# Non-canonical spatial organization of heterochromatin in mouse preimplantation embryos

Amélie Bonnet-Garnier, Katia Ancelin

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Amélie Bonnet-Garnier, Katia Ancelin. Non-canonical spatial organization of heterochromatin in mouse preimplantation embryos. Reproduction [Cambridge], 2025, 169 (2), 10.1530/REP-24-0271. hal-04988933

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Non-canonical spatial organization of heterochromatin in
mammalian preimplantation embryos

<table><tr><td>Journal:</td><td>Reproduction</td></tr><tr><td>Manuscript ID</td><td>Draft</td></tr><tr><td>mstype:</td><td>Invited review</td></tr><tr><td>Date Submitted by the Author:</td><td>n/a</td></tr><tr><td>Complete List of Authors:</td><td>Bonnet-Garnier, Amelie; INRAE, UMR 1198 BREED Ancelin, Katia; Institut Cochin, Institut Cochin</td></tr><tr><td>Keywords:</td><td>Embryo development, Gene regulation, Fertilization, Developmental biology</td></tr></table>

SCHOLARONE™
Manuscripts
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Non-canonical spatial organization of heterochromatin in mammalian

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preimplantation embryos

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Amélie Bonnet-Garnier ¹,²# and Katia Ancelin ³#

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¹ Université Paris-Saclay, UVSQ, INRAE, BREED; 78350, Jouy-en-Josas, France.

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² Ecole Nationale Vétérinaire d'Alfort, BREED; 94700, Maisons-Alfort, France.

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³ Université Paris Cité, CNRS, Inserm, Institut Cochin, F-75014 Paris, France

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# to whom correspondence should be addressed: amelie.bonnet-garnier@inrae.fr and
katia.ancelin@inserm.fr

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Abstract

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Upon fertilization in mammals, the chromatin packaging of both parental genomes undergoes profound remodeling. Sperm DNA is primarily packed with protamines, whereas oocyte DNA is arrested in metaphase II of meiosis in the form of compacted chromosomes. The two parental chromatin exhibit substantial epigenetic differences and their reprogramming post-fertilization relies on various factors present in the oocyte cytoplasm which will replace protamines for histones and complete meiosis (for review see (Du et al., 2022)). This intensive remodeling is crucial for the zygote to acquire a totipotent state, supporting the development of this new organism, while preserving parental epigenetic memory. Subsequent blastomere cleavages lead to transition to a pluripotent state with the first differentiation at the Blastocyst stage. Concomitantly, the embryonic genome activation (EGA) enables the embryo to switch from the pool of maternal RNAs and proteins by transcribing its own genome. Its timing varies across species (e.g. at 2-cell stage for mouse, 4-cell stage for pig, and human, and 8-cell stage for rabbit and cow) (Madeja et al., 2019).

EGA is vital for embryonic development and occurs in distinct waves, with species-specific timing (for review (Ko, 2016; Madeja et al., 2019)). In mice, the initial minor wave starts at the zygote stage and is rather promiscuous with transcription occurring throughout the entire genome, including repetitive sequences (Fadloun et al., 2013; Abe et al., 2015). The minor wave is followed by the major wave at the 2-cell stage, which is marked by a rapid increase in transcript synthesis. Proper EGA regulation ensures the timely transcription of the correct gene subsets during preimplantation development.

The accessibility of DNA to transcription factors and transcription machinery is dictated by the compaction of chromatin. Euchromatin, being less condensed, is transcriptionally permissive, whereas heterochromatin corresponds to the condensed fraction of the genome, and is non-permissive to transcription (for review see (Janssen et al., 2018)). Heterochromatin is further subdivided into constitutive heterochromatin, which is mostly identical across cell types and contains most repetitive fraction of the genome (e.g. satellite repeats at centromeres; subtelomeric and telomeric repeats at the extremities of chromosomes), and facultative
heterochromatin which is more cell-specific and contains developmentally regulated repressed regions. Euchromatin is enriched in histone post-translational modifications that promote chromatin openness such as acetylation for example at lysine 9 and 27 (refereed as H3K9 or K27 ac) and methylation (e.g. H3K4me), notably at promoters and enhancers of active genes, whereas heterochromatin is enriched in repressive marks such as H3K9me3, H4K20me3 in the case of constitutive heterochromatin, or H3K27me3 and H2AK119Ub in the case of facultative heterochromatin, with or without DNA methylation (Janssen et al., 2018). These types of chromatin also adopt different nuclear locations. Euchromatin is mostly found in the inner part of the nucleus, whereas heterochromatin is predominantly found at the nuclear periphery or close to nucleoli.

Apart from the tridimensional (3D) compaction state of chromatin, increasing evidence supports the idea that the organization of genomic regions within the nuclear space coincides with genome regulation, including its transcriptional status. This phenomenon, referred to as Spatial Genome Organization (termed SGO in this review), takes into account the 3D conformation of genomic regions, as well as their nuclear location, with respect to their nature (e.g. unique loci versus repetitive sequences) or specific nuclear compartments (e.g. nucleoli, nuclear periphery) (Borsos and Torres-Padilla, 2016; Crosetto and Bienko, 2020). SGO is sequence dependent and cell-type specific and can vary during development, aging, and is frequently altered in pathological contexts. The formation of a totipotent embryo entails large-scale reorganization of SGO, especially for constitutive heterochromatin, with variation depending on the parental genomic origin and the transition to pluripotency as the first two cell cleavages occur (Santos et al., 2005; Martin et al., 2006a; Payne et al., 2021). Recent research has intensely focused on the relationship between SGO, in particular for heterochromatin, and the sequential waves of EGA. More generally, the understanding between developmental regulation of SGO, in particular at heterochromatin, and its function in early embryonic progression could help for a better comprehension of SGO changes of diseases as well as for during cell reprogramming.
This review first summarizes the fundamental features of nuclear organization, focusing on heterochromatin structure and function in somatic and germ cells. Next, the specificities of heterochromatin remodeling following fertilization are highlighted in different mammalian species, with presentation of molecular mechanisms when known. Finally, the review will explore how the unique SGO in mice might shape the transcriptome of a newly formed embryo, particularly concerning H3K27me3 dependent imprinting.

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## I- Differentiated cells: key features of nuclear organization

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The study of genetic material packaging in the nucleus began over a century ago using light microscopy with the pioneering observations of T.Boveri and C.Rabl on nematodes and amphibian cells respectively (for review see (Cremer and Cremer, 2010)). Since then a range of techniques has been developed to understand genome organization at multiple scales within the nucleus, considering cell-to-cell heterogeneity across tissues and developmental stages (Bouwman et al., 2022). This section describes the common features of SGO, with a specific focus on heterochromatin.

