Associating Cellular Epigenetic Models with Human Phenotypes

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EPIGENETICS loci changing DNA methylation consistently OPINION in multiple individuals but also that these changes select for specific genes within Associating cellular epigenetic the genome that may have importance for disease risk or aetiology. Despite what sound like positive results models with human phenotypes of these EWAS approaches, we7,8 and others9,10 have noted that this typical EWAS Tuuli Lappalainen and John M. Greally approach suffers from multiple problems. Abstract | Epigenetic association studies have been carried out to test the These problems can be grouped into two categories: the robust identification of hypothesis that environmental perturbations trigger cellular reprogramming, with molecular (usually DNA methylation) downstream effects on cellular function and phenotypes. There have now been differences associated with the phenotype numerous studies of the potential molecular mediators of epigenetic changes by studied, and the interpretability of these epigenome-wide association studies (EWAS). However, a challenge for the field is results. The robustness issue is the typical the interpretation of the results obtained. We describe a second-generation EWAS focus of reviews addressing the problems of designing and carrying out EWAS7,9,10 approach, which focuses on the possible cellular models of epigenetic and usually includes discussion of cohort perturbations, studied by rigorous analysis and interpretation of genomic data. selection, statistical approaches used when Thus refocused, epigenetics research aligns with the field of functional genomics dealing with high-dimensional data and to provide insights into environmental and genetic influences on phenotypic caution about the technical artefacts that 11–13 variation in humans. can occur in all genome-wide studies . In this Opinion article, we focus instead on the issue of interpretability of even The first association of epigenetic processes described as epigenetic5,6 (BOX 1) and could the highest confidence DNA methylation with human disease occurred in the early thus be a molecular mediator propagating changes. Although our focus is on DNA 1980s when it was found that the DNA the memory of past cellular perturbations. methylation, as the most commonly studied of cancer cells had less 5‑methylcytosine Taking advantage of new assays to regulatory mechanism in human EWAS, the than normal counterpart cells1,2 and that interrogate increasing proportions of lessons apply to the broadening group of within this globally hypomethylated the genome, DNA methylation studies transcriptional regulators studied in human cancer genome the promoters of tumour were carried out on samples of cells from diseases. suppressor genes had paradoxically individuals affected by the phenotype of First, at the core of the interpretability increased DNA methylation3. The increased interest, comparing these profiles with concern are the often vague definitions DNA methylation of promoters of tumour comparable samples from individuals and terminologies used when discussing suppressor genes provided a reasonable lacking the phenotype. The genome-wide epigenetics, the biological hypothesis mechanism for silencing of those genes association study (GWAS) was used as being studied and the potential cellular and for their contribution to increased a model in these surveys, but instead of and molecular processes mediating the cellular growth. These observations raised looking at the relative allele frequencies phenotypic changes, as we discuss in the intriguing possibility that events other of DNA variants, these epigenome-wide detail below. Second, DNA methylation than DNA sequence mutations were association studies (EWAS) tested whether the can change in response to a diverse range occurring in cancer and caused cells to DNA methylation at individual or groups of of influences and does not solely reflect become chronically altered in terms of adjacent cytosines in the genome differed a chronic alteration of transcriptional transcriptional regulation. systematically in those with the phenotype regulation in response to a perturbation. The question then arose whether similar of interest. For example, a well-recognized source cellular changes were taking place in disease EWAS has become a fast-growing area of variability of DNA methylation is the phenotypes other than cancer. Of particular of research7. The typical results published presence of systematic differences in interest were those in which environmental for EWAS are those of significant changes cell-subtype proportions between the groups exposures of various types were linked to in DNA methylation of modest degree, tested14,15. DNA sequence polymorphism is altered disease susceptibility, in particular substantially less than the potential also increasingly recognized to have a very those occurring a long time before the maximum change of 100%. It is generally powerful influence on transcriptional phenotype emerged. The observation that possible to infer associations between loci regulation, estimated to account for DNA methylation could be transmitted from changing DNA methylation and the likely a substantial proportion of differences of parent to daughter cells4 had prompted this functions of proteins encoded by nearby DNA methylation between individuals. transcriptional regulatory mechanism to be genes, indicating that not only are specific Twin studies show the heritability of DNA

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Box 1 | A history of the word ‘epigenetics’ people who have already developed a phenotype. It has now been shown that DNA The adjective ‘epigenetic’ is derived from ‘epigenesis’, a theory of embryology that describes methylation in peripheral blood leukocytes specialized tissue structures developing from non-specialized precursors. Although the need for can be altered in response to increased such a theory is perhaps puzzling today, it was the counter to the preceding theory of body mass index25,26 or altered blood lipid ‘preformationism’, which instead proposed that development started with a miniature version 27 of the organism in a gamete. Aristotle (384–322 bc) was an early voice dissenting from the theory of profiles . Reverse causation occurring preformationism, having made observations of development in chickens that were inconsistent at the molecular level occurs when DNA with a preformationism process. With the development of microscopy in the 17th century, it was methylation is altered by transcription confirmed that mature organs were formed from structurally different precursors, referred to as an through the locus28,29, reflecting rather than emerging property of the developing organ, or epigenesis. causing the transcriptional differences In the 20th century, Waddington combined training in Drosophila melanogaster genetics (in the observed. laboratory of T. H. Morgan) and embryology, which involved