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The RNA modification N 6 -methyladenosine (m 6 A) has critical roles in many biological processes 1 , 2 . However, the function of m 6 A in the early phase of mammalian development remains poorly understood. Here we show that the m 6 A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m 6 A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m 6 A RNAs act upstream of SETDB1 to repress retrotransposons and Dux , the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m 6 A RNA and YTHDC1 in chromatin modification and retrotransposon repression. N 6 -methyladenosine RNA and its reader YTHDC1 serve as a bridge to silencing retrotransposons through the RNA derived from these retrotransposons in mouse ES cells.

Key Points Transposable elements (TEs) are increasingly recognized as a potent source of regulatory sequences in eukaryotic genomes The selfish replication cycle of TEs drove the evolution of finely tuned regulatory activities that favoured their propagation and has predisposed them to be co-opted for the regulation of host genes There is a growing number of examples of TE-derived sequences that have been co-opted to regulate important biological processes in organismal development and physiology Dysfunction of TE-derived regulatory sequences is also emerging as a potential driver of diseases including cancer and autoimmunity Functional genomics and genome-editing technologies herald an exciting era for understanding the biological effect of TEs Transposable elements (TEs) are widely known for their deleterious consequences of selfish propagation and mutagenesis. However, as described in this Review, TEs also provide hosts with rich, beneficial gene-regulatory machinery in the form of regulatory DNA elements and TE-derived gene products. The authors highlight the diverse regulatory contributions of TEs to organismal physiology and pathology, provide a framework for responsibly assigning functional roles to TEs and offer visions for the future. Transposable elements (TEs) are a prolific source of tightly regulated, biochemically active non-coding elements, such as transcription factor-binding sites and non-coding RNAs. Many recent studies reinvigorate the idea that these elements are pervasively co-opted for the regulation of host genes. We argue that the inherent genetic properties of TEs and the conflicting relationships with their hosts facilitate their recruitment for regulatory functions in diverse genomes. We review recent findings supporting the long-standing hypothesis that the waves of TE invasions endured by organisms for eons have catalysed the evolution of gene-regulatory networks. We also discuss the challenges of dissecting and interpreting the phenotypic effect of regulatory activities encoded by TEs in health and disease.

The SMAD2 and SMAD3 protein interactome links TGFβ signalling to diverse effectors including m 6 A methyltransferase, which has a role in regulating differentiation of human pluripotent stem cells. SMAD interactions in stem cells TGFβ signalling is involved in many different physiological processes, including embryonic development, but there is no complete description of the tasks accomplished by SMAD2/3 proteins, the downstream effectors of TGFβ signals. Ludovic Vallier and colleagues describe the full set of interactions of SMAD2/3 in human pluripotent stem cells. They find that these interactions are linked to diverse molecular processes in addition to transcription. SMAD2/3 also regulates post-transcriptional modification of messenger RNA through regulation of N 6 -methyladenosine (m 6 A) deposition onto transcripts that are involved in early fate decisions. This is carried out during differentiation to modulate the stability of the transcript. These findings link extracellular signalling cues to post-transcriptional regulations of fate regulators. The TGFβ pathway has essential roles in embryonic development, organ homeostasis, tissue repair and disease 1 , 2 . These diverse effects are mediated through the intracellular effectors SMAD2 and SMAD3 (hereafter SMAD2/3), whose canonical function is to control the activity of target genes by interacting with transcriptional regulators 3 . Therefore, a complete description of the factors that interact with SMAD2/3 in a given cell type would have broad implications for many areas of cell biology. Here we describe the interactome of SMAD2/3 in human pluripotent stem cells. This analysis reveals that SMAD2/3 is involved in multiple molecular processes in addition to its role in transcription. In particular, we identify a functional interaction with the METTL3–METTL14–WTAP complex, which mediates the conversion of adenosine to N 6 -methyladenosine (m 6 A) on RNA 4 . We show that SMAD2/3 promotes binding of the m 6 A methyltransferase complex to a subset of transcripts involved in early cell fate decisions. This mechanism destabilizes specific SMAD2/3 transcriptional targets, including the pluripotency factor gene NANOG , priming them for rapid downregulation upon differentiation to enable timely exit from pluripotency. Collectively, these findings reveal the mechanism by which extracellular signalling can induce rapid cellular responses through regulation of the epitranscriptome. These aspects of TGFβ signalling could have far-reaching implications in many other cell types and in diseases such as cancer 5 .

