Studying lineage decision-making in vitro: emerging concepts and novel tools.

Correct and timely lineage decisions are critical for normal embryonic development and homeostasis of adult tissues. Therefore, the search for fundamental principles that underlie lineage decision-making lies at the heart of developmental biology. Here, we review attempts to understand lineage decision-making as the interplay of single-cell heterogeneity and gene regulation. Fluctuations at the single-cell level are an important driving force behind cell-state transitions and the creation of cell-type diversity. Gene regulatory networks amplify such fluctuations and define stable cell types. They also mediate the influence of signaling inputs on the lineage decision. In this review, we focus on insights gleaned from in vitro differentiation of embryonic stem cells. We discuss emerging concepts, with an emphasis on transcriptional regulation, dynamical aspects of differentiation, and functional single-cell heterogeneity. We also highlight some novel tools to study lineage decision-making in vitro.

The developmental control of transposable elements and the evolution of higher species.

Transposable elements (TEs) account for at least 50% of the human genome. They constitute essential motors of evolution through their ability to modify genomic architecture, mutate genes and regulate gene expression. Accordingly, TEs are subject to tight epigenetic control during the earliest phases of embryonic development via histone and DNA methylation. Key to this process is recognition by sequence-specific RNA- and protein-based repressors. Collectively, these mediators are responsible for silencing a very broad range of TEs in an evolutionarily dynamic fashion. As a consequence, mobile elements and their controllers exert a marked influence on transcriptional networks in embryonic stem cells and a variety of adult tissues. The emerging picture is not that of a simple arms race but rather of a massive and sophisticated enterprise of TE domestication for the evolutionary benefit of the host.

The nature of embryonic stem cells.

Mouse embryonic stem (ES) cells perpetuate in vitro the broad developmental potential of naïve founder cells in the preimplantation embryo. ES cells self-renew relentlessly in culture but can reenter embryonic development seamlessly, differentiating on schedule to form all elements of the fetus. Here we review the properties of these remarkable cells. Arising from the stability, homogeneity, and equipotency of ES cells, we consider the concept of a pluripotent ground state. We evaluate the authenticity of ES cells in relation to cells in the embryo and examine their utility for dissecting mechanisms that confer pluripotency and that execute fate choice. We summarize current knowledge of the transcription factor circuitry that governs the ES cell state and discuss the opportunity to expose molecular logic further through iterative computational modeling and experimentation. Finally, we present a perspective on unresolved questions, including the challenge of deriving ground state pluripotent stem cells from non-rodent species.

Resolving the 3D Landscape of Transcription-Linked Mammalian Chromatin Folding.

Whereas folding of genomes at the large scale of epigenomic compartments and topologically associating domains (TADs) is now relatively well understood, how chromatin is folded at finer scales remains largely unexplored in mammals. Here, we overcome some limitations of conventional 3C-based methods by using high-resolution Micro-C to probe links between 3D genome organization and transcriptional regulation in mouse stem cells. Combinatorial binding of transcription factors, cofactors, and chromatin modifiers spatially segregates TAD regions into various finer-scale structures with distinct regulatory features including stripes, dots, and domains linking promoters-to-promoters (P-P) or enhancers-to-promoters (E-P) and bundle contacts between Polycomb regions. E-P stripes extending from the edge of domains predominantly link co-expressed loci, often in the absence of CTCF and cohesin occupancy. Acute inhibition of transcription disrupts these gene-related folding features without altering higher-order chromatin structures. Our study uncovers previously obscured finer-scale genome organization, establishing functional links between chromatin folding and gene regulation.

Cdk1 Controls Global Epigenetic Landscape in Embryonic Stem Cells.

The cyclin-dependent kinase 1 (Cdk1) drives cell division. To uncover additional functions of Cdk1, we generated knockin mice expressing an analog-sensitive version of Cdk1 in place of wild-type Cdk1. In our study, we focused on embryonic stem cells (ESCs), because this cell type displays particularly high Cdk1 activity. We found that in ESCs, a large fraction of Cdk1 substrates is localized on chromatin. Cdk1 phosphorylates many proteins involved in epigenetic regulation, including writers and erasers of all major histone marks. Consistent with these findings, inhibition of Cdk1 altered histone-modification status of ESCs. High levels of Cdk1 in ESCs phosphorylate and partially inactivate Dot1l, the H3K79 methyltransferase responsible for placing activating marks on gene bodies. Decrease of Cdk1 activity during ESC differentiation de-represses Dot1l, thereby allowing coordinated expression of differentiation genes. These analyses indicate that Cdk1 functions to maintain the epigenetic identity of ESCs.

