Dynamic Roles of Signaling Pathways in Maintaining Pluripotency of Mouse and Human Embryonic Stem Cells.

Culturing of mouse and human embryonic stem cells (ESCs) in vitro was a major breakthrough in the field of stem cell biology. These models gained popularity very soon mainly due to their pluripotency. Evidently, the ESCs of mouse and human origin share typical phenotypic responses due to their pluripotent nature, such as self-renewal capacity and potency. The conserved network of core transcription factors regulates these responses. However, significantly different signaling pathways and upstream transcriptional networks regulate expression and activity of these core pluripotency factors in ESCs of both the species. In fact, ample evidence shows that a pathway, which maintains pluripotency in mouse ESCs, promotes differentiation in human ESCs. In this review, we discuss the role of canonical signaling pathways implicated in regulation of pluripotency and differentiation particularly in mouse and human ESCs. We believe that understanding these distinct and at times-opposite mechanisms-is critical for the progress in the field of stem cell biology and regenerative medicine.

Determinants of minor satellite RNA function in chromosome segregation in mouse embryonic stem cells.

The centromere is a fundamental higher-order structure in chromosomes ensuring their faithful segregation upon cell division. Centromeric transcripts have been described in several species and suggested to participate in centromere function. However, low sequence conservation of centromeric repeats appears inconsistent with a role in recruiting highly conserved centromeric proteins. Here, we hypothesized that centromeric transcripts may function through a secondary structure rather than sequence conservation. Using mouse embryonic stem cells (ESCs), we show that an imbalance in the levels of forward or reverse minor satellite (MinSat) transcripts leads to severe chromosome segregation defects. We further show that MinSat RNA adopts a stem-loop secondary structure, which is conserved in human α-satellite transcripts. We identify an RNA binding region in CENPC and demonstrate that MinSat transcripts function through the structured region of the RNA. Importantly, mutants that disrupt MinSat secondary structure do not cause segregation defects. We propose that the conserved role of centromeric transcripts relies on their secondary RNA structure.

Analysis of long-range chromatin contacts, compartments and looping between mouse embryonic stem cells, lens epithelium and lens fibers.

BACKGROUND: Nuclear organization of interphase chromosomes involves individual chromosome territories, "open" and "closed" chromatin compartments, topologically associated domains (TADs) and chromatin loops. The DNA- and RNA-binding transcription factor CTCF together with the cohesin complex serve as major organizers of chromatin architecture. Cellular differentiation is driven by temporally and spatially coordinated gene expression that requires chromatin changes of individual loci of various complexities. Lens differentiation represents an advantageous system to probe transcriptional mechanisms underlying tissue-specific gene expression including high transcriptional outputs of individual crystallin genes until the mature lens fiber cells degrade their nuclei. RESULTS: Chromatin organization between mouse embryonic stem (ES) cells, newborn (P0.5) lens epithelium and fiber cells were analyzed using Hi-C. Localization of CTCF in both lens chromatins was determined by ChIP-seq and compared with ES cells. Quantitative analyses show major differences between number and size of TADs and chromatin loop size between these three cell types. In depth analyses show similarities between lens samples exemplified by overlaps between compartments A and B. Lens epithelium-specific CTCF peaks are found in mostly methylated genomic regions while lens fiber-specific and shared peaks occur mostly within unmethylated DNA regions. Major differences in TADs and loops are illustrated at the ~ 500 kb Pax6 locus, encoding the critical lens regulatory transcription factor and within a larger ~ 15 Mb WAGR locus, containing Pax6 and other loci linked to human congenital diseases. Lens and ES cell Hi-C data (TADs and loops) together with ATAC-seq, CTCF, H3K27ac, H3K27me3 and ENCODE cis-regulatory sites are shown in detail for the Pax6, Sox1 and Hif1a loci, multiple crystallin genes and other important loci required for lens morphogenesis. The majority of crystallin loci are marked by unexpectedly high CTCF-binding across their transcribed regions. CONCLUSIONS: Our study has generated the first data on 3-dimensional (3D) nuclear organization in lens epithelium and lens fibers and directly compared these data with ES cells. These findings generate novel insights into lens-specific transcriptional gene control, open new research avenues to study transcriptional condensates in lens fiber cells, and enable studies of non-coding genetic variants linked to cataract and other lens and ocular abnormalities.