This year marks the tenth anniversary of the generation of induced pluripotent stem cells (iPSCs) by transcription factor-mediated somatic cell reprogramming. Takahashi and Yamanaka portray the path towards this ground-breaking discovery and discuss how, since then, research has focused on understanding the mechanisms underlying iPSC generation and on translating such advances to the clinic. The past 10 years have seen great advances in our ability to manipulate cell fate, including the induction of pluripotency in vitro to generate induced pluripotent stem cells (iPSCs). This process proved to be remarkably simple from a technical perspective, only needing the host cell and a defined cocktail of transcription factors, with four factors — octamer-binding protein 3/4 (OCT3/4), SOX2, Krüppel-like factor 4 (KLF4) and MYC (collectively referred to as OSKM) — initially used. The mechanisms underlying transcription factor-mediated reprogramming are still poorly understood; however, several mechanistic insights have recently been obtained. Recent years have also brought significant progress in increasing the efficiency of this technique, making it more amenable to applications in the fields of regenerative medicine, disease modelling and drug discovery.

CRISPR/Cas9 has revolutionized our ability to engineer genomes and conduct genome-wide screens in human cells 1 – 3 . Whereas some cell types are amenable to genome engineering, genomes of human pluripotent stem cells (hPSCs) have been difficult to engineer, with reduced efficiencies relative to tumour cell lines or mouse embryonic stem cells 3 – 13 . Here, using hPSC lines with stable integration of Cas9 or transient delivery of Cas9-ribonucleoproteins (RNPs), we achieved an average insertion or deletion (indel) efficiency greater than 80%. This high efficiency of indel generation revealed that double-strand breaks (DSBs) induced by Cas9 are toxic and kill most hPSCs. In previous studies, the toxicity of Cas9 in hPSCs was less apparent because of low transfection efficiency and subsequently low DSB induction 3 . The toxic response to DSBs was P53/TP53 -dependent, such that the efficiency of precise genome engineering in hPSCs with a wild-type P53 gene was severely reduced. Our results indicate that Cas9 toxicity creates an obstacle to the high-throughput use of CRISPR/Cas9 for genome engineering and screening in hPSCs. Moreover, as hPSCs can acquire P53 mutations 14 , cell replacement therapies using CRISPR/Cas9-enginereed hPSCs should proceed with caution, and such engineered hPSCs should be monitored for P53 function. CRISPR–Cas9-induced DNA damage triggers p53 to limit the efficiency of gene editing in human pluripotent cells.

We recently derived mouse expanded potential stem cells (EPSCs) from individual blastomeres by inhibiting the critical molecular pathways that predispose their differentiation. EPSCs had enriched molecular signatures of blastomeres and possessed developmental potency for all embryonic and extra-embryonic cell lineages. Here, we report the derivation of porcine EPSCs, which express key pluripotency genes, are genetically stable, permit genome editing, differentiate to derivatives of the three germ layers in chimeras and produce primordial germ cell-like cells in vitro. Under similar conditions, human embryonic stem cells and induced pluripotent stem cells can be converted, or somatic cells directly reprogrammed, to EPSCs that display the molecular and functional attributes reminiscent of porcine EPSCs. Importantly, trophoblast stem-cell-like cells can be generated from both human and porcine EPSCs. Our pathway-inhibition paradigm thus opens an avenue for generating mammalian pluripotent stem cells, and EPSCs present a unique cellular platform for translational research in biotechnology and regenerative medicine. Gao, Nowak-Imialek, Chen et al. generate porcine and human stem cells that possess expanded developmental potency for both embryonic and extra-embryonic cell lineages.

Totipotency is the ability of a single cell to give rise to all of the differentiated cell types that build the conceptus, yet how to capture this property in vitro remains incompletely understood. Defining totipotency relies on a variety of assays of variable stringency. Here, we describe criteria to define totipotency. We explain how distinct criteria of increasing stringency can be used to judge totipotency by evaluating candidate totipotent cell types in mice, including early blastomeres and expanded or extended pluripotent stem cells. Our data challenge the notion that expanded or extended pluripotent states harbour increased totipotent potential relative to conventional embryonic stem cells under in vitro and in vivo conditions. Posfai, Schell, Janiszewski et al. assess candidate totipotent stem cells with in vitro and in vivo assays of increasing stringency to evaluate their developmental potential and lineage contributions.