Genomic Profiling by ALaP-Seq Reveals Transcriptional Regulation by PML Bodies through DNMT3A Exclusion.

The promyelocytic leukemia (PML) body is a phase-separated nuclear structure physically associated with chromatin, implying its crucial roles in genome functions. However, its role in transcriptional regulation is largely unknown. We developed APEX-mediated chromatin labeling and purification (ALaP) to identify the genomic regions proximal to PML bodies. We found that PML bodies associate with active regulatory regions across the genome and with ∼300 kb of the short arm of the Y chromosome (YS300) in mouse embryonic stem cells. The PML body association with YS300 is essential for the transcriptional activity of the neighboring Y-linked clustered genes. Mechanistically, PML bodies provide specific nuclear spaces that the de novo DNA methyltransferase DNMT3A cannot access, resulting in the steady maintenance of a hypo-methylated state at Y-linked gene promoters. Our study underscores a new mechanism for gene regulation in the 3D nuclear space and provides insights into the functional properties of nuclear structures for genome function.

TET1 is a tumor suppressor of hematopoietic malignancy.

The methylcytosine dioxygenase TET1 ('ten-eleven translocation 1') is an important regulator of 5-hydroxymethylcytosine (5hmC) in embryonic stem cells. The diminished expression of TET proteins and loss of 5hmC in many tumors suggests a critical role for the maintenance of this epigenetic modification. Here we found that deletion of Tet1 promoted the development of B cell lymphoma in mice. TET1 was required for maintenance of the normal abundance and distribution of 5hmC, which prevented hypermethylation of DNA, and for regulation of the B cell lineage and of genes encoding molecules involved in chromosome maintenance and DNA repair. Whole-exome sequencing of TET1-deficient tumors revealed mutations frequently found in non-Hodgkin B cell lymphoma (B-NHL), in which TET1 was hypermethylated and transcriptionally silenced. Our findings provide in vivo evidence of a function for TET1 as a tumor suppressor of hematopoietic malignancy.

Reference Maps of human ES and iPS cell variation enable high-throughput characterization of pluripotent cell lines.

The developmental potential of human pluripotent stem cells suggests that they can produce disease-relevant cell types for biomedical research. However, substantial variation has been reported among pluripotent cell lines, which could affect their utility and clinical safety. Such cell-line-specific differences must be better understood before one can confidently use embryonic stem (ES) or induced pluripotent stem (iPS) cells in translational research. Toward this goal we have established genome-wide reference maps of DNA methylation and gene expression for 20 previously derived human ES lines and 12 human iPS cell lines, and we have measured the in vitro differentiation propensity of these cell lines. This resource enabled us to assess the epigenetic and transcriptional similarity of ES and iPS cells and to predict the differentiation efficiency of individual cell lines. The combination of assays yields a scorecard for quick and comprehensive characterization of pluripotent cell lines.

Identification of a dynamic gene regulatory network required for pluripotency factor-induced reprogramming of mouse fibroblasts and hepatocytes.

The generation of induced pluripotent stem cells (iPSCs) from somatic cells provides an excellent model to study mechanisms of transcription factor-induced global alterations of the epigenome and genome function. Here, we have investigated the early transcriptional events of cellular reprogramming triggered by the co-expression of Oct4, Sox2, Klf4, and c-Myc (OSKM) in mouse embryonic fibroblasts (MEFs) and mouse hepatocytes (mHeps). In this analysis, we identified a gene regulatory network composed of nine transcriptional regulators (9TR; Cbfa2t3, Gli2, Irf6, Nanog, Ovol1, Rcan1, Taf1c, Tead4, and Tfap4), which are directly targeted by OSKM, in vivo. Functional studies using single and double shRNA knockdowns of any of these factors caused disruption of the network and dramatic reductions in reprogramming efficiency, indicating that this network is essential for the induction and establishment of pluripotency. We demonstrate that the stochastic co-expression of 9TR network components occurs in a remarkably small number of cells, approximating the percentage of terminally reprogrammed cells as a result of dynamic molecular events. Thus, the early DNA-binding patterns of OSKM and the subsequent probabilistic co-expression of essential 9TR components in subpopulations of cells undergoing reprogramming steer the reconstruction of a gene regulatory network marking the transition to pluripotency.