microsurgical manipulations and grafting As a result of these issues, we have of embryos in tissue culture. His focus was on the regulators of cellular differentiation during proposed that no EWAS to date, which development, attempting to understand how cells changed their differentiation fates, responding to includes our own studies, can be said to inductive effects evoked by humoral factors acting on cells with different competence to respond. be fully interpretable8. The goal of this He described these effects in terms of what he called the epigenetic landscape67,98, depicted in terms of a hillside within which a valley branches out into a continuously bifurcating delta of valleys, down article is to focus on how this situation which a ball could be imagined to roll, representing cell differentiation and distinctive fates (FIG. 1a). can be improved and how the concerns of Waddington’s goal was to bridge the gap between the two fields of embryology and genetics99, vaguely defined terminology and biological with the downhill slope the process of epigenesis and the delta of valleys of cell fates formed because hypotheses, confounding factors and of the influence of genes. His goal to reconcile the embryologists’ model of epigenesis and the ambiguous causation can be addressed. potential role of genetic influences in development was reflected by his describing the cell fate Although we focus on humans and on landscape as epigenetic, fusing the words epigenesis and genetic. diseases other than cancer, and on the Waddington’s idea was re‑interpreted in 1958 by Nanney, who made the assumption that analysis of DNA methylation rather than epigenetic systems were those responsible for cellular memory, whether differentiation in differences in gene expression or chromatin a vertebrate cell or the memory of a prior exposure to galactoside by Escherichia coli33. In the states between cases and controls, many of 1970s, this idea of cellular memory as an epigenetic property of cells was taken a step further by both Riggs5 and Holliday6, who noted that DNA methylation offered a potential transcriptional these insights should apply more broadly. regulatory mechanism that could be maintained through cell division. The further re‑interpretation We propose that, although we currently by Riggs and Holliday described epigenetic properties more in terms of Nanney’s definition face substantial challenges in generating focused on cellular memory and less attributable to Waddington’s model reconciling the model of results that are interpretable in terms of epigenesis with genetics, what could be described today as the field of developmental genetics. their underlying biological models, these By describing DNA methylation as epigenetic, the definition shifted from a cellular to a molecular challenges all appear to be surmountable, focus, involving processes that are heritable in daughter chromatids following cell division. allowing unprecedented characterization 100 101 The two models of genomic imprinting and X chromosome inactivation share the of the sources and mechanisms underlying characteristics of, first, silencing of one of a pair of alleles and, second, occurring early during phenotypic variation in humans in this development (during gametogenesis or early embryogenesis, respectively) and persisting through post-GWAS era. the lifetime. The findings that DNA methylation and other transcriptional regulatory mechanisms were distinctively organized on the active and silent alleles, subsequently allied with the explosion of new assays for studying such regulatory processes genome-wide, prompted the latest The complex definition of epigenetics definition of epigenetics. In this definition, epigenetics is back-translated to its imagined original Some of the current debate about the Greek roots, “the inheritance of variation (-genetics) above and beyond (epi-) changes in the DNA applications, interpretation and study sequence” (REF. 102). We describe this as the ‘epi+genetics’ definition, encompassing nearly design of EWAS is because of the extremely all regulatory differences between cells, interchangeable with the generic description vague definition of the word ‘epigenetics’. ‘transcriptional regulatory’, which is probably a more appropriate term to use in most cases of current In BOX 1 we describe the evolution of the use of the adjective epigenetic. At present, the cellular inheritance aspect of the definition of Riggs use of this term, which is also the focus of and Holliday is often implied but not explicitly tested, whereas the original Nanney and Waddington some excellent reviews30,31. What is apparent emphases on cellular memory and cellular properties involved in development have been lost. from this historical summary is that the definition has changed to reflect what we could study at the time: the early definitions methylation to be between 18%16 and also known as mQTLs) that have identified were proposed in an era when we were 37%17, whereas studies in human HapMap tens of thousands of CpG dinucleotides at limited to testing cellular developmental lymphoblastoid cell lines (LCLs) estimated which DNA methylation levels are affected events during morphogenesis, whereas more 22–63% of the DNA methylation variability by common genetic variation in human recent definitions are derived from the major to be due to DNA sequence polymorphism18, populations18,20,22–24. However, many EWAS advances in understanding the biochemistry a study of a three-generation family do not measure or account for genetic of transcriptional regulation and by increasing this estimate to 80%19, and more effects on DNA methylation. A third issue is computational biologists using genome-wide recent studies estimating DNA sequence reverse causation. If the starting hypothesis data from assays studying these regulators, in polymorphism effects to account for is that DNA methylation changes are part each case treating transcriptional regulatory 13.9% of DNA methylation variability in of a chronic alteration in transcriptional processes as a type of information separate monocytes20 and 65.7% in a study of various regulation leading to the phenotype studied, from and controlling the function of DNA cell types21. The importance of genetic it is often not be possible to find conclusive sequence. This transition over time from effects is also emphasized by studies of DNA evidence for this hypothesis in the usual developmental biology to molecular biology methylation quantitative trait loci (meQTLs; cross-sectional study design that includes and informatics is substantial, making it

