Simultaneous profiling of transcriptome and DNA methylome from a single cell.
ABSTRACT: Single-cell transcriptome and single-cell methylome technologies have become powerful tools to study RNA and DNA methylation profiles of single cells at a genome-wide scale. A major challenge has been to understand the direct correlation of DNA methylation and gene expression within single-cells. Due to large cell-to-cell variability and the lack of direct measurements of transcriptome and methylome of the same cell, the association is still unclear.Here, we describe a novel method (scMT-seq) that simultaneously profiles both DNA methylome and transcriptome from the same cell. In sensory neurons, we consistently identify transcriptome and methylome heterogeneity among single cells but the majority of the expression variance is not explained by proximal promoter methylation, with the exception of genes that do not contain CpG islands. By contrast, gene body methylation is positively associated with gene expression for only those genes that contain a CpG island promoter. Furthermore, using single nucleotide polymorphism patterns from our hybrid mouse model, we also find positive correlation of allelic gene body methylation with allelic expression.Our method can be used to detect transcriptome, methylome, and single nucleotide polymorphism information within single cells to dissect the mechanisms of epigenetic gene regulation.
Project description:Embryonic stem cells (ESCs) consist of a population of self-renewing cells displaying extensive phenotypic and functional heterogeneity. Research towards the understanding of the epigenetic mechanisms underlying the heterogeneity among ESCs is still in its initial stage. Key issues, such as how to identify cell-subset specifically methylated loci and how to interpret the biological meanings of methylation variations remain largely unexplored. To fill in the research gap, we implemented a computational pipeline to analyze single-cell methylome and to perform an integrative analysis with single-cell transcriptome data. According to the origins of variation in DNA methylation, we determined the genomic loci associated with allelic-specific methylation or asymmetric DNA methylation, and explored a beta mixture model to infer the genomic loci exhibiting cell-subset specific methylation (CSM). We observed that the putative CSM loci in ESCs are significantly enriched in CpG island (CGI) shelves and regions with histone marks for promoter and enhancer, and the genes hosting putative CSM loci show wide-ranging expression among ESCs. More interestingly, the putative CSM loci may be clustered into co-methylated modules enriching the binding motifs of distinct sets of transcription factors. Taken together, our study provided a novel tool to explore single-cell methylome and transcriptome to reveal the underlying transcriptional regulatory networks associated with epigenetic heterogeneity of ESCs.
Project description:Oocyte maturation is a coordinated process that is tightly linked to reproductive potential. A better understanding of gene regulation during human oocyte maturation will not only answer an important question in biology, but also facilitate the development of in vitro maturation technology as a fertility treatment. We generated single-cell transcriptome and used our previously published single-cell methylome data from human oocytes at different maturation stages to investigate how genes are regulated during oocyte maturation, focusing on the potential regulatory role of non-CpG methylation. DNMT3B, a gene encoding a key non-CpG methylation enzyme, is one of the 1,077 genes upregulated in mature oocytes, which may be at least partially responsible for the increased non-CpG methylation as oocytes mature. Non-CpG differentially methylated regions (DMRs) between mature and immature oocytes have multiple binding motifs for transcription factors, some of which bind with DNMT3B and may be important regulators of oocyte maturation through non-CpG methylation. Over 98% of non-CpG DMRs locate in transposable elements, and these DMRs are correlated with expression changes of the nearby genes. Taken together, this data indicates that global non-CpG hypermethylation during oocyte maturation may play an active role in gene expression regulation, potentially through the interaction with transcription factors.
Project description:DNA methylation can control some CpG-poor genes but unbiased studies have not found a consistent genome-wide association with gene activity outside of CpG islands or shores possibly due to use of cell lines or limited bioinformatics analyses. We performed reduced representation bisulfite sequencing (RRBS) of rat dorsal root ganglia encompassing postmitotic primary sensory neurons (n = 5, r > 0.99; orthogonal validation p < 10(-19)). The rat genome suggested a dichotomy of genes previously reported in other mammals: low CpG content (< 3.2%) promoter (LCP) genes and high CpG content (? 3.2%) promoter (HCP) genes. A genome-wide integrated methylome-transcriptome analysis showed that LCP genes were markedly hypermethylated when repressed, and hypomethylated when active with a 40% difference in a broad region at the 5' of the transcription start site (p < 10(-87) for -6000 bp to -2000 bp, p < 10(-73) for -2000 bp to +2000 bp, no difference in gene body p = 0.42). HCP genes had minimal TSS-associated methylation regardless of transcription status, but gene body methylation appeared to be lost in repressed HCP genes. Therefore, diametrically opposite methylome-transcriptome associations characterize LCP and HCP genes in postmitotic neural tissue in vivo.