The coupling of X-chromosome inactivation to pluripotency.

X-chromosome inactivation, or the silencing of one X chromosome that occurs initially in the female somatic four-cell-stage embryo, is reversed during embryonic development first at the time of inner cell mass formation and again during formation of germ cell precursors. Such X-chromosome reactivation in the mouse implies the silencing of the Xist gene and the transcription of its antisense partner, Tsix, from both X chromosomes. In murine embryonic stem cells, both genes are under the transcriptional control of a series of critical pluripotency factors, namely, OCT3/4, NANOG, SOX2, KLF4, C-MYC and REX1. Although the inactive/active status of the two X chromosomes present in female human embryonic stem cells remains controversial, the reactivation of X-chromosome inactivation seems to be a signature for the naive pluripotent state.

Genetically Encoded Tools for Optical Dissection of the Mammalian Cell Cycle.

Eukaryotic cells spend most of their life in interphase of the cell cycle. Understanding the rich diversity of metabolic and genomic regulation that occurs in interphase requires the demarcation of precise phase boundaries in situ. Here, we report the properties of two genetically encoded fluorescence sensors, Fucci(CA) and Fucci(SCA), which enable real-time monitoring of interphase and cell-cycle biology. We re-engineered the Cdt1-based sensor from the original Fucci system to respond to S phase-specific CUL4Ddb1-mediated ubiquitylation alone or in combination with SCFSkp2-mediated ubiquitylation. In cultured cells, Fucci(CA) produced a sharp triple color-distinct separation of G1, S, and G2, while Fucci(SCA) permitted a two-color readout of G1 and S/G2. Fucci(CA) applications included tracking the transient G1 phase of rapidly dividing mouse embryonic stem cells and identifying a window for UV-irradiation damage in S phase. These results show that Fucci(CA) is an essential tool for quantitative studies of interphase cell-cycle regulation.

Id genes are essential for early heart formation.

Deciphering the fundamental mechanisms controlling cardiac specification is critical for our understanding of how heart formation is initiated during embryonic development and for applying stem cell biology to regenerative medicine and disease modeling. Using systematic and unbiased functional screening approaches, we discovered that the Id family of helix-loop-helix proteins is both necessary and sufficient to direct cardiac mesoderm formation in frog embryos and human embryonic stem cells. Mechanistically, Id proteins specify cardiac cell fate by repressing two inhibitors of cardiogenic mesoderm formation-Tcf3 and Foxa2-and activating inducers Evx1, Grrp1, and Mesp1. Most importantly, CRISPR/Cas9-mediated ablation of the entire Id (Id1-4) family in mouse embryos leads to failure of anterior cardiac progenitor specification and the development of heartless embryos. Thus, Id proteins play a central and evolutionarily conserved role during heart formation and provide a novel means to efficiently produce cardiovascular progenitors for regenerative medicine and drug discovery applications.

LncPRESS1 Is a p53-Regulated LncRNA that Safeguards Pluripotency by Disrupting SIRT6-Mediated De-acetylation of Histone H3K56.

Recent evidence suggests that lncRNAs play an integral regulatory role in numerous functions, including determination of cellular identity. We determined global expression (RNA-seq) and genome-wide profiles (ChIP-seq) of histone post-translational modifications and p53 binding in human embryonic stem cells (hESCs) undergoing differentiation to define a high-confidence set of 40 lncRNAs, which are p53 transcriptional targets. We focused on lncRNAs highly expressed in pluripotent hESCs and repressed by p53 during differentiation to identify lncPRESS1 as a p53-regulated transcript that maintains hESC pluripotency in concert with core pluripotency factors. RNA-seq of hESCs depleted of lncPRESS1 revealed that lncPRESS1 controls a gene network that promotes pluripotency. Further, we found that lncPRESS1 physically interacts with SIRT6 and prevents SIRT6 chromatin localization, which maintains high levels of histone H3K56 and H3K9 acetylation at promoters of pluripotency genes. In summary, we describe a p53-regulated, pluripotency-specific lncRNA that safeguards the hESC state by disrupting SIRT6 activity.

