ZFP281 controls transcriptional and epigenetic changes promoting mouse pluripotent state transitions via DNMT3 and TET1.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates epiblast development in vivo during the peri-implantation period of mouse embryo development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events that occur during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knockin cell models, we identify the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, which is dependent on the formation of R-loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Multifaceted SOX2-chromatin interaction underpins pluripotency progression in early embryos.

Pioneer transcription factors (TFs), such as OCT4 and SOX2, play crucial roles in pluripotency regulation. However, the master TF-governed pluripotency regulatory circuitry was largely inferred from cultured cells. In this work, we investigated SOX2 binding from embryonic day 3.5 (E3.5) to E7.5 in the mouse. In E3.5 inner cell mass (ICM), SOX2 regulates the ICM-trophectoderm program but is dispensable for opening global enhancers. Instead, SOX2 occupies preaccessible enhancers in part opened by early-stage expressing TFs TFAP2C and NR5A2. SOX2 then widely redistributes when cells adopt naive and formative pluripotency by opening enhancers or poising them for rapid future activation. Hence, multifaceted pioneer TF-enhancer interaction underpins pluripotency progression in embryos, including a distinctive state in E3.5 ICM that bridges totipotency and pluripotency.

ZFP281 coordinates DNMT3 and TET1 for transcriptional and epigenetic control in pluripotent state transitions.

The progression from naive through formative to primed in vitro pluripotent stem cell states recapitulates the development of the epiblast in vivo during the peri-implantation period of mammalian development. Activation of the de novo DNA methyltransferases and reorganization of transcriptional and epigenetic landscapes are key events occurring during these pluripotent state transitions. However, the upstream regulators that coordinate these events are relatively underexplored. Here, using Zfp281 knockout mouse and degron knock-in cell models, we uncover the direct transcriptional activation of Dnmt3a/3b by ZFP281 in pluripotent stem cells. Chromatin co-occupancy of ZFP281 and DNA hydroxylase TET1, dependent on the formation of R loops in ZFP281-targeted gene promoters, undergoes a "high-low-high" bimodal pattern regulating dynamic DNA methylation and gene expression during the naïive-formative-primed transitions. ZFP281 also safeguards DNA methylation in maintaining primed pluripotency. Our study demonstrates a previously unappreciated role for ZFP281 in coordinating DNMT3A/3B and TET1 functions to promote pluripotent state transitions.

Evolutionary epigenomic analyses in mammalian early embryos reveal species-specific innovations and conserved principles of imprinting.

While mouse remains the most popular model, the conservation of parental-to-embryonic epigenetic transition across mammals is poorly defined. Through analysis of oocytes and early embryos in human, bovine, porcine, rat, and mouse, we revealed remarkable species-specific innovations as no single animal model fully recapitulates the human epigenetic transition. In rodent oocytes, transcription-dependent DNA methylation allows methylation of maternal imprints but not intergenic paternal imprints. Unexpectedly, prevalent DNA hypermethylation, paralleled by H3K36me2/3, also occurs in nontranscribed regions in porcine and bovine oocytes, except for megabase-long "CpG continents (CGCs)" where imprinting control regions preferentially reside. Broad H3K4me3 and H3K27me3 domains exist in nonhuman oocytes, yet only rodent H3K27me3 survives beyond genome activation. Coincidently, regulatory elements preferentially evade H3K27me3 in rodent oocytes, and failure to do so causes aberrant embryonic gene repression. Hence, the diverse mammalian innovations of parental-to-embryonic transition center on a delicate "to-methylate-or-not" balance in establishing imprints while protecting other regulatory regions.

Role of CxxC-finger protein 1 in establishing mouse oocyte epigenetic landscapes.