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### 1- Radial arrangement of chromosomes

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Early studies, using DNA fluorescence *in situ* hybridization (DNA-FISH) on 3D preserved nuclei revealed that interphase chromosomes are not randomly distributed, and occupy defined areas called chromosome territories (CT). This feature is conserved across species (Cremer and Cremer, 2010). During cell differentiation, specific genomic regions can relocate outside of their territories, and these structural changes in chromosome organization are correlated with specific transcriptional programs (Chambeyron and Bickmore, 2004; Chaumeil et al., 2006). The position of individual chromosomes within the nucleus is influenced by anchoring to the nuclear periphery or to specific compartments (e.g. the nucleolus) and this
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chromosome radiality depends on various factors, including chromosome size and gene
density (Crosetto and Bienko, 2020; Bouwman *et al.*, 2022).

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Here, we discuss the importance of genomic regions positioning with respect to
heterochromatin.

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2- Heterochromatin positioning within the nucleus

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In the nucleus, the heterochromatin (e.g. compacted and inactive regions of the genome) is localized in three distinct compartments: either at the nuclear periphery or around the nucleolus or can aggregate to form specific 3D structures (called chromocenters in mouse but not so well defined in other mammals). This spatial segregation of heterochromatic from euchromatic regions reinforce a separation between transcriptionnally inactive and active chromatin which can contribute to the silencing of loci by locate them to heterochromatic regions. The mechanisms that induce or maintain this segregation has been studied mainly in differentiated cells.

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Hence nuclear membrane proteins (e.g. Lamins) are implicated in tethering heterochromatin (both facultative and constitutive) to the nuclear periphery and are part of the repressive mechanisms involved. For example in cells of mammalian females, the inactive X chromosome locates close to the nuclear lamina (called the Barr body) and in human cells, a subset of pericentric and telomeric regions also (van Steensel and Belmont, 2017). On the other hand, H3K9me2 allows anchoring of genomic regions associated with Lamin proteins, called Lamina-associated domains (LAD), to the nuclear envelope and serves as an epigenetic memory for guidance after mitosis (Poleshko et al., 2019). Without this bookmarking or in the absence of Lamin A/C or B, some LADs change location after mitosis and associate with the nucleolus as nucleolus-associated domains (NADs) (van Steensel and Belmont, 2017), meaning that nucleolus periphery can, like the nucleus periphery, act as a

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magnet for heterochromatin. Similarly, the genomic regions linked to the nucleoli are relocated
towards the periphery of the nucleus when the nucleolus is disrupted (Politz et al., 2016).

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The nucleolus is a membrane-less compartment, constituted of three sub-regions dedicated
to ribosomal genes (rDNA) transcription (fibrillar center), ribosomal RNA processing (dense
fibrillar center) and their folding / maturation with ribosomal proteins (granular center). So why
is the nucleolus involved in heterochromatin spatial organization? The most compelling
hypothesis to date is that the substantial quantity (about half of the repeats) of silenced rDNA
serves as a foundation for the heterochromatinization of adjacent sequences. Indeed,
disrupting the silencing of rDNA during the differentiation of embryonic stem cells (ESCs),
impairs the heterochromatin clustering at the nucleolar periphery (Gupta and Santoro, 2020).

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Lastly, the repetitive fraction of the genome consists of pericentromeric, centromeric and
telomeric regions. These regions are enriched with specific epigenetic marks such as
H3K9me2/3, H4K20me3 and DNA methylation, and specific readers (like ChromoDomain
proteins, CBX1 or CBX2) maintaining a closed state across all cell types (Janssen et al., 2018).
These regions have a key role in ensuring genome stability and aggregate into chromocenters,
that can be detected by DNA FISH or immunofluorescence. Upon differentiation, the number
of clusters can increase in size and reduce in number, with a tendency to localize at the nuclear
periphery (Mayer et al., 2005). Of note, the most peculiar SGO described, in highly
differentiated cells, is the single central and large chromocenter of the rod photoreceptors cell
nucleus (Borsos M and Torres-Padilla, 2016).

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The 3D organization and compaction of heterochromatin are crucial for maintaining a
repressive transcriptional state, with genes located at its vicinity being often silent, and can
vary depending on the cell-state and its potency state (totipotent, pluripotent or differentiated)

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157 3- Hierarchical organization of the genome

158 The mapping of *cis* interactions of genomic sequences across the entire genome using Hi-C,
159 a Chromosome Conformation Capture technique, has revolutionized our understanding of the
160 3D organization of genomes at the subchromosomal level (Lieberman-Aiden *et al.*, 2009). Hi-
161 C approaches have confirmed that chromosomes occupy distinct territories during interphase
162 and that chromatin organizes into megabased-sized active (A) or inactive (B) compartments,
163 typically associated with euchromatin and heterochromatin respectively. Additionally,
164 chromatin is partitioned into smaller domains (<1 Mb), that have been named TADs (for
165 Topologically Associating Domains) which contain chromatin loops (Dixon *et al.*, 2012; Nora
166 *et al.*, 2012). The compartment and domain layers of chromatin folding rely on two
167 independent mechanisms: the chromatin state defines the compartment scale whereas
168 architectural proteins, such as cohesin, condensin or the CTCF, are involved in TAD formation
169 via loop extrusion (Schwarzer *et al.*, 2017). The interplay between these two independent
170 modes of chromatin folding is believed to regulate transitions in 3D organization of the
171 genome, with spatial separation for different portions, contributing altogether to regulate gene
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173 Studies of chromatin topology have revealed its crucial role in genome regulation, including
174 gene expression. For instance, genes within the same TAD share regulatory elements (such
175 as enhancers) that facilitate their co-regulation. Recent studies have shown that pathogenic
176 mutations can alter the structure of these domains, leading to a compromised gene regulation
177 and disease (Bompadre and Andrey, 2019).

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180 II- Gametes: non-canonical nuclear organization

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182 In mammals, gametogenesis is a unique differentiation process marked by drastic, sex-
183 dependent restructuring of the genome topology, culminating in meiotic divisions that halve
the chromosome number. A hallmark of meiosis is the recombination between homologous chromosomes following chromosome pairing and synapsis. This process requires a transient polarized nuclear arrangement called the bouquet configuration, where all telomeres first attach to the nuclear envelope and slide along, allowing the free ends of the chromosomes to search for their homologous pairs, before clustering at one pole of the nucleus during the zygotene stage. For a comprehensive outline of gamete SGO, readers are referred to (Zhang et al., 2022). Altogether, these studies have revealed that nuclear organization is highly dynamic and sex-specific during mouse gamete differentiation, and is often referred to as typical or non-canonical to highlight its unconventional features. Here, we focus on key differences in heterochromatin organization between male and female gametes, when information is available, and is mostly based on the mouse model.

