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Expression and phase separation potential of heterochromatin proteins during early mouse development

2019; Springer Nature; Volume: 20; Issue: 12 Linguagem: Inglês

10.15252/embr.201947952

1469-3178

Manuel Guthmann, Adam Burton, Maria‐Elena Torres‐Padilla,

RNA modifications and cancer

Report7 November 2019Open Access Expression and phase separation potential of heterochromatin proteins during early mouse development Manuel Guthmann Manuel Guthmann Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany Faculty of Biology, Ludwig-Maximilians Universität, München, Germany Search for more papers by this author Adam Burton Adam Burton Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany Faculty of Biology, Ludwig-Maximilians Universität, München, Germany Search for more papers by this author Maria-Elena Torres-Padilla Corresponding Author Maria-Elena Torres-Padilla [email protected] orcid.org/0000-0002-1020-2074 Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany Faculty of Biology, Ludwig-Maximilians Universität, München, Germany Search for more papers by this author Manuel Guthmann Manuel Guthmann Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany Faculty of Biology, Ludwig-Maximilians Universität, München, Germany Search for more papers by this author Adam Burton Adam Burton Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany Faculty of Biology, Ludwig-Maximilians Universität, München, Germany Search for more papers by this author Maria-Elena Torres-Padilla Corresponding Author Maria-Elena Torres-Padilla [email protected] orcid.org/0000-0002-1020-2074 Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany Faculty of Biology, Ludwig-Maximilians Universität, München, Germany Search for more papers by this author Author Information Manuel Guthmann1,2, Adam Burton1,2 and Maria-Elena Torres-Padilla *,1,2 1Institute of Epigenetics and Stem Cells (IES), Helmholtz Zentrum München, München, Germany 2Faculty of Biology, Ludwig-Maximilians Universität, München, Germany *Corresponding author. Tel: +49 89 3187 3317; E-mail: [email protected] EMBO Reports (2019)20:e47952https://doi.org/10.15252/embr.201947952 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract In most eukaryotes, constitutive heterochromatin is associated with H3K9me3 and HP1α. The latter has been shown to play a role in heterochromatin formation through liquid–liquid phase separation. However, many other proteins are known to regulate and/or interact with constitutive heterochromatic regions in several species. We postulate that some of these heterochromatic proteins may play a role in the regulation of heterochromatin formation by liquid–liquid phase separation. Indeed, an analysis of the constitutive heterochromatin proteome shows that proteins associated with constitutive heterochromatin are significantly more disordered than a random set or a full nucleome set of proteins. Interestingly, their expression begins low and increases during preimplantation development. These observations suggest that the preimplantation embryo is a useful model to address the potential role for phase separation in heterochromatin formation, anticipating exciting research in the years to come. Synopsis Proteins of the constitutive heterochromatin proteome tend to contain intrinsically disordered regions. Their expression correlates with chromocenter formation and reduced cellular plasticity during mouse pre-implantation development. Proteins associated with constitutive heterochromatin display a higher disorder score than a random or nuclear set of proteins. The expression of genes associated with constitutive heterochromatin increases during pre-implantation development. The pre-implantation embryo is a useful model to address potential roles for phase-separation in heterochromatin formation. Introduction In eukaryotes, around 145 basepairs of DNA are wrapped around octamers of the four canonical histones H2A, H2B, H3 and H4 to form the nucleosome. The nucleosome is the building block of the chromatin, which in addition includes other chromatin-associated proteins that bind nucleosomes and also the linker histone H1. Functionally, chromatin has been traditionally divided into two categories: hetero- and euchromatin 1, which were first recognised cytologically by Emil Heitz 2. Heterochromatin appeared as regions of the nucleus that do not decondense after mitosis, which he considered to be a non-functional part of the genome. Nowadays, the definition of heterochromatin has broadened to include features such as (i) histone modifications such as histone 3 lysine 9 trimethylation (H3K9me3), H3K27me3, DNA methylation and potentially also H3K56me3 3, 4; (ii) a (mostly) transcriptionally silent state; (iii) a late replicating nature; (iv) an electron-dense and condensed state in electron microscopy 5, and more recently (v) a higher resistance to