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Funder: The Marks lab is supported by an NWO-XS grant, Funder: M.V. is part of the Oncode Institute, which is partly funded by the Dutch Cancer Society, Funder: D.W.Z. is part of the Oncode Institute, which is partly funded by the Dutch Cancer Society, Human naive pluripotent stem cells have unrestricted lineage potential. Underpinning this property, naive cells are thought to lack chromatin-based lineage barriers. However, this assumption has not been tested. Here we define the chromatin-associated proteome, histone post-translational modifications and transcriptome of human naive and primed pluripotent stem cells. Our integrated analysis reveals differences in the relative abundance and activities of distinct chromatin modules. We identify a strong enrichment of polycomb repressive complex 2 (PRC2)-associated H3K27me3 in the chromatin of naive pluripotent stem cells and H3K27me3 enrichment at promoters of lineage-determining genes, including trophoblast regulators. PRC2 activity acts as a chromatin barrier restricting the differentiation of naive cells towards the trophoblast lineage, whereas inhibition of PRC2 promotes trophoblast-fate induction and cavity formation in human blastoids. Together, our results establish that human naive pluripotent stem cells are not epigenetically unrestricted, but instead possess chromatin mechanisms that oppose the induction of alternative cell fates.

Human cholangiocyte organoids show great promise for regenerative therapies and in vitro modeling of bile duct development and diseases. However, the cystic organoids lack the branching morphology of intrahepatic bile ducts (IHBDs). Here, we report establishing human branching cholangiocyte organoid (BRCO) cultures. BRCOs self-organize into complex tubular structures resembling the IHBD architecture. Single-cell transcriptomics and functional analysis showed high similarity to primary cholangiocytes, and importantly, the branching growth mimics aspects of tubular development and is dependent on JAG1/NOTCH2 signaling. When applied to cholangiocarcinoma tumor organoids, the morphology changes to an in vitro morphology like primary tumors. Moreover, these branching cholangiocarcinoma organoids (BRCCAOs) better match the transcriptomic profile of primary tumors and showed increased chemoresistance to gemcitabine and cisplatin. In conclusion, BRCOs recapitulate a complex process of branching morphogenesis in vitro. This provides an improved model to study tubular formation, bile duct functionality, and associated biliary diseases.

Epigenetic profiling by chromatin immunoprecipitation followed by sequencing (ChIP-seq) has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks, and for screening for biomarkers. However, the ChIP-seq protocol for low-input samples is laborious and time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelization. To enable standardized, automated ChIP-seq profiling of low-input samples, we constructed microfluidic PDMS-based plates capable of performing 24 sensitive ChIP reactions within 30 min of hands-on time and 4.5 h of machine-running time. These disposable plates can be conveniently loaded into a widely available controller for pneumatics and thermocycling. In light of the plug and play (PnP) ChIP plates and workflow, we named our procedure PnP-ChIP-seq. We show high-quality ChIP-seq on hundreds to a few thousand of cells for all six post-translational histone modifications that are included in the International Human Epigenome Consortium set of reference epigenomes. PnP-ChIP-seq robustly detects epigenetic differences on promoters and enhancers between naive and more primed mouse embryonic stem cells (mESCs). Furthermore, we used our platform to generate epigenetic profiles of rare subpopulations of mESCs that resemble the two-cell stage of embryonic development. PnP-ChIP-seq allows nonexpert laboratories worldwide to conveniently run robust, standardized ChIP-seq, whereas its high throughput, consistency, and sensitivity pave the way toward large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.

Epigenetic enzymes are critically involved in gene regulation during lineage commitment. In this issue of Stem Cell Reports, Zhu et al. (2022) unravel extensive redundancy between subunits of the epigenetic regulatory Polycomb Repressive Complex 1 using a systematic knockout strategy in mouse embryonic stem cells.

Summary Polycomb Repressive Complex 2 (PRC2) plays an essential role in gene repression during development, catalyzing H3 lysine 27 trimethylation (H3K27me3). MTF2 in the PRC2.1 sub-complex, and JARID2 in PRC2.2, are central in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). To investigate how PRC2.1 and PRC2.2 cooperate, we combined Polycomb mutant mESCs with chemical inhibition of binding to H3K27me3. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, which are mutually reinforced. Whereas PRC2.1 recruitment is mediated by MTF2 binding to DNA, JARID2-containing PRC2.2 recruitment is more dependent on PRC1. Both recruitment axes are supported by core subunit EED binding to H3K27me3, but EED inhibition exhibits a more pronounced effect in Jarid2 null cells. Finally, we show that PRC1 and PRC2 enhance reciprocal binding. Together, these data disentangle the interdependent interactions that are important for PRC2 recruitment., Graphical Abstract, Highlights • Systematic analysis of PRC2 genomic binding in mouse ESCs • Mutations and chemical inhibition uncover roles of EED and other interactions • Contributions of MTF2 to PRC2.1 binding and of JARID2 and PRC1 to PRC2.2 binding • PRC1, PRC2.1, and PRC2.2 are all mutually interdependent for binding to chromatin, Veenstra and Marks and colleagues systematically analyze the contributions of Polycomb Repressive Complexes PRC2.1, PRC2.2, and PRC1 in ESCs, using null mutations in combination with chemical inhibition of EED, the core subunit that interacts with H3K27me3. The mutations and the EED inhibition uncover the functional overlap, and the compensatory and highly interdependent binding of PRC2.1, PRC2.2, and PRC1.