1- Constitutive heterochromatin

During spermatogenesis, chromatin is highly reorganized with most histones being gradually replaced by protamines, resulting in a highly compact structure within the sperm head. Only a few regions remain associated with histones / nucleosomes and their genomic locations are still under investigation (Gold et al., 2018). Until recently, the SGO of heterochromatin in sperm was described as a single large cluster of centromeric and pericentromeric regions in the nucleus center, with telomeres attached to the basal part of the sperm head in mice (van der Heijden et al., 2006), rat (Meyer-Ficca et al., 1998) and human (Haaf and Ward, 1995). However this organization has been recently challenged and warrants more analysis, indeed an alternative organization suggesting a lower clustering of centromeres and a random distribution of telomeres throughout the nucleus has been proposed in human (Acloque et al., 2013; Ioannou et al., 2017) and pig. At least in pig species, it is the homology in satellite sequences that leads the chromosomes clustering in chromocenters.

During oogenesis, at the end of the follicles stage formation, fully-grown oocytes become transcriptionally silent concomitantly with a drastic change in the global organization of the
chromatin. In growing oocytes, chromatin is either decondensed in a configuration called Non-Surrounded Nucleolus (NSN) or condensed around a dense fibrillar sphere known as the Nucleolus like body (NLB - inherited from the former active nucleolus) in a configuration called surrounded nucleolus (SN) (Tan et al., 2009). The NSN configuration of GV chromatin is present in all mammalian species studied so far (mouse, rat, monkey, human, rabbit, cow, ferret, cat, pig and horse - refer to (Luciano et al., 2014) for review). Oocytes must exit from the NSN configuration to acquire full meiotic competence, and assume the SN or equivalent configuration and terminate gene transcription to acquire competence for early embryonic development. In mice, the fertilized oocytes in SN configuration are the only ones able to sustain proper development (Zuccotti et al., 2002). The proportion of NSN versus SN changes with ages, with the latest becoming predominant in older mice (Bonnet-Garnier et al., 2012). In NSN configuration, constitutive heterochromatin (major and minor satellites sequences) clusters in chromocenters whereas in SN configuration, these sequences form a ring around the NLB along with ribosomal genes (Zuccotti et al., 2002; Bonnet-Garnier et al., 2012). So far (to the best of our knowledge), no information is available regarding the SGO of satellite repeated sequences in GV oocytes of other mammals species than mice although the link between this transition and the cytoplasmic maturity seems to be conserved in several other mammalian species (Bogolyubova and Bogolyubov, 2020).

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2- Sex biased general genome organization: TADs versus "TAD-like" dependent on Polycomb.

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In mouse spermatozoa, TADs are found but with longer-distances interactions (>15Mb), consistent with the dense packaging of the paternal genome (for review see (Gold et al., 2018)). On the contrary, human sperm lacks typical TADs, but show a strong A/B compartmentalization, underlying divergence in mammal (Chen et al., 2019).

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New methods using low input material have improved our understanding of the structural organization of the genome in specific tissues, even when they are only available in small
amounts, as in the case of the female germ line, with fewer oocytes to recover compared to
sperm for the male gametes (reviewed in (Bouwman et al., 2022)). To date, only mouse female
germline have been characterized for its 3D chromatin organization, and has revealed
important differences with male germline. Mature oocytes at the germinal vesicle (GV) stage
in mice display structural domains termed PADs (Polycomb Associating Domains) (Du et al.,
2020). They do not have the same genomic locations as structural domains described as
TADs in mESCs, and are characterized by their chromatin enrichment in H3K27me3 marks
dependent of the Polycomb PRC2 complex (Du et al., 2020). These female germline specific
structural domains are regulate by independent mechanisms from TADs found in somatic cells
and likely also in sperm (see part IV).

### III- Preimplantation embryos: shifting from non-canonical to canonical spatial genome organization

The dynamics of nuclear organization following sperm-oocyte fusion were initially tracked by staining the DNA (e.g DAPI), or the most repetitive fractions of the genome (centromeres, telomeres), versus chromosome-specific regions by DNA FISH, or chromosome territories (painting). These studies revealed an atypical, or "non-canonical" embryonic genome configuration, which resolves as the embryo transitions from totipotency to pluripotency (Mayer et al., 2000; Martin et al., 2006a; Probst et al., 2007; Koehler et al., 2009; Aguirre-Lavin et al., 2012). Recent low-input or single-cell HiC techniques provided a deeper understanding of the nuclear changes and embryonic genome restructuring in mammalian embryos. This section outlines the shift from non-canonical to canonical organization, in particular at heterochromatin, during the first cell cleavages mostly in mouse, or other mammals when available.
268 1- Constitutive heterochromatin dynamics

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270 1-1 Organization at the nuclear periphery

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272 Mouse zygotes and 2-cell stage embryos demonstrate significant global genome spatial
273 repositioning at the nuclear periphery with LADs formed de novo rather than inherited from
274 gametes (Borsos et al., 2019). Each parental genome establishes LADs with distinctive
275 dynamics, independently of DNA replication, but their structure converges after the 8-cell
276 stage. At these early stages, mouse telomeres and centromeres are also found clustering
277 close to the periphery, but each gathering at opposite side off the nucleus, stretching the
278 chromosomes, consistent with a nuclear configuration originally described by (Cremer and
279 Cremer, 2010; Aguirre-Lavin et al., 2012; Payne et al., 2021). This peculiar polarity of the
280 nucleus is likely lost as the embryo progress toward the blastocyst stage, maybe as with
281 maturation at the nuclear lamina progress (see below). Other specific association of telomeres
282 to internal structures such as NADs has not been tested to date in early mammalian embryos.

