating for low coverage, incomplete conversion and sequencing errors. AVAILABILITY AND IMPLEMENTATION: BEAT is freely available as part of Bioconductor at www.bioconductor.org/packages/devel/bioc/html/BEAT.html. The package is distributed under the GNU Lesser General Public License 3.0.

Epigenetic factors in aging and longevity.

Epigenetics refers to phenotypic changes caused by mechanisms that are unrelated to changes in the underlying DNA sequence, most notably chromatin remodeling driven by histone modifications, and DNA methylation. Such variation is transmitted by cell division, but generally not passed on through the germ line. An increasing body of evidence supports a role for epigenetic changes in the etiology of aging and its associated disease sequelae. Here, we review the role of epigenetics in aging and longevity with a focus on DNA methylation. Increased understanding of those aging-related processes that are driven by epigenetic mechanisms will allow for the development of novel epigenetic-based diagnostic, preventive, and therapeutic strategies for age-related diseases.

Global, integrated analysis of methylomes and transcriptomes from laser capture microdissected bronchial and alveolar cells in human lung.

Gene regulatory analysis of highly diverse human tissues in vivo is essentially constrained by the challenge of performing genome-wide, integrated epigenetic and transcriptomic analysis in small selected groups of specific cell types. Here we performed genome-wide bisulfite sequencing and RNA-seq from the same small groups of bronchial and alveolar cells isolated by laser capture microdissection from flash-frozen lung tissue of 12 donors and their peripheral blood T cells. Methylation and transcriptome patterns differed between alveolar and bronchial cells, while each of these epithelia showed more differences from mesodermally-derived T cells. Differentially methylated regions (DMRs) between alveolar and bronchial cells tended to locate at regulatory regions affecting promoters of 4,350 genes. A large number of pathways enriched for these DMRs including GTPase signal transduction, cell death, and skeletal muscle. Similar patterns of transcriptome differences were observed: 4,108 differentially expressed genes (DEGs) enriched in GTPase signal transduction, inflammation, cilium assembly, and others. Prioritizing using DMR-DEG regulatory network, we highlighted genes, e.g., ETS1, PPARG, and RXRG, at prominent alveolar vs. bronchial cell discriminant nodes. Our results show that multi-omic analysis of small, highly specific cells is feasible and yields unique physiologic loci distinguishing human lung cell types in situ.

Development of a Method to Implement Whole-Genome Bisulfite Sequencing of cfDNA from Cancer Patients and a Mouse Tumor Model.

The goal of this study was to develop a method for whole genome cell-free DNA (cfDNA) methylation analysis in humans and mice with the ultimate goal to facilitate the identification of tumor derived DNA methylation changes in the blood. Plasma or serum from patients with pancreatic neuroendocrine tumors or lung cancer, and plasma from a murine model of pancreatic adenocarcinoma was used to develop a protocol for cfDNA isolation, library preparation and whole-genome bisulfite sequencing of ultra low quantities of cfDNA, including tumor-specific DNA. The protocol developed produced high quality libraries consistently generating a conversion rate >98% that will be applicable for the analysis of human and mouse plasma or serum to detect tumor-derived changes in DNA methylation.

Genome-wide, Single-Cell DNA Methylomics Reveals Increased Non-CpG Methylation during Human Oocyte Maturation.

The establishment of DNA methylation patterns in oocytes is a highly dynamic process marking gene-regulatory events during fertilization, embryonic development, and adulthood. However, after epigenetic reprogramming in primordial germ cells, how and when DNA methylation is re-established in developing human oocytes remains to be characterized. Here, using single-cell whole-genome bisulfite sequencing, we describe DNA methylation patterns in three different maturation stages of human oocytes. We found that while broad-scale patterns of CpG methylation have been largely established by the immature germinal vesicle stage, localized changes continue into later development. Non-CpG methylation, on the other hand, undergoes a large-scale, generalized remodeling through the final stage of maturation, with the net overall result being the accumulation of methylation as oocytes mature. The role of the genome-wide, non-CpG methylation remodeling in the final stage of oocyte maturation deserves further investigation.

Single-cell genome-wide bisulfite sequencing uncovers extensive heterogeneity in the mouse liver methylome.

