Biology subjects

Reik, W.

Publications and source records attributed to Reik, W..

At least 19 recordsLinked to original sources

bioRxiv2026

Dynamic TET1 repression coordinates activation of macroH2A.2-poised bivalent genes during neuroectoderm differentiation

Cell state transitions involve changes in gene expression programs in conjunction with epigenetic regulation occurring in chromatin. Bivalent chromatin, marked by overlapping active H3K4me3 and repressive H3K27me3, poises key developmental genes during pluripotency for cell type-specific activation during differentiation. However, the molecular mechanisms that activate bivalent genes during lineage commitment remain poorly understood. Here, we elucidate how the cytosine dioxygenase TET1 promotes neuroectoderm differentiation through dynamic chromatin binding and a non-catalytic repressor function. Through a hypothesis-free screen for TET1 chromatin complex partners, we find that TET1 physically interacts with macroH2A.2 and H2A.X on chromatin. Integrated multi-omic analyses reveal that the non-catalytic repressor function of TET1 and a novel activating function of the H2A variant macroH2A.2 together promote a poised transcriptional state within bivalent chromatin during pluripotency. Upon pluripotency exit, TET1 disengages from macroH2A.2-marked early neuroectoderm genes to permit their activation, while remaining bound as a repressor at mesoendoderm and late neural genes to prevent alternative cell state transitions. Furthermore, TET1 retention at accessible primed enhancers supports neural program activation. Together, our findings reveal a noncanonical functional interplay within bivalent chromatin between TET1 and macroH2A.2 that maintains a poised state during pluripotency and predicts selective gene activation during neuroectoderm specification.

developmental biology↗

bioRxiv2026

Cell-to-cell variability and gain of methylation at polycomb CpG islands as a hallmark of aging

Aging is a complex multifactorial process that affects cellular function and tissue homeostasis over time. Despite extensive research, the molecular mechanisms driving cellular aging remain poorly understood1,2. Many studies have focused on changes in DNA methylation as an indicator of aging3. In particular, the degree of methylation at polycomb CpG islands has been shown to be predictive of phenotypic changes associated with aging4,5. Since many age-related pathological processes, are thought to be of single-cell origin (e.g. cancer), we questioned whether polycomb DNA methylation also occurs preferentially in a subset of cells within the overall population. Using single-cell whole-genome methylation data from multiple ages and tissues, we identify Average Polycomb CpG Methylation as a hallmark of cellular aging. This revealed that aging occurs at varying rates within specific cells, with faster proliferating cells showing accelerated levels. Gene expression analysis in "young" and "old" single cells identified changes in immune response, translation regulation, tumorigenesis, neurodegeneration and other cellular processes associated with aging. These results challenge traditional models of homogeneous cellular aging and suggest that aging itself is a highly individualized process at the single-cell level that may be driven by programmed changes in polycomb CpG island DNA methylation.

genomics↗

bioRxiv2026

A Physics-based Model Reveals Mechanisms of Epigenetic Memory

In multi-cellular eukaryotic organisms, cell type and specific functional identity are defined by the epigenetic patterning of chemical modifications to DNA and chromatin that modulate the expression and silencing of specific genes. When a cell divides, histones containing important epigenetic marks are distributed between the two daughter strands leading to a temporary dilution of epigenetic information and cell identity. In this work we introduce a physics-based model of epigenetic memory that explains how cells restore and maintain H3K9me3 and H3K27me3 histone methylation patterning after cell division. We demonstrate that emergence and maintenance of the epigenetic program is driven by an evolved mechanism that makes use of the biophysics of polymers, phase condensates and enzymatic activity. We validate our model via genome-wide epigenetic time-course simulation and comparison to experimental epigenetic data from multiple donors, multiple cell types, and for multiple epigenetic marks. Finally, we use our model as a conceptual framework to understand cellular reprogramming by hypothesizing that these processes first contend with and later utilize somatic epigenetic maintenance programs.