Following implantation, the naive pluripotent epiblast of the mouse blastocyst generates a rosette, undergoes lumenogenesis and forms the primed pluripotent egg cylinder, which is able to generate the embryonic tissues. How pluripotency progression and morphogenesis are linked and whether intermediate pluripotent states exist remain controversial. We identify here a rosette pluripotent state defined by the co-expression of naive factors with the transcription factor OTX2. Downregulation of blastocyst WNT signals drives the transition into rosette pluripotency by inducing OTX2. The rosette then activates MEK signals that induce lumenogenesis and drive progression to primed pluripotency. Consequently, combined WNT and MEK inhibition supports rosette-like stem cells, a self-renewing naive-primed intermediate. Rosette-like stem cells erase constitutive heterochromatin marks and display a primed chromatin landscape, with bivalently marked primed pluripotency genes. Nonetheless, WNT induces reversion to naive pluripotency. The rosette is therefore a reversible pluripotent intermediate whereby control over both pluripotency progression and morphogenesis pivots from WNT to MEK signals. Neagu, van Genderen and Escudero et al. show that simultaneous inhibition of WNT and MEK signalling maintains a naive-primed intermediate pluripotency state in vitro, which displays features of the mouse embryonic rosette.

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DNA methylation has long been considered the primary epigenetic mediator of genomic imprinting in mammals. Recent epigenetic profiling during early mouse development revealed the presence of domains of trimethylation of lysine 27 on histone H3 (H3K27me3) and chromatin compaction specifically at the maternally derived allele, independent of DNA methylation. Within these domains, genes are exclusively expressed from the paternally derived allele. This novel mechanism of noncanonical imprinting plays a key role in the development of mouse extraembryonic tissues and in the regulation of imprinted X-chromosome inactivation, highlighting the importance of parentally inherited epigenetic histone modifications. Here, we discuss the mechanisms underlying H3K27me3-mediated noncanonical imprinting in perspective of the dynamic chromatin landscape during early mouse development and explore evolutionary origins of noncanonical imprinting.

The epidermal compartment of the skin is regenerated constantly by proliferation of epidermal keratinocytes. Differentiation of a subset of these keratinocytes allows the epidermis to retain its barrier properties. Regulation of keratinocyte fate—whether to remain proliferative or terminally differentiate—is complex and not fully understood. The objective of our study was to assess if DNA methylation changes contribute to the regulation of keratinocyte fate. We employed genome‐wide MethylationEPIC beadchip array measuring approximately 850 000 probes combined with RNA sequencing of in vitro cultured non‐differentiated and terminally differentiated adult human primary keratinocytes. We did not observe a correlation between methylation status and transcriptome changes. Moreover, only two differentially methylated probes were detected, of which one was located in the TRIM29 gene. Although TRIM29 knock‐down resulted in lower expression levels of terminal differentiation genes, these changes were minor. From these results, we conclude that—in our in vitro experimental setup—it is unlikely that changes in DNA methylation have an important regulatory role in terminal keratinocyte differentiation.

Reprogramming requires resetting the epigenome toward a pluripotent chromatin state. A new chemical screen identifies epigenetic and signaling roadblocks for reprogramming of human somatic cells, with the inhibition of these roadblocks resulting in a more permissive epigenome for reprogramming.

Clusters of enhancers, referred as to super-enhancers (SEs), control the expression of cell identity genes. The organisation of these clusters, and how they are remodelled upon developmental transitions remain poorly understood. Here, we report the existence of two types of enhancer units within SEs typified by distinctive CpG methylation dynamics in embryonic stem cells (ESCs). We find that these units are either prone for decommissioning or remain constitutively active in epiblast stem cells (EpiSCs), as further established in the peri-implantation epiblast in vivo. Mechanistically, we show a pivotal role for ESRRB in regulating the activity of ESC-specific enhancer units and propose that the developmentally regulated silencing of ESRRB triggers the selective inactivation of these units within SEs. Our study provides insights into the molecular events that follow the loss of ESRRB binding, and offers a mechanism by which the naive pluripotency transcriptional programme can be partially reset upon embryo implantation., Clusters of enhancers, called super-enhancers (SEs), control the expression of cell identity genes. Here, the authors identify two types of enhancer units within SEs in ESCs, characterised by distinctive CpG methylation dynamics, and find that ESRRB regulates the enhancer units decommissioned at exit from naive pluripotency.