sonication 6. Heterochromatin can be further broadly divided into constitutive heterochromatin—which is located at centromeric and telomeric regions, as well as at most repeat elements throughout most eukaryotic genomes—and facultative heterochromatin, which harbours the H3K27me3 mark and often localises to temporally or spatially regulated genes 5. Over the last two decades, a rather unified model for constitutive heterochromatin establishment has emerged whereby the Suv39h1/h2 (Su(var)9-1) enzymes initiate a feedback cascade by catalysing H3K9me3, which in turns recruits heterochromatin protein 1 (HP1) proteins, primarily through their chromodomain 7-9. Downstream recruitment of Suv420h1/h2 (Su(var)4-20) reinforces a heterochromatic loop by catalysing H4K20me3 10, while as yet unknown enzymes deposit H3K64me3 11. Subsequent recruitment of Suv39h1/h2 by both HP1 and H3K9me3 enables spreading and amplification of the heterochromatin domain. In addition, RNA-mediated interactions of HP1 and the Su(var) enzymes themselves have also been implicated in maintaining constitutive heterochromatin in mouse, human and yeast 12-15. However, relatively little is known about the mechanisms that direct heterochromatin formation in vivo, at the beginning of development. It has recently been suggested that heterochromatin can form by phase separation through the local accumulation of HP1α 16, 17. Phase-separated compartments appear as immiscible liquid droplets that emerge through multivalent, weak interactions between biological polymers, which can be either proteins or nucleic acids 18, 19. Multivalent interactions can be provided by intrinsically disordered domains (IDRs) or structured domains. Liquid droplets can undergo fission, coalesce into larger droplets and relax to their original spherical shape after shear stress 20, 21. Since the discovery that P granules form by liquid–liquid phase separation in the Caenorhabditis elegans germline around 10 years ago, many studies have shown that several membrane-less organelles may in fact form through phase separation 22-26. These include the nucleolus, which has physical properties of a phase-separated liquid-like droplet formed of several immiscible liquid sub-compartments 21, 27, but also stress granules and paraspeckles 28, 29 as well as cajal bodies 23. More recently, some studies have also suggested a role for phase separation in transcription initiation, by facilitating the recruitment of the transcriptional machinery 30-35. Similarly, liquid–liquid phase separation was suggested to play a role in facultative heterochromatin formation by enabling the assembly of the polycomb repressive complex 1 36. In the phase-separation-based model for constitutive heterochromatin formation 16, 17, 37, the binding of HP1α to H3K9me3 would lead to a local increase in HP1α concentration, which in turn would nucleate a phase-separated compartment that could then grow and fuse, enabling the formation of constitutive heterochromatin. The liquid–liquid phase separation biophysical properties would also explain the selective exclusion of certain proteins from these heterochromatin compartments. In such a model, exclusion from domains may be due to the inability to interact with phase-inclusive components, but it can also result from the emergent biophysical properties of the domain. However, a recent report shows that IDR-rich liquid condensates tend to exclude chromatin, which is at odds with the proposed growth and fusion of phase-separated heterochromatin compartments. In fact, when promoting droplet formation at heterochromatin using a synthetic “CasDrop” approach, condensates appear at the periphery of such regions 38. Thus, these conceptual frameworks to understand the formation and physical properties of heterochromatic genomic regions are still in their early days, and have not yet incorporated all the additional proteins known to be present at constitutive heterochromatin, and which may therefore play a role in regulating heterochromatin establishment. How and whether these mechanisms operate in the early mammalian embryo at the onset of epigenetic reprogramming are unknown. Even though heterochromatin has been extensively studied, little is known about its biophysical properties as well as the mechanisms that underlie heterochromatin formation, as opposed to maintenance, in vivo. Here, we have undertaken an analysis to investigate the properties of heterochromatin-associated proteins and their potential to phase separate as well as their expression pattern at the earliest developmental stages in the mouse embryo. Finally, we propose possible avenues for addressing phase separation as a potential mechanism for heterochromatin formation at the beginning of development. Results and Discussion Several mass spectrometry studies have been carried out in mammalian cells to better understand the pathways involved in constitutive