Epigenetic reprogramming in the transition from pluripotency to totipotency.

Mammalian development commences with the zygote, which can differentiate into both embryonic and extraembryonic tissues, a capability known as totipotency. Only the zygote and embryos around zygotic genome activation (ZGA) (two-cell embryo stage in mice and eight-cell embryo in humans) are totipotent cells. Epigenetic modifications undergo extremely extensive changes during the acquisition of totipotency and subsequent development of differentiation. However, the underlying molecular mechanisms remain elusive. Recently, the discovery of mouse two-cell embryo-like cells, human eight-cell embryo-like cells, extended pluripotent stem cells and totipotent-like stem cells with extra-embryonic developmental potential has greatly expanded our understanding of totipotency. Experiments with these in vitro models have led to insights into epigenetic changes in the reprogramming of pluri-to-totipotency, which have informed the exploration of preimplantation development. In this review, we highlight the recent findings in understanding the mechanisms of epigenetic remodeling during totipotency capture, including RNA splicing, DNA methylation, chromatin configuration, histone modifications, and nuclear organization.

Pursuing totipotency: authentic totipotent stem cells in culture.

Totipotent stem cells are transiently occurring in vivo cells that can form all cell types of the embryo including placenta, with their in vitro counterparts being actively pursued. Subsequently, totipotent-like cells are established with variable robustness and biological relevance. Here, we summarize current progress on capturing these cells in culture.

CTCF is a barrier for 2C-like reprogramming.

Totipotent cells have the ability to generate embryonic and extra-embryonic tissues. Interestingly, a rare population of cells with totipotent-like potential, known as 2 cell (2C)-like cells, has been identified within ESC cultures. They arise from ESC and display similar features to those found in the 2C embryo. However, the molecular determinants of 2C-like conversion have not been completely elucidated. Here, we show that the CCCTC-binding factor (CTCF) is a barrier for 2C-like reprogramming. Indeed, forced conversion to a 2C-like state by the transcription factor DUX is associated with DNA damage at a subset of CTCF binding sites. Depletion of CTCF in ESC efficiently promotes spontaneous and asynchronous conversion to a 2C-like state and is reversible upon restoration of CTCF levels. This phenotypic reprogramming is specific to pluripotent cells as neural progenitor cells do not show 2C-like conversion upon CTCF-depletion. Furthermore, we show that transcriptional activation of the ZSCAN4 cluster is necessary for successful 2C-like reprogramming. In summary, we reveal an unexpected relationship between CTCF and 2C-like reprogramming.

Mouse totipotent stem cells captured and maintained through spliceosomal repression.

Since establishment of the first embryonic stem cells (ESCs), in vitro culture of totipotent cells functionally and molecularly comparable with in vivo blastomeres with embryonic and extraembryonic developmental potential has been a challenge. Here we report that spliceosomal repression in mouse ESCs drives a pluripotent-to-totipotent state transition. Using the splicing inhibitor pladienolide B, we achieve stable in vitro culture of totipotent ESCs comparable at molecular levels with 2- and 4-cell blastomeres, which we call totipotent blastomere-like cells (TBLCs). Mouse chimeric assays combined with single-cell RNA sequencing (scRNA-seq) demonstrate that TBLCs have a robust bidirectional developmental capability to generate multiple embryonic and extraembryonic cell lineages. Mechanically, spliceosomal repression causes widespread splicing inhibition of pluripotent genes, whereas totipotent genes, which contain few short introns, are efficiently spliced and transcriptionally activated. Our study provides a means for capturing and maintaining totipotent stem cells.

Evaluating totipotency using criteria of increasing stringency.

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.

TRF2-mediated telomere protection is dispensable in pluripotent stem cells.

In mammals, telomere protection is mediated by the essential protein TRF2, which binds chromosome ends and ensures genome integrity1,2. TRF2 depletion results in end-to-end chromosome fusions in all cell types that have been tested so far. Here we find that TRF2 is dispensable for the proliferation and survival of mouse embryonic stem (ES) cells. Trf2-/- (also known as Terf2) ES cells do not exhibit telomere fusions and can be expanded indefinitely. In response to the deletion of TRF2, ES cells exhibit a muted DNA damage response that is characterized by the recruitment of γH2AX-but not 53BP1-to telomeres. To define the mechanisms that control this unique DNA damage response in ES cells, we performed a CRISPR-Cas9-knockout screen. We found a strong dependency of TRF2-null ES cells on the telomere-associated protein POT1B and on the chromatin remodelling factor BRD2. Co-depletion of POT1B or BRD2 with TRF2 restores a canonical DNA damage response at telomeres, resulting in frequent telomere fusions. We found that TRF2 depletion in ES cells activates a totipotent-like two-cell-stage transcriptional program that includes high levels of ZSCAN4. We show that the upregulation of ZSCAN4 contributes to telomere protection in the absence of TRF2. Together, our results uncover a unique response to telomere deprotection during early development.