During oogenesis, oocytes gain competence and subsequently undergo meiotic maturation and prepare for embryonic development; trimethylated histone H3 on lysine-4 (H3K4me3) mediates a wide range of nuclear events during these processes. Oocyte-specific knockout of CxxC-finger protein 1 (CXXC1, also known as CFP1) impairs H3K4me3 accumulation and causes changes in chromatin configurations. This study investigated the changes in genomic H3K4me3 landscapes in oocytes with Cxxc1 knockout and the effects on other epigenetic factors such as the DNA methylation, H3K27me3, H2AK119ub1 and H3K36me3. H3K4me3 is overall decreased after knocking out Cxxc1, including both the promoter region and the gene body. CXXC1 and MLL2, which is another histone H3 methyltransferase, have nonoverlapping roles in mediating H3K4 trimethylation during oogenesis. Cxxc1 deletion caused a decrease in DNA methylation levels and affected H3K27me3 and H2AK119ub1 distributions, particularly at regions with high DNA methylation levels. The changes in epigenetic networks implicated by Cxxc1 deletion were correlated with the transcriptional changes in genes in the corresponding genomic regions. This study elucidates the epigenetic changes underlying the phenotypes and molecular defects in oocytes with deleted Cxxc1 and highlights the role of CXXC1 in orchestrating multiple factors that are involved in establishing the appropriate epigenetic states of maternal genome.

Formative pluripotent stem cells show features of epiblast cells poised for gastrulation.

The pluripotency of mammalian early and late epiblast could be recapitulated by naïve embryonic stem cells (ESCs) and primed epiblast stem cells (EpiSCs), respectively. However, these two states of pluripotency may not be sufficient to reflect the full complexity and developmental potency of the epiblast during mammalian early development. Here we report the establishment of self-renewing formative pluripotent stem cells (fPSCs) which manifest features of epiblast cells poised for gastrulation. fPSCs can be established from different mouse ESCs, pre-/early-gastrula epiblasts and induced PSCs. Similar to pre-/early-gastrula epiblasts, fPSCs show the transcriptomic features of formative pluripotency, which are distinct from naïve ESCs and primed EpiSCs. fPSCs show the unique epigenetic states of E6.5 epiblast, including the super-bivalency of a large set of developmental genes. Just like epiblast cells immediately before gastrulation, fPSCs can efficiently differentiate into three germ layers and primordial germ cells (PGCs) in vitro. Thus, fPSCs highlight the feasibility of using PSCs to explore the development of mammalian epiblast.

The landscape of RNA Pol II binding reveals a stepwise transition during ZGA.

Zygotic genome activation (ZGA) is the first transcription event in life1. However, it is unclear how RNA polymerase is engaged in initiating ZGA in mammals. Here, by developing small-scale Tn5-assisted chromatin cleavage with sequencing (Stacc-seq), we investigated the landscapes of RNA polymerase II (Pol II) binding in mouse embryos. We found that Pol II undergoes 'loading', 'pre-configuration', and 'production' during the transition from minor ZGA to major ZGA. After fertilization, Pol II is preferentially loaded to CG-rich promoters and accessible distal regions in one-cell embryos (loading), in part shaped by the inherited parental epigenome. Pol II then initiates relocation to future gene targets before genome activation (pre-configuration), where it later engages in full transcription elongation upon major ZGA (production). Pol II also maintains low poising at inactive promoters after major ZGA until the blastocyst stage, coinciding with the loss of promoter epigenetic silencing factors. Notably, inhibition of minor ZGA impairs the Pol II pre-configuration and embryonic development, accompanied by aberrant retention of Pol II and ectopic expression of one-cell targets upon major ZGA. Hence, stepwise transition of Pol II occurs when mammalian life begins, and minor ZGA has a key role in the pre-configuration of transcription machinery and chromatin for genome activation.

CFP1-dependent histone H3K4 trimethylation in murine oocytes facilitates ovarian follicle recruitment and ovulation in a cell-nonautonomous manner.

CxxC-finger protein 1 (CFP1)-mediated trimethylated histone H3 at lysine-4 (H3K4me3) during oocyte development enables the oocyte genome to establish the competence to generate a new organism. Nevertheless, it remains unclear to which extent this epigenetic modification forms an instructive component of ovarian follicle development. We investigated the ovarian functions using an oocyte-specific Cxxc1 knockout mouse model, in which the H3K4me3 accumulation is downregulated in oocytes of developing follicles. CFP1-dependent H3K4 trimethylation in oocytes was necessary to maintain the expression of key paracrine factors and to facilitate the communication between an oocyte and the surrounding granulosa cells. The distinct gene expression patterns in cumulus cells within preovulatory follicles were disrupted by the Cxxc1 deletion in oocytes. Both follicle growth and ovulation were compromised after CFP1 deletion, because Cxxc1 deletion in oocytes indirectly impaired essential signaling pathways in granulosa cells that mediate the functions of follicle-stimulating hormone and luteinizing hormone. Therefore, CFP1-regulated epigenetic modification of the oocyte genome influences the responses of ovarian follicles to gonadotropin in a cell-nonautonomous manner.

