Hongcang Gu

Genome-scale DNA methylation maps of pluripotent and differentiated cells

DNA methylation is essential for normal development and has been implicated in many pathologies including cancer. Our knowledge about the genome-wide distribution of DNA methylation, how it changes during cellular differentiation and how it relates to histone methylation and other chromatin modifications in mammals remains limited. Here we report the generation and analysis of genome-scale DNA methylation profiles at nucleotide resolution in mammalian cells. Using high-throughput reduced representation bisulphite sequencing and single-molecule-based sequencing, we generated DNA methylation maps covering most CpG islands, and a representative sampling of conserved non-coding elements, transposons and other genomic features, for mouse embryonic stem cells, embryonic-stem-cell-derived and primary neural cells, and eight other primary tissues. Several key findings emerge from the data. First, DNA methylation patterns are better correlated with histone methylation patterns than with the underlying genome sequence context. Second, methylation of CpGs are dynamic epigenetic marks that undergo extensive changes during cellular differentiation, particularly in regulatory regions outside of core promoters. Third, analysis of embryonic-stem-cell-derived and primary cells reveals that ‘weak’ CpG islands associated with a specific set of developmentally regulated genes undergo aberrant hypermethylation during extended proliferation in vitro, in a pattern reminiscent of that reported in some primary tumours. More generally, the results establish reduced representation bisulphite sequencing as a powerful technology for epigenetic profiling of cell populations relevant to developmental biology, cancer and regenerative medicine.

Reference Maps of Human ES and iPS Cell Variation Enable High-Throughput Characterization of Pluripotent Cell Lines

The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

Charting a dynamic DNA methylation landscape of the human genome

DNA methylation is a defining feature of mammalian cellular identity and is essential for normal development. Most cell types, except germ cells and pre-implantation embryos, display relatively stable DNA methylation patterns, with 70–80% of all CpGs being methylated. Despite recent advances, we still have a limited understanding of when, where and how many CpGs participate in genomic regulation. Here we report the in-depth analysis of 42 whole-genome bisulphite sequencing data sets across 30 diverse human cell and tissue types. We observe dynamic regulation for only 21.8% of autosomal CpGs within a normal developmental context, most of which are distal to transcription start sites. These dynamic CpGs co-localize with gene regulatory elements, particularly enhancers and transcription-factor-binding sites, which allow identification of key lineage-specific regulators. In addition, differentially methylated regions (DMRs) often contain single nucleotide polymorphisms associated with cell-type-related diseases as determined by genome-wide association studies. The results also highlight the general inefficiency of whole-genome bisulphite sequencing, as 70–80% of the sequencing reads across these data sets provided little or no relevant information about CpG methylation. To demonstrate further the utility of our DMR set, we use it to classify unknown samples and identify representative signature regions that recapitulate major DNA methylation dynamics. In summary, although in theory every CpG can change its methylation state, our results suggest that only a fraction does so as part of coordinated regulatory programs. Therefore, our selected DMRs can serve as a starting point to guide new, more effective reduced representation approaches to capture the most informative fraction of CpGs, as well as further pinpoint putative regulatory elements.

A unique regulatory phase of DNA methylation in the early mammalian embryo

DNA methylation is highly dynamic during mammalian embryogenesis. It is broadly accepted that the paternal genome is actively depleted of 5-methylcytosine at fertilization, followed by passive loss that reaches a minimum at the blastocyst stage. However, this model is based on limited data, and so far no base-resolution maps exist to support and refine it. Here we generate genome-scale DNA methylation maps in mouse gametes and from the zygote through post-implantation. We find that the oocyte already exhibits global hypomethylation, particularly at specific families of long interspersed element 1 and long terminal repeat retroelements, which are disparately methylated between gametes and have lower methylation values in the zygote than in sperm. Surprisingly, the oocyte contributes a unique set of differentially methylated regions (DMRs)—including many CpG island promoters—that are maintained in the early embryo but are lost upon specification and absent from somatic cells. In contrast, sperm-contributed DMRs are largely intergenic and become hypermethylated after the blastocyst stage. Our data provide a genome-scale, base-resolution timeline of DNA methylation in the pre-specified embryo, when this epigenetic modification is most dynamic, before returning to the canonical somatic pattern.