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unsurprising that there is currently room for broad definition that we describe as interest and then molecular studies should major differences of opinion about the most epi+genetics (BOX 1) is that it equates be designed that can test this cellular appropriate use of this term. ‘epigenetic’ with ‘transcriptional regulatory’ hypothesis, as discussed in detail below. This molecular processes, encompassing any shifts the emphasis productively, defining Cellular reprogramming. Rather than non-DNA candidate genomic regulator epigenetic properties being those of cells discussing the relative merits of different that distinguishes cells, including a diverse and not equated loosely with a broad range definitions of epigenetics, acknowledging range of chromatin properties and small of molecular transcriptional regulators, in their wide range of historical roots (BOX 1), RNAs. However, it is not reasonable to effect returning the definition of epigenetics we prefer to focus on the most useful infer that all molecular genomic regulators to its original roots. An advantage of an definition for epigenetics when carrying out have the ability to mediate cellular memory emphasis on cellular epigenetic properties association studies with phenotypes and the and thus be capable of mediating cellular is that it is likely to be simpler to develop implications of such definitions in terms reprogramming. For example, unlike testable hypotheses focused on cell states and of the underlying biological models tested plants, animals do not appear to have fates, which can then be explored in terms of by these studies. Although such epigenetic RNA-dependent RNA polymerases38, the diverse molecular mediators involved in association studies are frequently proposed making it difficult to understand how transcriptional regulation. or published without first defining a clear small RNAs could by themselves propagate hypothesis about what is happening to cells effects over multiple rounds of animal cell Refocusing on TFs being tested, it is probably reasonable to division. However, small RNAs should be The current epi+genetics definition, as we generalize that most such studies aim to test reasonable reporters of the effects of cellular describe it, encompassing all kinds of putative how different environmental conditions reprogramming, and it is plausible that by transcriptional regulators has one surprisingly or perturbations lead to changes in the inducing changes of chromatin states they universal omission. A successful EWAS properties of one or more cell types that may exert effects beyond the period of their typically defines loci where DNA methylation are associated with the development of the own presence in a cell lineage39. The second differs in multiple individuals with the phenotype. A specific example of this is challenge is that the molecular mechanisms phenotype compared with individuals without the Developmental Origins of Health and that have been shown to mediate cellular the phenotype. Disease (DOHaD) hypothesis32 of early memory are not limited to this function. developmental events, such as perturbations For example, DNA methylation has been of the nutritional state in utero, having convincingly shown to associate with Glossary a considerable effect on disease risk much classical paradigms of epigenetic regulation later in life. This implicitly refers to the such as imprinting and X chromosome Canalization The maintenance of a cell and its progeny within 33 40–42 definition of epigenetics by Nanney , which inactivation , but this does not imply a differentiation lineage. The term refers to the canal-like focuses on cellular memory of past events that DNA methylation occurring elsewhere structures depicted by Waddington in his epigenetic affecting their later functional state and to in the genome is always propagating an landscape, which he described not as canals but as the proposed plasticity of cell states during epigenetic cellular memory. creodes, a neologism from biblical Greek words meaning ‘necessary’ and ‘path’. development34–36. Overall, the cellular model underlying epigenetic association Redefining epigenetic properties as those of Epigenetic studies can generally be described as one cells. We currently describe many molecular We define an epigenetic property as that of a cell, of cellular reprogramming, akin to, but genomic processes as epigenetic, whether mediated by genomic regulators, conferring on the cell the much more limited in degree, than the heritable or not. This laxity of definition ability to remember a past event. cellular reprogramming that occurs in allows us to make the logical leap that any Epigenome-wide association studies the production of induced pluripotent changes we see of these transcriptional (EWAS). Studies of the epigenome for nonrandom stem cells37. We make the case below that regulators in a population of cells are association of a difference in organization of a genomic transcription factors (TFs) are likely to mediating a cellular memory of prior regulator, comparing individuals with a phenotype with individuals lacking the phenotype. The epigenome is itself have a major mechanistic role in mediating perturbation. In turn, this allows an EWAS defined as the genome-wide distribution of transcriptional the chronic alterations of transcriptional to be founded upon the hypothesis that by regulators believed to mediate the memory of past regulation within a canonical cell type identifying differences in any of this broad cellular events. sought in a typical EWAS. A more overt group of molecular epigenetic regulators, focus on the idea of cellular reprogramming we have identified a mediator of cellular Genome-wide association study (GWAS). A study that looks for association between genetic should be of value in designing and reprogramming. This leads to a problem variation and a high-level trait such as disease or interpreting EWAS, whereas currently of interpretation of the completed EWAS. biomarker across individuals, typically scanning millions of this cellular model is generally not stated It should be emphasized that it is perfectly genetic variants genome-wide for association signals. explicitly as the hypothesis being tested, reasonable to use any of the broad range Polycreodism either in descriptions of EWAS designs or in of candidate transcriptional regulators as A systematic variability of cell fate decisions to create reports of the results of the studies. reflective of cellular reprogramming, but a distinctive repertoire of cells in a tissue. without any requirement that they mediate Equating epigenetics with molecular this process. Quantitative trait loci processes. In an EWAS, evidence supporting As an alternative approach, we propose (QTLs). Loci in the genome at which genetic variation is associated with molecular variation across individuals. For changes in cellular reprogramming is the following: the EWAS should be based example, individuals with a particular single nucleotide sought by testing molecular characteristics initially on clearly defining a model for variant have altered expression levels of a gene (eQTL), of that cell type. The first problem that the cellular events that are believed to be altered DNA methylation (meQTL; also known as mQTL) or emerges from the use of the current, occurring to mediate the phenotype of altered chromatin state (chromQTL).