Epigenomic analysis of gastrulation identifies a unique chromatin state for primed pluripotency.

Around implantation, the epiblast (Epi) transits from naïve to primed pluripotency, before giving rise to the three germ layers. How chromatin is reconfigured during this developmental window remains poorly understood. We performed a genome-wide investigation of chromatin landscapes during this period. We find that enhancers in ectoderm are already pre-accessible in embryonic day 6.5 (E6.5) Epi when cells enter a primed pluripotent state. Unexpectedly, strong trimethylation of histone H3 at lysine 4 (H3K4me3) emerges at developmental gene promoters in E6.5 Epi and positively correlates with H3K27me3, thus establishing bivalency. These genes also show enhanced spatial interactions. Both the strong bivalency and spatial clustering are virtually absent in preimplantation embryos and are markedly reduced in fate-committed lineages. Finally, we show that KMT2B is essential for establishing bivalent H3K4me3 at E6.5 but becomes partially dispensable later. Its deficiency leads to impaired activation of developmental genes and subsequent embryonic lethality. Thus, our data characterize lineage-specific chromatin reconfiguration and a unique chromatin state for primed pluripotency.

Multi-omics profiling of mouse gastrulation at single-cell resolution.

Formation of the three primary germ layers during gastrulation is an essential step in the establishment of the vertebrate body plan and is associated with major transcriptional changes1-5. Global epigenetic reprogramming accompanies these changes6-8, but the role of the epigenome in regulating early cell-fate choice remains unresolved, and the coordination between different molecular layers is unclear. Here we describe a single-cell multi-omics map of chromatin accessibility, DNA methylation and RNA expression during the onset of gastrulation in mouse embryos. The initial exit from pluripotency coincides with the establishment of a global repressive epigenetic landscape, followed by the emergence of lineage-specific epigenetic patterns during gastrulation. Notably, cells committed to mesoderm and endoderm undergo widespread coordinated epigenetic rearrangements at enhancer marks, driven by ten-eleven translocation (TET)-mediated demethylation and a concomitant increase of accessibility. By contrast, the methylation and accessibility landscape of ectodermal cells is already established in the early epiblast. Hence, regulatory elements associated with each germ layer are either epigenetically primed or remodelled before cell-fate decisions, providing the molecular framework for a hierarchical emergence of the primary germ layers.

In vitro culture of cynomolgus monkey embryos beyond early gastrulation.

Gastrulation is a key event in embryonic development when the germ layers are specified and the basic animal body plan is established. The complexities of primate gastrulation remain a mystery because of the difficulties in accessing primate embryos at this stage. Here, we report the establishment of an in vitro culture (IVC) system that supports the continuous development of cynomolgus monkey blastocysts beyond early gastrulation up to 20 days after fertilization. The IVC embryos highly recapitulated the key events of in vivo early postimplantation development, including segregation of the epiblast and hypoblast, formation of the amniotic and yolk sac cavities, appearance of the primordial germ cells, and establishment of the anterior-posterior axis. Single-cell RNA-sequencing analyses of the IVC embryos provide information about lineage specification during primate early postimplantation development. This system provides a platform with which to explore the characteristics and mechanisms of early postimplantation embryogenesis in primates with possible conservation of cell movements and lineages in human embryogenesis.

SETD2 regulates the maternal epigenome, genomic imprinting and embryonic development.

The oocyte epigenome plays critical roles in mammalian gametogenesis and embryogenesis. Yet, how it is established remains elusive. Here, we report that histone-lysine N-methyltransferase SETD2, an H3K36me3 methyltransferase, is a crucial regulator of the mouse oocyte epigenome. Deficiency in Setd2 leads to extensive alterations of the oocyte epigenome, including the loss of H3K36me3, failure in establishing the correct DNA methylome, invasion of H3K4me3 and H3K27me3 into former H3K36me3 territories and aberrant acquisition of H3K4me3 at imprinting control regions instead of DNA methylation. Importantly, maternal depletion of SETD2 results in oocyte maturation defects and subsequent one-cell arrest after fertilization. The preimplantation arrest is mainly due to a maternal cytosolic defect, since it can be largely rescued by normal oocyte cytosol. However, chromatin defects, including aberrant imprinting, persist in these embryos, leading to embryonic lethality after implantation. Thus, these data identify SETD2 as a crucial player in establishing the maternal epigenome that in turn controls embryonic development.

