The Epigenome in Pluripotency and Differentiation
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Review Review The epigenome in pluripotency and differentiation The ability to culture pluripotent stem cells and direct their differentiation into Rathi D Thiagarajan1, Robert specific cell types in vitro provides a valuable experimental system for modeling Morey1 & Louise C Laurent*1 1 pluripotency, development and cellular differentiation. High-throughput profiling of Department of Reproductive Medicine, The University of California, San Diego, the transcriptomes and epigenomes of pluripotent stem cells and their differentiated La Jolla, CA, USA derivatives has led to identification of patterns characteristic of each cell type, *Author for correspondence: discovery of new regulatory features in the epigenome and early insights into the [email protected] complexity of dynamic interactions among regulatory elements. This work has also revealed potential limitations of the use of pluripotent stem cells as in vitro models of developmental events, due to epigenetic variability among different pluripotent stem cell lines and epigenetic instability during derivation and culture, particularly at imprinted and X-inactivated loci. This review focuses on the two most well-studied epigenetic mechanisms, DNA methylation and histone modifications, within the context of pluripotency and differentiation. Keywords: differentiation • DNA methylation • epigenome • histone modification • imprinting • pluripotency • sequencing • stem cells • X inactivation Early mammalian development involves genetic mechanisms in the regulation of the precise orchestration of gene expression in transcriptome, and recognition that changes a spatial and temporal manner in order to in the epigenome during differentiation can establish cell lineage fate. Starting from a point to genomic features that play key roles totipotent state, cells fated to the embryonic in the differentiation process [1] . lineages pass through a pluripotent state and In virtually every cell type and organ sys- then branch off into the germline and the tem, normal differentiation and development three germ cell lineages: the ectoderm, meso- is associated with characteristic changes derm and endoderm. Multipotent progenitor in epigenetic patterns, which allow for the cells in these major lineages then differentiate establishment and stable maintenance of a further to produce more than 200 specialized wide variety of cellular phenotypes without cell types in the fully developed organism. alterations to the genome (recently reviewed The differentiation process is accompanied in [2]). In terms of disease, epigenetic aber- by changes in the transcriptome, and much rations have been shown to result in devel- has been learned about the signals that govern opmental abnormalities, degenerative disease it by performing gene expression analyses of and cancer (also reviewed in [3–6])[7–9]. In the differentiating and differentiated cells. Such most inclusive definition, epigenetics is the experiments have demonstrated the critical study of mechanisms that change gene activ- role that transcription factors play in the regu- ity without altering the DNA sequence, and lation of temporal and spatial gene expression thereby include not only DNA methylation programs by binding to cis-regulatory regions and histone modifications, but also transcrip- in response to environmental cues. There is tion factors and noncoding RNAs. In fact, an increasing appreciation of the role of epi- there has been rather extensive debate regard- part of 10.2217/EPI.13.80 © 2014 Future Medicine Ltd Epigenomics (2014) 6(1), 121–137 ISSN 1750–1911 121 Review Thiagarajan, Morey & Laurent ing the definition of epigenetics, and in particular how noncoding RNAs (as we have recently written a review heritable a mark must be to be considered epigenetic. on the role of miRNAs in pluripotency [19]). Recent advances in the fields of cell biology and epig- enomics have demonstrated that many epigenomic The epigenome in mammalian development marks are less stable than previously believed, and that DNA methylation is an essential mechanism that has certain sequences of epigenetic events that were once been shown to play a key role in both gene regulation thought to be irreversible (e.g., progression from plu- in the context of genomic imprinting, X chromosome ripotent or multipotent stem cells to fully differenti- inactivation and regulation of some autosomal nonim- ated mature cell types) can in fact be reversed using printed genes and preservation of genomic stability. It cellular reprogramming techniques [10–12] . is becoming increasingly clear that DNA methylation Human pluripotent stem cells (PSCs) have tremen- at different genomic loci is regulated by several differ- dous