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These DNA methylation changes therefore X inactivation53. Polycomb group proteins original epigenetic landscape67 (FIG. 1a), occur in a sequence-specific manner. were first recognized in Drosophila this model can be shown as the emergence However, the enzymes responsible for melanogaster to have the properties of a minor, mosaic subpopulation of DNA methylation and other changes often of maintaining long-term silencing of distinctively altered cells occurring to slightly associated with gene regulation (such homeotic genes during development54,55. expand an existing canal (or creode, as as histone modifications) do not have In mammalian cells, the histone H3 lysine 27 Waddington described them67) that depicts DNA-binding domains with the ability to trimethylation (H3K27me3) modification a canonical cell type (FIG. 1b). This model target restricted sets of sequences in this way. deposited by the PRC2 complex is enriched includes cellular reprogramming events Targeting transcriptional regulatory events at genes encoding the TFs that could that lead to regulatory changes of a variable to specific DNA sequences in mammalian potentially transdifferentiate the cell56. number of genes, without identifiably genomes typically involves the activities of The processes that target Polycomb to changing the overall cell type, as discussed TFs. TFs have all of the properties required specific loci to confer the memories of in further detail below. This cellular to direct transcriptional regulation43, to prior cell states appear to involve TFs in reprogramming model can also be applied mediate environmental influences44 D. melanogaster 57–61, but in mammals the to stable genomic regulatory changes in and to maintain cellular memory45, doing sequence-specific targeting of Polycomb is long-lived postmitotic cells, which removes so in a sequence-specific way46. Possibly less well understood. In addition to a role the need for its molecular mediators to be because of their large numbers and the for TFs, long non-coding RNAs (lncRNAs) heritable through cell division. technical challenges in mapping binding of a and pre-existing histone modifications also particular TF throughout the genome47, they appear to contribute, with CG dinucleotide- Polycreodism. However, there is a are understudied when looking for mediators richness of target sequences being another second cellular model to consider. When of cell perturbations and memory. Another feature of mammalian Polycomb targets the cellular reprogramming event occurs factor is probably the assumed relationship (as previously reviewed in REFS 62,63). in differentiating cells as a response to between TF binding and DNA methylation, Altogether many of the specific mechanisms perturbation, during embryonic or later the likely preconception being that DNA of the Polycomb complex and its inheritance development, it may influence cell fate methylation directs TF binding and not vice through DNA replication are unknown64, decisions, leading to differences in the versa. However, recent studies of the dynamic Polycomb appears to have a general role repertoire of cell subtypes formed within changes in both TF binding and DNA in the canalization of cell fate decisions in a tissue, but without necessarily involving methylation in differentiating embryonic Waddington’s epigenetic landscape model. changes in the molecular characteristics stem cells support a model in which We can therefore regard the mediators of of any of the mature cells composing the TF binding induces the local loss memory of past cellular states as involving differentiated tissue. We describe this as of DNA methylation in many situations48,49. TFs regulating targeting to specific polycreodism — the variation in proportions DNA methylation assays can therefore be sequences, with a role for lncRNAs in of cell types formed during development as regarded as a way of footprinting TF binding some cases and with Polycomb involved to a response to a perturbation. The depiction patterns in the mammalian genome. However, mediate long-term silencing events. The of this model is different from that of the there are situations in which the presence of examples of X chromosome inactivation cellular reprogramming affecting more DNA methylation exerts a primary role to and genomic imprinting also involve TFs differentiated cells, with no change in the influence TF binding50, making it difficult to at early stages, with Yy1 helping to establish location or structure of each creode, but generalize about the complex interrelationship X chromosome inactivation52, whereas a change in their relative depths (FIG. 1c). of these molecular events. Zfp57 has a comparable role in selecting This model of variability of cell fate decisions loci for genomic imprinting51. As each of was proposed by Waddington as a means by The role of TFs in paradigms of epigenetic these TFs bind or function elsewhere in which genetic variation or environmental regulation. Returning to the major the genome at loci not undergoing these changes could influence development67. This paradigms of epigenetic events in the paradigmatically epigenetic regulatory polycreodism model is of particular interest genome, both genomic imprinting51 and processes65,66, the point is reinforced that the for DOHaD studies. Although such studies X chromosome inactivation52 involve mediators of memory of past cellular events have generally focused on testing the cellular specific TFs functioning at an early stage do not act exclusively to confer what could reprogramming model, long-term memory as part of a multi-component process. be called the epigenetic properties of the of a perturbation during development can However, once established, the two alleles cell. Understanding the precise molecular also be created by an altered repertoire of with distinct transcriptional regulatory mechanisms conferring cellular memory in cells in an organ or system that can become states are identically exposed to a repertoire different contexts is a key requirement in fixed and persist throughout the lifespan of TFs but function in a manner that is epigenetics research and should be open to of the individual. In practice, it should be dependent on their pre-existing states. the possibility that TFs are involved. stressed that the distinction between the Therefore, although TFs have a primary role cellular reprogramming and polycreodism in establishing transcriptional regulatory Cellular epigenetic models models will be limited by our ability to define patterns, there are other mechanisms to Cellular reprogramming. As described and distinguish cell types, and that testing mediate a memory of prior cellular events above, the typical, current cellular for polycreodism in an EWAS-like study that can overcome the ongoing presence of model of epigenetic perturbation is the design can be very challenging. Despite these TFs. Major candidates for mediating this reprogramming of one or more cell types caveats — which are discussed further below resistance to TF activity are the Polycomb in individuals with a phenotype, compared — we view polycreodism as a fundamentally repressive complexes (PRCs), with PRC2 with the same cells in individuals without important cellular model of human disease recruited at an early stage of mammalian the phenotype. As depicted by Waddington’s that should be more widely considered.

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a b c Epigenesis

Genetic Cellular reprogramming Polycreodism | Figure 1 Epigenetic landscapes of cell reprogramming and cell-fate within an existing creode of differentiated cells intoNature which Reviews a small | Geneticsminority changes. The cell fate decisions made during development are repre- of cells move but retain the apparent identity of cells of the original creode. sented by Waddington’s epigenetic landscape, which consists of a ball c | The deepening of one creode depicts the increasing likelihood of cells rolling down a hill within channels (‘creodes’), with bifurcations represent- ending up in that differentiated state. Such variability in creode use ing differentiation choices. a | In this lineage decision, the cell has an equal between individuals is described in this Opinion article as polycreodism likelihood of ending up in one of two different creodes. b | Cellular re and represents an alternative model for perturbations affecting programming changes the epigenetic landscape, in which the emergence genomic regulation during development, with potential phenotypic of a mosaic subset of a canonical cell type is represented by a new channel consequences.