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284 1-2 Spatial organization in regards with Nucleolar Precursor Bodies (NPBs)

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286 In mice, centromeric and pericentromeric heterochromatin is centrally located in very early
287 zygotic pronuclei (PN), as shown by staining of their chromatin with HP1b or by DNA FISH
288 (Adenot et al., 1997; Martin et al., 2006b; Merico et al., 2007; Probst et al., 2007; Aguirre-Lavin
289 et al., 2012). In mid to late zygotic stages, pericentromeric sequences form a ring surrounding
290 the Nucleolar Precursor Bodies (NPBs) which are electron-dense, DNA-free and membrane-
291 less nuclear areas specific to mice early embryonic stages (1-cell to 8-cell stages) (Figure 1).
292 From the 2-cell to the 4-cell stage, the association of major satellite sequences with NPBs
293 decreases as chromocenters form at the time of EGA, adopting a somatic-like spatial
294 organization from the 8-cell to the blastocyst stage (Martin et al., 2006b; Aguirre-Lavin et al.,
295 2012). As the spatial reorganization of repeated sequences at the time of EGA has also been
demonstrated in rabbit (Bonnet-Garnier et al., 2018), we can assume that SGO of pericentromeric sequences is conserved among mammals although it remains to be demonstrated in human, cow or pig species. These changes in SGO have been correlated with success in somatic nuclear transfers, suggesting that they are critical for development (Maalouf et al., 2009). Microsurgical removal in the oocyte of the nucleolus-like bodies (e.g. NLBs) leads to random centromere localization after fertilization and developmental arrest at the 2-cell stage (Fulka and Aoki, 2016; Benc et al., 2021). The targeting of satellite sequences to the nuclear periphery and away from NPBs similarly results in developmental defects (Jachowicz et al., 2013). Altogether, these results suggest a functional role for NPBs in heterochromatin organization after fertilization (Padeken and Heun, 2014), and a correlation between SGO with developmental potential of the newly formed embryos.

1-3 Specific epigenetic markers of chromocenter during early development

There is a clear parental asymmetry in epigenetic marks at the zygotic stage in mice, bovine and rabbit species, with H3K9me3 being the major marker for maternal pericentromeres inherited from the oocytes enrichment in H3K9me3 (Pichugin et al., 2010; Bonnet-Garnier et al., 2018), whereas Polycomb marks are added de novo at the paternal pericentromeres thanks to the enrichment in cPRC1 components like Rnf2/Ring1A or CBX2 (Puschendorf et al., 2008; Tardat et al., 2015). These differences can be also related to a faster reorganization of the paternal PN with incorporation first of H3.3 by DAXX / ATRX (Liu et al., 2020). This parental asymmetry is maintained at the 2-cell stage in bovine and rabbit embryos meaning that both parental genomes are still spatially segregated (Pichugin et al., 2010; Bonnet-Garnier et al., 2018) and then at the 8-cell stage (when the EGA occurs in these species), H3K9me3 form highly enriched chromocenters) in both species. In mice early embryos, H3K27me3 accumulates progressively at chromocenters until the 2-cell stage and at the16-cell stage, all of them are enriched with both H3K9me3 and H3K27me3 marks. This co-labelling of
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chromocenters is specific of embryonic cells up to the Blastocyst stage or in naive pluripotent

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ESCs (Pailles *et al.*, 2022).

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2- Organization of facultative heterochromatin

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2-1 Transient and H3K27me3-enriched structural domains before the 4-cell stage in mouse.

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When comparing HiC contact maps of somatic cells (e.g. mESCs) with data from low input or

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single-cell HiC data in mouse, it appears that genomic interaction numbers are low in zygote

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and 2-cell stage embryos, and generally increase in number after in 8-cell stage onwards (Du

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*et al.*, 2017, 2020; Flyamer *et al.*, 2017; Ke *et al.*, 2017; Collombet *et al.*, 2020). However,

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structural domains identical to PADs found in mature oocytes and demarcated by H3K27me3-

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enriched chromatin transiently appear after fertilization until the 4-cell stage, resembling

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compartment-like interactions (Figure 1) (Collombet *et al.*, 2020; Du *et al.*, 2020). However,

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their smaller size scale might explain why they were not detected as A/B compartments in

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earlier studies (Du *et al.*, 2017; Flyamer *et al.*, 2017; Ke *et al.*, 2017). Rather, these early

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structural domains might correspond to those detected in single nucleus HiC studies for the

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zygote stage when using aggregated data to detect chromatin interactions over the entire

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genome rather than at any given loci (Flyamer *et al.*, 2017; Gassler *et al.*, 2017). Currently,

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these PADs are not reported in human and pig, the two other mammalian species tested (Chen

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*et al.*, 2019; Li *et al.*, 2020), but it might reflect the differences in HiC analysis. Alternatively,

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their absence might be linked with the divergence in early facultative heterochromatin

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composition based on H3K27me3 patterns across different mammalian species, particularly

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after fertilization (Xia *et al.*, 2019; Zhang *et al.*, 2019; Lu *et al.*, 2021; Bu *et al.*, 2022; Zhou *et

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*et al.*, 2023). H3K27me3 broad domains, coinciding with Polycomb structural domains, seem to

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disappear faster after fertilization in cow, human and pig embryos compared to mouse,

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although the absence of parental origin tracking, or the embryo status themselves

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(parthenogenotes in pig, 3N in human) might override possible conclusions concerning the
persistence of H3K27me3 associated domains. An independent study using human morula
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embryos and allelic profiling for gene expression along with H3K27me3 pattern actually report
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the existence of H3K27me3 enriched maternal domains after EGA, coinciding with paternally
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biased gene expression, similar to rodents (Zhang et al., 2019). Therefore, it might be
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premature to draw too strict conclusion about the lack of conserved features in early genome
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organization with the few mammalian species tested, and juxtaposition of HiC heatmaps with
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H3K27me3 pattern might be a first step in this understanding.
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LADs are another hallmark of SGO at the start of development, and are present as early as
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the zygote stage (Borsos et al., 2019). However they shift away from the nuclear periphery at
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the 2C stage coinciding with the strengthening of PADs (Borsos et al., 2019; Collombet et al.,
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2020). Mapping their common genomic locations could provide a better understanding of
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genome reprogramming in early embryos, in particular at heterochromatin.
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2-2 Case of the paternal X during preimplantation development in mice.
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X chromosome inactivation (XCI) achieves gene dosage compensation between XY and XX
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individuals in mammals via the acquisition of facultative heterochromatin features on one of
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the 2Xs in female (random choice). XCI begins with Xist expression from one of the 2Xs, which
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encodes for a lncRNA, that will coat the X from which it is expressed in cis which will trigger
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chromatin changes for gene silencing (for review see (Loda et al., 2022)). In mouse female
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preimplantation embryos, Xist is expressed from the paternal X (Xp), leading to paternal XCI.
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While the paternal X often localizes near the nucleolus, the importance of this nuclear
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localization for its heterochromatin formation and silencing remains untested (Namekawa et
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al., 2010).
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Structural changes of the inactive X have been extensively studied in differentiating cells. HiC
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analysis across mammalian species shows that genomic interactions on the inactive X (Xi)
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occur over longer distances, compacting the Xi into two megadomains, erasing most TADs
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379 (for review see (Loda et al., 2022). Interestingly, during preimplantation developmental stages, the Xp lacks a bipartite structure, unlike differentiated cells (Collombet et al., 2020), and it is not known when latter specific conformation is achieved in vivo. In culture cells, inactive X-linked genes reside within the Xist coated territory of the Xi, and active genes that escape inactivation are positioned outside, accessible to transcription machinery (Chaumeil et al., 2006). Similarly, at the blastocyst stage, the Xp, mostly silent, is more compact than its active counterpart (the maternal X, Xm), with inactive genes centrally located within the chromosome territory (Collombet et al., 2020). Gene inactivation occurred progressively from the 4-cell to the blastocyst stage on the Xp (Borensztein et al., 2017). While TADs are formed progressively on the Xm and Xp after the EGA, the genes subjected to the earliest silencing on the Xp exhibit TAD loss after they are silenced (Collombet et al., 2020). This observation suggests that the loss of TADs follows or accompanies gene silencing, but it may not be a direct cause of transcriptional repression.