Transcriptome Analysis by RNA Sequencing of Mouse Embryonic Stem Cells Stocked on International Space Station for 1584 Days in Frozen State after Culture on the Ground.

As a space project, in "Stem Cells" by the Japan Aerospace Exploration Agency (JAXA), frozen mouse ES cells were stored on the International Space Station (ISS) in the Minus Eighty Degree Laboratory Freezer for ISS (MELFI) for 1584 days. After taking these cells back to the ground, the cells were thawed and cultured, and their gene expressions were comprehensively analyzed using RNA sequencing in order to elucidate the early response of the cells to long-time exposure to space radiation consisting of various ionized particles. The comparisons of gene expression involved in double-stranded break (DSB) repair were examined. The expressions of most of the genes that were involved in homologous recombination (HR) and non-homologous end joining (NHEJ) were not significantly changed between the ISS-stocked cells and ground-stocked control cells. However, the transcription of Trp53inp1 (tumor protein 53 induced nuclear protein-1), Cdkn1a (p21), and Mdm2 genes increased in ISS-stocked cells as well as Fe ion-irradiated cells compared to control cells. This suggests that accumulated DNA damage caused by space radiation exposure would activate these genes, which are involved in cell cycle arrest for repair and apoptosis in a p53-dependent or -independent manner, in order to prevent cells with damaged genomes from proliferating and forming tumors.

Rbm46 inhibits reactive oxygen species in mouse embryonic stem cells through modulating BNIP3-mediated mitophagy.

Embryonic stem cells (ESCs) exhibit a metabolic preference for glycolysis over oxidative phosphorylation to meet their substantial adenosine triphosphate (ATP) demands during self-renewal. This metabolic choice inherently maintains low mitochondrial activity and minimal reactive oxygen species (ROS) generation. Nonetheless, the intricate molecular mechanisms governing the restraint of ROS production and the mitigation of cellular damage remain incompletely elucidated. In this study, we reveal the pivotal role of RNA-binding motif protein 46 (RBM46) in ESCs, acting as a direct post transcriptional regulator of ROS levels by modulating BCL2/adenovirus E1B 19 kDa protein-interacting protein 3 (Bnip3) mRNA expression. Rbm46 knockout lead to diminished mitochondrial autophagy, culminating in elevated ROS within ESCs, disrupting the delicate balance required for healthy self-renewal. These findings provide insights into a novel mechanism governing ROS regulation in ESCs.

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway hubs as mouse ES cells exit pluripotency.

Enhancers bind transcription factors, chromatin regulators, and non-coding transcripts to modulate the expression of target genes. Here, we report 3D genome structures of single mouse ES cells as they are induced to exit pluripotency and transition through a formative stage prior to undergoing neuroectodermal differentiation. We find that there is a remarkable reorganization of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. In the formative state, genes important for pluripotency exit establish contacts with emerging enhancers within these multiway hubs, suggesting that the structural changes we have observed may play an important role in modulating transcription and establishing new cell identities.

In Vitro Generation of Megakaryocytes from Engineered Mouse Embryonic Stem Cells.

The in vitro differentiation of pluripotent stem cells into desired lineages enables mechanistic studies of cell transitions into more mature states that can provide insights into the design principles governing cell fate control. We are interested in reprogramming pluripotent stem cells with synthetic gene circuits to drive mouse embryonic stem cells (mESCs) down the hematopoietic lineage for the production of megakaryocytes, the progenitor cells for platelets. Here, we describe the methodology for growing and differentiating mESCs, in addition to inserting a transgene to observe its expression throughout differentiation. This entails four key methods: (1) growing and preparing mouse embryonic fibroblasts for supporting mESC growth and expansion, (2) growing and preparing OP9 feeder cells to support the differentiation of mESCs, (3) the differentiation of mESCs into megakaryocytes, and (4) utilizing an integrase-mediated docking site to insert transgenes for their stable integration and expression throughout differentiation. Altogether, this approach demonstrates a streamline differentiation protocol that emphasizes the reprogramming potential of mESCs that can be used for future mechanistic and therapeutic studies of controlling cell fate outcomes.

Synthetic reversed sequences reveal default genomic states.