BACKGROUND: Transmission fidelity of CpG DNA methylation patterns is not foolproof, with error rates from less than 1 to well over 10 % per CpG site, dependent on preservation of the methylated or unmethylated state and the type of sequence. This suggests a fairly high chance of errors. However, the consequences of such errors in terms of cell-to-cell variation have never been demonstrated by experimentally measuring intra-tissue heterogeneity in an adult organism. RESULTS: We employ single-cell DNA methylomics to analyze heterogeneity of genome-wide 5-methylcytosine (5mC) patterns within mouse liver. Our results indicate a surprisingly high level of heterogeneity, corresponding to an average epivariation frequency of approximately 3.3 %, with regions containing H3K4me1 being the most variable and promoters and CpG islands the most stable. Our data also indicate that the level of 5mC heterogeneity is dependent on genomic features. We find that non-functional sites such as repeat elements and introns are mostly unstable and potentially functional sites such as gene promoters are mostly stable. CONCLUSIONS: By employing a protocol for whole-genome bisulfite sequencing of single cells, we show that the liver epigenome is highly unstable with an epivariation frequency in DNA methylation patterns of at least two orders of magnitude higher than somatic mutation frequencies.

The dark side of circulating nucleic acids.

Free circulating or cell-free DNA (cfDNA), possibly from dying cells that release their contents into the blood as they break down, have become of major interest as a source for noninvasive diagnostics. Recent work demonstrated the uptake of human cfDNA in mouse cells in vitro and in vivo, accompanied by the activation of a cellular DNA damage response (DDR) and the appearance of apoptotic proteins in the host cells. By acting as a source of mobile genetic elements, cfDNA could be a continuous source of DNA mutagenesis of healthy cells in the body throughout life, promoting progressive cellular aging in vivo. As such, cfDNA may causally contribute to multiple aging-related diseases, such as cancer, diabetes, and Alzheimer's disease.

DNA methylome and transcriptome sequencing in human ovarian granulosa cells links age-related changes in gene expression to gene body methylation and 3'-end GC density.

Diminished ovarian function occurs early and is a primary cause for age-related decline in female fertility; however, its underlying mechanism remains unclear. This study investigated the roles that genome and epigenome structure play in age-related changes in gene expression and ovarian function, using human ovarian granulosa cells as an experimental system. DNA methylomes were compared between two groups of women with distinct age-related differences in ovarian functions, using both Methylated DNA Capture followed by Next Generation Sequencing (MethylCap-seq) and Reduced Representation Bisulfite Sequencing (RRBS); their transcriptomes were investigated using mRNA-seq. Significant, non-random changes in transcriptome and DNA methylome features are observed in human ovarian granulosa cells as women age and their ovarian functions deteriorate. The strongest correlations between methylation and the age-related changes in gene expression are not confined to the promoter region; rather, high densities of hypomethylated CpG-rich regions spanning the gene body are preferentially associated with gene down-regulation. This association is further enhanced where CpG regions are localized near the 3'-end of the gene. Such features characterize several genes crucial in age-related decline in ovarian function, most notably the AMH (Anti-Müllerian Hormone) gene. The genome-wide correlation between the density of hypomethylated intragenic and 3'-end regions and gene expression suggests previously unexplored mechanisms linking epigenome structure to age-related physiology and pathology.

High preservation of CpG cytosine methylation patterns at imprinted gene loci in liver and brain of aged mice.

A gradual loss of the correct patterning of 5-methyl cytosine marks in gene promoter regions has been implicated in aging and age-related diseases, most notably cancer. While a number of studies have examined DNA methylation in aging, there is no consensus on the magnitude of the effects, particularly at imprinted loci. Imprinted genes are likely candidate to undergo age-related changes because of their demonstrated plasticity in utero, for example, in response to environmental cues. Here we quantitatively analyzed a total of 100 individual CpG sites in promoter regions of 11 imprinted and non-imprinted genes in liver and cerebral cortex of young and old mice using mass spectrometry. The results indicate a remarkably high preservation of methylation marks during the aging process in both organs. To test if increased genotoxic stress associated with premature aging would destabilize DNA methylation we analyzed two DNA repair defective mouse models showing a host of premature aging symptoms in liver and brain. However, also in these animals, at the end of their life span, we found a similarly high preservation of DNA methylation marks. We conclude that patterns of DNA methylation in gene promoters of imprinted genes are surprisingly stable over time in normal, postmitotic tissues and that the multiple documented changes with age are likely to involve exceptions to this pattern, possibly associated with specific cellular responses to age-related changes other than genotoxic stress.