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393 These studies further highlight the extreme remodeling of the parental genome organization, particularly at early facultative heterochromatin, occurring along with the establishment of matured embryonic transcription during mammalian preimplantation development. The 394 molecular mechanisms underlying these transient and unique chromatin structures are only 395 beginning to be understood, while the functional implications for these changes in 396 development warrant further investigations. 397

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401 **IV- Insights into molecular mechanisms of heterochromatin remodeling in** 402 **preimplantation mouse embryos.** 403

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404 In this section, we will delve into the molecular mechanisms underlying the dynamic 405 reorganization of heterochromatin and its strong parental asymmetry in early mammalian
406 embryos. In particular, we will highlight factors in mouse with defined roles in SGO, or else
407 elaborate on their putative functions (Table 1 and 2).

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1-Components and regulators of constitutive heterochromatin in early development

410 Maintenance of pericentric heterochromatin and its reestablishment on the paternal genome
411 post-fertilization relies on the coordinated actions of various factors such as H3K9me2/3
412 readers (e.g., HP1 and ATRX), histone remodelers (including histone methyltransferases
413 SUV39H1, SUV39H2, SUV4-20, and Dot1l), and histone chaperones (HIRA, DAXX, and
414 CAF1) (Table 1). Imaging analysis of chromocenter clustering during the early post-fertilization
415 stages using satellite sequence staining has yet to be conducted for most factors in mutant
416 conditions. What is currently known is that expression of a catalytically inactive DOTL1 caused
417 precocious formation of chromocenters, but without association with the sole remaining NPB,
418 as well as developmental arrest at the 2-cell stage (Ooga et al., 2013). These results clearly
419 show that heterochromatin regulators are involved in chromocenter formation, leading to
420 speculation that other factors are involved in the targeting of constitutive heterochromatin to
421 NLBs in SN oocytes and NPBs in zygotic pronuclei, with potential functional implications for
422 developmental potential (Table 1).

423 Heterochromatin organization has been linked to phase separation, a process involving
424 separation between material with different physical properties (for review see (Hildebrand and
425 Dekker, 2020)). After fertilization, mouse heterochromatin undergoes phase separation
426 immediately: pericentromeric heterochromatin exhibits liquid-like properties at the 2-cell stage,
427 before the transformation into chromocenters at the 4-cell stage and initiation of transcriptional
428 silencing (Guthmann et al., 2019).

429 Concerning the nuclear lamina, ectopic expression of KDM5B, an H3K4me3 demethylase,
430 hinders the formation of paternal LADs (Borsos et al., 2019). These results highlight the
unconventional regulation of heterochromatin during early mouse development, which seems
to be dependent on a classic mark of euchromatin, H3K4me3, for its spatial organization.

433

434 2-Regulators for Polycomb structural domains and the transition to TADs

435

436 Genomic interactions within TADs are formed through loop extrusion, driven by the cohesin
437 complex composed of the subunit SMC1 and 2, which acts as "sliding" ring along the
438 chromatin (for review see (Davidson and Peters, 2021)). Alternatively, compaction at the
439 compartment level, is independent of the cohesin machinery and is proposed to be determined
440 by chromatin state (Hildebrand and Dekker, 2020).

441 Current models propose that obstacles to the cohesin complex partly governs the size of the
442 extruded loop. CTCF, a zinc finger DNA-binding factor with a long standing role in gene
443 dosage regulation, has been pinpointed as one such barrier. While structural domains
444 emerging after the 8-cell stage in mouse embryos display CTCF binding sites at their
445 boundaries, the earliest post-fertilization structural domains enriched in Polycomb chromatin
446 lack these motifs (Collombet et al., 2020). Depleting mouse embryos of CTCF does not
447 prevent TAD formation in blastocysts, although some are lost, with mild gene expression
448 changes (Andreu et al., 2022) (Table 2). However, in human embryos, CTCF is strictly
449 required for establishment of the TADs (Chen et al., 2019) implying that usage of the same
500 regulators of short distance genomic interactions might be different among mammals.

451 When the cohesin complex itself is targeted, through SCC1 loss, Polycomb dependent
452 domains strengthened their inter-domain interactions in mature oocytes (Du et al., 2020) in
453 agreement with an antagonist role of the cohesin complex on compartment formation. Other
454 studies focused on 3D genome organization after fertilization by altering the cohesin complex
455 or factors known for their regulatory roles at its engagement/disengagement (see Table 2). As
456 such, WAPL is known to open the cohesin ring and provoke an exit for the extruded DNA,
457 while MCM is part of the replication fork machinery that has been shown to impede cohesin
458 sliding (Davidson and Peters, 2021). Independent maternal loss of SCC1, or WAPL, or MCM
or CTCF alter the balance between loop extrusions and formation of compartment-like structures (Dequeker et al., 2022; Gassler et al., 2017). Loss of "compartment" organization in WAPL absence and increased compartment-like without SCC1 suggests that the eviction of the cohesin complex is crucial for this layer of genome organization immediately after fertilization, and might therefore play important roles in antagonizing Polycomb domain formation. Indeed these 3D organization is dependent on the chromatin state (H3K27me3 broad domains), and resemble compartment levels (Collombet et al., 2020; Du et al., 2020). These results suggest that the loop extrusion machinery and the PRC2 complex might be engaged in a tug of war, and their relative abundance may determine genome-folding dynamics after fertilization and up to the 4-cell stage.