N6-methyladenosine (m6A) depletion regulates pluripotency exit by activating signaling pathways in embryonic stem cells.

N6-methyladenosine (m6A) deposition on messenger RNA (mRNA) controls embryonic stem cell (ESC) fate by regulating the mRNA stabilities of pluripotency and lineage transcription factors (TFs) [P. J. Batista et al., Cell Stem Cell 15, 707-719 (2014); Y. Wang et al., Nat. Cell Biol. 16, 191-198 (2014); and S. Geula et al., Science 347, 1002-1006 (2015)]. If the mRNAs of these two TF groups become stabilized, it remains unclear how the pluripotency or lineage commitment decision is implemented. We performed noninvasive quantification of Nanog and Oct4 TF protein levels in reporter ESCs to define cell-state dynamics at single-cell resolution. Long-term single-cell tracking shows that immediate m6A depletion by Mettl3 knock-down in serum/leukemia inhibitory factor supports both pluripotency maintenance and its departure. This is mediated by differential and opposing signaling pathways. Increased FGF5 mRNA stability activates pErk, leading to Nanog down-regulation. FGF5-mediated coactivation of pAkt reenforces Nanog expression. In formative stem cells poised toward differentiation, m6A depletion activates both pErk and pAkt, increasing the propensity for mesendodermal lineage induction. Stable m6A depletion by Mettl3 knock-out also promotes pErk activation. Higher pErk counteracts the pluripotency exit delay exhibited by stably m6A-depleted cells upon differentiation. At single-cell resolution, we illustrate that decreasing m6A abundances activates pErk and pAkt-signaling, regulating pluripotency departure.

Disabling de novo DNA methylation in embryonic stem cells allows an illegitimate fate trajectory.

Genome remethylation is essential for mammalian development but specific reasons are unclear. Here we examined embryonic stem (ES) cell fate in the absence of de novo DNA methyltransferases. We observed that ES cells deficient for both Dnmt3a and Dnmt3b are rapidly eliminated from chimeras. On further investigation we found that in vivo and in vitro the formative pluripotency transition is derailed toward production of trophoblast. This aberrant trajectory is associated with failure to suppress activation of Ascl2Ascl2 encodes a bHLH transcription factor expressed in the placenta. Misexpression of Ascl2 in ES cells provokes transdifferentiation to trophoblast-like cells. Conversely, Ascl2 deletion rescues formative transition of Dnmt3a/b mutants and improves contribution to chimeric epiblast. Thus, de novo DNA methylation safeguards against ectopic activation of Ascl2 However, Dnmt3a/b-deficient cells remain defective in ongoing embryogenesis. We surmise that multiple developmental transitions may be secured by DNA methylation silencing potentially disruptive genes.

Developmental HCN channelopathy results in decreased neural progenitor proliferation and microcephaly in mice.

The development of the cerebral cortex relies on the controlled division of neural stem and progenitor cells. The requirement for precise spatiotemporal control of proliferation and cell fate places a high demand on the cell division machinery, and defective cell division can cause microcephaly and other brain malformations. Cell-extrinsic and -intrinsic factors govern the capacity of cortical progenitors to produce large numbers of neurons and glia within a short developmental time window. In particular, ion channels shape the intrinsic biophysical properties of precursor cells and neurons and control their membrane potential throughout the cell cycle. We found that hyperpolarization-activated cyclic nucleotide-gated cation (HCN) channel subunits are expressed in mouse, rat, and human neural progenitors. Loss of HCN channel function in rat neural stem cells impaired their proliferation by affecting the cell-cycle progression, causing G1 accumulation and dysregulation of genes associated with human microcephaly. Transgene-mediated, dominant-negative loss of HCN channel function in the embryonic mouse telencephalon resulted in pronounced microcephaly. Together, our findings suggest a role for HCN channel subunits as a part of a general mechanism influencing cortical development in mammals.

A common molecular logic determines embryonic stem cell self-renewal and reprogramming.