biophysics↗

bioRxiv2025

A transcriptional clock of the human pluripotency transition

Developmental timing differs strikingly between mammals. All embryonic and some placental lineages emerge from the pluripotent epiblast in a temporally defined sequence, lasting about two weeks in the human embryo, but only two days in mice. Moreover, the order of lineage segregation and gene expression differ between the species. We used human pluripotent stem cells to recapitulate this window of epiblast development in vitro. Simultaneous profiling of gene expression and chromatin accessibility in single cells revealed a robust, autonomous switch between cell states during this process. We reconstructed the integrative gene regulatory network (GRN) of transcription factors (TFs) and signalling molecules. Notably, this revealed a transcriptional cascade including temporally close positive and distant inhibitory connections. We suggest that this cascade acts as a transcriptional clock and governs the directionality, timing and intrinsic decisions during epiblast development. From individual gene interactions, we derived a mechanistic mathematical model of the transcriptional clock of pluripotency that closely reproduced gene expression dynamics and identified key regulatory connections. Moreover, our model revealed a bistable switch governing the transition, thus translating the GRN inference into an interpretable mechanism. Strikingly, the GRN model derived for humans predicted the acceleration of developmental timing in mice when initialised with mouse-specific expression patterns, yet still leading to a human-like expression state. Therefore, TF levels explain developmental timing, whereas the architecture of the network defines its trajectory. Together, our work provides novel conceptual insights into the intrinsic mechanisms of developmental transitions in the human embryo. HIGHLIGHTSO_LIThe human pluripotency transition is guided by an intrinsic developmental programme C_LIO_LIA cascade of transcription factors defines directionality and timing of the pluripotency transition through positive and negative interactions C_LIO_LITranscription factor levels explain developmental timing C_LIO_LIThe architecture of the gene regulatory network defines its trajectory C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/643129v1_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@1faf1aaorg.highwire.dtl.DTLVardef@10053borg.highwire.dtl.DTLVardef@1c76aacorg.highwire.dtl.DTLVardef@12c7b89_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

A Spatiotemporal Atlas of Mouse Gastrulation and Early Organogenesis to Explore Axial Patterning and Project In Vitro Models onto In Vivo Space

At the onset of murine gastrulation, pluripotent epiblast cells migrate through the primitive streak, generating mesodermal and endodermal precursors, while the ectoderm arises from the remaining epiblast. Together, these germ layers establish the body plan, defining major body axes and initiating organogenesis. Although comprehensive single cell transcriptional atlases of dissociated mouse embryos across embryonic stages have provided valuable insights during gastrulation, the spatial context for cell differentiation and tissue patterning remain underexplored. In this study, we employed spatial transcriptomics to measure gene expression in mouse embryos at E6.5 and E7.5 and integrated these datasets with previously published E8.5 spatial transcriptomics1 and a scRNA-seq2 atlas spanning E6.5 to E9.5. This approach resulted in a comprehensive spatiotemporal atlas, comprising over 150,000 cells with 88 refined cell type annotations as well as genome-wide transcriptional imputation during mouse gastrulation and early organogenesis. The atlas facilitates exploration of gene expression dynamics along anterior-posterior and dorsal-ventral axes at cell type, tissue, and organismal scales, revealing insights into mesodermal fate decisions within the primitive streak. Moreover, we developed a bioinformatics pipeline to project additional scRNA-seq datasets into a spatiotemporal framework and demonstrate its utility by analysing cardiovascular models of gastrulation3. To maximise impact, the atlas is publicly accessible via a user-friendly web portal empowering the wider developmental and stem cell biology communities to explore mechanisms of early mouse development in a spatiotemporal context.