heterochromatin maintenance and integrity. Most of them focused on the identification of proteins that bind H3K9me3 using peptides or modified nucleosomes pulldowns 39-41 or chromatin immunoprecipitation 42-44. More recently, heterochromatin proteins have been identified by mass spectrometry of the sonication-resistant fraction of the chromatin 6. Functionally, however, much of our knowledge on heterochromatin stems from genetic screens in model organisms including Schizosaccharomyces pombe, C. elegans and Drosophila melanogaster 45-47. In Drosophila, position-effect variegation analyses have identified proteins important for heterochromatin maintenance and/or spreading 48. Likewise, genetic screens in S. pombe have uncovered genes involved in heterochromatin integrity using a pericentromeric insertion of the ade6+ reporter for example 49. In C. elegans, many repressors have been identified in screens for defects in vulva development or nuclear peripheral localisation 46, 50. In an effort to identify the most relevant protein components of constitutive heterochromatin—and thereby potential proteins that may promote heterochromatin phase separation—we undertook a bioinformatic analysis, initially based on 7 mass spectrometry studies performed in mammalian cells 6, 39-44. We focused primarily on H3K9me3 as a proxy for constitutive heterochromatin, since it is its most prevalent mark across most, albeit not all, eukaryotes. We selected proteins as heterochromatic based on their ability to bind H3K9me3-modified peptides, H3K9me3-modified nucleosomes with and without methylated DNA, or to their presence in the sonication-resistant fraction of the chromatin. Our analysis of all these studies revealed 672 proteins identified as heterochromatic by at least one study, with 148 of these proteins being present in more than one study (Table EV1). To increase stringency in our selection, we then explored the conservation across evolution of the proteins identified by mass spectrometry. For this, we searched for the ortholog genes encoding the 672 proteins in Danio rerio, S. pombe, D. melanogaster and C. elegans. Our results show that 205 (31%) genes had orthologs in all the species that we investigated. In addition, 36 (24%) of the 148 genes coding for the proteins found in more than one mass spectrometry study had orthologs in all species (Table EV1). Among these, 36 genes are the well-characterised Cbx1, Cbx3 and Cbx5, which encode the three mammalian HP1 isoforms known to bind H3K9me3 and to play a role in constitutive heterochromatin maintenance and/or spreading. We thus speculate that a thorough investigation of the remaining 33 genes will lead to the discovery of other proteins that may play a role in constitutive heterochromatin. Because a biochemical identification does not necessarily imply that these proteins and their corresponding orthologs functionally regulate heterochromatin formation and/or maintenance, we mined our results against datasets derived from previous genetic screens. This was possible in three species (S. pombe, D. melanogaster and C. elegans) but not in D. rerio, as we were unable to find publicly available compilations of screens in this species 46, 48, 49. Interestingly, we found very little overlap between the 672 proteins identified based on the biochemical studies performed with mammalian cell culture models, and the genetic screens across other model organisms. In fact, only Cbx1, Cbx3 and Cbx5 were common across all datasets and species. This raises interesting questions, as to whether non-“core” heterochromatin proteins in different species may be important to potentially specify additional heterochromatin features. Alternatively, redundancy could potentially prevent identification of proteins in in vivo screens. Due to the small number of hits obtained through the analysis of genetic screenings, we decided to perform our downstream analyses below on the common 148 proteins identified from the biochemical studies, which, for simplicity, will be referred to as heterochromatic proteins hereafter. The physical properties of phase separation and heterochromatin Membrane-less organelles are thought to form through the nucleation of protein and nucleic acid scaffolds, which will be enriched in the phase-separated compartment, compared with the surrounding solution 20. A key parameter determining the composition of the droplet is the scaffold's concentration 51. The scaffold proteins that mediate phase separation often contain IDRs, thought to be important for nucleating liquid droplets 29, 52-55. However, IDRs can be present in “nucleating” components as well as “recruited” components. Most attention in the field has been devoted to IDRs, but it is important to keep in mind that structured domains may also contribute to phase separation. IDRs are structural features of protein domains, which are often found in linker regions between folded