H3K27me3 at pericentromeric heterochromatin is a defining feature of the early mouse blastocyst.

Early mouse development is characterized by structural and epigenetic changes while cells progress towards differentiation. At blastocyst stage, the segregation of the three primordial lineages is accompanied by establishment of differential patterns of DNA methylation and post-translational modifications of histones, such as H3K27me3. Here, we analysed the dynamics of H3K27me3 at pericentromeric heterochromatin (PCH) during early development. We also followed the localization of EZH2 and BEND3, previously shown in ESCs to drive PRC2 to hypomethylated PCH. We show that the location of H3K27me3 at PCH, in addition to H3K9me3, is a defining feature of embryonic cells in vivo. Moreover, it may play an important role in structuring PCH and preserving genomic integrity at a time of globally relaxed chromatin. At peri-implantation stages, while DNA methylation is still low, EZH2 and then H3K27me3, leave PCH in epiblast progenitors at the time of their spatial segregation from primitive endoderm cells, while BEND3 remains there up to implantation. The comparison with stem cells (ESCs and TSCs) reveals that the epigenetic marks (i.e. H3K9me3 and H3K27me3) of PCH are reset during in vitro derivation and only partially restored thereafter. This highlights possible divergences between in vitro and "in embryo" epigenetic regulation regarding constitutive heterochromatin.

Integrated multi-omics reveal polycomb repressive complex 2 restricts human trophoblast induction.

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.

PRC1 uncomplexed.

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.

Human branching cholangiocyte organoids recapitulate functional bile duct formation.

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.

There is another: H3K27me3-mediated genomic imprinting.

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.

Cholangiocyte organoids from human bile retain a local phenotype and can repopulate bile ducts in vitro.

The well-established 3D organoid culture method enabled efficient expansion of cholangiocyte-like cells from intrahepatic (IHBD) and extrahepatic bile duct (EHBD) tissue biopsies. The extensive expansion capacity of these organoids enables various applications, from cholangiocyte disease modelling to bile duct tissue engineering. Recent research demonstrated the feasibility of culturing cholangiocyte organoids from bile, which was minimal-invasive collected via endoscopic retrograde pancreaticography (ERCP). However, a detailed analysis of these bile cholangiocyte organoids (BCOs) and the cellular region of origin was not yet demonstrated. In this study, we characterize BCOs and mirror them to the already established organoids initiated from IHBD- and EHBD-tissue. We demonstrate successful organoid-initiation from extrahepatic bile collected from gallbladder after resection and by ERCP or percutaneous transhepatic cholangiopathy from a variety of patients. BCOs initiated from these three sources of bile all show features similar to in vivo cholangiocytes. The regional-specific characteristics of the BCOs are reflected by the exclusive expression of regional common bile duct genes (HOXB2 and HOXB3) by ERCP-derived BCOs and gallbladder-derived BCOs expressing gallbladder-specific genes. Moreover, BCOs have limited hepatocyte-fate differentiation potential compared to intrahepatic cholangiocyte organoids. These results indicate that organoid-initiating cells in bile are likely of local (extrahepatic) origin and are not of intrahepatic origin. Regarding the functionality of organoid initiating cells in bile, we demonstrate that BCOs efficiently repopulate decellularized EHBD scaffolds and restore the monolayer of cholangiocyte-like cells in vitro. Bile samples obtained through minimally invasive procedures provide a safe and effective alternative source of cholangiocyte organoids. The shedding of (organoid-initiating) cholangiocytes in bile provides a convenient source of organoids for regenerative medicine.

A plug and play microfluidic platform for standardized sensitive low-input chromatin immunoprecipitation.

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.

Terminal keratinocyte differentiation in vitro is associated with a stable DNA methylome.

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.

STARR-seq identifies active, chromatin-masked, and dormant enhancers in pluripotent mouse embryonic stem cells.

BACKGROUND: Enhancers are distal regulators of gene expression that shape cell identity and control cell fate transitions. In mouse embryonic stem cells (mESCs), the pluripotency network is maintained by the function of a complex network of enhancers, that are drastically altered upon differentiation. Genome-wide chromatin accessibility and histone modification assays are commonly used as a proxy for identifying putative enhancers and for describing their activity levels and dynamics. RESULTS: Here, we applied STARR-seq, a genome-wide plasmid-based assay, as a read-out for the enhancer landscape in "ground-state" (2i+LIF; 2iL) and "metastable" (serum+LIF; SL) mESCs. This analysis reveals that active STARR-seq loci show modest overlap with enhancer locations derived from peak calling of ChIP-seq libraries for common enhancer marks. We unveil ZIC3-bound loci with significant STARR-seq activity in SL-ESCs. Knock-out of Zic3 removes STARR-seq activity only in SL-ESCs and increases their propensity to differentiate towards the endodermal fate. STARR-seq also reveals enhancers that are not accessible, masked by a repressive chromatin signature. We describe a class of dormant, p53 bound enhancers that gain H3K27ac under specific conditions, such as after treatment with Nocodazol, or transiently during reprogramming from fibroblasts to pluripotency. CONCLUSIONS: In conclusion, loci identified as active by STARR-seq often overlap with those identified by chromatin accessibility and active epigenetic marking, yet a significant fraction is epigenetically repressed or display condition-specific enhancer activity.

Two Functional Axes of Feedback-Enforced PRC2 Recruitment in Mouse Embryonic Stem Cells.

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.

In vitro capture and characterization of embryonic rosette-stage pluripotency between naive and primed states.

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.

Dynamic CpG methylation delineates subregions within super-enhancers selectively decommissioned at the exit from naive pluripotency.

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.

Critical Role for P53 in Regulating the Cell Cycle of Ground State Embryonic Stem Cells.

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.

Untargeted histone profiling during naive conversion uncovers conserved modification markers between mouse and human.

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.

The Complexity of PRC2 Subcomplexes.

Polycomb repressive complex 2 (PRC2) is a multisubunit protein complex essential for the development of multicellular organisms. Recruitment of PRC2 to target genes, followed by deposition and propagation of its catalytic product histone H3 lysine 27 trimethylation (H3K27me3), are key to the spatiotemporal control of developmental gene expression. Recent breakthrough studies have uncovered unexpected roles for substoichiometric PRC2 subunits in these processes. Here, we elaborate on how the facultative PRC2 subunits regulate catalytic activity, locus-specific PRC2 binding, and propagation of H3K27me3, and how this affects chromatin structure, gene expression, and cell fate.

A Mass Spectrometry Survey of Chromatin-Associated Proteins in Pluripotency and Early Lineage Commitment.

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.

Publisher Correction: Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency.

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.

Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of 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.