developmental biology↗

bioRxiv2024

Collective dynamics of DNA methylation during ageing

Ageing is the decline of physiological function over time. Statistical models termed ageing clocks can predict chronological and biological age from the longitudinal time evolution of DNA methylation. Here, we show that DNA methylation ageing is also manifest in how pairs of genomic loci evolve with respect to each other with age. Using sequencing data from a range of tissues in mouse we show that genomic correlations in DNA methylation during ageing are characterised by an enrichment of correlations between sites that are roughly 500 bp apart. We trace the origin of this behaviour to collective dynamics in the boundaries of CpG islands. We derive a simple, biophysical model that explains the origin due to a tilt in the competition between methylating and demethylating processes and an ensuing wetting-like phenomenon. Using Chip-seq data, we argue for a molecular mechanism based on a PRC2-dependent dilution of H3K27me3 during ageing. Our work gives a new perspective on epigenetic ageing, highlighting the importance of correlative as opposed to longitudinal dynamics.

genomics↗

bioRxiv2024

Using CRISPR barcoding as a molecular clock to capture dynamic processes at single-cell resolution

Biological processes are fundamentally dynamic, yet existing methods for capturing these temporal changes are limited. We present scDynaBar, a novel approach that integrates CRISPR-Cas9 dynamic barcoding with single-cell sequencing to enable the recording of temporal cellular events. In this system, genetic barcodes accumulate mutations over a 25-day period and then are sequenced together with the transcriptome of each single cell. We propose that this gradual accumulation of genetic diversity can be exploited to create an ordered record of a cellular event. We apply this approach to track the transition from a pluripotent state to a two-cell (2C)-like state in mouse embryonic stem cells (mESCs). The results provide compelling evidence for the transient nature of the 2C-like state. Additionally, our system shows consistent mutation rates across diverse cell types in a mouse gastruloid model, underscoring its robustness and versatility across various biological contexts. This technique not only improves our ability to study single-cell dynamics but also creates new opportunities for recording other temporal signals--in other words, using dynamic barcoding as a molecular clock in individual cells. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=74 SRC="FIGDIR/small/618192v2_ufig1.gif" ALT="Figure 1"> View larger version (19K): org.highwire.dtl.DTLVardef@1665361org.highwire.dtl.DTLVardef@ec3114org.highwire.dtl.DTLVardef@8a3123org.highwire.dtl.DTLVardef@a0235f_HPS_FORMAT_FIGEXP M_FIG C_FIG

cell biology↗

bioRxiv2024

Epigenetic priming of embryonic enhancer elements coordinates developmental gene networks

Embryonic development requires the accurate spatiotemporal execution of cell lineage-specific gene expression programs, which are controlled by transcriptional enhancers. Developmental enhancers adopt a primed chromatin state prior to their activation; however how this primed enhancer state is established, maintained, and how it affects the regulation of developmental gene networks remains poorly understood. Here, we use comparative multi-omic analyses of human and mouse early embryonic development to identify subsets of post-gastrulation lineage-specific enhancers which are epigenetically primed ahead of their activation, marked by the histone modification H3K4me1 within the epiblast. We show that epigenetic priming occurs at lineage-specific enhancers for all three germ layers, and that epigenetic priming of enhancers confers lineage-specific regulation of key developmental gene networks. Surprisingly in some cases, lineage-specific enhancers are epigenetically marked already in the zygote, weeks before their activation during lineage specification. Moreover, we outline a generalisable strategy to use naturally occurring human genetic variation to delineate important sequence determinants of primed enhancer function. Our findings identify an evolutionarily conserved program of enhancer priming and begin to dissect the temporal dynamics and mechanisms of its establishment and maintenance during early mammalian development.

developmental biology↗

bioRxiv2024

Eomes directs the formation of spatially and functionally diverse extra-embryonic hematovascular tissues

During mouse gastrulation, extraembryonic mesoderm (ExEM) contributes to the extraembryonic yolk sac (YS) and allantois, both of which are essential for successful gestation. Although the genetic networks coordinating intra-embryonic mesodermal subtype specification are well-studied, the mechanisms driving ExEM diversification are poorly understood. Here, we reveal that embryoid body in vitro differentiation generates two distinct lineages of mesodermal cells matching YS and allantois respectively. Combining in vitro models with in vivo chimeric embryo analysis, we discover that Eomesodermin (Eomes) regulates the formation of a subset of YS-fated ExEM but is dispensable for allantois formation. Furthermore, simultaneous disruption of Eomes and T impedes the specification of any YS or allantois mesoderm, indicating compensatory roles for T during allantois formation when Eomes is disrupted. Our study highlights previously unrecognized functional and mechanistic diversity in ExEM diversification and endothelial development and introduces a tractable EB model to dissect the signaling pathways and transcriptional networks driving the formation of key extraembryonic tissues.