Epigenetic profiling by ChIP-Seq has become a powerful tool for genome-wide identification of regulatory elements, for defining transcriptional regulatory networks and for screening for biomarkers. However, the ChIP-Seq protocol for low-input samples is laborious, time-consuming and suffers from experimental variation, resulting in poor reproducibility and low throughput. Although prototypic microfluidic ChIP-Seq platforms have been developed, these are poorly transferable as they require sophisticated custom-made equipment and in-depth microfluidic and ChIP expertise, while lacking parallelisation. To enable standardized, automated ChIP-Seq profiling of low-input samples, we constructed PDMS-based plates containing microfluidic Integrated Fluidic Circuits capable of performing 24 sensitive ChIP reactions within 30 minutes hands-on time. These disposable plates can conveniently be loaded into a widely available controller for pneumatics and thermocycling, making the ChIP-Seq procedure Plug and Play (PnP). We demonstrate high-quality ChIP-seq on hundreds to few thousands of cells for multiple widely-profiled post-translational histone modifications, together allowing genome-wide identification of regulatory elements. As proof of principle, we managed to generate high-quality epigenetic profiles of rare totipotent subpopulations of mESCs using our platform. In light of the ready-to-go ChIP plates and the automated workflow, we named our procedure PnP-ChIP-Seq. PnP-ChIP-Seq allows non-expert labs worldwide to conveniently run robust, standardized ChIP-Seq, while its high-throughput, consistency and sensitivity paves the way towards large-scale profiling of precious sample types such as rare subpopulations of cells or biopsies.Reviewer link to dataAll sequencing data has been submitted to the NCBI GEO database. Reviewer link: http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=klwnocicrpaxrkv&acc=GSE120673

Summary Mouse embryonic stem cells (ESCs) grown in serum-supplemented conditions are characterized by an extremely short G1 phase due to the lack of G1-phase control. Concordantly, the G1-phase-specific P53-P21 pathway is compromised in serum ESCs. Here, we provide evidence that P53 is activated upon transition of serum ESCs to their pluripotent ground state using serum-free 2i conditions and that is required for the elongated G1 phase characteristic of ground state ESCs. RNA sequencing and chromatin immunoprecipitation sequencing analyses reveal that P53 directly regulates the expression of the retinoblastoma (RB) protein and that the hypo-phosphorylated, active RB protein plays a key role in G1-phase control. Our findings suggest that the P53-P21 pathway is active in ground state 2i ESCs and that its role in the G1-checkpoint is abolished in serum ESCs. Taken together, the data reveal a mechanism by which inactivation of P53 can lead to loss of RB and uncontrolled cell proliferation., Highlights • The P53-P21 pathway is activated upon adaptation of ESCs to their pluripotent ground state. • P53 is required for the elongated G1-phase characteristic to 2i ESCs. • P53 binds the promoter and activates Rb1 expression., Compared with somatic cells, mouse embryonic stem cells (ESCs) grown in serum-rich conditions display a shortened G1 phase and lack activity of the P53-P21 pathway. Stunnenberg and colleagues show that P53 is activated upon adaptation of ESCs to their pluripotent ground state in serum-free conditions. P53 modulates elongation of the G1 phase not via P21, but through transcriptional activation of the RB protein.

Recent progress has enabled the conversion of primed human embryonic stem cells (hESCs) to the naive state of pluripotency, resembling the well-characterized naive mouse ESCs (mESCs). However, a thorough histone epigenetic characterization of this conversion process is currently lacking, while its likeness to the mouse model has not been clearly established. Here, we profile the histone epigenome of hESCs during conversion in a time-resolved experimental design, using an untargeted mass spectrometry-based approach. In total, 23 histone post-translational modifications (hPTMs) changed significantly over time. H3K27Me3 was the most prominently increasing marker hPTM in naive hESCs. This is in line with previous reports in mouse, prompting us to compare all the shared hPTM fold changes between mouse and human, revealing a set of conserved hPTM markers for the naive state. Principally, we present the first roadmap of the changing human histone epigenome during the conversion of hESCs from the primed to the naive state. This further revealed similarities with mouse, which hint at a conserved mammalian epigenetic signature of the ground state of pluripotency.