Empirical support for the polycreodism this micronutrient deficiency during differences in the proportions of cell model comes from some fascinating development also predisposes to renal subtypes between cases and controls recent examples of developmental disease later in life. would be reflected by changes in DNA perturbations. One such observation Perturbations of cell fate decisions acting methylation across a large number of loci. has been made about micronutrient through TFs are not limited to the effects However, this has legitimately been thought deficiency during mouse development of micronutrients. Endocrine-disrupting of as an influence merely confounding as a mechanism for later susceptibility chemicals (EDCs) modify normal endocrine the interpretation of the results of a to an adult asthma-like phenotype. system function and represent a major study testing the cellular reprogramming A retinoid-deficient environment during area of interest in epigenetics research71. hypothesis14,15,75. Differences in cell-type embryonic days 9.5–14.5 in a mouse One well-studied example of EDCs is the proportions between cases and controls model was shown to result in increased organotin family, members of which are used are often thought of as consequences or amounts of smooth muscle around the as pesticides and in the manufacture of poly‑ correlates of the phenotype rather than airways in mice that appeared otherwise vinylchloride (PVC) pipes. In utero exposure causes and are eliminated analytically to the healthy68. Pulmonary function tests of mice or Xenopus laevis to the organotin greatest extent possible in EWAS to avoid carried out on these mice in adulthood tributyltin chloride leads to accumulation false-positive findings14,15,75,76. However, revealed increased airway resistance68, of fat from birth to adulthood, leading such well-intentioned rigour applied to the representing the non-inflammatory part these compounds to be called obesogens72. retinoid-deficient mouse example described of a phenotype resembling human reactive Mechanistically, tributyltin appears to above would eliminate the disease-mediating airways disease or asthma. A very similar function at least partially through the outcome of the developmental perturbation: phenotype of increased smooth muscle peroxisome proliferator-activated receptor-γ the accumulation of smooth muscle around the airway has also been found (PPARγ) and the retinoid X receptor surrounding the airways of the lung. The in mice exposed to a vitamin D‑deficient (RXR)72, causing mesenchymal stem cells polycreodism cell model is certainly tenable environment in utero69. Both vitamin A, to differentiate preferentially into the in any phenotype in which the alteration of the dietary precursor of retinoic acid, and adipocyte lineage73. Again, the phenotypic the repertoire of cells forming the organ may vitamin D are essential micronutrients effect appears to be mediated by a change in be mechanistically important in causing the that, in combination with retinoid X choice of lineage commitment rather than phenotype. receptors, function as nuclear receptors. requiring the reprogramming of a specific These nuclear receptors represent cell type. Such a change in the proportions The new challenge of defining a cell type well-known examples of TFs that regulate of cell subtypes forming an organ or a Successfully carrying out a study associating gene expression and when deficient have system and contributing to disease risk is epigenetic properties, however defined, with the unexpected effect of causing changes a familiar model for immunologists, who a phenotype can therefore be appreciated in cell-type proportions within a tissue have made many observations linking to be intimately related to definition during development through altered cell immune repertoire formation and risk of and quantification of the cell subtypes fate decisions. The phenotypic effects of inflammatory or autoimmune diseases74. present in samples tested. Not only is vitamin A deficiency during development this needed to overcome the influence may not be limited to pulmonary Polycreodism and cell-subtype adjustment of systematic changes in cell-subtype manifestations: mild retinoid deficiency strategies. Variability of cell fate potential proportions as a confounding influence during pregnancy is also associated with is never the cellular model tested in on DNA methylation studies14,15,77 but also decreased numbers of nephrons in the current EWAS. Within the heterogeneous to generate evidence supporting a cellular kidneys70, supporting the possibility that tissues unavoidably sampled in EWAS, reprogramming versus a polycreodism

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cellular model. However, there is a key issue is a phenotype-associated change in the CD4+ memory T cells to consider: how do we define a cell type proportion of a mosaic subset of cells with CD4+ memory antiviral T cells in the first place? Is what we call cellular distinctive DNA methylation, at sites where CD4+ naive T cells reprogramming flexibly modifying the DNA methylation is different between the function of a canonical cell type, or are cell subtypes present. 1.0 cell identities discrete and discontinuous78, Many EWAS to date have made efforts so that cellular reprogramming generates to adjust for the confounding effects of outcomes comparable to those occurring cell-subtype composition, which is essential 0.8 during cell fate determination that result in for identifying cellular reprogramming polycreodism? events. Some studies use cell sorting or selection based on surface markers to limit 0.6 Three levels of cell-type definition. cell-type diversity, although we have shown Cell types can be thought of as being that this is not by itself sufficient to generate distinguishable at three levels. The populations that are homogeneous in terms 0.4 traditional approach with lowest of molecular regulators81. The computational resolution is based on histology, which approaches that aim to infer cell-subtype Cell-type proportion involves looking for morphological proportions are divided into those that differences. By using different types use reference DNA methylation data from 0.2 of markers, histologically identical the cell subtypes that are believed to be cells can be discriminated further. For present14, and those that assume covariation example, although lymphocytes look very of changes in DNA methylation at multiple 0 homogeneous by histology, they can be loci in the genome to reflect the effects of Age 0yr Age 40yr Age 90yr subdivided using cell surface markers a presumed number of latent cell subtypes Figure 2 | Preliminary data from scRNA-seq 75,76 Nature Reviews | Genetics into numerous different B cell and T cell present . However, even after carrying out analysis. We show the results of deconvolution subpopulations. However, the new era of these corrective measures, the residual DNA of bulk cell population RNA sequencing (RNA- single-cell transcriptomics has revealed methylation changes have continued to be seq) data, using CD4 + T cell subpopulations a much finer level of resolution at the of only modest degree. This indicates that detected in a reference single-cell RNA-seq molecular level, discriminating beyond either cellular reprogramming is affecting (scRNA-seq) resource. Specifically, we used refer- even cell surface marker classifications a mosaic subpopulation of cells or that ence scRNA-seq data from 33,000 peripheral and uncovering an increasing number of these analyses have remained vulnerable to blood mononuclear cells (PBMCs) from the cell types and subtypes79. The resolution the influences of cell subtype, which may 10x Genomics Chromium v1 system (see 10x Genomics reference single-cell