developmental biology↗

bioRxiv2023

Maternal SMARCA5 is required for major ZGA in mouse embryos

Zygotic genome activation (ZGA) in mice takes place in two waves, a minor wave in the one-cell embryo, and a major wave at the two-cell stage, both accompanied by global transcriptional and epigenetic reprogramming. However, the orchestration of these reprogramming events by maternal factors deposited in the oocyte is not yet entirely understood. We and others have recently shown that epigenetic modifiers such as SMARCA5 (the main ATPase in ISWI complexes) can initiate the ZGA transcriptional programme in vitro. So far, the role of SMARCA5 in ZGA in vivo has not been addressed, as constitutive knock-out mice lacking SMARCA5 are not viable. We have overcome this limitation by using the targeted protein-depletion system Trim Away to degrade SMARCA5 in early zygotes. We further harnessed the power of single cell multi-omics (scNMT-seq) and showed that in the absence of SMARCA5, major ZGA genes fail to be upregulated at the two-cell stage. This is explained by the lower accessibility and disrupted nucleosome positioning at their promoters and distal regulatory regions, compared to wild-type embryos. In contrast, we show that global chromatin accessibility at the two-cell stage is higher in SMARCA5 depleted embryos compared to control embryos, and this is accompanied by other global structural changes involving heterochromatic regions. Our results show that SMARCA5 has a global regulatory role at the two-cell stage, which includes the control of ZGA gene promoters and distal regulatory regions.

developmental biology↗

bioRxiv2023

Primordial germ cell DNA demethylation and development require DNA translesion synthesis.

Mutations in DNA damage response (DDR) factors are associated with human infertility, which affects up to 15% of the population. It remains unclear if the role of DDR is solely in meiosis. One pathway implicated in human fertility is DNA translesion synthesis (TLS), which allows replication impediments to be bypassed. We find that TLS is essential for pre-meiotic germ cell development in the embryo. Loss of the central TLS component, REV1, significantly inhibits the induction of human PGC-like cells (hPGCLCs). This is recapitulated in mice, where deficiencies in TLS initiation (Rev1-/- or PcnaK164R/K164R) or extension (Rev7-/-) result in a >150-fold reduction in the number of primordial germ cells (PGCs) and complete sterility. In contrast, the absence of TLS does not impact the growth, function, or homeostasis of somatic tissues. Surprisingly, we find a complete failure in both activation of the germ cell transcriptional program and in DNA demethylation, a critical step in germline epigenetic reprogramming. Our findings show that for normal fertility, DNA repair is required not only for meiotic recombination but for progression through the earliest stages of germ cell development in mammals.

developmental biology↗

bioRxiv2023

Single cell DNA methylation ageing in mouse blood

Ageing is the accumulation of changes and overall decline of the function of cells, organs and organisms over time. At the molecular and cellular level, the concept of biological age has been established and biomarkers of biological age have been identified, notably epigenetic DNA-methylation based clocks. With the emergence of single-cell DNA methylation profiling methods, the possibility to study biological age of individual cells has been proposed, and a first proof-of-concept study, based on limited single cell datasets mostly from early developmental origin, indicated the feasibility and relevance of this approach to better understand organismal changes and cellular ageing heterogeneity. Here we generated a large single-cell DNA methylation and matched transcriptome dataset from mouse peripheral blood samples, spanning a broad range of ages (10-101 weeks of age). We observed that the number of genes expressed increased at older ages, but gene specific changes were small. We next developed a robust single cell DNA methylation age predictor (scEpiAge), which can accurately predict age in a broad range of publicly available datasets, including very sparse data and it also predicts age in single cells. Interestingly, the DNA methylation age distribution is wider than technically expected in 19% of single cells, suggesting that epigenetic age heterogeneity is present in vivo and may relate to functional differences between cells. In addition, we observe differences in epigenetic ageing between the major blood cell types. Our work provides a foundation for better single-cell and sparse data epigenetic age predictors and highlights the significance of cellular heterogeneity during ageing. Highlights- Model to estimate DNA methylation age in single cells - Large multi-omics dataset of single cells from murine blood - Epigenetic age deviations from chronological age are greater than technical expected from technical variability - Number of genes expressed increases with chronological and epigenetic age

