DNA methylation in mammalian development and disease.

The DNA methylation field has matured from a phase of discovery and genomic characterization to one seeking deeper functional understanding of how this modification contributes to development, ageing and disease. In particular, the past decade has seen many exciting mechanistic discoveries that have substantially expanded our appreciation for how this generic, evolutionarily ancient modification can be incorporated into robust epigenetic codes. Here, we summarize the current understanding of the distinct DNA methylation landscapes that emerge over the mammalian lifespan and discuss how they interact with other regulatory layers to support diverse genomic functions. We then review the rising interest in alternative patterns found during senescence and the somatic transition to cancer. Alongside advancements in single-cell and long-read sequencing technologies, the collective insights made across these fields offer new opportunities to connect the biochemical and genetic features of DNA methylation to cell physiology, developmental potential and phenotype.

Integrative Analyses of Human Reprogramming Reveal Dynamic Nature of Induced Pluripotency.

Induced pluripotency is a promising avenue for disease modeling and therapy, but the molecular principles underlying this process, particularly in human cells, remain poorly understood due to donor-to-donor variability and intercellular heterogeneity. Here, we constructed and characterized a clonal, inducible human reprogramming system that provides a reliable source of cells at any stage of the process. This system enabled integrative transcriptional and epigenomic analysis across the human reprogramming timeline at high resolution. We observed distinct waves of gene network activation, including the ordered re-activation of broad developmental regulators followed by early embryonic patterning genes and culminating in the emergence of a signature reminiscent of pre-implantation stages. Moreover, complementary functional analyses allowed us to identify and validate novel regulators of the reprogramming process. Altogether, this study sheds light on the molecular underpinnings of induced pluripotency in human cells and provides a robust cell platform for further studies. PAPERCLIP.

Autonomous transposons tune their sequences to ensure somatic suppression.

Transposable elements (TEs) are a major constituent of human genes, occupying approximately half of the intronic space. During pre-messenger RNA synthesis, intronic TEs are transcribed along with their host genes but rarely contribute to the final mRNA product because they are spliced out together with the intron and rapidly degraded. Paradoxically, TEs are an abundant source of RNA-processing signals through which they can create new introns1, and also functional2 or non-functional chimeric transcripts3. The rarity of these events implies the existence of a resilient splicing code that is able to suppress TE exonization without compromising host pre-mRNA processing. Here we show that SAFB proteins protect genome integrity by preventing retrotransposition of L1 elements while maintaining splicing integrity, via prevention of the exonization of previously integrated TEs. This unique dual role is possible because of L1's conserved adenosine-rich coding sequences that are bound by SAFB proteins. The suppressive activity of SAFB extends to tissue-specific, giant protein-coding cassette exons, nested genes and Tigger DNA transposons. Moreover, SAFB also suppresses LTR/ERV elements in species in which they are still active, such as mice and flies. A significant subset of splicing events suppressed by SAFB in somatic cells are activated in the testis, coinciding with low SAFB expression in postmeiotic spermatids. Reminiscent of the division of labour between innate and adaptive immune systems that fight external pathogens, our results uncover SAFB proteins as an RNA-based, pattern-guided, non-adaptive defence system against TEs in the soma, complementing the RNA-based, adaptive Piwi-interacting RNA pathway of the germline.

Self-patterning of human stem cells into post-implantation lineages.

Investigating human development is a substantial scientific challenge due to the technical and ethical limitations of working with embryonic samples. In the face of these difficulties, stem cells have provided an alternative to experimentally model inaccessible stages of human development in vitro1-13. Here we show that human pluripotent stem cells can be triggered to self-organize into three-dimensional structures that recapitulate some key spatiotemporal events of early human post-implantation embryonic development. Our system reproducibly captures spontaneous differentiation and co-development of embryonic epiblast-like and extra-embryonic hypoblast-like lineages, establishes key signalling hubs with secreted modulators and undergoes symmetry breaking-like events. Single-cell transcriptomics confirms differentiation into diverse cell states of the perigastrulating human embryo14,15 without establishing placental cell types, including signatures of post-implantation epiblast, amniotic ectoderm, primitive streak, mesoderm, early extra-embryonic endoderm, as well as initial yolk sac induction. Collectively, our system captures key features of human embryonic development spanning from Carnegie stage16 4-7, offering a reproducible, tractable and scalable experimental platform to understand the basic cellular and molecular mechanisms that underlie human development, including new opportunities to dissect congenital pathologies with high throughput.

Molecular features of cellular reprogramming and development.