From pluripotency to totipotency: an experimentalist's guide to cellular potency.

Embryonic stem cells (ESCs) are derived from the pre-implantation mammalian blastocyst. At this point in time, the newly formed embryo is concerned with the generation and expansion of both the embryonic lineages required to build the embryo and the extra-embryonic lineages that support development. When used in grafting experiments, embryonic cells from early developmental stages can contribute to both embryonic and extra-embryonic lineages, but it is generally accepted that ESCs can give rise to only embryonic lineages. As a result, they are referred to as pluripotent, rather than totipotent. Here, we consider the experimental potential of various ESC populations and a number of recently identified in vitro culture systems producing states beyond pluripotency and reminiscent of those observed during pre-implantation development. We also consider the nature of totipotency and the extent to which cell populations in these culture systems exhibit this property.

On transposons and totipotency.

Our perception of the role of the previously considered 'selfish' or 'junk' DNA has been dramatically altered in the past 20 years or so. A large proportion of this non-coding part of mammalian genomes is repetitive in nature, classified as either satellites or transposons. While repetitive elements can be termed selfish in terms of their amplification, such events have surely been co-opted by the host, suggesting by itself a likely altruistic function for the organism at the subject of such natural selection. Indeed numerous examples of transposons regulating the functional output of the host genome have been documented. Transposons provide a powerful framework for large-scale relatively rapid concerted regulatory activities with the ability to drive evolution. Mammalian totipotency has emerged as one key stage of development in which transposon-mediated regulation of gene expression has taken centre stage in the past few years. During this period, large-scale (epigenetic) reprogramming must be accomplished in order to activate the host genome. In mice and men, one particular element murine endogenous retrovirus with leucine tRNA primer (MERVL) (and its counterpart human ERVL (HERVL)) appears to have acquired roles as a key driving force in this process. Here, I will discuss and interpret the current knowledge and its implications regarding the role of transposons, particularly of long interspersed nuclear elements (LINE-1s) and endogenous retroviruses (ERVs), in the regulation of totipotency. This article is part of a discussion meeting issue 'Crossroads between transposons and gene regulation'.

SETDB1-Mediated Cell Fate Transition between 2C-Like and Pluripotent States.

Known as a histone H3K9 methyltransferase, SETDB1 is essential for embryonic development and pluripotent inner cell mass (ICM) establishment. However, its function in pluripotency regulation remains elusive. In this study, we find that under the "ground state" of pluripotency with two inhibitors (2i) of the MEK and GSK3 pathways, Setdb1-knockout fails to induce trophectoderm (TE) differentiation as in serum/LIF (SL), indicating that TE fate restriction is not the direct target of SETDB1. In both conditions, Setdb1-knockout activates a group of genes targeted by SETDB1-mediated H3K9 methylation, including Dux. Notably, Dux is indispensable for the reactivation of 2C-like state genes upon Setdb1 deficiency, delineating the mechanistic role of SETDB1 in totipotency restriction. Furthermore, Setdb1-null ESCs maintain pluripotent marker (e.g., Nanog) expression in the 2i condition. This "ground state" Setdb1-null population undergoes rapid cell death by activating Ripk3 and, subsequently, RIPK1/RIPK3-dependent necroptosis. These results reveal the essential role of Setdb1 between totipotency and pluripotency transition.

Derivation and Characterization of a Pig Embryonic-Stem-Cell-Derived Exocrine Pancreatic Cell Line.