RNA-seq data), to distinguish cell types depends not only not have been recognized in the lack of from which we identified 31 cell subtypes using on the cell-type diversity of studied tissues molecular genomic level of resolution. the Seurat software84 and selected 156 genes but also on technical factors, with greater that are differentially expressed between three capacity to discriminate cell subtypes when Using single-cell sequencing to measure major CD4+ T cell subtypes. We then used the more cells are tested and deeper sequencing cell subtypes. Single-cell sequencing has Cibersort software with default settings85 to is carried out. For these technical reasons, quickly emerged as a powerful approach to apply the expression signatures of these 156 and sometimes also because of biological characterize cell-subtype composition at genes for estimation of the proportions of the continuity between cell types, the patterns higher resolution and accuracy than by using three CD4+ T cell types in a public RNA -seq + of single cell profiles can sometimes previous strategies, providing direct insights data set of bulk CD4 T cells in individuals of dif- defy easy classifications into discrete into the functional state of each cell and ferent ages (see Gene Expression Omnibus accession number GSE65515)86. We note the categories79,80. allowing characterization of cell populations age-associated shift from naive to memory T cells that are not well captured by cell sorting. and the ability of scRNA-seq to distinguish Current EWAS cell-subtype adjustment Although single-cell bisulfite sequencing multiple CD4+ T memory cell types. DNA methyl- strategies. The potential influence of technologies are being developed to allow ation studies of these cells would probably have unrecognized cell-subtype diversity is DNA methylation analysis at the single-cell shown moderate changes between samples and apparent from the results of EWAS studies level82,83, by far the most mature and scalable age groups, reflecting these unrecognized, themselves. In every EWAS to date, without single-cell technology is single cell RNA mosaic cell-subtype changes. known exception, statistically significant sequencing (scRNA-seq). We show its use associations have been based on modest for cell-type composition quantification in changes of DNA methylation levels between FIG. 2 with a fairly simple analysis. details and data sources, see FIG. 2). The cases and controls. DNA methylation cannot We used reference scRNA-seq data from analysis shows a robust signal of the known change to such a limited extent within an 33,000 peripheral blood mononuclear cells shift from naive towards memory CD4+ individual cell, as the cytosine on a pair of (PBMCs) to identify differentially expressed T cell types with age87, although we note that chromosomes can be methylated genes between four major CD4+ T cell the proportions of the memory CD4+ T cell on neither (representing 0% methylation), subtypes84, and we then used the expression subpopulations were sensitive to the choice both (representing 100% methylation) signatures of these 156 genes for estimation and processing of the reference data sets. or (relatively rarely) one of the alleles of the proportions of the four CD4+ T cell This analysis shows that although (representing 50% methylation). Thus, types in a public RNA-seq data set of bulk decomposition analyses based on scRNA-seq a modest change in DNA methylation of, CD4+ T cells in individuals of different are not yet fully mature, selection using the for example, 20% can only occur if there ages85,86 (for additional methodological CD4 surface marker alone is not enough

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to enrich a uniform cell population for are responsible for modified regulatory the sample collection challenges, but are interpretable epigenetic association studies properties and function of cells, without essential to enable the discovery of smaller and that cell-subtype proportions can vary changing the overall cell type. effects that may be biologically interesting substantially, even between individuals and, importantly, allow more advanced of the same age. It could be argued that Study subjects and biospecimens. statistical modelling and correction of a large scRNA-seq is not essential to quantify Cross-sectional study designs commonly number of covariates, as discussed below. CD4+ T cell subtypes, as they can be used in EWAS — typically case–control The choice of tissue sampled needs a clear quantified by flow cytometry, but as studies — are affected by reverse causation, biological justification. Is the hypothesis scRNA-seq has even revealed cell types for because any association between a cellular that the cells in this tissue mediate the which surface markers are not known79 phenotype such as DNA methylation and phenotype, or do they report an exposure and can be applied to tissues for which a disease phenotype can be a result of that causes the phenotype in a different flow cytometric markers are less developed causality in either direction. One way to tissue? If the cells are likely to merely reflect than haematopoietic cells it looks to be define causality is by revealing the temporal the consequences of an existing phenotype, a promising approach to apply in functional development of cellular changes using they may be suitable for biomarker use but genomics studies in general. We conclude longitudinal sampling, with other analytical not for testing for cellular reprogramming. that scRNA-seq now allows fine-grained options discussed further below. The Furthermore, it is likely to be a more analysis and correction for cell-type necessary sample size depends on cell-type informative approach if the cells being composition in epigenetic association diversity, biological models and the desired tested have been found to have a distinctive studies and that emerging single-cell analysis. Large numbers of samples increase phenotype. Although samples are always epigenomic assays will provide an additional layer of information not only for cell-subtype | analysis but also for studying mechanisms of Box 2 Testing for cellular reprogramming gene regulation at the cellular level. We divide these guidelines into two parts: those focused on revealing high-confidence differences between the groups studied and those specifically needed to allow interpretation of the results as The second-generation EWAS involving cellular reprogramming. Defining the hypothesis. We propose several Revealing high-confidence differences approaches and practices for improving the Design of EWAS. Questions at the outset of an epigenome-wide association study (EWAS) should interpretability of EWAS, which we refer include whether the specific hypothesis is best tested with cross-sectional, longitudinal or twin to collectively as the second-generation cohorts. Can the cell type tested be proposed to have a biologically realistic link to the exposure EWAS. The first step is basic: to define and/or phenotype? Is the cohort size adequate, not only for the primary association but also for the hypothesis being tested. This article the correction of other confounding factors (such as methylation quantitative trait loci (meQTLs))? emphasizes the need to clarify the proposed What are the analytical approaches and resources in place to handle large, multidimensional data sets? What is the plan for validating the findings? cellular epigenetic hypothesis being tested as the priority, as discussed above, indicating Replication. Significant findings should be replicated in an independent cohort to protect against how cellular changes are mechanistically spurious findings. It should be noted that biases might replicate across data sets. causal to a disease. In