Differentiating somatic cells are progressively restricted to specialized functions during ontogeny, but they can be experimentally directed to form other cell types, including those with complete embryonic potential. Early nuclear reprogramming methods, such as somatic cell nuclear transfer (SCNT) and cell fusion, posed significant technical hurdles to precise dissection of the regulatory programmes governing cell identity. However, the discovery of reprogramming by ectopic expression of a defined set of transcription factors, known as direct reprogramming, provided a tractable platform to uncover molecular characteristics of cellular specification and differentiation, cell type stability and pluripotency. We discuss the control and maintenance of cellular identity during developmental transitions as they have been studied using direct reprogramming, with an emphasis on transcriptional and epigenetic regulation.

DOT1L promotes spermatid differentiation by regulating expression of genes required for histone-to-protamine replacement.

Unique chromatin remodeling factors orchestrate dramatic changes in nuclear morphology during differentiation of the mature sperm head. A crucial step in this process is histone-to-protamine exchange, which must be executed correctly to avoid sperm DNA damage, embryonic lethality and male sterility. Here, we define an essential role for the histone methyltransferase DOT1L in the histone-to-protamine transition. We show that DOT1L is abundantly expressed in mouse meiotic and postmeiotic germ cells, and that methylation of histone H3 lysine 79 (H3K79), the modification catalyzed by DOT1L, is enriched in developing spermatids in the initial stages of histone replacement. Elongating spermatids lacking DOT1L fail to fully replace histones and exhibit aberrant protamine recruitment, resulting in deformed sperm heads and male sterility. Loss of DOT1L results in transcriptional dysregulation coinciding with the onset of histone replacement and affecting genes required for histone-to-protamine exchange. DOT1L also deposits H3K79me2 and promotes accumulation of elongating RNA Polymerase II at the testis-specific bromodomain gene Brdt. Together, our results indicate that DOT1L is an important mediator of transcription during spermatid differentiation and an indispensable regulator of male fertility.

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.

Epigenetic regulator function through mouse gastrulation.

During ontogeny, proliferating cells become restricted in their fate through the combined action of cell-type-specific transcription factors and ubiquitous epigenetic machinery, which recognizes universally available histone residues or nucleotides in a context-dependent manner1,2. The molecular functions of these regulators are generally well understood, but assigning direct developmental roles to them is hampered by complex mutant phenotypes that often emerge after gastrulation3,4. Single-cell RNA sequencing and analytical approaches have explored this highly conserved, dynamic period across numerous model organisms5-8, including mouse9-18. Here we advance these strategies using a combined zygotic perturbation and single-cell RNA-sequencing platform in which many mutant mouse embryos can be assayed simultaneously, recovering robust  morphological and transcriptional information across a panel of ten essential regulators. Deeper analysis of central Polycomb repressive complex (PRC) 1 and 2 components indicates substantial cooperativity, but distinguishes a dominant role for PRC2 in restricting the germline. Moreover, PRC mutant phenotypes emerge after gross epigenetic and transcriptional changes within the initial conceptus prior to gastrulation. Our experimental framework may eventually lead to a fully quantitative view of how cellular diversity emerges using an identical genetic template and from a single totipotent cell.

DOT1L bridges transcription and heterochromatin formation at mammalian pericentromeres.

Repetitive DNA elements are packaged in heterochromatin, but many require bursts of transcription to initiate and maintain long-term silencing. The mechanisms by which these heterochromatic genome features are transcribed remain largely unknown. Here, we show that DOT1L, a conserved histone methyltransferase that modifies lysine 79 of histone H3 (H3K79), has a specialized role in transcription of major satellite repeats to maintain pericentromeric heterochromatin and genome stability. We find that H3K79me3 is selectively enriched relative to H3K79me2 at repetitive elements in mouse embryonic stem cells (mESCs), that DOT1L loss compromises pericentromeric satellite transcription, and that this activity involves possible coordination between DOT1L and the chromatin remodeler SMARCA5. Stimulation of transcript production from pericentromeric repeats by DOT1L participates in stabilization of heterochromatin structures in mESCs and cleavage-stage embryos and is required for preimplantation viability. Our findings uncover an important role for DOT1L as a bridge between transcriptional activation of repeat elements and heterochromatin stability, advancing our understanding of how genome integrity is maintained and how chromatin state is set up during early development.

Molecular recording of mammalian embryogenesis.

Ontogeny describes the emergence of complex multicellular organisms from single totipotent cells. This field is particularly challenging in mammals, owing to the indeterminate relationship between self-renewal and differentiation, variation in progenitor field sizes, and internal gestation in these animals. Here we present a flexible, high-information, multi-channel molecular recorder with a single-cell readout and apply it as an evolving lineage tracer to assemble mouse cell-fate maps from fertilization through gastrulation. By combining lineage information with single-cell RNA sequencing profiles, we recapitulate canonical developmental relationships between different tissue types and reveal the nearly complete transcriptional convergence of endodermal cells of extra-embryonic and embryonic origins. Finally, we apply our cell-fate maps to estimate the number of embryonic progenitor cells and their degree of asymmetric partitioning during specification. Our approach enables massively parallel, high-resolution recording of lineage and other information in mammalian systems, which will facilitate the construction of a quantitative framework for understanding developmental processes.

