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Proximity biotinylation workflows typically require CRISPR-based genetic manipulation of target cells. To overcome this bottleneck, we fused the TurboID proximity biotinylation enzyme to Protein A. Upon target cell permeabilization, the ProtA-Turbo enzyme can be targeted to proteins or post-translational modifications of interest using bait-specific antibodies. Addition of biotin then triggers bait-proximal protein biotinylation. Biotinylated proteins can subsequently be enriched from crude lysates and identified by mass spectrometry. We demonstrate this workflow by targeting Emerin, H3K9me3 and BRG1. Amongst the main findings, our experiments reveal that the essential protein FLYWCH1 interacts with a subset of H3K9me3-marked (peri)centromeres in human cells. The ProtA-Turbo enzyme represents an off-the-shelf proximity biotinylation enzyme that facilitates proximity biotinylation experiments in primary cells and can be used to understand how proteins cooperate in vivo and how this contributes to cellular homeostasis and disease., Proximity biotinylation is a powerful tool to profile interactomes, but it requires genetic engineering of the target protein. Here, the authors develop a proximity biotinylation enzyme that can be directed to the target using antibodies, enabling interactome profiling of endogenous proteins or PTMs.

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.

Understanding how chromatin is regulated is essential to fully grasp genome biology, and establishing the locus-specific protein composition is a major step toward this goal. Here we explain why the isolation and analysis of a specific chromatin segment are technically challenging, independently of the method. We then describe the published strategies and discuss their advantages and limitations. We conclude by discussing why significant technology developments are required to unambiguously describe the composition of small single loci.

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.

SummarySwitch-like cyclin-dependent kinase (CDK)-1 activation is thought to underlie the abruptness of mitotic onset, but how CDKs can simultaneously phosphorylate many diverse substrates is unknown, and direct evidence for such phosphorylation dynamics in vivo is lacking. Here, we analysed protein phosphorylation states in single Xenopus embryos throughout synchronous cell cycles. Over a thousand phosphosites were dynamic in vivo, and assignment of cell cycle phases using egg extracts revealed hundreds of S-phase phosphorylations. Targeted phosphoproteomics in single embryos showed switch-like mitotic phosphorylation of diverse protein complexes. The majority of cell cycle-regulated phosphosites occurred in CDK consensus motifs, and 72% located to intrinsically disordered regions. Dynamically phosphorylated proteins, and documented substrates of cell cycle kinases, are significantly more disordered than phosphoproteins in general. Furthermore, 30-50% are components of membraneless organelles. Our results suggest that phosphorylation of intrinsically disordered proteins by cell cycle kinases, particularly CDKs, allows switch-like mitotic cellular reorganisation.

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.

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.

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 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

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.