Pervasive transcriptional activity is observed across diverse species. The genomes of extant organisms have undergone billions of years of evolution, making it unclear whether these genomic activities represent effects of selection or 'noise'1-4. Characterizing default genome states could help understand whether pervasive transcriptional activity has biological meaning. Here we addressed this question by introducing a synthetic 101-kb locus into the genomes of Saccharomyces cerevisiae and Mus musculus and characterizing genomic activity. The locus was designed by reversing but not complementing human HPRT1, including its flanking regions, thus retaining basic features of the natural sequence but ablating evolved coding or regulatory information. We observed widespread activity of both reversed and native HPRT1 loci in yeast, despite the lack of evolved yeast promoters. By contrast, the reversed locus displayed no activity at all in mouse embryonic stem cells, and instead exhibited repressive chromatin signatures. The repressive signature was alleviated in a locus variant lacking CpG dinucleotides; nevertheless, this variant was also transcriptionally inactive. These results show that synthetic genomic sequences that lack coding information are active in yeast, but inactive in mouse embryonic stem cells, consistent with a major difference in 'default genomic states' between these two divergent eukaryotic cell types, with implications for understanding pervasive transcription, horizontal transfer of genetic information and the birth of new genes.

AGO1 controls protein folding in mouse embryonic stem cell fate decisions.

Argonaute (AGO) proteins are evolutionarily conserved RNA-binding proteins that control gene expression through the small RNAs they interact with. Whether AGOs have regulatory roles independent of RNAs, however, is unknown. Here, we show that AGO1 controls cell fate decisions through facilitating protein folding. We found that in mouse embryonic stem cells (mESCs), while AGO2 facilitates differentiation via the microRNA (miRNA) pathway, AGO1 controls stemness independently of its binding to small RNAs. We determined that AGO1 specifically interacts with HOP, a co-chaperone for the HSP70 and HSP90 chaperones, and enhances the folding of a set of HOP client proteins with intrinsically disordered regions. This AGO1-mediated facilitation of protein folding is important for maintaining stemness in mESCs. Our results demonstrate divergent functions between AGO1 and AGO2 in controlling cellular states and identify an RNA-independent function of AGO1 in controlling gene expression and cell fate decisions.

Di-n-butyl phthalate promotes the neural differentiation of mouse embryonic stem cells through neurogenic differentiation 1.

An understanding of the risk of gene deletion and mutation posed by endocrine-disrupting chemicals (EDCs) is necessary for the identification of etiological reagents for many human diseases. Therefore, the characterization of the genetic traits caused by developmental exposure to EDCs is an important research subject. A new regenerative approach using embryonic stem cells (ESCs) holds promise for the development of stem-cell-based therapies and the identification of novel therapeutic agents against human diseases. Here, we focused on the characterization of the genetic traits and alterations in pluripotency/stemness triggered by phthalate ester derivatives. Regarding their in vitro effects, we reported the abilities of ESCs regarding proliferation, cell-cycle control, and neural ectoderm differentiation. The expression of their stemness-related genes and their genetic changes toward neural differentiation were examined, which led to the observation that the tumor suppressor gene product p53/retinoblastoma protein 1 and its related cascades play critical functions in cell-cycle progression, cell death, and neural differentiation. In addition, the expression of neurogenic differentiation 1 was affected by exposure to di-n-butyl phthalate in the context of cell differentiation into neural lineages. The nervous system is one of the most sensitive tissues to exposure to phthalate ester derivatives. The present screening system provides a good tool for studying the mechanisms underlying the effects of EDCs on the developmental regulation of humans and rodents, especially on the neuronal development of ESCs.

ATG5 attenuates inflammatory signaling in mouse embryonic stem cells to control differentiation.

Attenuated inflammatory response is a property of embryonic stem cells (ESCs). However, the underlying mechanisms are unclear. Moreover, whether the attenuated inflammatory status is involved in ESC differentiation is also unknown. Here, we found that autophagy-related protein ATG5 is essential for both attenuated inflammatory response and differentiation of mouse ESCs and that attenuation of inflammatory signaling is required for mouse ESC differentiation. Mechanistically, ATG5 recruits FBXW7 to promote ubiquitination and proteasome-mediated degradation of ß-TrCP1, resulting in the inhibition of nuclear factor κB (NF-κB) signaling and inflammatory response. Moreover, differentiation defects observed in ATG5-depleted mouse ESCs are due to ß-TrCP1 accumulation and hyperactivation of NF-κB signaling, as loss of ß-TrCP1 and inhibition of NF-κB signaling rescued the differentiation defects. Therefore, this study reveals a previously uncharacterized mechanism maintaining the attenuated inflammatory response in mouse ESCs and further expands the understanding of the biological roles of ATG5.