Polycomb Repressive Complex 2 (PRC2) plays an essential role in development by catalysing trimethylation of histone H3 lysine 27 (H3K27me3), resulting in gene repression. PRC2 consists of two sub-complexes, PRC2.1 and PRC2.2, in which the PRC2 core associates with distinct ancillary subunits such as MTF2 and JARID2, respectively. Both MTF2, present in PRC2.1, and JARID2, present in PRC2.2, play a role in core PRC2 recruitment to target genes in mouse embryonic stem cells (mESCs). However, it remains unclear how these distinct sub-complexes cooperate to establish Polycomb domains. Here, we combine a range of Polycomb mutant mESCs with chemical inhibition of PRC2 catalytic activity, to systematically dissect their relative contributions to PRC2 binding to target loci. We find that PRC2.1 and PRC2.2 mediate two distinct paths for recruitment, with mutually reinforced binding. Part of the cross-talk between PRC2.1 and PRC2.2 occurs via their catalytic product H3K27me3, which is bound by the PRC2 core-subunit EED, thereby mediating a positive feedback. Strikingly, removal of either JARID2 or H3K27me3 only has a minor effect on PRC2 recruitment, whereas their combined ablation largely attenuates PRC2 recruitment. This strongly suggests an unexpected redundancy between JARID2 and EED-H3K27me3-mediated recruitment of PRC2. Furthermore, we demonstrate that all core PRC2 recruitment occurs through the combined action of MTF2-mediated recruitment of PRC2.1 to DNA and PRC1-mediated recruitment of JARID2-containing PRC2.2. Both axes of binding are supported by EED-H3K27me3 positive feedback, but to a different degree. Finally, we provide evidence that PRC1 and PRC2 mutually reinforce reciprocal binding. Together, these data disentangle the interdependent and cooperative interactions between Polycomb complexes that are important to establish Polycomb repression at target sites.HighlightsSystematic analysis of Polycomb complex binding to target loci in mESCs using null mutations and chemical inhibition.PRC1, PRC2.1 and PRC2.2 are all mutually dependent for binding to chromatin, mediated in part by H3K27me3.PRC2.1 recruitment is dependent on MTF2PRC2.2 recruitment by JARID2 is dependent on PRC1 and largely redundant with recruitment by H3K27me3

Background Genomic imprinting, resulting in parent-of-origin specific gene expression, plays a critical role in mammalian development. Here, we apply allele-specific RNA-seq on isogenic B6D2F1 mice to assay imprinted genes in tissues from early embryonic tissues between E3.5 and E7.25 and in pluripotent cell lines to evaluate maintenance of imprinted gene expression. For the cell lines, we include embryonic stem cells (ESCs) and epiblast stem cells (EpiSCs) derived from fertilized embryos and from embryos obtained after nuclear transfer (NT) or parthenogenetic activation (PGA). Results As homozygous genomic regions of PGA-derived cells are not compatible with allele-specific RNA-seq, we developed an RNA-seq-based genotyping strategy allowing identification of informative heterozygous regions. Global analysis shows that proper imprinted gene expression as observed in embryonic tissues is largely lost in the ESC lines included in this study, which mainly consisted of female ESCs. Differentiation of ESC lines to embryoid bodies or NPCs does not restore monoallelic expression of imprinted genes, neither did reprogramming of the serum-cultured ESCs to the pluripotent ground state by the use of 2 kinase inhibitors. Fertilized EpiSC and EpiSC-NT lines largely maintain imprinted gene expression, as did EpiSC-PGA lines that show known paternally expressed genes being silent and known maternally expressed genes consistently showing doubled expression. Notably, two EpiSC-NT lines show aberrant silencing of Rian and Meg3, two critically imprinted genes in mouse iPSCs. With respect to female EpiSC, most of the lines displayed completely skewed X inactivation suggesting a (near) clonal origin. Conclusions Altogether, our analysis provides a comprehensive overview of imprinted gene expression in pluripotency and provides a benchmark to allow identification of cell lines that faithfully maintain imprinted gene expression and therefore retain full developmental potential. Electronic supplementary material The online version of this article (10.1186/s13072-019-0259-8) contains supplementary material, which is available to authorized users.

International audience; The pluripotent ground state is defined as a basal state free of epigenetic restrictions, which influence lineage specification. While naive embryonic stem cells (ESCs) can be maintained in a hypomethylated state with open chromatin when grown using two small-molecule inhibitors (2i)/leukemia inhibitory factor (LIF), in contrast to serum/LIF-grown ESCs that resemble early post-implantation embryos, broader features of the ground-state pluripotent epigenome are not well understood. We identified epigenetic features of mouse ESCs cultured using 2i/LIF or serum/LIF by proteomic profiling of chromatin-associated complexes and histone modifications. Polycomb-repressive complex 2 (PRC2) and its product H3K27me3 are highly abundant in 2i/LIF ESCs, and H3K27me3 is distributed genome-wide in a CpG-dependent fashion. Consistently, PRC2-deficient ESCs showed increased DNA methylation at sites normally occupied by H3K27me3 and increased H4 acetylation. Inhibiting DNA methylation in PRC2-deficient ESCs did not affect their viability or transcriptome. Our findings suggest a unique H3K27me3 configuration protects naive ESCs from lineage priming, and they reveal widespread epigenetic crosstalk in ground-state pluripotency.

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

Pluripotency can be captured in vitro in the form of Embryonic Stem Cells (ESCs). These ESCs can be either maintained in the unrestricted "naïve" state of pluripotency, adapted to developmentally more constrained "primed" pluripotency or differentiated towards each of the three germ layers. Epigenetic protein complexes and transcription factors have been shown to specify and instruct transitions from ESCs to distinct cell states. In this study, proteomic profiling of the chromatin landscape by chromatin enrichment for proteomics (ChEP) is used in mouse naive pluripotent ESCs, primed pluripotent Epiblast stem cells (EpiSCs), and cells in early stages of differentiation. A comprehensive overview of epigenetic protein complexes associated with the chromatin is provided and proteins associated with the maintenance and loss of pluripotency are identified. The data reveal major compositional alterations of epigenetic complexes during priming and differentiation of naïve pluripotent ESCs. These results contribute to the understanding of ESC differentiation and provide a framework for future studies of lineage commitment of ESCs.