Project description:DNA methylation plays a major role in the epigenetic regulation of gene expression. Although a few DNA methylation profiling studies of porcine genome which is one of the important biomedical models for human diseases have been reported, the available data are still limited. We tried to study methylation patterns of diverse pig tissues as a study of the International Swine Methylome Consortium to generate the swine reference methylome map to extensively evaluate the methylation profile of the pig genome at a single base resolution. We generated and analysed the DNA methylome profiles of five different tissues and a cell line originated from pig. On average, 39.85 and 62.1% of cytosine and guanine dinucleotides (CpGs) of CpG islands and 2 kb upstream of transcription start sites were covered, respectively. We detected a low rate (an average of 1.67%) of non-CpG methylation in the six samples except for the neocortex (2.3%). The observed global CpG methylation patterns of pigs indicated high similarity to other mammals including humans. The percentage of CpG methylation associated with gene features was similar among the tissues but not for a 3D4/2 cell line. Our results provide essential information for future studies of the porcine epigenome.
Project description:Using next-generation sequencing to decipher methylome and transcriptome and underlying molecular mechanisms contributing to rheumatoid arthritis (RA) for improving future therapies, we performed methyl-seq and RNA-seq on peripheral blood mononuclear cells (PBMCs) from RA subjects and normal donors. Principal component analysis and hierarchical clustering revealed distinct methylation signatures in RA with methylation aberrations noted across chromosomes. Methylation alterations varied with CpG features and genic characteristics. Typically, CpG islands and CpG shores were hypermethylated and displayed the greatest methylation variance. Promoters were hypermethylated and enhancers/gene bodies were hypomethylated, with methylation variance associated with expression variance. RA genetically associated genes preferentially displayed differential methylation and differential expression or interacted with differentially methylated and differentially expressed genes. These differentially methylated and differentially expressed genes were enriched with several signaling pathways and disease categories. 10 genes (CD86, RAB20, XAF1, FOLR3, LTBR, KCNH8, DOK7, PDGFA, PITPNM2, CELSR1) with concomitantly differential methylation in enhancers/promoters/gene bodies and differential expression in B cells were validated. This integrated analysis of methylome and transcriptome identified novel epigenetic signatures associated with RA and highlighted the interaction between genetics and epigenetics in RA. These findings help our understanding of the pathogenesis of RA and advance epigenetic studies in regards to the disease.
Project description:AIMS:Antenatal hypoxia negatively impacts foetal heart development, and increases the risk of heart disease later in life. The molecular mechanisms remain largely elusive. Here, we conducted a genome-wide analysis to study the impact of antenatal hypoxia on DNA methylome and transcriptome profiling in foetal and adult offspring hearts. METHODS AND RESULTS:Pregnant rats were treated with normoxia or hypoxia (10.5% O2) from Day 15 to Day 21 of gestation. Hearts were isolated from near-term foetuses and 5-month-old male and female offsprings, and DNA methylome and RNA-seq were performed. Methylome data shows a sharp dip in CpG methylation centred at the transcription start site (TSS). CpG islands (CGIs) and CpG island shores (CGSs) within 10?kb upstream of the TSS are hypomethylated, compared with CGIs and CGSs within gene bodies. Combining transcriptome, data indicate an inverse relation between gene expression and CpG methylation around the TSS. Of interest, antenatal hypoxia induces opposite changes in methylation patterns in foetal and adult hearts, with hypermethylation in the foetus and hypomethylation in the adult. Also, there is significant sex dimorphism of changes in gene expression patterns in the adult offspring heart. Notably, pathway analysis indicates that enrichment of inflammation-related pathways are significantly greater in the adult male heart than those in the female heart. CONCLUSION:Our study provides an initial framework and new insights into foetal hypoxia-mediated epigenetic programming of pro-inflammatory phenotype in the heart development, linking antenatal stress, and developmental programming of heart vulnerability to disease later in life.