OBJECTIVES: The aim of this study was to identify an epithelial cell line isolated from the spontaneous differentiation of totipotent pig epiblast cells. METHODS: PICM-31 and its colony-cloned derivative cell line, PICM-31A, were established from the culture and differentiation of an epiblast mass isolated from an 8-day-old pig blastocyst. The cell lines were analyzed by transmission electron microscopy, marker gene expression, and mass spectroscopy-based proteomics. RESULTS: The PICM-31 cell lines were continuously cultured and could be successively colony cloned. They spontaneously self-organized into acinarlike structures. Transmission electron microscopy indicated that the cell lines' cells were epithelial and filled with secretory granules. Candidate gene expression analysis of the cells showed an exocrine pancreatic profile that included digestive enzyme expression, for example, carboxypeptidase A1, and expression of the fetal marker, α-fetoprotein. Pancreatic progenitor marker expression included pancreatic and duodenal homeobox 1, NK6 homeobox 1, and pancreas-specific transcription factor 1a, but not neurogenin 3. Proteomic analysis of cellular proteins confirmed the cells' production of digestive enzymes and showed that the cells expressed cytokeratins 8 and 18. CONCLUSIONS: The PICM-31 cell lines provide in vitro models of fetal pig pancreatic exocrine cells. They are the first demonstration of continuous cultures, that is, cell lines, of nontransformed pig pancreas cells.

Single-nucleus Hi-C reveals unique chromatin reorganization at oocyte-to-zygote transition.

Chromatin is reprogrammed after fertilization to produce a totipotent zygote with the potential to generate a new organism. The maternal genome inherited from the oocyte and the paternal genome provided by sperm coexist as separate haploid nuclei in the zygote. How these two epigenetically distinct genomes are spatially organized is poorly understood. Existing chromosome conformation capture-based methods are not applicable to oocytes and zygotes owing to a paucity of material. To study three-dimensional chromatin organization in rare cell types, we developed a single-nucleus Hi-C (high-resolution chromosome conformation capture) protocol that provides greater than tenfold more contacts per cell than the previous method. Here we show that chromatin architecture is uniquely reorganized during the oocyte-to-zygote transition in mice and is distinct in paternal and maternal nuclei within single-cell zygotes. Features of genomic organization including compartments, topologically associating domains (TADs) and loops are present in individual oocytes when averaged over the genome, but the presence of each feature at a locus varies between cells. At the sub-megabase level, we observed stochastic clusters of contacts that can occur across TAD boundaries but average into TADs. Notably, we found that TADs and loops, but not compartments, are present in zygotic maternal chromatin, suggesting that these are generated by different mechanisms. Our results demonstrate that the global chromatin organization of zygote nuclei is fundamentally different from that of other interphase cells. An understanding of this zygotic chromatin 'ground state' could potentially provide insights into reprogramming cells to a state of totipotency.

Evolutionary origin and functional divergence of totipotent cell homeobox genes in eutherian mammals.

BACKGROUND: A central goal of evolutionary biology is to link genomic change to phenotypic evolution. The origin of new transcription factors is a special case of genomic evolution since it brings opportunities for novel regulatory interactions and potentially the emergence of new biological properties. RESULTS: We demonstrate that a group of four homeobox gene families (Argfx, Leutx, Dprx, Tprx), plus a gene newly described here (Pargfx), arose by tandem gene duplication from the retinal-expressed Crx gene, followed by asymmetric sequence evolution. We show these genes arose as part of repeated gene gain and loss events on a dynamic chromosomal region in the stem lineage of placental mammals, on the forerunner of human chromosome 19. The human orthologues of these genes are expressed specifically in early embryo totipotent cells, peaking from 8-cell to morula, prior to cell fate restrictions; cow orthologues have similar expression. To examine biological roles, we used ectopic gene expression in cultured human cells followed by high-throughput RNA-seq and uncovered extensive transcriptional remodelling driven by three of the genes. Comparison to transcriptional profiles of early human embryos suggest roles in activating and repressing a set of developmentally-important genes that spike at 8-cell to morula, rather than a general role in genome activation. CONCLUSIONS: We conclude that a dynamic chromosome region spawned a set of evolutionarily new homeobox genes, the ETCHbox genes, specifically in eutherian mammals. After these genes diverged from the parental Crx gene, we argue they were recruited for roles in the preimplantation embryo including activation of genes at the 8-cell stage and repression after morula. We propose these new homeobox gene roles permitted fine-tuning of cell fate decisions necessary for specification and function of embryonic and extra-embryonic tissues utilised in mammalian development and pregnancy.

Totipotency in the absence of CAF-I: unhindered choices when the chaperone is out.