The mechanisms underlying enhancer activation and the extent to which enhancer-promoter rewiring contributes to spatiotemporal gene expression are not well understood. Using integrative and time-resolved analyses we show that the extensive transcriptome and epigenome resetting during the conversion between 'serum' and '2i' states of mouse embryonic stem cells (ESCs) takes place with minimal enhancer-promoter rewiring that becomes more evident in primed-state pluripotency. Instead, differential gene expression is strongly linked to enhancer activation via H3K27ac. Conditional depletion of transcription factors and allele-specific enhancer analysis reveal an essential role for Esrrb in H3K27 acetylation and activation of 2i-specific enhancers. Restoration of a polymorphic ESRRB motif using CRISPR-Cas9 in a hybrid ESC line restores ESRRB binding and enhancer H3K27ac in an allele-specific manner but has no effect on chromatin interactions. Our study shows that enhancer activation in serum- and 2i-ESCs is largely driven by transcription factor binding and epigenetic marking in a hardwired network of chromatin interactions.

The ground state of pluripotency is defined as a minimal unrestricted epigenetic state as present in the Inner Cell Mass. Mouse embryonic stem cells (ESCs) grown in a defined serum-free medium with two kinase inhibitors (“2i ESCs”) have been postulated to reflect ground-state pluripotency, whereas ESCs grown in the presence of serum (“serum ESCs”) share more similarities with post-implantation epiblast cells. Pluripotency results from an intricate interplay between cytoplasmic, nuclear and chromatin-associated proteins. Here, we perform quantitative subcellular proteomics to gain insight in the molecular mechanisms sustaining the pluripotent states reflected by 2i and serum ESCs. We describe a full SILAC workflow and quality controls for proteomic comparison of 2i and serum ESCs, allowing subcellular proteomics of the cytoplasm, nucleoplasm and chromatin. The obtained quantitative information revealed increased levels of naïve pluripotency factors on the chromatin of 2i ESCs. Surprisingly, the cytoplasmic proteome suggests that 2i and serum ESCs utilize distinct metabolic programs, which include upregulation of free radical buffering by the glutathione pathway in 2i ESCs. Through induction of intracellular radicals, we show that the altered metabolic environment renders 2i ESCs less sensitive to oxidative stress. Altogether, this work provides novel insights into the proteomic landscape underlying ground state pluripotency. Keywords: Embryonic stem cells, Metabolism, Naïve pluripotency, SILAC

During early mammalian development, transient pools of pluripotent cells emerge that can be immortalised upon stem cell derivation. The pluripotent state, ‘naïve’ or ‘primed’, depends on the embryonic stage and derivation conditions used. Here we analyse the temporal gene expression patterns of mouse, cattle and porcine embryos at stages that harbour different types of pluripotent cells. We document conserved and divergent traits in gene expression, and identify predictor genes shared across the species that are associated with pluripotent states in vivo and in vitro. Amongst these are the pluripotency-linked genes Klf4 and Lin28b. The novel genes discovered include naïve- (Spic, Scpep1 and Gjb5) and primed-associated (Sema6a and Jakmip2) genes as well as naïve to primed transition genes (Dusp6 and Trip6). Both Gjb5 and Dusp6 play a role in pluripotency since their knockdown results in differentiation and downregulation of key pluripotency genes. Our interspecies comparison revealed new insights of pluripotency, pluripotent stem cell identity and a new molecular criterion for distinguishing between pluripotent states in various species, including human., Summary: Interspecies comparison of mouse, bovine and pig embryos revealed conserved genes which distinguish between naïve and primed pluripotency states, including in human. Some of these genes interfere with the pluripotency network and lead to differentiation.

In the version of the article originally published, extra lines were displayed in Fig. 7. Fig. 7a contained a solid black line that extended into panel b, and Fig. 7c contained two extra scale bars on the left. These have been removed from the figure. The errors have been corrected in the HTML and PDF versions of the article.

Polycomb-mediated repression of gene expression is essential for development, with a pivotal role played by trimethylation of histone H3 lysine 27 (H3K27me3), which is deposited by Polycomb Repressive Complex 2 (PRC2). The mechanism by which PRC2 is recruited to target genes has remained largely elusive, particularly in vertebrates. Here we demonstrate that MTF2, one of the three vertebrate homologs of Drosophila melanogaster Polycomblike, is a DNA-binding, methylation-sensitive PRC2 recruiter in mouse embryonic stem cells. MTF2 directly binds to DNA and is essential for recruitment of PRC2 both in vitro and in vivo. Genome-wide recruitment of the PRC2 catalytic subunit EZH2 is abrogated in Mtf2 knockout cells, resulting in greatly reduced H3K27me3 deposition. MTF2 selectively binds regions with a high density of unmethylated CpGs in a context of reduced helix twist, which distinguishes target from non-target CpG islands. These results demonstrate instructive recruitment of PRC2 to genomic targets by MTF2.