cell biology↗

bioRxiv2023

Dynamic DNA methylation turnover at the exit of pluripotency epigenetically primes gene regulatory elements for hematopoietic lineage specification

Epigenetic mechanisms govern developmental cell fate decisions, but how DNA methylation coordinates with chromatin structure and three-dimensional DNA folding to enact cell-type specific gene expression programmes remains poorly understood. Here, we use mouse embryonic stem and epiblast-like cells deficient for 5-methyl cytosine or its oxidative derivatives (5-hydroxy-, 5-formyl- and 5-carboxy-cytosine) to dissect the gene regulatory mechanisms that control cell lineage specification at the exit of pluripotency. Genetic ablation of either DNA methyltransferase (Dnmt) or Ten-eleven-translocation (Tet) activity yielded largely distinct sets of dysregulated genes, revealing divergent transcriptional defects upon perturbation of individual branches of the DNA cytosine methylation cycle. Unexpectedly, we found that disrupting DNA methylation or oxidation interferes with key enhancer features, including chromatin accessibility, enhancer-characteristic histone modifications, and long-range chromatin interactions with putative target genes. In addition to affecting transcription of select genes in pluripotent stem cells, we observe impaired enhancer priming, including a loss of three-dimensional interactions, at regulatory elements associated with key lineage-specifying genes that are required later in development, as we demonstrate for the key hematopoietic genes Klf1 and Lyl1. Consistently, we observe impaired transcriptional activation of blood genes during embryoid body differentiation of knockout cells. Our findings identify a novel role for the dynamic turnover of DNA methylation at the exit of pluripotency to establish and maintain chromatin states that epigenetically prime enhancers for later activation during developmental cell diversification. HighlightsO_LIWe perform a detailed epigenetic characterisation of the mouse embryonic stem cell (ESC) to epiblast-like cell (EpiLC) transition in wild type, Tet triple-knockout (TKO) and Dnmt TKO lines and develop a novel clustering approach to interrogate the data. C_LIO_LITet TKO reduces H3K4me1 and H3K27ac levels across enhancer elements upon pluripotency exit whilst Dnmt TKO affects only H3K4me1 levels, suggesting a novel role for oxidative derivatives in H3K4me1 deposition. C_LIO_LITet TKO and Dnmt TKO affect enhancer priming in EpiLCs which is associated with failure to upregulate hematopoietic genes upon differentiation. C_LIO_LILong-range chromosomal interactions between primed enhancers and their target genes are weakened in both Dnmt and Tet TKO. C_LI

molecular biology↗

bioRxiv2022

Inter-gastruloid heterogeneity revealed by single cell transcriptomics time course: implications for organoid based perturbation studies