Embryonal totipotent cells can produce both embryonic and extraembryonic tissues and can generate whole organisms. In mice this level of genome plasticity is preserved in the 2-cell embryos, but is absent in embryonic cells from later stages of development. Recently it has been demonstrated that totipotent-like cells spontaneously appear in embryonic stem cell cultures and that the depletion of the histone chaperone Chromatin Assembly Factor I (CAF-I) increases the abundance of 2cell-like cells. On the other hand, earlier studies have demonstrated that CAF-I is necessary for epigenetic conversions at the telomeres of S. cerevisiae. This commentary proposes that the absence of CAF-I confers totipotency of embryonic cells and that its activation triggers chromatin changes that reset the epigenome toward cell differentiation.

Mapping the journey from totipotency to lineage specification in the mouse embryo.

Understanding the past is to understand the present. Mammalian life, with all its complexity comes from a humble beginning of a single fertilized egg cell. Achieving this requires an enormous diversification of cellular function, the majority of which is generated through a series of cellular decisions during embryogenesis. The first decisions are made as the embryo prepares for implantation, a process that will require specialization of extra-embryonic lineages while preserving an embryonic one. In this mini-review, we will focus on the mouse as a mammalian model and discuss recent advances in the decision making process of the early embryo.

Chromatin dynamics in the regulation of cell fate allocation during early embryogenesis.

Following fertilization, gametes undergo epigenetic reprogramming in order to revert to a totipotent state. How embryonic cells subsequently acquire their fate and the role of chromatin dynamics in this process are unknown. Genetic and experimental embryology approaches have identified some of the players and morphological changes that are involved in early mammalian development, but the exact events underlying cell fate allocation in single embryonic cells have remained elusive. Experimental and technological advances have recently provided novel insights into chromatin dynamics and nuclear architecture in single cells; these insights have reshaped our understanding of the mechanisms underlying cell fate allocation and plasticity in early mammalian development.

Piwi Proteins and piRNAs step onto the systems biology stage.

Animal germ cells are totipotent because they maintain a highly unique and specialized epigenetic state for its genome. To accomplish this, germ cells express a rich repertoire of specialized RNA-binding protein complexes such as the Piwi proteins and Piwi-interacting RNAs (piRNAs): a germ-cell branch of the RNA interference (RNAi) phenomenon which includes microRNA and endogenous small interfering RNA pathways. Piwi proteins and piRNAs are deeply conserved in animal evolution and play essential roles in fertility and regeneration. Molecular mechanisms for how these ribonucleoproteins act upon the transcriptome and genome are only now coming to light with the application of systems-wide approaches in both invertebrates and vertebrates. Systems biology studies on invertebrates have revealed that transcriptional and heritable silencing is a main mechanism driven by Piwi proteins and piRNA complexes. In vertebrates, Piwi-targeting mechanisms and piRNA biogenesis have progressed, while the discovery that the nuclease activity of Piwi protein is essential for vertebrate germ cell development but not completely required in invertebrates highlights the many complexities of this pathway in different animals. This review recounts how recent systems-wide approaches have rapidly accelerated our appreciation for the broad reach of the Piwi pathway on germline genome regulation and what questions facing the field await to be unraveled.

Changes in histone methylation and acetylation during microspore reprogramming to embryogenesis occur concomitantly with Bn HKMT and Bn HAT expression and are associated with cell totipotency, proliferation, and differentiation in Brassica napus.

In response to stress treatments, microspores can be reprogrammed to become totipotent cells that follow an embryogenic pathway producing haploid and double-haploid embryos which are important biotechnological tools in plant breeding. Recent studies have revealed the involvement of DNA methylation in regulating this process, but no information is available on the role of histone modifications in microspore embryogenesis. Histone modifications are major epigenetic marks controlling gene expression during plant development and in response to environmental changes. Lysine methylation of histones, accomplished by histone lysine methyltransferases (HKMTs), can occur on different lysine residues, with histone H3K9 methylation being mainly associated with transcriptionally silenced regions. In contrast, histone H3 and H4 acetylation is carried out by histone acetyltransferases (HATs) and is associated with actively transcribed genes. In this work, we analyzed 3 different histone epigenetic marks: dimethylation of H3K9 (H3K9me2) and acetylation of H3 and H4 (H3Ac and H4Ac) during microspore embryogenesis in Brassica napus by Western blot and immunofluorescence assays. The expression patterns of histone methyltransferase BnHKMT and histone acetyltransferase BnHAT genes have also been analyzed by qPCR. Our results revealed different spatial and temporal distribution patterns for methylated and acetylated histone variants during microspore embryogenesis and their similarity with the expression profiles of BnHKMT and BnHAT, respectively. The data presented suggest the participation of H3K9me2 and HKMT in embryo cell differentiation and heterochromatinization events, whereas H3Ac, H4Ac, and HAT would be involved in transcriptional activation, totipotency, and proliferation events during cell reprogramming and embryo development.