Mouse embryonic stem (ES) cells are a popular model system to study biological processes, though uncovering recessive phenotypes requires inactivat- ing both alleles. Building upon resources from the International Knockout Mouse Consortium (IKMC), we developed a targeting vector for second allele in- activation in conditional-ready IKMC ‘knockout-first’ ES cell lines. We applied our technology to sev- eral epigenetic regulators, recovering bi-allelic tar- geted clones with a high efficiency of 60% and used Flp recombinase to restore expression in two null cell lines to demonstrate how our system confirms causality through mutant phenotype reversion. We designed our strategy to select against re-targeting the ‘knockout-first’ allele and identify essential genes in ES cells, including the histone methyltransferase Setdb1. For confirmation, we exploited the flexibil- ity of our system, enabling tamoxifen inducible con- ditional gene ablation while controlling for genetic background and tamoxifen effects. Setdb1 ablated ES cells exhibit severe growth inhibition, which is not rescued by exogenous Nanog expression or cultur- ing in naive pluripotency ‘2i’ media, suggesting that the self-renewal defect is mediated through pluripo- tency network independent pathways. Our strategy to generate null mutant mouse ES cells is applicable to thousands of genes and repurposes existing IKMC Intermediate Vectors.

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naïve pluripotency. Here, we examine the initial transition process. The ES cell population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naïve status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naïve pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state display features of early post-implantation epiblast and are distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naïve cells transition to a distinct formative phase of pluripotency preparatory to lineage priming., Summary: Extinction of mouse embryonic stem cell identity upon differentiation immediately follows collapse of the naïve pluripotency transcription factor circuitry and precedes upregulation of lineage-specific factors.

Transit amplification (TA), a state of combined, rapid proliferative expansion and differentiation of stem cell-descendants, remains poorly defined at the molecular level. The Polycomb Repressive Complex 1 (PRC1) protein BMI1 has been localized to TA compartments, yet its exact role in TA is unclear. PRC1 proteins control gene expression, cell proliferation and DNA-damage repair. Coordination of such DNA-templated activities during TA is predicted to be crucial to support DNA replication and differentiation-associated transcriptional programming. We here examined whether chondrogenesis provides a relevant biological context for synchronized coordination of these chromatin-based tasks by BMI1. Taking advantage of a prominently featuring TA-phase during chondrogenesis in vitro and in vivo, we here report that TA is completely dependent on intact PRC1 function. BMI1-depleted chondrogenic progenitors rapidly accumulate double strand DNA breaks during DNA replication, present massive non-H3K27me3-directed transcriptional deregulation and fail to undergo chondrogenic TA. Genome-wide accumulation of Topoisomerase 2α and Geminin suggests a model in which PRC1 synchronizes replication and transcription during rapid chondrogenic progenitor expansion. Our combined data reveals for the first time a vital cell-autonomous role for PRC1 during chondrogenesis. We provide evidence that chondrocyte hyper-replication and hypertrophy represent a unique example of programmed senescence in vivo. These findings provide new perspectives on PRC1 function in development and disease., The authors received financial support from the European Molecular Biology Organization (Germany; www.embl.de) ASTF5-2009 (F.S.) and the René Vogels foundation (F.S.); grant BFU2010-18146 (MINECO, MV) and Oncocycle program S2010/BMD2470 (Comunidad de Madrid, MV); the Dutch Science Organization (ZonMW-NWO; www.NWO.nl) VIDI grant 864.12.007 (H.M.); VIDI grant 016.046.362 (J.W.V.); Netherlands Reuma foundation (The Netherlands; www.ruemafonds.nl) grant LLP14 (J.W.V., L. van Rhijn, Maastricht, The Netherlands) and a transnational University of Limburg grant (J.W.V., B.W.G.).

A myriad of diseases is caused or characterized by alteration of epigenetic patterns, including changes in DNA methylation, post-translational histone modifications, or chromatin structure. These changes of the epigenome represent a highly interesting layer of information for disease stratification and for personalized medicine. Traditionally, epigenomic profiling required large amounts of cells, which are rarely available with clinical samples. Also, the cellular heterogeneity complicates analysis when profiling clinical samples for unbiased genome-wide biomarker discovery. Recent years saw great progress in miniaturization of genome-wide epigenomic profiling, enabling large-scale epigenetic biomarker screens for disease diagnosis, prognosis, and stratification on patient-derived samples. All main genome-wide profiling technologies have now been scaled down and/or are compatible with single-cell readout, including: (i) Bisulfite sequencing to determine DNA methylation at base-pair resolution, (ii) ChIP-Seq to identify protein binding sites on the genome, (iii) DNaseI-Seq/ATAC-Seq to profile open chromatin, and (iv) 4C-Seq and HiC-Seq to determine the spatial organization of chromosomes. In this review we provide an overview of current genome-wide epigenomic profiling technologies and main technological advances that allowed miniaturization of these assays down to single-cell level. For each of these technologies we evaluate their application for future biomarker discovery. We will focus on (i) compatibility of these technologies with methods used for clinical sample preservation, including methods used by biobanks that store large numbers of patient samples, and (ii) automation of these technologies for robust sample preparation and increased throughput.