Zbed3 participates in the subcortical maternal complex and regulates the distribution of organelles.

We previously identified a subcortical maternal complex (SCMC) that is essential for early embryogenesis and female fertility in mice. However, the molecular mechanism by which the SCMC affects female fertility remains largely uncharacterized. Here, we report that a novel maternal protein, zinc finger BED-type containing 3 (Zbed3), participates in the SCMC. Depletion of maternal Zbed3 results in reduced fecundity of females, because of the impaired and delayed development in a proportion of mutant embryos. The loss of maternal Zbed3 results in asymmetric zygotic division and abnormal distributions of organelles in the affected oocytes and zygotes, similar to the phenotypes observed in females with disrupted core SCMC genes. Further investigation revealed that these phenotypes are associated with disrupted dynamics of microtubules and/or formation of cytoplasmic lattices (CPLs). The stability and localization of Zbed3 depend on, but are not required for, the formation of the SCMC. Thus, our data suggest Zbed3 as one of downstream proteins mediating SCMC functions and provide further insights into the roles of the SCMC and CPLs in female fertility.

Dynamic epigenomic landscapes during early lineage specification in mouse embryos.

In mammals, all somatic development originates from lineage segregation in early embryos. However, the dynamics of transcriptomes and epigenomes acting in concert with initial cell fate commitment remains poorly characterized. Here we report a comprehensive investigation of transcriptomes and base-resolution methylomes for early lineages in peri- and postimplantation mouse embryos. We found allele-specific and lineage-specific de novo methylation at CG and CH sites that led to differential methylation between embryonic and extraembryonic lineages at promoters of lineage regulators, gene bodies, and DNA-methylation valleys. By using Hi-C experiments to define chromatin architecture across the same developmental period, we demonstrated that both global demethylation and remethylation in early development correlate with chromatin compartments. Dynamic local methylation was evident during gastrulation, which enabled the identification of putative regulatory elements. Finally, we found that de novo methylation patterning does not strictly require implantation. These data reveal dynamic transcriptomes, DNA methylomes, and 3D chromatin landscapes during the earliest stages of mammalian lineage specification.

Allelic reprogramming of 3D chromatin architecture during early mammalian development.

In mammals, chromatin organization undergoes drastic reprogramming after fertilization. However, the three-dimensional structure of chromatin and its reprogramming in preimplantation development remain poorly understood. Here, by developing a low-input Hi-C (genome-wide chromosome conformation capture) approach, we examined the reprogramming of chromatin organization during early development in mice. We found that oocytes in metaphase II show homogeneous chromatin folding that lacks detectable topologically associating domains (TADs) and chromatin compartments. Strikingly, chromatin shows greatly diminished higher-order structure after fertilization. Unexpectedly, the subsequent establishment of chromatin organization is a prolonged process that extends through preimplantation development, as characterized by slow consolidation of TADs and segregation of chromatin compartments. The two sets of parental chromosomes are spatially separated from each other and display distinct compartmentalization in zygotes. Such allele separation and allelic compartmentalization can be found as late as the 8-cell stage. Finally, we show that chromatin compaction in preimplantation embryos can partially proceed in the absence of zygotic transcription and is a multi-level hierarchical process. Taken together, our data suggest that chromatin may exist in a markedly relaxed state after fertilization, followed by progressive maturation of higher-order chromatin architecture during early development.

Trp-Asp (WD) Repeat Domain 1 Is Essential for Mouse Peri-implantation Development and Regulates Cofilin Phosphorylation.

Trp-Asp (WD) repeat domain 1 (WDR1) is a highly conserved actin-binding protein across all eukaryotes and is involved in numerous actin-based processes by accelerating Cofilin severing actin filament. However, the function and the mechanism of WDR1 in mammalian early development are still largely unclear. We now report that WDR1 is essential for mouse peri-implantation development and regulates Cofilin phosphorylation in mouse cells. The disruption of maternal WDR1 does not obviously affect ovulation and female fertility. However, depletion of zygotic WDR1 results in embryonic lethality at the peri-implantation stage. In WDR1 knock-out cells, we found that WDR1 regulates Cofilin phosphorylation. Interestingly, WDR1 is overdosed to regulate Cofilin phosphorylation in mouse cells. Furthermore, we showed that WDR1 interacts with Lim domain kinase 1 (LIMK1), a well known phosphorylation kinase of Cofilin. Altogether, our results provide new insights into the role and mechanism of WDR1 in physiological conditions.