Epigenetic restriction of extraembryonic lineages mirrors the somatic transition to cancer.

In mammals, the canonical somatic DNA methylation landscape is established upon specification of the embryo proper and subsequently disrupted within many cancer types. However, the underlying mechanisms that direct this genome-scale transformation remain elusive, with no clear model for its systematic acquisition or potential developmental utility. Here, we analysed global remethylation from the mouse preimplantation embryo into the early epiblast and extraembryonic ectoderm. We show that these two states acquire highly divergent genomic distributions with substantial disruption of bimodal, CpG density-dependent methylation in the placental progenitor. The extraembryonic epigenome includes specific de novo methylation at hundreds of embryonically protected CpG island promoters, particularly those that are associated with key developmental regulators and are orthologously methylated across most human cancer types. Our data suggest that the evolutionary innovation of extraembryonic tissues may have required co-option of DNA methylation-based suppression as an alternative to regulation by Polycomb-group proteins, which coordinate embryonic germ-layer formation in response to extraembryonic cues. Moreover, we establish that this decision is made deterministically, downstream of promiscuously used-and frequently oncogenic-signalling pathways, via a novel combination of epigenetic cofactors. Methylation of developmental gene promoters during tumorigenesis may therefore reflect the misappropriation of an innate trajectory and the spontaneous reacquisition of a latent, developmentally encoded epigenetic landscape.

Loss of DNA methyltransferase activity in primed human ES cells triggers increased cell-cell variability and transcriptional repression.

Maintenance of pluripotency and specification towards a new cell fate are both dependent on precise interactions between extrinsic signals and transcriptional and epigenetic regulators. Directed methylation of cytosines by the de novo methyltransferases DNMT3A and DNMT3B plays an important role in facilitating proper differentiation, whereas DNMT1 is essential for maintaining global methylation levels in all cell types. Here, we generated single-cell mRNA expression data from wild-type, DNMT3A, DNMT3A/3B and DNMT1 knockout human embryonic stem cells and observed a widespread increase in cellular and transcriptional variability, even with limited changes in global methylation levels in the de novo knockouts. Furthermore, we found unexpected transcriptional repression upon either loss of the de novo methyltransferase DNMT3A or the double knockout of DNMT3A/3B that is further propagated upon differentiation to mesoderm and ectoderm. Taken together, our single-cell RNA-sequencing data provide a high-resolution view into the consequences of depleting the three catalytically active DNMTs in human pluripotent stem cells.

DNA methylation dynamics of the human preimplantation embryo.

In mammals, cytosine methylation is predominantly restricted to CpG dinucleotides and stably distributed across the genome, with local, cell-type-specific regulation directed by DNA binding factors. This comparatively static landscape is in marked contrast with the events of fertilization, during which the paternal genome is globally reprogrammed. Paternal genome demethylation includes the majority of CpGs, although methylation remains detectable at several notable features. These dynamics have been extensively characterized in the mouse, with only limited observations available in other mammals, and direct measurements are required to understand the extent to which early embryonic landscapes are conserved. We present genome-scale DNA methylation maps of human preimplantation development and embryonic stem cell derivation, confirming a transient state of global hypomethylation that includes most CpGs, while sites of residual maintenance are primarily restricted to gene bodies. Although most features share similar dynamics to those in mouse, maternally contributed methylation is divergently targeted to species-specific sets of CpG island promoters that extend beyond known imprint control regions. Retrotransposon regulation is also highly diverse, and transitions from maternally to embryonically expressed elements. Together, our data confirm that paternal genome demethylation is a general attribute of early mammalian development that is characterized by distinct modes of epigenetic regulation.

DNA methylation dynamics during in vivo differentiation of blood and skin stem cells.

DNA methylation is a mechanism of epigenetic regulation that is common to all vertebrates. Functional studies underscore its relevance for tissue homeostasis, but the global dynamics of DNA methylation during in vivo differentiation remain underexplored. Here we report high-resolution DNA methylation maps of adult stem cell differentiation in mouse, focusing on 19 purified cell populations of the blood and skin lineages. DNA methylation changes were locus specific and relatively modest in magnitude. They frequently overlapped with lineage-associated transcription factors and their binding sites, suggesting that DNA methylation may protect cells from aberrant transcription factor activation. DNA methylation and gene expression provided complementary information, and combining the two enabled us to infer the cellular differentiation hierarchy of the blood lineage directly from genome-scale data. In summary, these results demonstrate that in vivo differentiation of adult stem cells is associated with small but informative changes in the genomic distribution of DNA methylation.