After the publication of this work [1], we noticed there was an error in Fig. 5 where −1,0 and 1 are incorrectly displayed in the y-axis in panel b. Please see the corrected Fig. 5 below. We apologize for this error. Fig. 5 llele-specific RNA-seq on three NPC lines identifies three distal regions of genes that escape XCI. a Ratio of Xi/Xa (y-axis; for each of the three NPC lines sorted from highest to lowest) for genes showing a log2 ratio of at least −5. We set ...

Embryonic germ cells (EGC) are cultured pluripotent cells derived from primordial germ cells (PGC). This study explored the possibility of establishing porcine EGC from domestic breeds and Yucatan mini pigs using embryos at Days 17-24 of gestation. In vitro culture of PGC from both pooled and individual embryos resulted in the successful derivation of putative EGC lines from Days 20 to 24 with high efficiency. RT-PCR showed that gene expression among all 31 obtained cell lines was very similar, and only minor changes were detected during in vitro passaging of the cells. Genome-wide RNA-Seq expression profiling showed no expression of the core pluripotency markers OCT4, SOX2, and NANOG, although most other pluripotency genes were expressed at levels comparable to those of mouse embryonic stem cells (ESC). Moreover, germ-specific genes such as BLIMP1 retained their expression. Functional annotation clustering of the gene expression pattern of the putative EGC suggests partial differentiation toward endo/mesodermal lineages. The putative EGC were able to form embryoid bodies in suspension culture and to differentiate into epithelial-like, mesenchymal-like, and neuronal-like cells. However, their injection into immunodeficient mice did not result in teratoma formation. Our results suggest that the PGC-derived cells described in this study are EGC-like, but seem to be multipotent rather than pluripotent cells. Nevertheless, the thorough characterization of these cells in this study, and especially the identification of various genes and pathways involved in pluripotency by RNA-Seq, will serve as a rich resource for further derivation of porcine EGC.

Variable genomic loci have been employed in a number of molecular epidemiology studies of white spot syndrome virus (WSSV), but it is unknown which loci are suitable molecular markers for determining WSSV spread on different spatiotemporal scales. Although previous work suggests that multiple introductions of WSSV occurred in central Vietnam, it is largely uncertain how WSSV was introduced and subsequently spread. Here, we evaluate five variable WSSV DNA loci as markers of virus spread on an intermediate (i.e. regional) scale, and develop a detailed and statistically supported model for the spread of WSSV. The genotypes of 17 WSSV isolates from along the coast of Vietnam – nine of which were newly characterized in this study – were analysed to obtain sufficient samples on an intermediate scale and to allow statistical analysis. Only the ORF23/24 variable region is an appropriate marker on this scale, as geographically proximate isolates show similar deletion sizes. The ORF14/15 variable region and variable-number tandem repeat (VNTR) loci are not useful as markers on this scale. ORF14/15 may be suitable for studying larger spatiotemporal scales, whereas VNTR loci are probably suitable for smaller scales. For ORF23/24, there is a clear pattern in the spatial distribution of WSSV: the smallest genomic deletions are found in central Vietnam, and larger deletions are found in the south and the north. WSSV genomic deletions tend to increase over time with virus spread in cultured shrimp, and our data are therefore congruent with the hypothesis that WSSV was introduced in central Vietnam and then radiated out.

Background During early embryonic development, one of the two X chromosomes in mammalian female cells is inactivated to compensate for a potential imbalance in transcript levels with male cells, which contain a single X chromosome. Here, we use mouse female embryonic stem cells (ESCs) with non-random X chromosome inactivation (XCI) and polymorphic X chromosomes to study the dynamics of gene silencing over the inactive X chromosome by high-resolution allele-specific RNA-seq. Results Induction of XCI by differentiation of female ESCs shows that genes proximal to the X-inactivation center are silenced earlier than distal genes, while lowly expressed genes show faster XCI dynamics than highly expressed genes. The active X chromosome shows a minor but significant increase in gene activity during differentiation, resulting in complete dosage compensation in differentiated cell types. Genes escaping XCI show little or no silencing during early propagation of XCI. Allele-specific RNA-seq of neural progenitor cells generated from the female ESCs identifies three regions distal to the X-inactivation center that escape XCI. These regions, which stably escape during propagation and maintenance of XCI, coincide with topologically associating domains (TADs) as present in the female ESCs. Also, the previously characterized gene clusters escaping XCI in human fibroblasts correlate with TADs. Conclusions The gene silencing observed during XCI provides further insight in the establishment of the repressive complex formed by the inactive X chromosome. The association of escape regions with TADs, in mouse and human, suggests that TADs are the primary targets during propagation of XCI over the X chromosome. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0698-x) contains supplementary material, which is available to authorized users.