Recent advances in organoid and genome editing technologies are allowing for perturbation experiments at an unprecedented scale. However, before doing such experiments it is important to understand the gene expression profile in each of the organoids cells, as well as how much heterogeneity there is between individual organoids. Here we characterise an organoid model of mouse gastrulation called gastruloids using single cell RNA-sequencing of individual organoids at half-day intervals between day 3 and day 5 of differentiation (roughly corresponding to E6.5-E8.75 in vivo). Our study reveals multiple differentiation trajectories that have hitherto not been characterised in gastruloids. Intriguingly, we observe that individual gastruloids displayed a strong bias towards producing either mesodermal (largely somitic) or ectodermal (specifically neural) cell types. This bifurcation is already seen at the earliest sampled time point, and is characterised by increased activity of WNT-associated pathways in mesodermally-biased gastruloids as compared to neurally-biased gastruloids. Notably, at day 5, mesodermal gastruloids show an increase in the proportion of neural cells, while neural gastruloids do not produce notably more mesodermal cells. This is in line with previous studies on how the balance between these cell types is regulated. We demonstrate using in silico simulations that without proper understanding of the inter-organoid heterogeneity, perturbation experiments have either very high false positive or negative rates, depending on the statistical model used. Thus in future studies, modelling of inter-organoid heterogeneity will be crucial when designing organoid-based perturbation studies. HighlightsO_LIA single cell RNA-sequencing time course of day 3 to day 5 mouse gastruloids reveals multiple mesodermal and neural differentiation trajectories hitherto uncharacterised in gastruloids C_LIO_LISingle gastruloid, single cell RNA-sequencing of mouse gastruloids reveals that gastruloids are either mesodermally- or neurally-biased C_LIO_LIThe two classes of gastruloid arise from differences in response strength to the WNT-agonist chiron C_LIO_LIAt day 5, mesodermal gastruloids start making more neural cells, while neural gastruloids do not make more mesodermal cells, aligning with previously studied in vivo feedback loops C_LIO_LIWe show using simulations that understanding interorganoid heterogeneity is a crucial consideration in the design and analysis of well-powered organoid-based perturbation studies C_LI

genomics↗

bioRxiv2022

Decoding gene regulation in the mouse embryo using single-cell multi-omics

Following gastrulation, the three primary germ layers develop into the major organs in a process known as organogenesis. Single-cell RNA sequencing has enabled the profiling of the gene expression dynamics of these cell fate decisions, yet a comprehensive map of the interplay between transcription factors and cis-regulatory elements is lacking, as are the underlying gene regulatory networks. Here we generate a multi-omics atlas of mouse early organogenesis by simultaneously profiling gene expression and chromatin accessibility from tens of thousands of single cells. We develop a computational method to leverage the multimodal readouts to predict transcription factor binding events in cis-regulatory elements, which we then use to infer gene regulatory networks that underpin lineage commitment events. Finally, we show that these models can be used to generate in silico predictions of the effect of transcription factor perturbations. We validate this experimentally by showing that Brachyury is essential for the differentiation of neuromesodermal progenitors to somitic mesoderm fate by priming cis-regulatory elements. The data set can be interactively explored at https://www.bioinformatics.babraham.ac.uk/shiny/shiny_multiome_organogenesis/

developmental biology↗

bioRxiv2022

Multi-omic rejuvenation of naturally aged tissues by a single cycle of transient reprogramming

The expression of the pluripotency factors OCT4, SOX2, KLF4 and MYC (OSKM) can convert somatic differentiated cells into pluripotent stem cells in a process known as reprogramming. Notably, cycles of brief OSKM expression do not change cell identity but can reverse markers of aging in cells and extend longevity in progeroid mice. However, little is known about the mechanisms involved. Here, we have studied changes in the DNA methylome, transcriptome and metabolome in naturally aged mice subject to a single period of transient OSKM expression. We found that this is sufficient to reverse DNA methylation changes that occur upon aging in the pancreas, liver, spleen and blood. Similarly, we observed reversion of transcriptional changes, especially regarding biological processes known to change during aging. Finally, some serum metabolites altered with aging were also restored to young levels upon transient reprogramming. These observations indicate that a single period of OSKM expression can drive epigenetic, transcriptomic and metabolomic changes towards a younger configuration in multiple tissues and in the serum.

cell biology↗

bioRxiv2021

The distinct effects of MEK and GSK3 inhibition upon the methylome and transcriptome of mouse embryonic stem cells