During differentiation and reprogramming, new cell identities are generated by reconfiguration of gene regulatory networks. Here, we combined automated formal reasoning with experimentation to expose the logic of network activation during induction of naïve pluripotency. We find that a Boolean network architecture defined for maintenance of naïve state embryonic stem cells (ESC) also explains transcription factor behaviour and potency during resetting from primed pluripotency. Computationally identified gene activation trajectories were experimentally substantiated at single-cell resolution by RT-qPCR Contingency of factor availability explains the counterintuitive observation that Klf2, which is dispensable for ESC maintenance, is required during resetting. We tested 124 predictions formulated by the dynamic network, finding a predictive accuracy of 77.4%. Finally, we show that this network explains and predicts experimental observations of somatic cell reprogramming. We conclude that a common deterministic program of gene regulation is sufficient to govern maintenance and induction of naïve pluripotency. The tools exemplified here could be broadly applied to delineate dynamic networks underlying cell fate transitions.

SUMOylation coordinates BERosome assembly in active DNA demethylation during cell differentiation.

During active DNA demethylation, 5-methylcytosine (5mC) is oxidized by TET proteins to 5-formyl-/5-carboxylcytosine (5fC/5caC) for replacement by unmethylated C by TDG-initiated DNA base excision repair (BER). Base excision generates fragile abasic sites (AP-sites) in DNA and has to be coordinated with subsequent repair steps to limit accumulation of genome destabilizing secondary DNA lesions. Here, we show that 5fC/5caC is generated at a high rate in genomes of differentiating mouse embryonic stem cells and that SUMOylation and the BER protein XRCC1 play critical roles in orchestrating TDG-initiated BER of these lesions. SUMOylation of XRCC1 facilitates physical interaction with TDG and promotes the assembly of a TDG-BER core complex. Within this TDG-BERosome, SUMO is transferred from XRCC1 and coupled to the SUMO acceptor lysine in TDG, promoting its dissociation while assuring the engagement of the BER machinery to complete demethylation. Although well-studied, the biological importance of TDG SUMOylation has remained obscure. Here, we demonstrate that SUMOylation of TDG suppresses DNA strand-break accumulation and toxicity to PARP inhibition in differentiating mESCs and is essential for neural lineage commitment.

LIN28A loss of function is associated with Parkinson's disease pathogenesis.

Parkinson's disease (PD) is neurodegenerative movement disorder characterized by degeneration of midbrain-type dopamine (mDA) neurons in the substantia nigra (SN). The RNA-binding protein Lin28 plays a role in neuronal stem cell development and neuronal differentiation. In this study, we reveal that Lin28 conditional knockout (cKO) mice show degeneration of mDA neurons in the SN, as well as PD-related behavioral deficits. We identify a loss-of-function variant of LIN28A (R192G substitution) in two early-onset PD patients. Using an isogenic human embryonic stem cell (hESC)/human induced pluripotent stem cell (hiPSC)-based disease model, we find that the Lin28 R192G variant leads to developmental defects and PD-related phenotypes in mDA neuronal cells that can be rescued by expression of wild-type Lin28A. Cell transplantation experiments in PD model rats show that correction of the LIN28A variant in the donor patient (pt)-hiPSCs leads to improved behavioral phenotypes. Our data link LIN28A to PD pathogenesis and suggest future personalized medicine targeting this variant in patients.

Metabolic regulation of pluripotency and germ cell fate through α-ketoglutarate.

An intricate link is becoming apparent between metabolism and cellular identities. Here, we explore the basis for such a link in an in vitro model for early mouse embryonic development: from naïve pluripotency to the specification of primordial germ cells (PGCs). Using single-cell RNA-seq with statistical modelling and modulation of energy metabolism, we demonstrate a functional role for oxidative mitochondrial metabolism in naïve pluripotency. We link mitochondrial tricarboxylic acid cycle activity to IDH2-mediated production of alpha-ketoglutarate and through it, the activity of key epigenetic regulators. Accordingly, this metabolite has a role in the maintenance of naïve pluripotency as well as in PGC differentiation, likely through preserving a particular histone methylation status underlying the transient state of developmental competence for the PGC fate. We reveal a link between energy metabolism and epigenetic control of cell state transitions during a developmental trajectory towards germ cell specification, and establish a paradigm for stabilizing fleeting cellular states through metabolic modulation.