domains as well as in post-translational modification sites, lack a unique three-dimensional structure and tend to have low-complexity sequences 56, 57. IDRs are thought to drive liquid–liquid phase separation by forming multivalent interactions through their amino acid side chains 19. We asked whether the heterochromatin proteins that we identified have a higher propensity to exhibit disorder properties or IDRs. To characterise the potential of the 148 proteins to contribute to heterochromatin phase separation, we generated disorder estimates for them using two prediction algorithms, PONDR-VLXT 58 and IUPRED 59. IUPRED and PONDR take into account the context of individual amino acids to calculate disorder scores for each amino acid in a given protein context. The predicted scores are thus presented as percentage disorder, mean disorder and length of disordered segments. The results obtained with both predictors were not always similar. However, the tendency was the same, and therefore, we averaged the results obtained with both algorithms. Heterochromatin proteins displayed a significantly higher disorder score, as compared to either a random group of total proteins or nuclear proteins of the same size (median = 0.47, compared with 0.31 and 0.37, respectively; Fig 1A). The median percentage length of disordered domains, measured as percentage of amino acids of the total protein length, was 44% (Fig 1A), which is similar to the percentages calculated for the proteome of several phase-separated membrane-less organelles and is higher than the value for organised structures such as the proteasome 60. In addition, the percentage of the protein (length) containing disordered domains was also significantly higher compared with a random (22%) or the nuclear (30%) set of proteins, indicating that heterochromatin proteins are more disordered than a random set of proteins or compared with nuclear proteins in general. Interestingly, not only the percentage of amino acids within disorder domains but also the length of disorder domains was significantly higher in the heterochromatin group of proteins (Fig 1A). Of note, heterochromatin proteins tend to be longer, compared with both groups of proteins, but also when compared with a set of global chromatin proteins or of DNA-binding proteins (Fig 1B). The comparisons with the proteins constituting the nuclear protein groups clearly show that the subset of heterochromatin proteins displays features consistent with higher disorder scores. Figure 1. Analysis of the disorder content of the selected heterochromatin proteins Analysis of three factors to measure disorder behaviour using both the PONDR-VLXT and IUPRED predictors. In the left panel, the disorder score per protein. In the centre panel, the percentage of predicted disorder per protein. In the right panel, the lengths of the predicted disordered regions for each protein set (length of disordered segments (> 30 a.a.)). For length of disordered regions, segments shorter than 30 amino acids were removed (based on Forma-Kay et al 56 and Ward et al 105). The 148 heterochromatin proteins were compared with control protein sets of the same number generated from random sampling of chromatin, nuclear, DNA binding or total proteomes. The dotted lines correspond to the median value for the distributions shown. *P ≤ 0.05 and ns > 0.05 by two-sided unpaired Wilcoxon rank-sum test. Length in amino acids of the proteins analysed in the indicated groups. The 148 heterochromatin proteins were compared with control protein sets of the same number generated from random sampling of chromatin, nuclear, DNA binding or total proteomes. The dotted lines correspond to the median value for the distributions shown. *P ≤ 0.05 and ns > 0.05 by two-sided unpaired Wilcoxon rank-sum test. Download figure Download PowerPoint We then asked whether this feature is exclusive to heterochromatin proteins or whether chromatin proteins in general and DNA-binding proteins possess IDRs as well. For this, we calculated disorder scores, overall percentage (in a.a.) disorder and length of disorder segments for these two additional groups of proteins. Interestingly, our analyses revealed that proteins with the potential to bind DNA and chromatin have a higher disorder score as calculated using IUPRED and PONDR-VLXT predictors, as well as higher overall percentage disorder score, compared with a random set of proteins, or to nuclear proteins (Fig 1A). We conclude that the specific part of the nucleome, which constitutes the chromatin and has the ability to bind DNA, has a higher potential to phase separate, based on IDR constitution. To further assess the possible phase separation propensity of the 148 proteins, we used a different predictor for phase separation based on potential planar protein–protein contacts 61 (not shown). In fact, 38 of them were predicted to have a propensity to reversibly and dynamically