SETD3 regulates endoderm differentiation of mouse embryonic stem cells through canonical Wnt signaling pathway.

With self-renewal and pluripotency features, embryonic stem cells (ESCs) provide an invaluable tool to investigate early cell fate decisions. Pluripotency exit and lineage commitment depend on precise regulation of gene expression that requires coordination between transcription (TF) and chromatin factors in response to various signaling pathways. SET domain-containing 3 (SETD3) is a methyltransferase that can modify histones in the nucleus and actin in the cytoplasm. Through an shRNA screen, we previously identified SETD3 as an important factor in the meso/endodermal lineage commitment of mouse ESCs (mESC). In this study, we identified SETD3-dependent transcriptomic changes during endoderm differentiation of mESCs using time-course RNA-seq analysis. We found that SETD3 is involved in the timely activation of the endoderm-related gene network. The canonical Wnt signaling pathway was one of the markedly altered signaling pathways in the absence of SETD3. The assessment of Wnt transcriptional activity revealed a significant reduction in Setd3-deleted (setd3∆) mESCs coincident with a decrease in the nuclear pool of the key TF ß-catenin level, though no change was observed in its mRNA or total protein level. Furthermore, a proximity ligation assay (PLA) found an interaction between SETD3 and ß-catenin. We were able to rescue the differentiation defect by stably re-expressing SETD3 or activating the canonical Wnt signaling pathway by changing mESC culture conditions. Our results suggest that alterations in the canonical Wnt pathway activity and subcellular localization of ß-catenin might contribute to the endoderm differentiation defect of setd3∆ mESCs.

Tet-mediated DNA methylation dynamics affect chromosome organization.

DNA Methylation is a significant epigenetic modification that can modulate chromosome states, but its role in orchestrating chromosome organization has not been well elucidated. Here we systematically assessed the effects of DNA Methylation on chromosome organization with a multi-omics strategy to capture DNA Methylation and high-order chromosome interaction simultaneously on mouse embryonic stem cells with DNA methylation dioxygenase Tet triple knock-out (Tet-TKO). Globally, upon Tet-TKO, we observed weakened compartmentalization, corresponding to decreased methylation differences between CpG island (CGI) rich and poor domains. Tet-TKO could also induce hypermethylation for the CTCF binding peaks in TAD boundaries and chromatin loop anchors. Accordingly, CTCF peak generally weakened upon Tet-TKO, which results in weakened TAD structure and depletion of long-range chromatin loops. Genes that lost enhancer-promoter looping upon Tet-TKO showed DNA hypermethylation in their gene bodies, which may compensate for the disruption of gene expression. We also observed distinct effects of Tet1 and Tet2 on chromatin organization and increased DNA methylation correlation on spatially interacted fragments upon Tet inactivation. Our work showed the broad effects of Tet inactivation and DNA methylation dynamics on chromosome organization.

ZFP982 confers mouse embryonic stem cell characteristics by regulating expression of Nanog, Zfp42, and Dppa3.

BACKGROUND: Understanding the genetic underpinnings of protein networks conferring stemness is of broad interest for basic and translational research. METHODS: We used multi-omics analyses to identify and characterize stemness genes, and focused on the zinc finger protein 982 (Zfp982) that regulates stemness through the expression of Nanog, Zfp42, and Dppa3 in mouse embryonic stem cells (mESC). RESULTS: Zfp982 was expressed in stem cells, and bound to chromatin through a GCAGAGKC motif, for example near the stemness genes Nanog, Zfp42, and Dppa3. Nanog and Zfp42 were direct targets of ZFP982 that decreased in expression upon knockdown and increased upon overexpression of Zfp982. We show that ZFP982 expression strongly correlated with stem cell characteristics, both on the transcriptional and morphological levels. Zfp982 expression decreased with progressive differentiation into ecto-, endo- and mesodermal cell lineages, and knockdown of Zfp982 correlated with morphological and transcriptional features of differentiated cells. Zfp982 showed transcriptional overlap with members of the Hippo signaling pathway, one of which was Yap1, the major co-activator of Hippo signaling. Despite the observation that ZFP982 and YAP1 interacted and localized predominantly to the cytoplasm upon differentiation, the localization of YAP1 was not influenced by ZFP982 localization. CONCLUSIONS: Together, our study identified ZFP982 as a transcriptional regulator of early stemness genes, and since ZFP982 is under the control of the Hippo pathway, underscored the importance of the context-dependent Hippo signals for stem cell characteristics.