Dnmt3a is essential for hematopoietic stem cell differentiation

Loss of the de novo DNA methyltransferases Dnmt3a and Dnmt3b in embryonic stem cells obstructs differentiation; however, the role of these enzymes in somatic stem cells is largely unknown. Using conditional ablation, we show that Dnmt3a loss progressively impairs hematopoietic stem cell (HSC) differentiation over serial transplantation, while simultaneously expanding HSC numbers in the bone marrow. Dnmt3a-null HSCs show both increased and decreased methylation at distinct loci, including substantial CpG island hypermethylation. Dnmt3a-null HSCs upregulate HSC multipotency genes and downregulate differentiation factors, and their progeny exhibit global hypomethylation and incomplete repression of HSC-specific genes. These data establish Dnmt3a as a critical participant in the epigenetic silencing of HSC regulatory genes, thereby enabling efficient differentiation.

Comparison of sequencing-based methods to profile DNA methylation and identification of monoallelic epigenetic modifications

Analysis of DNA methylation patterns relies increasingly on sequencing-based profiling methods. The four most frequently used sequencing-based technologies are the bisulfite-based methods MethylC-seq and reduced representation bisulfite sequencing (RRBS), and the enrichment-based techniques methylated DNA immunoprecipitation sequencing (MeDIP-seq) and methylated DNA binding domain sequencing (MBD-seq). We applied all four methods to biological replicates of human embryonic stem cells to assess their genome-wide CpG coverage, resolution, cost, concordance and the influence of CpG density and genomic context. The methylation levels assessed by the two bisulfite methods were concordant (their difference did not exceed a given threshold) for 82% for CpGs and 99% of the non-CpG cytosines. Using binary methylation calls, the two enrichment methods were 99% concordant and regions assessed by all four methods were 97% concordant. We combined MeDIP-seq with methylation-sensitive restriction enzyme (MRE-seq) sequencing for comprehensive methylome coverage at lower cost. This, along with RNA-seq and ChIP-seq of the ES cells enabled us to detect regions with allele-specific epigenetic states, identifying most known imprinted regions and new loci with monoallelic epigenetic marks and monoallelic expression.

Quantitative comparison of genome-wide DNA methylation mapping technologies

DNA methylation plays a key role in regulating eukaryotic gene expression. Although mitotically heritable and stable over time, patterns of DNA methylation frequently change in response to cell differentiation, disease and environmental influences. Several methods have been developed to map DNA methylation on a genomic scale. Here, we benchmark four of these approaches by analyzing two human embryonic stem cell lines derived from genetically unrelated embryos and a matched pair of colon tumor and adjacent normal colon tissue obtained from the same donor. Our analysis reveals that methylated DNA immunoprecipitation sequencing (MeDIP-seq), methylated DNA capture by affinity purification (MethylCap-seq), reduced representation bisulfite sequencing (RRBS) and the Infinium HumanMethylation27 assay all produce accurate DNA methylation data. However, these methods differ in their ability to detect differentially methylated regions between pairs of samples. We highlight strengths and weaknesses of the four methods and give practical recommendations for the design of epigenomic case-control studies.DNA methylation plays a key role in regulating eukaryotic gene expression. Although mitotically heritable and stable over time, patterns of DNA methylation frequently change in response to cell differentiation, disease and environmental influences. Several methods have been developed to map DNA methylation on a genomic scale. Here, we benchmark four of these approaches by analyzing two human embryonic stem cell lines derived from genetically unrelated embryos and a matched pair of colon tumor and adjacent normal colon tissue obtained from the same donor. Our analysis reveals that methylated DNA immunoprecipitation sequencing (MeDIP-seq), methylated DNA capture by affinity purification (MethylCap-seq), reduced representation bisulfite sequencing (RRBS) and the Infinium HumanMethylation27 assay all produce accurate DNA methylation data. However, these methods differ in their ability to detect differentially methylated regions between pairs of samples. We highlight strengths and weaknesses of the four methods and give practical recommendations for the design of epigenomic case-control studies.