potential for in vitro modeling of development and ent mechanisms and involves the interplay between disease due to their self-renewal properties and their DNA methyltransferases (DNMTs), DNA dioxygen- ability to differentiate into all cell lineages in the body. ases, cytidine deaminases and various DNA-binding Human embryonic stem cells (hESCs) [13], which are proteins. These processes are particularly active during derived from the inner cell mass of blastocyst-stage pre- early development, and evidence is growing that they implantation embryos, are considered the gold standard are important for the establishment and stabilization of PSCs. Two other types of PSCs have been produced: the pluripotent state. induced pluripotent stem cells (iPSCs), which are gen- DNMT1 is the primary maintenance DNA meth- erated from somatic cells by overexpression of a small yltransferase, and is responsible for converting the number of reprogramming factors [10,11]; and somatic hemi-methylated CpG dinucleotides that are gener- cell nuclear transfer ESCs (SCNT-ESCs), which are ated during DNA synthesis into fully methylated derived from embryos resulting from the introduc- CpGs. DNMT3A and DNMT3B are de novo DNA tion of a somatic cell nucleus into an enucleated oocyte methyltransferases, which can establish new DNA [14] . In the blastocyst, the pluripotent state represented methylation at completely unmethylated CpG sites, as by the cells in the inner cell mass is transient. In vitro well as methylate cytosines located at non-CpG sites. however, the pluripotent state as represented by PSCs Maintenance of DNA methyltranferase activity has can be artificially maintained for an indefinite period been shown to be critical for development. Mice with of time. This greatly increases the utility of the cells, homozygous mutations in Dnmt1, resulting in partial but also makes them susceptible to genomic and epig- to complete loss of Dnmt1 function, were embry- enomic aberrations [15–18]. It is therefore important to onic lethal by midgestation and were associated with assess how such aberrations may impact the efficacy of imprinting defects [20]. Mouse Dnmt3a knockouts PSCs in accurately modeling development and disease, are postnatal lethal and associated with imprinting and serving as sources of material for cell therapy. defects [21], while Dnmt3b knockouts are lethal in the The dynamic interplay between the transcriptome late embryonic period and showed defects in methyla- and epigenome warrants implementation of genome- tion of endogenous viral and satellite DNA sequences wide approaches and high-throughput technologies to [21] . Dnmt3a/b double knockouts have a more severe identify and characterize mechanisms and molecular phenotype, dying at midgestation, indicating that factors that regulate genome function during develop- there is partial redundancy between these two DNA ment, lineage-specification and disease pathology. In this methyltransferases [20,22]. article, we describe the current genome-wide profiling It has been shown that the absence of all three DNA platforms and large-scale consortiums that have utilized methyltransferases in mouse TKO ESCs results in these technologies to understand the epigenome, and near-total demethylation of genomic DNA, but does also review the existing literature with an emphasis on not affect self-renewal, expression of pluripotency publications that have utilized genome-wide approaches markers or global chromatin structure [23]. However, to describe: the epigenetic landscape of pluripotency in contrast to wild-type mouse ESCs or mouse ESCs and cellular differentiation, as modeled by undifferenti- lacking Dnmt1 only, the mouse TKO ESCs do not ated and differentiated PSCs; differences among differ- stably differentiate when induced to form embryoid ent types of PSCs; differences between in vitro main- bodies, and readily revert to a pluripotent phenotype tained ESCs and cells in the developing embryo; and when returned to culture conditions favorable for the dynamic changes in the epigenome that occur dur- undifferentiated PSCs [24]. These results indicate that ing reprogramming. We will focus on two of the most DNA methylation is dispensable for maintenance of well-studied epigenetic mechanisms: DNA methylation the undifferentiated state, but is necessary for lineage and histone modifications, and will only briefly touch on commitment. 122 Epigenomics (2014) 6(1) future science group The epigenome in pluripotency & differentiation Review Differential methylation of CpG sites in CpG may be critical for establishing the DNA methylation islands [25] and those distributed more sparsely across imprints during oogenesis [33,34]. the genome is established during development by How imprinted loci retain their parent-of-origin spe- global demethylation