biomarker studies Interpretation of the results in light of cellular reprogramming in which the question of causality is not Accounting for sources of variability. Cell-type composition, genetic variation and transcription of interest, many components of sec‑ should be measured in all subjects. In data creation and analysis, technical artefacts and batch ond-generation EWAS will apply, but much effects need to be considered and controlled for. As much quantitative information as possible more straightforward study designs may should be captured about other factors that are associated with the phenotype and possibly with the molecular trait (for example, metadata such as age, sex and diet) to avoid spurious correlations. be sufficient as interpretability is not the priority. Of the cellular epigenetic models, Analysis. Testing for association between phenotype and molecular traits is fairly straightforward the hypothesis of cellular reprogramming per se, but normalization and correction of confounders adds substantial complexity. It is essential is broadly amenable to EWAS-like study for epigenetic association studies to establish statistical quality control tools and best practices. A quantile–quantile plot of P‑values (the most classical genome-wide association study (GWAS) designs that use observational molecular quality control plot) is extremely informative about the distribution of effects genome-wide: few data of people with appropriate phenotype significant loci with the vast majority of the data following the null hypothesis would pinpoint and environmental exposure data, with specific potentially reprogrammed loci, whereas inflated P‑values genome-wide are a sign of the caveats and opportunities discussed extremely widespread association with the phenotype, or (more likely) confounders that have below. By contrast, the hypothesis been insufficiently accounted for. underlying the polycreodism model is not Interpretation. High-confidence changes in a molecular characteristic such as DNA methylation as straightforward to test because it is likely can indicate several interesting underlying processes. The confounding variables are by to be very difficult to distinguish whether themselves of major potential value in understanding processes leading to phenotypes, so that the change in cell proportions is the cause or when a DNA methylation change is discarded because it is secondary to, for example, transcription consequence of the phenotype. Approaches through that locus, what has been revealed is a gene or non-coding RNA expression difference to study causal polycreodism are likely that is nonrandomly associated with the individuals with the phenotype and is thus potentially to include animal models, such as those involved in mediating the phenotype. It is worth starting with the view that all associations are due discussed above, but these methods are not to events other than cellular reprogramming, and by systematically excluding everything yet established. Therefore, in this section, we attributable to confounding factors any remaining changes can be defined as indicative of cellular focus on the usual cellular reprogramming reprogramming. It remains important to be cautious about inferring causality even at this stage: extensive downstream studies will be required to test causality for an association, which should BOX 2 model (summarized in ), with studies not be a requirement of an EWAS. aiming to pinpoint loci in the genome that

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going to be heterogeneous in terms of genomic regulators that are markers of loci generated presents computational challenges. cell subtypes, even after purification81, it likely to drive cellular reprogramming. However, we believe that the costs involved is essential that the cell type of interest is Genome-wide assays testing regulatory are ultimately justified by the markedly as free from contaminating cell types as features of the genome are extremely diverse improved interpretability of results. possible to obtain maximum sensitivity to and numerous, raising the question of This combination of assays should have detect DNA methylation changes of modest whether some are better than others for greatly improved sensitivity and specificity degree and to facilitate correction for identifying cellular reprogramming. to identify cis-regulatory loci that differ cell-subtype diversity. To detect low-level mosaic changes within between cases and controls in all or a subset a cell population, DNA methylation studies of cells being studied, compared with Cellular subtype quantification. The point are inherently more sensitive than the broad traditional DNA methylation analyses has been stressed already, but a central group of chromatin immunoprecipitation carried out in isolation. As changes in DNA component in the testing of cellular (ChIP)-based assays, which are not nearly methylation and loci of open chromatin reprogramming models is determining as quantitative. As cellular reprogramming may be merely reporting alterations of TF the cell-subtype composition of samples as could be associated with the relocation of binding that represent the primary causal a major confounding variable. It appears cis-regulatory elements within the genome, molecular change, as discussed above, these that scRNA-seq represents a generally useful there is no a priori way of predicting assays can reveal the loci where TF binding approach, being both widely accessible and which loci to test, requiring that molecular is altered in association with a phenotype. fairly affordable, and could potentially be profiling be as comprehensive as possible The subsequent inference of the potential applied to tissues in which cell subtypes throughout the genome, rather than limited mechanisms of reprogramming involving have not been well studied. scRNA-seq to predefined loci. Whole-genome bisulfite these loci should include those potentially allows identification, discrimination and sequencing (WGBS) is the assay that allows mediated by TFs. One approach to identify quantification of cell subtypes present most potential cis-regulatory loci to be tested TFs that mediate cellular reprogramming in a sample. scRNA-seq may not need to for DNA methylation (5‑methylcytosine is to look for motifs that are significantly be carried out on every sample, but may and a smaller contribution by 5‑hydroxy enriched at the loci with high-confidence instead be performed on a limited subset methylcytosine)88. Parallel use of the assay changes in genomic regulation, which of samples to identify cell subtypes and for transposase-accessible chromatin with can help to identify known binding sites their distinctive gene expression profiles. high-throughput sequencing (ATAC-seq)89 for the one or more TFs that may be These scRNA-seq data can then be used to generates a comprehensive map of loci of regulating cellular reprogramming. This estimate through analytical approaches the open chromatin, which are the candidate can allow preliminary insights into the cell-type composition of samples on which cis-regulatory loci for that cell type and are upstream signalling involved in the cellular bulk RNA sequencing has also been carried valuable for pinpointing the locations where reprogramming process. out. Failing the availability of single-cell DNA methylation changes are more likely molecular studies, characterization of the to have regulatory effects. Furthermore, Interpreting the results. The loci that cell population with flow cytometry or at measuring gene expression by RNA-seq emerge from this stringent analysis looking least quantitative histology will allow some can be valuable for linking changes in the for cellular reprogramming should be degree of insight into