Cigarette smoke is the principal risk factor for chronic obstructive pulmonary disease (COPD). Multidrug resistance proteins, such as multidrug resistance-associated protein-1 (MRP1), P-glycoprotein (P-gp), and lung resistance-related protein (LRP), may protect against oxidative stress and toxic compounds generated by cigarette smoking. Expression of MRP1, P-gp, and LRP was evaluated in bronchial epithelium of two study groups of COPD patients and their controls and was associated with disease status and smoking history. In study group 1, MRP1, but not P-gp and LRP expression, was lower (p=0.029) in normal bronchial epithelium of COPD patients (n=11) compared to healthy controls (n=8). MRP1 expression was high in squamous metaplastic epithelium. When including expression in squamous metaplastic cells, MRP1 was still lower in total bronchial epithelium in the COPD group (p=0.038). In study group 2, expression of MRP1, but not of P-gp and LRP, was lower (p=0.047) in lung tissue of (very) severe COPD (n=10) vs mild to moderate COPD (n=9) patients. In conclusion, MRP1 expression was lower in bronchial biopsies of COPD patients than of healthy controls and was also lower in patients with severe COPD than with mild/moderate COPD. Our findings indicate that diminished MRP1 expression in normal bronchial epithelium is associated with COPD. The exact role in COPD pathogenesis is to be revealed by further functional studies.

White spot syndrome virus (WSSV), member of a. new virus family called Nimaviridae, is a major scourge in worldwide shrimp, cultivation. Geographical isolates of WSSV identified so far are very similar in morphology and proteome, and show little difference in restriction fragment length polymorphism (RFLP) pattern. We have mapped the genomic differences between three completely sequenced WSSV isolates, originating from Thailand (WSSV-TH), China (WSSV-CN) and Taiwan (WSSV-TW). Alignment of the genomic sequences of these geographical isolates revealed an overall nucleotide identity of 99.32%. The major difference among the three isolates is a deletion of approximately 13 kb (WSSV-TH) and 1 kb (WSSV-CN), present in the same genomic region, relative to WSSV-TW. A second difference involves a genetically variable region of about 750 bp. All other variations >2 bp between the three isolates are located in repeat regions along the genome. Except for the homologous regions (hrl, hr3, hr8 and hr9), these variable repeat regions are almost exclusively located in ORFs, of which the genomic repeat regions in ORF75, ORF94 and ORF125 can be used for PCR based classification of WSSV isolates in epidemiological studies. Furthermore, the comparison identified highly invariable genomic loci, which may be used for reliable monitoring of WSSV infections and for shrimp health certification.

White spot syndrome virus (WSSV) is a member of a new virus family (Nimaviridae) infecting crustaceans. The regulation of transcription of WSSV genes is largely unknown. Transcription of the major WSSV structural virion protein genes, vp28, vp26, vp24, vp19 and vp15, was studied to search for common promoter motifs for coordinate expression. The temporal expression of these genes and both 5' and 3' ends of the mRNA were determined, using infected crayfish gill tissue as a RNA source. RT-PCR showed that all five genes are expressed late in infection compared to the early ribonucleotide reductase large subunit gene. 5' RACE studies revealed a consensus late transcription initiation motif for only two of the five major virion protein genes. This motif was only found in one other upstream region of the putative translational start site of a gene with unknown function (ORF 158). No other conserved sequence motifs could be detected in the sequences surrounding the transcriptional start sites of the five major virion protein genes. All 5' ends were located about 25 nt downstream of an A/T rich sequence, including the consensus TATA-box sequence for vp15. The absence of a consensus motif is distinct from gene regulation of other large dsDNA viruses and suggests a unique regulation of WSSV transcription, in line with its unique taxonomic position.

Embryonic Stem Cells (ESCs) and Epiblast Stem Cells (EpiSCs) are the in vitro representatives of naïve and primed pluripotency, respectively. It is currently unclear how their epigenomes underpin the phenotypic and molecular characteristics of these distinct pluripotent states. Here, we performed a genome-wide comparison of DNA methylation between ESCs and EpiSCs by MethylCap-Seq. We observe that promoters are preferential targets for methylation in EpiSC compared to ESCs, in particular high CpG island promoters. This is in line with upregulation of the de novo methyltransferases Dnmt3a1 and Dnmt3b in EpiSC, and downregulation of the demethylases Tet1 and Tet2. Remarkably, the observed DNA methylation signature is specific to EpiSCs and differs from that of their in vivo counterpart, the postimplantation epiblast. Using a subset of promoters that are differentially methylated, we show that DNA methylation is established within a few days during in vitro outgrowth of the epiblast, and also occurs when ESCs are converted to EpiSCs in vitro. Once established, this methylation is stable, as ES-like cells obtained by in vitro reversion of EpiSCs display an epigenetic memory that only extensive passaging and sub-cloning are able to almost completely erase.