Current research shows that the two Polycomb machineries PRC1 and PRC2 may have independent developmental functions in SGO in mice (see Table 2). Ablation of PRC1 activity during oocyte growth leads to loss of PAD-type domains, proving that H2AK119Ub deposition is crucial for shaping chromatin condensation in the maternal lineage (Mei et al., 2021). In contrast, conditional deletion of PRC2 core subunit *Eed* inhibits Polycomb domains reformation between the zygote and the 2-cell stage but not in oocytes, indicating its importance for reestablishment after fertilization. However, it is important to note that this genetic deletion might have occurred when H3K27me3 maintenance no longer required PRC2, preventing us from entirely ruling out an earlier role for PRC2 in genome structure during germline formation.

One can also speculate that other components of the Polycomb machinery might be involved in SGO during early development. Recruitment of H3K27me3 readers such as CBX2, a PRC1 subunit, mediates gene silencing and phase separation forming condensates via its chromodomain (for review (Guo et al., 2021)). As CBX2 is present in zygotes and 2-cell stage embryos (Tardat et al., 2015), testing its role in the 3D genome organization would help understanding how transient hubs of gene silencing can appear in early development, possibly through the combination of Polycomb domain formation and long-range chromatin interactions
486 (see also Table 1). Recent studies identified EZHIP (EZH Inhibitory Protein) as a new factor
487 that restricts PRC2 activity in cancer and mouse germline (Pajtler et al., 2018; Hübner et al.,
488 2019; Jain et al., 2019; Piunti et al., 2019; Ragazzini et al., 2019). EZHIP has been proposed
489 to prevent PRC2 spreading by stabilizing a high-affinity H3K27me3-PRC2-EZHIP complex at
490 H3K27me3 enriched sites, impairing EZH1/2 catalytic activity, and blocking PRC2
491 redistribution (Jain et al., 2020). Whether EZHIP might be a crucial player in the formation or
492 constrain of H3K27me3 domains in female germline or after fertilization remains an important
493 question to test (Table 1).

494 Finally, active chromatin marks such as H3K36me2/3 and H3K4me3 can directly inhibit PRC2
495 catalytic activity (Schmitges et al., 2011; Guo et al., 2021). In oocytes, H2K27me3 domains
496 are anticorrelated with H3K36me3 and DNA methylation (Xu et al., 2019), and partially with
497 H3K4me3 (Zheng et al., 2016). Loss of SETD2-dependent H3K36me3 domains in the female
498 germline leads to the expansion of H3K27me3 chromatin after fertilization (Xu et al., 2019).
499 Thus, chromatin composition in the female germline and during early development instructs
500 for PRC2 recruitment and spreading, maintaining Polycomb domain boundaries, and
501 potentially the 3D organization. In favor of this model, a transient H3K4me3 enrichment
502 correlates with the weakening of most Polycomb structural domains at the 4-cell stage
503 (Collombet et al., 2020). Trithorax factors are known chromatin modifiers antagonizing
504 Polycomb machinery in gene expression regulation. The histone methyltransferase
505 KTM2B/MLL2 which targets H3K4 residues and is a member of the COMPASS/ Trithorax
506 complex is necessary for EGA as its maternal loss halts development before the 4C stage
507 (Andreu-Vieyra et al., 2010). Whether the transition from PADs to TADs also requires the
508 interplay between the two antagonist machinery Polycomb and Trithorax remains untested.
509

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512

V- Concluding remarks: emerging functions for SGO in early mammalian embryos.

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Increasing evidence concerns the role of nuclear organization as a crucial epigenetic layer for regulation of gene expression. For instance, telomere length variation in mouse and human embryos has recently been correlated with chromatin accessibility. These changes influence the dynamic of DUX4/Dux4 expression, a pioneer factor important for development (Zhang et al., 2023). Another aspect that received significant attention in the past decade is the dynamic transcription of heterochromatin during its reprogramming after fertilization. This section discusses recent findings linking the different types of heterochromatin and their transcription status with developmental progression in preimplantation embryos.

1- Noisy constitutive heterochromatin and its role in shaping the embryonic spatial organization

Several repetitive elements (e.g. transposable elements; tandem repeats) at different chromosome locations are highly transcribed during early development, mainly just after EGA until the morula stage (Peaston et al., 2004; Probst et al., 2010; Fadloun et al., 2013). During minor EGA, major satellite sequences are transcribed, and inhibiting their transcription leads not only to incomplete chromocenter formation but also developmental arrest at the 2-cell stage (Probst et al., 2010; Casanova et al., 2013). Transcription of satellite sequences begins with the forward strand in the paternal PN and reaches a peak at the 2-cell stage in the paternal genome, which lacks H3K9me3. This phenomenon suggests a need for RNA:DNA hybridization to recruit HP1 and SUV39H1 to pericentric heterochromatin after EGA. Consistently, knocking down major satellite repeats results in SUV39H1 proteins diffusing on chromatin, and SUV39H1 loss leads to upregulation of major satellite repeats in human ESCs (Camacho et al., 2017). The nuclear localization of pericentromeres is crucial for their transcriptional silencing after EGA. During their transition from NPBs to chromocenters, their expression profile changes, which is associated with embryonic developmental potential
540

(Casanova et al., 2013) and also with cloning success in somatic cell nuclear transfert
(Maalouf et al., 2009).

541

542

SGO of pericentromeric regions driven by ncRNA is not the sole spatial organization of the
genome specifically found during early embryonic development. A recent research, using 3D
DNA-FISH showed that rDNA sequences, which at the 2-cell stage are localized outside the
NPBs (Figure 1), begin to disperse within NPBs by the 4-cell stage when transcribed, reaching
maximum deployment at the 16-cell stage (Chebrout et al., 2022). This cloud-like organization
of nascent rRNA transcripts (47S) is unique to early embryonic stages as it disappears by the
morula stage, concomitantly with the first lineage differentiation. Of note, RNA Poll is involved
in producing single-stranded RNAs from centromeric and pericentromeric repetitive
sequences both in human (Camacho et al., 2017) and mice (Chebrout et al., 2022) and
inhibition of RNA Poll decreases transcript levels. We can speculate here that the spatial
organization of rDNA sequences and transcripts acts as a "permissive to transcription area"
possibly high jacked by repetitive sequences to be transcribed.