Inhibition of protein kinase C increases Prdm14 level to promote self-renewal of embryonic stem cells through reducing Suv39h-induced H3K9 methylation.

Inhibition of protein kinase C (PKC) efficiently promoted the self-renewal of embryonic stem cells (ESCs). However, information about the function of PKC inhibition remains lacking. Here, RNA-sequencing showed that the addition of Go6983 significantly inhibited the expression of de novo methyltransferases (Dnmt3a and Dnmt3b) and their regulator Dnmt3l, resulting in global hypomethylation of DNA in mouse ESCs. Mechanistically, PR domain-containing 14 (Prdm14), a site-specific transcriptional activator, partially contributed to Go6983-mediated repression of Dnmt3 genes. Administration of Go6983 increased Prdm14 expression mainly through the inhibition of PKCδ. High constitutive expression of Prdm14 phenocopied the ability of Go6983 to maintain` mouse ESC stemness in the absence of self-renewal-promoting cytokines. In contrast, the knockdown of Prdm14 eliminated the response to PKC inhibition and substantially impaired the Go6983-induced resistance of mouse ESCs to differentiation. Furthermore, liquid chromatography-mass spectrometry profiling and Western blotting revealed low levels of Suv39h1 and Suv39h2 in Go6983-treated mouse ESCs. Suv39h enzymes are histone methyltransferases that recognize dimethylated and trimethylated histone H3K9 specifically and usually function as transcriptional repressors. Consistently, the inhibition of Suv39h1 by RNA interference or the addition of the selective inhibitor chaetocin increased Prdm14 expression. Moreover, chromatin immunoprecipitation assay showed that Go6983 treatment led to decreased enrichment of dimethylation and trimethylation of H3K9 at the Prdm14 promoter but increased RNA polymerase Ⅱ binding affinity. Together, our results provide novel insights into the pivotal association between PKC inhibition-mediated self-renewal and epigenetic changes, which will help us better understand the regulatory network of stem cell pluripotency.

Interlinked bi-stable switches govern the cell fate commitment of embryonic stem cells.

The development of embryonic stem (ES) cells to extraembryonic trophectoderm and primitive endoderm lineages manifests distinct steady-state expression patterns of two key transcription factors-Oct4 and Nanog. How dynamically such kind of steady-state expressions are maintained remains elusive. Herein, we demonstrate that steady-state dynamics involving two bistable switches which are interlinked via a stepwise (Oct4) and a mushroom-like (Nanog) manner orchestrate the fate specification of ES cells. Our hypothesis qualitatively reconciles various experimental observations and elucidates how different feedback and feedforward motifs orchestrate the extraembryonic development and stemness maintenance of ES cells. Importantly, the model predicts strategies to optimize the dynamics of self-renewal and differentiation of embryonic stem cells that may have therapeutic relevance in the future.

Quiescence enables unrestricted cell fate in naive embryonic stem cells.

Quiescence in stem cells is traditionally considered as a state of inactive dormancy or with poised potential. Naive mouse embryonic stem cells (ESCs) can enter quiescence spontaneously or upon inhibition of MYC or fatty acid oxidation, mimicking embryonic diapause in vivo. The molecular underpinning and developmental potential of quiescent ESCs (qESCs) are relatively unexplored. Here we show that qESCs possess an expanded or unrestricted cell fate, capable of generating both embryonic and extraembryonic cell types (e.g., trophoblast stem cells). These cells have a divergent metabolic landscape comparing to the cycling ESCs, with a notable decrease of the one-carbon metabolite S-adenosylmethionine. The metabolic changes are accompanied by a global reduction of H3K27me3, an increase of chromatin accessibility, as well as the de-repression of endogenous retrovirus MERVL and trophoblast master regulators. Depletion of methionine adenosyltransferase Mat2a or deletion of Eed in the polycomb repressive complex 2 results in removal of the developmental constraints towards the extraembryonic lineages. Our findings suggest that quiescent ESCs are not dormant but rather undergo an active transition towards an unrestricted cell fate.