DNA methylation: roles in mammalian development.

DNA methylation is among the best studied epigenetic modifications and is essential to mammalian development. Although the methylation status of most CpG dinucleotides in the genome is stably propagated through mitosis, improvements to methods for measuring methylation have identified numerous regions in which it is dynamically regulated. In this Review, we discuss key concepts in the function of DNA methylation in mammals, stemming from more than two decades of research, including many recent studies that have elucidated when and where DNA methylation has a regulatory role in the genome. We include insights from early development, embryonic stem cells and adult lineages, particularly haematopoiesis, to highlight the general features of this modification as it participates in both global and localized epigenetic regulation.

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.

Epigenetic predisposition to reprogramming fates in somatic cells.

Reprogramming to pluripotency is a low-efficiency process at the population level. Despite notable advances to molecularly characterize key steps, several fundamental aspects remain poorly understood, including when the potential to reprogram is first established. Here, we apply live-cell imaging combined with a novel statistical approach to infer when somatic cells become fated to generate downstream pluripotent progeny. By tracing cell lineages from several divisions before factor induction through to pluripotent colony formation, we find that pre-induction sister cells acquire similar outcomes. Namely, if one daughter cell contributes to a lineage that generates induced pluripotent stem cells (iPSCs), its paired sibling will as well. This result suggests that the potential to reprogram is predetermined within a select subpopulation of cells and heritable, at least over the short term. We also find that expanding cells over several divisions prior to factor induction does not increase the per-lineage likelihood of successful reprogramming, nor is reprogramming fate correlated to neighboring cell identity or cell-specific reprogramming factor levels. By perturbing the epigenetic state of somatic populations with Ezh2 inhibitors prior to factor induction, we successfully modulate the fraction of iPSC-forming lineages. Our results therefore suggest that reprogramming potential may in part reflect preexisting epigenetic heterogeneity that can be tuned to alter the cellular response to factor induction.

A second wave of Notch signaling diversifies the intestinal secretory lineage.

The small intestine is well known for the function of its nutrient-absorbing enterocytes; yet equally critical for the maintenance of homeostasis is a diverse set of secretory cells, all of which are presumed to differentiate from the same intestinal stem cell. Despite major roles in intestinal function and health, understanding how the full spectrum of secretory cell types arises remains a longstanding challenge, largely due to their comparative rarity. Here, we investigate the fate specification of a rare and distinct population of small intestinal epithelial cells found in rats and humans but not mice: C FTR Hi gh E xpressers (CHEs). We use pseudotime trajectory analysis of single-cell RNA-seq data from rat intestinal jejunum to provide evidence that CHEs are specified along the secretory lineage and appear to employ a second wave of Notch-based signal transduction to distinguish these cells from other secretory cell types. We further validate the general order of transcription factors that direct these cells from unspecified progenitors within the crypt and experimentally demonstrate that Notch signaling is necessary to induce CHE fate both in vivo and in vitro . Our results suggest a model in which Notch is reactivated along the secretory lineage to specify the CHE population: a rare secretory cell type with putative functions in localized coordination of luminal pH and direct relevance to cystic fibrosis pathophysiology.

Reconstructing axial progenitor field dynamics in mouse stem cell-derived embryoids.

Embryogenesis requires substantial coordination to translate genetic programs to the collective behavior of differentiating cells, but understanding how cellular decisions control tissue morphology remains conceptually and technically challenging. Here, we combine continuous Cas9-based molecular recording with a mouse embryonic stem cell-based model of the embryonic trunk to build single-cell phylogenies that describe the behavior of transient, multipotent neuro-mesodermal progenitors (NMPs) as they commit into neural and somitic cell types. We find that NMPs show subtle transcriptional signatures related to their recent differentiation and contribute to downstream lineages through a surprisingly broad distribution of individual fate outcomes. Although decision-making can be heavily influenced by environmental cues to induce morphological phenotypes, axial progenitors intrinsically mature over developmental time to favor the neural lineage. Using these data, we present an experimental and analytical framework for exploring the non-homeostatic dynamics of transient progenitor populations as they shape complex tissues during critical developmental windows.

Genome-scale DNA methylation mapping of clinical samples at single-nucleotide resolution.

Bisulfite sequencing measures absolute levels of DNA methylation at single-nucleotide resolution, providing a robust platform for molecular diagnostics. We optimized bisulfite sequencing for genome-scale analysis of clinical samples: here we outline how restriction digestion targets bisulfite sequencing to hotspots of epigenetic regulation and describe a statistical method for assessing significance of altered DNA methylation patterns. Thirty nanograms of DNA was sufficient for genome-scale analysis and our protocol worked well on formalin-fixed, paraffin-embedded samples.