Higher chromatin mobility supports totipotency and precedes pluripotency in vivo.

The fusion of the gametes upon fertilization results in the formation of a totipotent cell. Embryonic chromatin is expected to be able to support a large degree of plasticity. However, whether this plasticity relies on a particular conformation of the embryonic chromatin is unknown. Moreover, whether chromatin plasticity is functionally linked to cellular potency has not been addressed. Here, we adapted fluorescence recovery after photobleaching (FRAP) in the developing mouse embryo and show that mobility of the core histones H2A, H3.1, and H3.2 is unusually high in two-cell stage embryos and decreases as development proceeds. The transition toward pluripotency is accompanied by a decrease in histone mobility, and, upon lineage allocation, pluripotent cells retain higher mobility than the differentiated trophectoderm. Importantly, totipotent two-cell-like embryonic stem cells also display high core histone mobility, implying that reprogramming toward totipotency entails changes in chromatin mobility. Our data suggest that changes in chromatin dynamics underlie the transitions in cellular plasticity and that higher chromatin mobility is at the nuclear foundations of totipotency.

Comparative quantitative proteomic analysis of embryogenic and non-embryogenic calli in maize suggests the role of oxylipins in plant totipotency.

Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In this study, maize immature zygotic embryos were used to generate embryogenic (EC) and non-embryogenic (NEC) calli. In order to compare proteomes of EC and NEC, two-dimensional electrophoresis (2-DE) in combination with mass spectrometry was used. This approach resulted into 361 quantified 2-DE spots out of which 44 were found statistically significantly differentially abundant between EC and NEC. Mass spectrometry provided the identity for 23 proteins that were classified into 8 metabolic categories. The most abundant were proteins associated with energy followed by proteins associated with disease and defense. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. BIOLOGICAL SIGNIFICANCE: Totipotency, the ability of somatic plant cell to generate whole plant through somatic embryogenesis, is still not well understood. In order to further advance understanding of this biological phenomenon, proteomes of embryogenic and non-embryogenic callus, derived from immature zygotic embryos of inbred maize line A19, were compared using 2-DE based proteomic technology. Based on the abundances of identified proteins in this and other studies, working model for plant totipotency was proposed. One aspect of this working model suggests that increased abundances of proteins associated with pyruvate biosynthesis and suppression of embryogenic genes might be responsible for differences between EC and NEC cells. Furthermore we speculate that the increased abundance of lipoxygenase in the NEC cells results in changes in the equilibrium levels of one or more signaling molecules and is at least partly responsible for somatic cell reprogramming during totipotency. This article is part of a Special Issue entitled: Environmental and structural proteomics.

Reprogramming in vivo produces teratomas and iPS cells with totipotency features.

Reprogramming of adult cells to generate induced pluripotent stem cells (iPS cells) has opened new therapeutic opportunities; however, little is known about the possibility of in vivo reprogramming within tissues. Here we show that transitory induction of the four factors Oct4, Sox2, Klf4 and c-Myc in mice results in teratomas emerging from multiple organs, implying that full reprogramming can occur in vivo. Analyses of the stomach, intestine, pancreas and kidney reveal groups of dedifferentiated cells that express the pluripotency marker NANOG, indicative of in situ reprogramming. By bone marrow transplantation, we demonstrate that haematopoietic cells can also be reprogrammed in vivo. Notably, reprogrammable mice present circulating iPS cells in the blood and, at the transcriptome level, these in vivo generated iPS cells are closer to embryonic stem cells (ES cells) than standard in vitro generated iPS cells. Moreover, in vivo iPS cells efficiently contribute to the trophectoderm lineage, suggesting that they achieve a more plastic or primitive state than ES cells. Finally, intraperitoneal injection of in vivo iPS cells generates embryo-like structures that express embryonic and extraembryonic markers. We conclude that reprogramming in vivo is feasible and confers totipotency features absent in standard iPS or ES cells. These discoveries could be relevant for future applications of reprogramming in regenerative medicine.