543

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547

548

549

550

551

552

553

554

555

Collectively, these findings confirm that single strand RNA from tandem repeated elements (major satellites, telomeres, rDNA, etc...) are essential for initiation of their spatial organization.
Additionally, these tandem repeated sequences take advantage of subnuclear specific environments (linked to liquid-liquid phase separation) to maintain their transcription and subsequently repress it. Surprisingly, if the spatial reorganization of these repeated elements is impaired for example if silenced, then the development is arrested. What are exactly the consequences on the minor or major activation of the genome need to be further investigated.

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2- Polycomb structural domains are parent specific and might regulate gene dosage at EGA.

564

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566

Early Polycomb-dependent domains in mouse embryos cover genomic regions with a parental bias (Collombet et al., 2020). The copy covered by H3K27me3 and presenting a Polycomb

567
structural domain (more often maternal than paternal) predominantly contains silent gene alleles, reflecting their repressive chromatin (Figure 2). The second parental allele of these genes, not embedded in the early structural domains, can be expressed, albeit often at a low level, in preimplantation embryos. These monoallelic genes are mostly associated with functions in terminal differentiation of specific tissues. In contrast, genes in structural domains emerging after the 4-cell stage exhibit a biallelic pattern, higher expression levels and often play pivotal roles in essential developmental processes. The unique and transient Polycomb structures are therefore associated with parent-specific gene expression modulation, raising questions about their role in developmental progression from totipotent to pluripotent embryos.

It is essential to determine if, for instance, they "sequester" non-essential genes to minimize transcription and preserve maternal resources from transcribing unnecessary new mRNAs.

This gene dosage modulation, via monoallelic expression, might directly contribute to pluripotency and developmental potential, as suggested by studies on the importance of the quantity of TLE3 product (a transcriptional co-repression) for ESCs differentiation or Nanog pattern (Miyanari and Torres-Padilla, 2012; Laing et al., 2015).

Recent studies in mice have identified genes subject to a novel non-canonical form of maternal imprinting, driven by histone modifications rather than DNA methylation (Inoue et al., 2017a).

This is the case for *Xist* in mice (Inoue et al., 2017b). Many maternal alleles of these genes adopt early Polycomb dependent domains and are imprinted through H2AK119Ub and H3K27me3 deposition respectively by PRC1 and PRC2 during oogenesis (Inoue et al., 2018; Collombet et al., 2020; Chen et al., 2021; Mei et al., 2021). Post-fertilization, H2AK119Ub is rapidly remodeled, equalizing between parental genomes. In contrast, H3K27me3 patterns persist, predominantly on the maternal genome, resulting in strong parental asymmetry, and maintaining non-canonical imprinting (Zheng et al., 2016; Inoue et al., 2017a, 2018). Depletion of H3K27me3 in oocytes leads to loss of non-canonical imprinting and reduced embryonic viability, primarily attributed to disrupted *Xist* expression pattern and aberrant X chromosome inactivation (Inoue et al., 2018; Matoba et al., 2022). The role of early 3D genome organization
in maintaining imprinting post-fertilization, alongside H3K27me3, remains unclear. Constraints
in H3K27me3 domain breadth likely define Polycomb-dependent 3D structures. Investigating
factors that counter H3K27me3 spreading, such as DNA methylation, H3K36me3, or EZHIP,
might reveal mechanisms independent of H3K27me3 itself. More generally if and how PRC2
*de novo* activity might be restrained after fertilization would be beneficial to understand how
parental epigenetic asymmetry might be perpetuated up to the blastocyst stage. As such,
mutant models with H3K27me3 equivalent parental enrichment and/or biallelic expression of
non-canonical imprinted genes could reveal the role of early genome organization in allele-
specific gene dosage. Unraveling if parent-specific dosage reduction benefits embryonic
development is now crucial and awaiting discovery.

In general, future research should focus on addressing the functional conservation of early
development SGO in other mammalian species. These are difficult questions that may require
alternative approaches, such as *in vitro* embryo based systems derived from stem cells, in
particular for non-human primate and human early development, and we expect these topics
to be intensively studied in the coming years.

611 **Declaration of interest,**

The author declare no conflicts of interest.

613 **Funding**

ABG is funded by the REVIVE Labex (Investissement d'Avenir, ANR-10-LABX-73) and
supported by the PHASE Department of the French National Research Institute for Agriculture,
Food and Environment (INRAE).

617 Author contribution statements

ABG and KA conceived and wrote the review. ABG obtained funds for open-access policy.
619 Acknowledgments

620 We thank Julie Chaumeil, Rafael Galupa, Michelle Halstead for their critical reading and
621 invaluable input.

622

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915 FIGURE LEGENDS

916 Figure 1: Transition in SGO during mouse embryonic development. Mouse preimplantation
917 development is schematized on the left, from zygote stage to blastocyst. The middle part
918 represents the localization changes for tandem repeats (satellites and rDNA). The right part
919 illustrates the 3D chromatin organization changes during the genome reprogramming, from
920 Polycomb structural domains to TADs. The model proposes that the SGO during early mouse
921 development shifts from a non-canonical to a canonical pattern during the first stages of life.

922 Figure 2: 3D chromatin organization and gene dosage. Changes in chromatin organization
923 affect the allelic choice for transcription.

924 Table 1: Factors that act at constitutive heterochromatin

925 Abbreviations: Mat cKO maternal conditional KO;;m+z maternal + zygotic; KD knock down;
926 GOF gain of function; Dm double mutant; PCH pericentric heterochromatin

927 Table 2: Factors that act for genome folding in early mouse development.

928 Abbreviations: Mat cKO maternal conditional KO; m+z maternal + zygotic; KD knock down;
929 GOF gain of function; Dm double mutant; PCH pericentric heterochromatin; nd not determined