Mouse embryonic stem cells (mESCs) were first cultured in vitro in serum-containing medium with leukaemia inhibitory factor, in which they exhibit heterogeneous expression of both pluripotency and some early differentiation markers. Over the last decade, however, it has become commonplace to grow mESCs with inhibitors of MEK and GSK3 signalling, which together elicit a more homogeneously naive state of pluripotency. Whilst 2i/L-cultured mESCs have been shown to be globally hypomethylated, a comprehensive understanding of the distinct effects of these signalling inhibitors upon the DNA methylome is still lacking. Here we carried out whole genome bisulphite and RNA sequencing of mESCs grown with MEK or GSK3 inhibition alone, including different time points and concentrations of MEK inhibitor treatment. This demonstrated that MEK inhibition causes a dose-dependent impairment of maintenance methylation via loss of UHRF1 protein, as well as rapid impairment of de novo methylation. In contrast, GSK3 inhibition triggers impairment of de novo methylation alone, and consequent hypomethylation is enriched at enhancers with a 2i/L-specific chromatin signature and coincides with upregulation of nearby genes. Our study provides detailed insights into the epigenetic and transcriptional impacts of inhibiting MEK or GSK3 signalling in mouse pluripotent cells. HighlightsO_LIMEK inhibition causes dose-dependent impairment of maintenance methylation via loss of UHRF1 protein, as well as impairment of de novo methylation. C_LIO_LIGSK3 inhibition triggers impairment of de novo methylation alone, which results in hypomethylation of enhancers and non-CGI promoters. C_LIO_LIEnhancers that are hypomethylated following GSK3 inhibition are enriched for 2i/L-specific pluripotency factor binding, TET2 and H3K4me1. C_LIO_LIEnhancer hypomethylation coincides with increased expression of nearby and Capture Hi-C linked genes. C_LI

developmental biology↗

bioRxiv2021

Modelling human zygotic genome activation in 8C-like cells in vitro

The remodelling of the epigenome and transcriptome of the fertilised oocyte to establish totipotency in the zygote and developing embryo is one of the most critical processes in mammalian embryogenesis. Zygotic or embryonic genome activation (ZGA, EGA) in the 2-cell embryo in mouse, and the 8-cell embryo in humans, constitutes the first major wave of transcription. Failure to initiate ZGA leads to developmental defects, and contributes to the high attrition rates of human pre-implantation embryos. Due to limitations in cell numbers and experimental tractability, the mechanisms that regulate human embryonic genome activation in the totipotent embryo remain poorly understood. Here we report the discovery of human 8-cell like cells (8CLCs) specifically among naive embryonic stem cells, but not primed pluripotent cells. 8CLCs express ZGA marker genes such as ZSCAN4, LEUTX and DUXA and their transcriptome closely resembles that of the 8-cell human embryo. 8-cell like cells reactivate 8-cell stage specific transposable elements such as HERVL and MLT2A1 and are characterized by upregulation of the DNA methylation regulator DPPA3. 8CLCs show reduced SOX2 protein, and can be identified based on expression of the novel ZGA-associated protein markers TPRX1 and H3.Y in vitro. Overexpression of the transcription factor DUX4. as well as spliceosome inhibition increase ZGA-like transcription and enhance TPRX1+ 8CLCs formation. Excitingly, the in vitro identified 8CLC marker proteins TPRX1 and H3.Y are also expressed in 8-cell human embryos at the time of genome activation and may thus be relevant in vivo. The discovery of 8CLCs provides a unique opportunity to model and manipulate human ZGA-like transcriptional programs in vitro, and might provide critical functional insights into one of the earliest events in human embryogenesis in vivo. HighlightsO_LIZGA markers and transposable elements are expressed in 8CLCs among naive human stem cells C_LIO_LIThe transcription factor DUX4 and spliceosome inhibition induce ZGA-like transcription C_LIO_LI8CLC marker proteins TPRX1 and H3.Y are expressed in nuclei of 8-cell human embryos C_LIO_LI8CLCs serve as a novel in vitro model for human ZGA C_LI

molecular biology↗