the cell-subtype chromatin to active gene expression and for expected to include those that cause the composition present in the samples tested. taking into account the reverse causation phenotype, changes associated with These estimates of cell-type proportions can effects on DNA methylation of transcription the phenotype but due to reverse causation then be included in association models to through a locus28,29. The RNA-seq data (in which the phenotype induces molecular remove their effect on EWAS findings, can also be used with reference single-cell changes in the cells8,25,27) and loci showing in order to hone in to the more limited transcriptomic information to estimate correlations with confounding factors number of loci at which regulatory changes cell-subtype proportions present, as that have not been recognized or fully indicative of cellular reprogramming events described earlier. Genotyping is also accounted for. We show some examples of are taking place. Finally, we note that even needed to account for the effects of DNA potential interpretations of certain types though it is possible to test for association sequence polymorphism on methylation of results in FIG. 3. Although the latter between cell-type proportions and the and other molecular phenotypes. The first class of loci can be reduced — but probably phenotype of interest, the necessary null step of this analysis is genetic association not entirely eliminated — by the approaches assumption is that the differences are a analysis to identify cis-meQTLs where described above, a cross-sectional design consequence rather than a cause of the a common genetic variant in the studied comparing individuals with and without phenotype. However, such information may population sample is associated with the a phenotype cannot on its own distinguish be useful for forming additional hypotheses DNA methylation level of a given CpG reverse causation effects from cellular of biological processes that contribute dinucleotide in cis. For sites with significant reprogramming8. Reverse causation effects to symptoms of a disease, or of possible meQTLs, the genotype can then be included can sometimes be biologically and medically polycreodism, which both require separate in the EWAS models as a covariate to interesting and may be informative about studies to unravel. account for the genetic source of variance. symptoms, progression and complications Generating these multiple data sets of a disease, but rarely represent the main Multiple complementary genome-wide presents challenges, as accounting for several reason for doing an EWAS. To detect reverse assays. Testing for the cellular covariates typically requires several hundreds causation, genetic analysis can make use reprogramming model requires not only of individuals to be analysed, the additional of the fact that genetic associations with estimates of cell-type composition but assays increase biospecimen type and phenotypes are always causal. Mendelian also the results of molecular assays testing volume requirements and the volume of data randomization is probably the best-known

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analytical approach for using genetic a 100 % DNA methylation and associations to infer causality relationships DNA methylation chromatin organization 90 reflecting TF occupancy between phenotypes . In addition, as 0 % mentioned above, molecular analysis of ATAC-seq peak longitudinal samples collected before and after the development of the phenotype can be informative about the temporal (Gene expression) order of events8. In the absence of evidence for reverse causation, following up the association found in an EWAS will probably require experiments with animal or cellular b DNA sequence variation models to confirm causality. This is also Increased altering TF occupancy, true for polycreodism hypotheses. Although DNA methylation with effects on DNA methylation and laborious, working out the causality chromatin organization of associations is valuable for a deeper biological understanding of how the phenotype came about and for developing (Gene expression) interventions for disease. A well-carried out EWAS of the cellular reprogramming model is a valuable step towards this goal, allowing Sequence variant the proposition of solid, testable hypotheses of causality. c Cell-subtype proportional changes reflected by Harvesting the confounders altered DNA methylation We envision that WGBS, ATAC-seq, and chromatin organization RNA-seq, scRNA-seq and genotyping of the samples in a second-generation EWAS represent a necessary panel of assays to (Gene expression) allow interpretation of the results obtained, although the pioneering empirical studies to test the value of this comprehensive approach are still lacking. We estimate that this panel of assays costs at least tenfold more per sample than a simple DNA methylation survey assay, a substantial d investment when carried out on a sizeable Cellular reprogramming involving targeted effects cohort. This cost increase is analogous to of Polycomb and TFs many early GWAS and genome-sequencing studies, in which initial small studies were found to be inadequate, requiring their own second-generation approaches involving substantially higher investments to achieve (Gene expression) scientific success with robust findings. We anticipate that a substantial proportion of the loci with DNA H3K27me3 methylation changes will be attributable Figure 3 | Interpreting the results of a second-generation EWAS. We show a few examples of how to to confounding variables. However, in a interpret the results of genome-wide assays of transcriptional regulators whenNature these Reviews are used | Genetics to char- second-generation EWAS, confounding acterize a locus of interest from an epigenome-wide association study (EWAS). a | A transcription factor variables can be not only corrected for but (TF)-centric perspective, in which low methylated regions (LMRs)49 and loci of open chromatin represent also harvested for additional biological the footprints of TFs (hexagons) binding at cis-regulatory elements, shown in the figure to have a positive insights. For example, reverse causation effect on gene expression. b | The presence of a DNA sequence variant within the locus that destabilizes because of transcription through a locus the binding of a TF and leads to loss of the group of TFs from the locus, with acquisition of DNA methyl- makes changes of DNA methylation ation, loss of open chromatin and loss of the partial stimulatory effect on gene expression. c | An altera- at that locus less likely to be driving tion in cell-subtype composition could be mistaken for true changes in DNA methylation and chromatin profiles within a cell type, for a locus that is distinctively organized in the contaminating cell subtype and cellular reprogramming, but indicates to a degree that is dependent on the proportion of contaminating cells. d | An example of how targeted a transcriptional difference associated Polycomb-mediated silencing to that locus could prevent TF binding, again leading to changes in DNA with the phenotype, which is potentially methylation, chromatin structure and gene expression, but now because of the type of cellular an insight into why the phenotype reprogramming event sought in many EWAS. Only when the competing models that result from developed. The data will also reveal gene confounding effects are excluded can this last model be predicted with confidence to be occurring. expression QTLs (eQTLs)91–93, meQTLs24 ATAC-seq, assay for transposase-accessible chromatin with high-throughput sequencing; H3K27me3, and chromatin state QTLs (chrQTLs)94 for histone H3 lysine 27 trimethylation.

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