self-associate. However, this predictor only takes the planar Pi-Pi interactions into consideration and further in-depth analysis of other interactions is typically required in order to better predict phase separation propensity. HP1α, for example, which is known to phase separate, was not present in this list of proteins predicted to self-associate, advocating the use of several features in parallel when making predictions for phase separation potential. Further to IDRs, interactions between amino acids with opposing charges as well as cation–pi interactions are likely to play a role in liquid droplet formation 54. Molecular interactions between positively charged amino acids with nucleic acids also certainly play a role in the establishment of membrane-less organelles enriched in RNA and RNA-binding proteins 55, 62. In agreement with the importance of electrostatic interactions between macromolecules with different charges, phosphorylation and acetylation have been shown to perturb phase separation and dissolve membrane-less organelles 62-65. Hydrophobic interactions have also been suggested to play an important role in phase separation 35, 66. Pi-Pi interactions between aromatic amino acids (Phe, Tyr, Trp and His) but also amino acids containing amide (Asn, Gln), carboxyl (Glu, Asp) or guanidinium (Arg) groups in their sidechain as well as amino acids with exposed backbone peptide bonds (Gly, Ser, Thr and Pro) are relevant for phase separation mediated by IDRs 61. Tyrosines and arginines have, for example, been shown to play a predominant role in the liquid droplet formation by the FUS family proteins 67. We thus undertook a more thorough analysis of all these features. For this, we aimed to generate a more restricted group of “bona fide” heterochromatin proteins, whose location in chromocentres and/or impact on heterochromatin functions have been validated by cell biological or genetic experiments. Specifically, we used a set of proteins identified as enriched at major satellites by PiCH in mouse embryonic stem cells 68. From these, we selected those proteins, which are lost from the major satellites upon Suv39h1/h2 depletion, and which had been identified as suppressors of variegation (Su(var)) and modifiers of murine metastable epialleles (Mommes). This led to a list of seven proteins: CBX1 (HP1β), CBX5 (HP1α), ATRX, UHRF1, DNMT1, SUV420H2 and SUV39H2 (Table EV2). Excepting SUV420H2 and SUV39H2, the remaining five proteins exhibited disorder scores and overall percentage disorder values higher than the median values of the random set and nuclear proteomes (Table EV2). We then expanded our analysis to other features indicative of a potential to phase separate, including IUPRED and FOLD disorder scores, presence of predicted prion-like domains, propensity for Pi-Pi contacts, fraction of charged residues and net charge per residues across each protein as well as hydrophobicity (Figs 2A–C and EV1A–E). In addition, to provide a relevant comparison, we performed the same analysis with the transcription factor FUS (Fig 2A), which has been shown to phase separate both in vitro and in vivo 67, 69. This uncovered, for example, a clear prion-like domain (PLD) in ATRX as well as high IUPRED scores in ATRX, but also in CBX5 (Fig 2B and C), as previously reported 17. Additionally, the N-terminal domain of SUV39H2, known to interact with RNA, exhibited also high IUPRED score (Fig EV1B). Interestingly, SUV39H2 is highly enriched in mouse zygotes 70, and therefore, the study of its role in heterochromatin formation, and potentially in phase separation, in vivo, should be an exciting research avenue. We find that the “bona fide” heterochromatin proteins contain various segments of high hydrophobicity and with a high fraction of charged residues (Figs 2A–C and EV1A–E), which could potentially favour phase separation. These features may be hard to interpret however, since they may not be sufficient per se to drive liquid–liquid phase separation, as recently shown for the FUS low-complexity domain 69. Overall, these analyses suggest that the “bona fide” heterochromatin proteins that we selected have additional features linked to the potential to phase separate. Figure 2. In-depth analysis of phase separation potential for FUS, CBX5 and ATRXThe analysis of regions of protein primary sequence potentially contributing to liquid–liquid phase separation for FUS, CBX5 and ATRX (see also Fig EV1) was implemented following the same methodology as published in Alberti et al 101. At the top, a schematic representation of the proteins is shown highlighting the different domains catalogued in UniProt. IUPRED; intrinsic disorder prediction using the IUPRED algorithm where values above 0.5 are considered disordered. PLD; prion-like domain prediction using the PLAAC algorithm where a value above 0.5 is considered a prion-like domain. FOLD; intrinsic disorder prediction with PLAAC (pink) or