Animals display substantial inter-species variation in the rate of embryonic development despite a broad conservation of the overall sequence of developmental events. Differences in biochemical reaction rates, including the rates of protein production and degradation, are thought to be responsible for species-specific rates of development 1 – 3 . However, the cause of differential biochemical reaction rates between species remains unknown. Here, using pluripotent stem cells, we have established an in vitro system that recapitulates the twofold difference in developmental rate between mouse and human embryos. This system provides a quantitative measure of developmental speed as revealed by the period of the segmentation clock, a molecular oscillator associated with the rhythmic production of vertebral precursors. Using this system, we show that mass-specific metabolic rates scale with the developmental rate and are therefore higher in mouse cells than in human cells. Reducing these metabolic rates by inhibiting the electron transport chain slowed down the segmentation clock by impairing the cellular NAD + /NADH redox balance and, further downstream, lowering the global rate of protein synthesis. Conversely, increasing the NAD + /NADH ratio in human cells by overexpression of the Lactobacillus brevis NADH oxidase Lb NOX increased the translation rate and accelerated the segmentation clock. These findings represent a starting point for the manipulation of developmental rate, with multiple translational applications including accelerating the differentiation of human pluripotent stem cells for disease modelling and cell-based therapies. An in vitro system that recapitulates temporal characteristics of embryonic development demonstrates that the different rates of mouse and human embryonic development stem from differences in metabolic rates and—further downstream—the global rate of protein synthesis.

Naive and primed pluripotent human embryonic stem cells bear transcriptional similarity to pre- and post-implantation epiblast and thus constitute a developmental model for understanding the pluripotent stages in human embryo development. To identify new transcription factors that differentially regulate the unique pluripotent stages, we mapped open chromatin using ATAC-seq and found enrichment of the activator protein-2 (AP2) transcription factor binding motif at naive-specific open chromatin. We determined that the AP2 family member TFAP2C is upregulated during primed to naive reversion and becomes widespread at naive-specific enhancers. TFAP2C functions to maintain pluripotency and repress neuroectodermal differentiation during the transition from primed to naive by facilitating the opening of enhancers proximal to pluripotency factors. Additionally, we identify a previously undiscovered naive-specific POU5F1 (OCT4) enhancer enriched for TFAP2C binding. Taken together, TFAP2C establishes and maintains naive human pluripotency and regulates OCT4 expression by mechanisms that are distinct from mouse. Pastor et al. demonstrate a role for TFAP2C in the promotion and maintenance of human naive pluripotency by facilitating the opening of enhancers close to pluripotency factors.

The epiblast (EPI) is the origin of all somatic and germ cells in mammals, and of pluripotent stem cells in vitro . To explore the ontogeny of human and primate pluripotency, here we perform comprehensive single-cell RNA sequencing for pre- and post-implantation EPI development in cynomolgus monkeys ( Macaca fascicularis ). We show that after specification in the blastocysts, EPI from cynomolgus monkeys (cyEPI) undergoes major transcriptome changes on implantation. Thereafter, while generating gastrulating cells, cyEPI stably maintains its transcriptome over a week, retains a unique set of pluripotency genes and acquires properties for ‘neuron differentiation’. Human and monkey pluripotent stem cells show the highest similarity to post-implantation late cyEPI, which, despite co-existing with gastrulating cells, bears characteristics of pre-gastrulating mouse EPI and epiblast-like cells in vitro . These findings not only reveal the divergence and coherence of EPI development, but also identify a developmental coordinate of the spectrum of pluripotency among key species, providing a basis for better regulation of human pluripotency in vitro . Using a single-cell sequencing analysis in monkey embryos, and comparing the genes expressed during early development in this species with those in mice and in human pluripotent stem cells, the authors define characteristics of pluripotency ontogeny across mammalian species. Species differences in developing pluripotent stem cells Using a single-cell-sequencing-based analysis in monkey embryos, and comparing the genes expressed during early development in this species and what is known from mouse and human studies, Mitinori Saitou and colleagues define characteristics of pluripotency ontogeny across mammalian species. They show that, surprisingly, monkey cells undergoing neuronal differentiation continue to express genes associated with pluripotency during gastrulation. The analysis also provides insights into the comparative properties of developmental-stage pluripotent stem cells in key species that will help to establish a basis for better regulation of human pluripotency in vitro .