Mitotic bookmarking redundancy by nuclear receptors in pluripotent cells.

Mitotic bookmarking transcription factors (TFs) are thought to mediate rapid and accurate reactivation after mitotic gene silencing. However, the loss of individual bookmarking TFs often leads to the deregulation of only a small proportion of their mitotic targets, raising doubts on the biological significance and importance of their bookmarking function. Here we used targeted proteomics of the mitotic bookmarking TF ESRRB, an orphan nuclear receptor, to discover a large redundancy in mitotic binding among members of the protein super-family of nuclear receptors. Focusing on the nuclear receptor NR5A2, which together with ESRRB is essential in maintaining pluripotency in mouse embryonic stem cells, we demonstrate conjoint bookmarking activity of both factors on promoters and enhancers of a large fraction of active genes, particularly those most efficiently reactivated in G1. Upon fast and simultaneous degradation of both factors during mitotic exit, hundreds of mitotic targets of ESRRB/NR5A2, including key players of the pluripotency network, display attenuated transcriptional reactivation. We propose that redundancy in mitotic bookmarking TFs, especially nuclear receptors, confers robustness to the reestablishment of gene regulatory networks after mitosis.

DNMT1 Y495C mutation interferes with maintenance methylation of imprinting control regions.

Hereditary Sensory and Autonomic Neuropathy Type 1E (HSAN1E) is a rare autosomal dominant neurological disorder due to missense mutations in DNA methyltransferase 1 (DNMT1). To investigate the nature of the dominant effect, we compared methylomes of transgenic R1wtDnmt1 and R1Dnmt1Y495C mouse embryonic stem cells (mESCs) overexpressing WT and the mutant mouse proteins respectively, with the R1 (wild-type) cells. In case of R1Dnmt1Y495C, 15 out of the 20 imprinting control regions were hypomethylated with transcript level dysregulation of multiple imprinted genes in ESCs and neurons. Non-imprinted regions, minor satellites, major satellites, LINE1 and IAP repeats were unaffected. These data mirror the specific imprinting defects associated with transient removal of DNMT1 in mESCs, deletion of the maternal-effect DNMT1o variant in preimplantation mouse embryos, and in part, reprogramming to naïve human iPSCs. This is the first DNMT1 mutation demonstrated to specifically affect Imprinting Control Regions (ICRs), and reinforces the differences in maintenance methylation of ICRs over non-imprinted regions. Consistent with nervous system abnormalities in the HSAN1E disorder and involvement of imprinted genes in normal development and neurogenesis, R1Dnmt1Y495C cells showed dysregulated pluripotency and neuron marker genes, and yielded more slender, shorter, and extensively branched neurons. We speculate that R1Dnmt1Y495C cells produce predominantly dimers containing mutant proteins, leading to a gradual and specific loss of ICR methylation during early human development.

DNA methylation restricts coordinated germline and neural fates in embryonic stem cell differentiation.

As embryonic stem cells (ESCs) transition from naive to primed pluripotency during early mammalian development, they acquire high DNA methylation levels. During this transition, the germline is specified and undergoes genome-wide DNA demethylation, while emergence of the three somatic germ layers is preceded by acquisition of somatic DNA methylation levels in the primed epiblast. DNA methylation is essential for embryogenesis, but the point at which it becomes critical during differentiation and whether all lineages equally depend on it is unclear. Here, using culture modeling of cellular transitions, we found that DNA methylation-free mouse ESCs with triple DNA methyltransferase knockout (TKO) progressed through the continuum of pluripotency states but demonstrated skewed differentiation abilities toward neural versus other somatic lineages. More saliently, TKO ESCs were fully competent for establishing primordial germ cell-like cells, even showing temporally extended and self-sustained capacity for the germline fate. By mapping chromatin states, we found that neural and germline lineages are linked by a similar enhancer dynamic upon exit from the naive state, defined by common sets of transcription factors, including methyl-sensitive ones, that fail to be decommissioned in the absence of DNA methylation. We propose that DNA methylation controls the temporality of a coordinated neural-germline axis of the preferred differentiation route during early development.