Preparation of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling

Genome-wide mapping of 5-methylcytosine is of broad interest to many fields of biology and medicine. A variety of methods have been developed, and several have recently been advanced to genome-wide scale using arrays and next-generation sequencing approaches. We have previously reported reduced representation bisulfite sequencing (RRBS), a bisulfite-based protocol that enriches CG-rich parts of the genome, thereby reducing the amount of sequencing required while capturing the majority of promoters and other relevant genomic regions. The approach provides single-nucleotide resolution, is highly sensitive and provides quantitative DNA methylation measurements. This protocol should enable any standard molecular biology laboratory to generate RRBS libraries of high quality. Briefly, purified genomic DNA is digested by the methylation-insensitive restriction enzyme MspI to generate short fragments that contain CpG dinucleotides at the ends. After end-repair, A-tailing and ligation to methylated Illumina adapters, the CpG-rich DNA fragments (40–220 bp) are size selected, subjected to bisulfite conversion, PCR amplified and end sequenced on an Illumina Genome Analyzer. Note that alignment and analysis of RRBS sequencing reads are not covered in this protocol. The extremely low input requirements (10–300 ng), the applicability of the protocol to formalin-fixed and paraffin-embedded samples, and the techniques single-nucleotide resolution extends RRBS to a wide range of biological and clinical samples and research applications. The entire process of RRBS library construction takes ∼9 d.

Reprogramming factor expression initiates widespread targeted chromatin remodeling.

Despite rapid progress in characterizing transcription factor-driven reprogramming of somatic cells to an induced pluripotent stem cell (iPSC) state, many mechanistic questions still remain. To gain insight into the earliest events in the reprogramming process, we systematically analyzed the transcriptional and epigenetic changes that occur during early factor induction after discrete numbers of divisions. We observed rapid, genome-wide changes in the euchromatic histone modification, H3K4me2, at more than a thousand loci including large subsets of pluripotency-related or developmentally regulated gene promoters and enhancers. In contrast, patterns of the repressive H3K27me3 modification remained largely unchanged except for focused depletion specifically at positions where H3K4 methylation is gained. These chromatin regulatory events precede transcriptional changes within the corresponding loci. Our data provide evidence for an early, organized, and population-wide epigenetic response to ectopic reprogramming factors that clarify the temporal order through which somatic identity is reset during reprogramming.

Transcriptional and epigenetic dynamics during specification of human embryonic stem cells.

Differentiation of human embryonic stem cells (hESCs) provides a unique opportunity to study the regulatory mechanisms that facilitate cellular transitions in a human context. To that end, we performed comprehensive transcriptional and epigenetic profiling of populations derived through directed differentiation of hESCs representing each of the three embryonic germ layers. Integration of whole-genome bisulfite sequencing, chromatin immunoprecipitation sequencing, and RNA sequencing reveals unique events associated with specification toward each lineage. Lineage-specific dynamic alterations in DNA methylation and H3K4me1 are evident at putative distal regulatory elements that are frequently bound by pluripotency factors in the undifferentiated hESCs. In addition, we identified germ-layer-specific H3K27me3 enrichment at sites exhibiting high DNA methylation in the undifferentiated state. A better understanding of these initial specification events will facilitate identification of deficiencies in current approaches, leading to more faithful differentiation strategies as well as providing insights into the rewiring of human regulatory programs during cellular transitions.