the PAPA (purple) algorithms and the fold index (yellow). Pi-Pi; phase separation predictor based on propensity for Pi-Pi contacts where a region of a protein is predicted to phase separate when its mean value is above 4. NCPR; net charge per residue and FCR; fraction of charged residues (sliding window of 5 using the localCIDER version 0.1.14). Hydro; hydrophobicity (sliding window of 9 using the Kyte and Doolittle scale). For FUS, the following domains or regions are depicted: QGSY, glutamine/glycine/serine/tyrosine-rich region (yellow); G-rich, glycine-rich region (green); RRM, RNA recognition motif domain (orange); RGG, arginine/glycine-rich region (brown); Zn, zinc finger domain (blue). For CBX5, the chromo (CD in orange) and the chromo shadow (CSD in yellow) domains are shown. For ATRX, the following domains or regions are depicted: ADD, ATRX-Dnmt3-Dnmt3L domain (orange); Zn, zinc finger domains (blue); HAB, helicase ATP binding (beige); NP: nucleotide (ATP) binding (red); HCT, helicase C-terminal (brown). Download figure Download PowerPoint Click here to expand this figure. Figure EV1. In-depth analysis of phase separation potential for the bona fide heterochromatin proteinsThe analysis of regions of protein primary sequence potentially contributing to liquid–liquid phase separation for CBX1, SUV39H2, SUV420H2, UHRF1 and DNMT1 was implemented following the same methodology as in Fig 2. For CBX1, the chromo (CD in orange) and the chromo shadow (CSD in yellow) domains are shown. For SUV39H2, the following domains or regions are depicted: CD, chromodomain (orange); Pre, Pre-SET domain (yellow); SET, SET domain (brown); Post, Post-SET domain (beige). For SUV420H2, the SET domain (brown). For UHRF1, the following domains or regions are depicted: Ubl, ubiquitin-like domain (orange); Tl1 and Tl2, Tudor-like 1 and 2 regions (brown); Zn, zinc finger domains (blue); YDG, YDG domain (yellow). For DNMT1, the following domains or regions are depicted: DMAP, DMAP-interaction domain (orange); NLS, nuclear localisation signal (red); Zn, zinc finger domain (blue); BAH1 and BAH2, bromo-adjacent homology 1 and 2 domains (brown); Mtase, SAM-dependent Mtase C5 type (yellow). Download figure Download PowerPoint The above biophysical and biochemical characteristics are in general used as a proxy to assess if a given molecular—and in some instances cellular—process could be explained by phase separation. However, they are only an indicator. In fact, local concentration and post-translational modifications are key. For example, in HP1α, phosphorylation is required for structural changes that promote phase separation 16. While such additional features should be taken into account, overall, our analysis reveals that several proteins associated biochemically with constitutive heterochromatin present characteristics of proteins within membrane-less organelles and some of them are predicted to phase separate. Establishment of heterochromatin in vivo A significant rearrangement and reprogramming of constitutive heterochromatin occurs during germ cell and subsequently early embryonic development 71, 72. During preimplantation development, H3K9me3 is dramatically decreased and re-established on both parental genomes, albeit with different temporal dynamics 73-75, while H4K20me3 and H3K64me3, two modifications downstream of H3K9me3 76, are both removed at the 2-cell stage and not re-established until post-implantation 11, 77. In addition, chromocentres only emerge from the late 2-cell stage onwards, while HP1α, the primary heterochromatin protein suggested to be responsible for its phase separation 16, 17, is not thought to be expressed during preimplantation development 78. We suggest that in order to understand the role of phase separation in heterochromatin function, it will be particularly revealing to describe the dynamics of phase-separated heterochromatin during these periods of development, when heterochromatin is dynamic. In addition, a clearer temporal correlation could be made between the known markers of heterochromatin and the phase-separated heterochromatin state. For example, which, if any, histone modifications or protein readers typical of classical constitutive heterochromatin (such as H3K9me3, H4K20me3 and HP1 isoforms) or features such as chromocentres, temporally and spatially correlate with the appearance of a phase-separated heterochromatic state? Can we predict phase transition occurrence during mouse preimplantation development? We reasoned that an analysis of the patterns of expression of heterochromatin proteins that we identified (Table EV1) during these stages of development, in combination with the knowledge of their predicted phase separation properties, can give a first forecast of the dynamics of phase-separated heterochromatin in mouse embryos. An analysis of publicly available RNAseq datasets 79 indicated a clear average upregulation of the gene