Project description:DNA methylation may be a stable epigenetic contributor to defining fat cell lineage.We performed reduced representation bisulfite sequencing (RRBS) and RNA-seq to depict a genome-wide integrative view of the DNA methylome and transcriptome during brown and white adipogenesis.Our analysis demonstrated that DNA methylation is a stable epigenetic signature for brown and white cell lineage before, during, and after differentiation. We identified 31 genes whose promoters were significantly differentially methylated between white and brown adipogenesis at all three time points of differentiation. Among them, five genes belong to the Hox family; their expression levels were anti-correlated with promoter methylation, suggesting a regulatory role of DNA methylation in transcription. Blocking DNA methylation with 5-Aza-cytidine increased the expression of these genes, with the most prominent effect on Hoxc10, a repressor of BAT marker expression.Our data suggest that DNA methylation may play an important role in lineage-specific development in adipocytes.
Project description:BACKGROUND: Cancer cells undergo massive alterations to their DNA methylation patterns that result in aberrant gene expression and malignant phenotypes. However, the mechanisms that underlie methylome changes are not well understood nor is the genomic distribution of DNA methylation changes well characterized. RESULTS: Here, we performed methylated DNA immunoprecipitation combined with high-throughput sequencing (MeDIP-seq) to obtain whole-genome DNA methylation profiles for eight human breast cancer cell (BCC) lines and for normal human mammary epithelial cells (HMEC). The MeDIP-seq analysis generated non-biased DNA methylation maps by covering almost the entire genome with sufficient depth and resolution. The most prominent feature of the BCC lines compared to HMEC was a massively reduced methylation level particularly in CpG-poor regions. While hypomethylation did not appear to be associated with particular genomic features, hypermethylation preferentially occurred at CpG-rich gene-related regions independently of the distance from transcription start sites. We also investigated methylome alterations during epithelial-to-mesenchymal transition (EMT) in MCF7 cells. EMT induction was associated with specific alterations to the methylation patterns of gene-related CpG-rich regions, although overall methylation levels were not significantly altered. Moreover, approximately 40% of the epithelial cell-specific methylation patterns in gene-related regions were altered to those typical of mesenchymal cells, suggesting a cell-type specific regulation of DNA methylation. CONCLUSIONS: This study provides the most comprehensive analysis to date of the methylome of human mammary cell lines and has produced novel insights into the mechanisms of methylome alteration during tumorigenesis and the interdependence between DNA methylome alterations and morphological changes.
Project description:The development of breast cancer metastasis is accompanied by dynamic transcriptome changes and dramatic alterations in nuclear and chromatin structure. The basis of these changes is incompletely understood. The DNA methylome of primary breast cancers contribute to transcriptomic heterogeneity and different metastatic behavior. Therefore we sought to characterize methylome remodeling during regional metastasis. We profiled the DNA methylome and transcriptome of 44 matched primary breast tumors and regional metastases. Striking subtype-specific patterns of metastasis-associated methylome remodeling were observed, which reflected the molecular heterogeneity of breast cancers. These divergent changes occurred primarily in CpG island (CGI)-poor areas. Regions of methylome reorganization shared by the subtypes were also observed, and we were able to identify a metastasis-specific methylation signature that was present across the breast cancer subclasses. These alterations also occurred outside of CGIs and promoters, including sequences flanking CGIs and intergenic sequences. Integrated analysis of methylation and gene expression identified genes whose expression correlated with metastasis-specific methylation. Together, these findings significantly enhance our understanding of the epigenetic reorganization that occurs during regional breast cancer metastasis across the major breast cancer subtypes and reveal the nature of methylome remodeling during this process.
Project description:DNA methylation is a frequently studied epigenetic modification due to its role in regulating gene expression and hence in biological processes and in determining phenotypic plasticity in organisms. Rudimentary DNA methylation patterns for some livestock species are publically available: among these, goat methylome deserves to be further explored.Genome-wide DNA methylation maps of the hypothalamus and ovary from Saanen goats were generated using Methyl-CpG binding domain protein sequencing (MBD-seq). Analysis of DNA methylation patterns indicate that the majority of methylation peaks found within genes are located gene body regions, for both organs. Analysis of the distribution of methylated sites per chromosome showed that chromosome X had the lowest number of methylation peaks. The X chromosome has one of the highest percentages of methylated CpG islands in both organs, and approximately 50% of the CpG islands in the goat epigenome are methylated in hypothalamus and ovary. Organ-specific Differentially Methylated Genes (DMGs) were correlated with the expression levels.The comparison between transcriptome and methylome in hypothalamus and ovary showed that a higher level of methylation is not accompanied by a higher gene suppression. The genome-wide DNA methylation map for two goat organs produced here is a valuable starting point for studying the involvement of epigenetic modifications in regulating goat reproduction performance.