930

931

932

933
<figure><img src="image_4.png" /><figcaption>Figure 1</figcaption></figure>
<figure><img src="image_5.png" /><figcaption>Figure 2</figcaption></figure>
<table><caption>Table 1.</caption><thead><tr><td>Factors</td><td>Methods</td><td>Studied stages</td><td>Technic</td><td>Role at chromatin</td><td>Observed functions/ phenotypes</td><td>Hypothesis for role in SGO of constitutive Heterochromatin</td><td>Study</td></tr></thead><tbody><tr><td>Ring1A/B</td><td>dm mat KO</td><td>zygote</td><td>IF</td><td>histone ubiquitination</td><td>recruitment of DAXX at PCH</td><td>promote chromocenter clustering?</td><td>Posfai et al 2012</td></tr><tr><td>KDM1A</td><td>mat cKO</td><td>2-cell</td><td>IF</td><td>histone demethylase</td><td>increased size for chromocenters</td><td>promote chromocenter clustering?</td><td>Ancelin et al 2016</td></tr><tr><td>SUV39H1</td><td>KD / GOF</td><td>zygote to 8-cell / Blasto</td><td>IF/DNA FISH</td><td>histone methylase</td><td>recruitment by HP1 at PCH after 8C stage</td><td>maintainance of chromocenter clustering ?</td><td>Burton et al 2020</td></tr><tr><td>DOT1L</td><td>GOF+inhibition</td><td>zygote + 2-cell</td><td>IF/DNA FISH</td><td>histone methylase</td><td>Early Major sat clustering (GoF)</td><td>promote chromocenter clustering?</td><td>Ooga et al 2013</td></tr><tr><td>ATRX</td><td>mat cKO</td><td>zygote</td><td>IF</td><td>binds to methylated histone</td><td>co-recruited with DAXX-HP1b (CBX1) at mat PCH</td><td>favor chromocenter formation and HP1 binding?</td><td>Liu et al 2020</td></tr><tr><td>DAXX</td><td>KO (m + z)</td><td>zygote + 2-cell</td><td>IF/DNA FISH</td><td>histone chaperonne</td><td>recruited by cPRC1-CBX2 at pat PCH</td><td>favor chromocenter formation and HP1 binding?</td><td>Liu et al 2020</td></tr><tr><td>CAF1</td><td>KO</td><td>zygote to 16-cell</td><td>IF</td><td>histone chaperonne</td><td>recruitment of HP1 at PCH</td><td>favor chromocenter formation and HP1 binding?</td><td>Houlard et al 2006</td></tr><tr><td>HIRA</td><td>mat cKO</td><td>zygote</td><td>IF</td><td>histone chaperonne</td><td>formation of paternal pronucleus</td><td>no role in chromocenter clustering but in rDNA repression?</td><td>Lin et al 2014</td></tr><tr><td>SETDB1</td><td>mat cKO</td><td>FGO</td><td>ChIP/IF</td><td>histone methylase</td><td>no information in that study</td><td>no role in Chromocenter clustering but in repeat silencing?</td><td>Kim et al 2016</td></tr><tr><td>SUV39H2</td><td>KD</td><td>zygote + 2-cell</td><td>IF</td><td>histone methylase</td><td>de novo H3K9me3 at pat PCH (late zygote)</td><td>favor chromocenter formation and HP1 binding?</td><td>Burton et al 2020</td></tr><tr><td>HP1b (CBX1)</td><td>mat cKO</td><td>zygote</td><td>IF</td><td>binds to methylated histone</td><td>Located at mat PCH from the GV oocyte</td><td>favor chromocenter formation through phase transition?</td><td>Tardat et al 2015</td></tr></tbody></table>
<table><caption>Table 2</caption><thead><tr><td>Factors</td><td>Methods</td><td>Experimental stage</td><td>Technic</td><td>Function at chromatin</td><td>Known roles in SGO during preimplantation</td><td>Proposed role in 3D chromatin organization</td><td>Study</td></tr></thead><tbody><tr><td>mSCC1</td><td>mat cKO</td><td>FGO</td><td>low input HiC</td><td>Cohesin complex</td><td>favors loop extrusion &amp; restricts compartimentalisation</td><td>restrains Polycomb structural domains formation?</td><td>Du et al 2020</td></tr><tr><td>mSCC1</td><td>mat cKO</td><td>zygote</td><td>sn HiC</td><td>Cohesin complex</td><td>favors loop extrusion &amp; restricts compartimentalisation</td><td>restrains Polycomb structural domains formation?</td><td>Gassler et al 2017</td></tr><tr><td>mWAPL</td><td>mat cKO</td><td>zygote</td><td>sn HiC</td><td>Cohesin release factor</td><td>restricts loop extrusion (reduced size loop)</td><td>favors Polycomb structural domains formation ?</td><td>Gassler et al 2017</td></tr><tr><td>mMCM</td><td>deposition inhibition</td><td>zygote</td><td>sn HiC</td><td>DNA replication</td><td>restricts loop extrusion (reduced size loop)</td><td>favors Polycomb structural domains formation?</td><td>Dequeker et al 2022</td></tr><tr><td>mCTCF</td><td>mat cKD</td><td>zygote</td><td>sn HiC</td><td>TF &amp; barrier to Cohesin</td><td>favors loop extrusion</td><td>restrains Polycomb structural domains formation?</td><td>Dequeker et al 2022</td></tr><tr><td>mCTCF</td><td>cKO (m+z)</td><td>blastocyst</td><td>se HiC</td><td>TF &amp; barrier to Cohesin</td><td>favors loop extrusion &amp; no impact compartimentalisation</td><td>act at late stage, not relevant to Polycomb structural domains</td><td>Andreu et al 2022</td></tr><tr><td>RING1A/B</td><td>mat cKO</td><td>FGO</td><td>low input HiC</td><td>H2AK119Ub</td><td>favor Polycomb structural domain &amp; compartment like</td><td>acts during germline formation?</td><td>Du et al 2020</td></tr><tr><td>EED</td><td>mat cKO</td><td>FGO+2 cell</td><td>low input HiC</td><td>H3K27 methylation</td><td>favor Polycomb structural domain &amp; compartment like</td><td>acts after fertilization?</td><td>Du et al 2020</td></tr><tr><td>MLL2/KMT2B</td><td>mat cKO</td><td>Germline + zygote</td><td>IF</td><td>H3K4 methylation</td><td>no information in that study</td><td>Restricts H3K27me3 domain &amp; favors Polycomb structural domain?</td><td>Andreu-Vieyra et al 2010</td></tr><tr><td>SETD2</td><td>mat cKO</td><td>FGO to blastocyst</td><td>ChIP seq / IF</td><td>H3K36 methylation</td><td>no information in that study</td><td>Restricts H3K27me3 domain &amp; favors Polycomb structural domain?</td><td>Xu et al 2019</td></tr><tr><td>EZHIP</td><td>KO</td><td>FGO</td><td>IF</td><td>PRC2 regulation</td><td>no information in that study</td><td>Restricts H3K27me3 domain &amp; favors Polycomb structural domain?</td><td>Ragazzini et al 2019</td></tr><tr><td>CBX2</td><td>GOF+inhibition</td><td>zygote</td><td>IF</td><td>bind to H3k27me3</td><td>no information in that study</td><td>Phase transition that could favor Polycomb structural domains?</td><td>Tardat et al 2015</td></tr></tbody></table>