Allelic reprogramming of the histone modification H3K4me3 in early mammalian development.

Histone modifications are fundamental epigenetic regulators that control many crucial cellular processes. However, whether these marks can be passed on from mammalian gametes to the next generation is a long-standing question that remains unanswered. Here, by developing a highly sensitive approach, STAR ChIP-seq, we provide a panoramic view of the landscape of H3K4me3, a histone hallmark for transcription initiation, from developing gametes to post-implantation embryos. We find that upon fertilization, extensive reprogramming occurs on the paternal genome, as H3K4me3 peaks are depleted in zygotes but are readily observed after major zygotic genome activation at the late two-cell stage. On the maternal genome, we unexpectedly find a non-canonical form of H3K4me3 (ncH3K4me3) in full-grown and mature oocytes, which exists as broad peaks at promoters and a large number of distal loci. Such broad H3K4me3 peaks are in contrast to the typical sharp H3K4me3 peaks restricted to CpG-rich regions of promoters. Notably, ncH3K4me3 in oocytes overlaps almost exclusively with partially methylated DNA domains. It is then inherited in pre-implantation embryos, before being erased in the late two-cell embryos, when canonical H3K4me3 starts to be established. The removal of ncH3K4me3 requires zygotic transcription but is independent of DNA replication-mediated passive dilution. Finally, downregulation of H3K4me3 in full-grown oocytes by overexpression of the H3K4me3 demethylase KDM5B is associated with defects in genome silencing. Taken together, these data unveil inheritance and highly dynamic reprogramming of the epigenome in early mammalian development.

Resetting Epigenetic Memory by Reprogramming of Histone Modifications in Mammals.

Polycomb group proteins and the related histone modification H3K27me3 can maintain the silencing of key developmental regulators and provide cellular memory. However, how such an epigenetic state is reprogrammed and inherited between generations is poorly understood. Using an ultra-sensitive approach, STAR ChIP-seq, we investigated H3K27me3 across 14 developmental stages along mouse gametogenesis and early development. Interestingly, highly pervasive H3K27me3 is found in regions depleted of transcription and DNA methylation in oocytes. Unexpectedly, we observed extensive loss of promoter H3K27me3 at Hox and other developmental genes upon fertilization. This is accompanied by global erasure of sperm H3K27me3 but inheritance of distal H3K27me3 from oocytes. The resulting allele-specific H3K27me3 patterns persist to blastocysts before being converted to canonical forms in postimplantation embryos, where both H3K4me3/H3K27me3 bivalent promoter marks are restored at developmental genes. Together, these data revealed widespread resetting of epigenetic memory and striking plasticity of epigenome during gametogenesis and early development.

The landscape of accessible chromatin in mammalian preimplantation embryos.

In mammals, extensive chromatin reorganization is essential for reprogramming terminally committed gametes to a totipotent state during preimplantation development. However, the global chromatin landscape and its dynamics in this period remain unexplored. Here we report a genome-wide map of accessible chromatin in mouse preimplantation embryos using an improved assay for transposase-accessible chromatin with high throughput sequencing (ATAC-seq) approach with CRISPR/Cas9-assisted mitochondrial DNA depletion. We show that despite extensive parental asymmetry in DNA methylomes, the chromatin accessibility between the parental genomes is globally comparable after major zygotic genome activation (ZGA). Accessible chromatin in early embryos is widely shaped by transposable elements and overlaps extensively with putative cis-regulatory sequences. Unexpectedly, accessible chromatin is also found near the transcription end sites of active genes. By integrating the maps of cis-regulatory elements and single-cell transcriptomes, we construct the regulatory network of early development, which helps to identify the key modulators for lineage specification. Finally, we find that the activities of cis-regulatory elements and their associated open chromatin diminished before major ZGA. Surprisingly, we observed many loci showing non-canonical, large open chromatin domains over the entire transcribed units in minor ZGA, supporting the presence of an unusually permissive chromatin state. Together, these data reveal a unique spatiotemporal chromatin configuration that accompanies early mammalian development.