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The mechanism by which DNA-damage affects self-renewal and pluripotency remains unclear. DNA damage and repair mechanisms have been largely elucidated in mutated cancer cells or simple eukaryotes, making valid interpretations on early development difficult. Here we show the impact of ionizing irradiation on the maintenance and early differentiation of mouse embryonic stem cells (ESCs). Our findings demonstrate that irradiation induces the upregulation of the p53 family genes, including p53, p63, and p73, resulting in elevated expression of the E3 ubiquitin ligases Itch and Trim32. Consequently, this impairs ESC maintenance by reducing the protein levels of key pluripotency transcription factors in both mouse ESCs and early embryos. Notably, our study reveals that irradiation-induced DNA damage leads to the recruitment of the BAF complex, causing it to dissociate from its binding sites on the target genes associated with the Yap, Wnt, and TGF-β pathways, thereby increasing signaling and promoting differentiation of ESCs into all three lineages. Importantly, pathway inhibition demonstrates that DNA damage accelerated ESC differentiation relies on Wnt and TGF-β, and is selectively dependent on p53 or p63/ p73 for mesoderm and endoderm respectively. Finally, our study reveals that p53 family proteins form complexes with effector proteins of key signaling pathways which actively contribute to ESC differentiation. In summary, this study uncovered a mechanism by which multiple differentiation signaling pathways converge on the p53 family genes to promote ESC differentiation and are impacted by exposure to ionizing radiation., Competing Interests: Declarations. Conflict of interests: There are no competing financial or nonfinancial interests regarding this work. Ethical approval: The study received approval from the Ethics Committee of Laboratory Animal Welfare in Tongji University (Ethics code: No.TJAB03221111). Consent for publication: Not applicable. Consent for publication: All the authors have consented to publication., (© 2025. The Author(s).)

Gene-expression noise can influence cell-fate choices across pathology and physiology. However, a crucial question persists: do regulatory proteins or pathways exist that control noise independently of mean expression levels? Our integrative approach, combining single-cell RNA sequencing with proteomics and regulator enrichment analysis, identifies 32 putative noise regulators. SON, a nuclear speckle-associated protein, alters transcriptional noise without changing mean expression levels. Furthermore, SON's noise control can propagate to the protein level. Long-read and total RNA sequencing shows that SON's noise control does not significantly change isoform usage or splicing efficiency. Moreover, SON depletion reduces state switching in pluripotent mouse embryonic stem cells and impacts their fate choice during differentiation. Collectively, we demonstrate a class of proteins that control noise orthogonally to mean expression levels. This work serves as a proof of concept that can identify other functional noise regulators throughout development and disease progression., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Nucleosome is the basic structural unit of the genome. During processes like DNA replication and gene transcription, the conformation of nucleosomes undergoes dynamic changes, including DNA unwrapping and rewrapping, as well as histone disassembly and assembly. However, the wrapping characteristics of nucleosomes across the entire genome, including region-specificity and their correlation with higher-order chromatin organization, remains to be studied. In this study, we investigate the wrapping length of DNA on nucleosomes across the whole genome using wrapping-seq. We discover that the chromatin of mouse ES cells forms Nucleosome Wrapping Domains (NRDs), which can also be observed in yeast and fly genomes. We find that the degree of nucleosome wrapping decreases after DNA replication and is promoted by transcription. Furthermore, we observe that nucleosome wrapping domains delineate Hi-C compartments and replication timing domains. In conclusion, we have unveiled a previously unrecognized domainization principle of the chromatin, encoded by nucleosome wrapping states., Competing Interests: Competing interests: The authors declare no competing interests., (© 2025. The Author(s).)

Background: X chromosome inactivation (XCI) is a female-specific process in which one X chromosome is silenced to balance X-linked gene expression between the sexes. XCI is initiated in early development by upregulation of the lncRNA Xist on the future inactive X (Xi). A subset of X-linked genes escape silencing and thus have higher expression in females, suggesting female-specific functions. One of these genes is the highly conserved gene Kdm6a, which encodes a histone demethylase that removes methyl groups at H3K27 to facilitate gene expression. KDM6A mutations have been implicated in congenital disorders such as Kabuki Syndrome, as well as in sex differences in development and cancer., Methods: Kdm6a was knocked out (KO) using CRISPR/Cas9 gene editing in hybrid female mouse embryonic stem (ES) cells derived either from a 129 × Mus castaneus (cast) cross or a BL6 x cast cross. In one of the lines a transcriptional stop signal inserted in Tsix results in completely skewed X silencing upon differentiation. The effects of both homozygous and heterozygous Kdm6a KO on Xist expression during the onset of XCI were measured by RT-PCR and RNA-FISH. Changes in gene expression and in H3K27me3 enrichment were investigated using allele-specific RNA-seq and Cut&Run, respectively. KDM6A binding to the Xist gene was characterized by Cut&Run., Results: We observed impaired upregulation of Xist and reduced coating of the Xi during early stages of differentiation in Kdm6a KO cells, both homozygous and heterozygous, suggesting a threshold effect of KDM6A. This was associated with aberrant overexpression of genes from the Xi after differentiation, indicating loss of X inactivation potency. Consistent with KDM6A having a direct role in Xist regulation, we found that the histone demethylase binds to the Xist promoter and KO cells show an increase in H3K27me3 at Xist, consistent with reduced expression., Conclusions: These results reveal a novel female-specific role for the X-linked histone demethylase, KDM6A in the initiation of XCI through histone demethylase-dependent activation of Xist during early differentiation. X chromosome inactivation is a female-specific mechanism that evolved to balance sex-linked gene dosage between females (XX) and males (XY) by silencing one X chromosome in females. X inactivation begins with the upregulation of the long noncoding RNA Xist on the future inactive X chromosome. While most genes become silenced on the inactive X chromosome some genes escape inactivation and thus have higher expression in females compared to males, suggesting that escape genes may have female-specific functions. One such gene encodes the histone demethylase KDM6A which function is to turn on gene expression by removing repressive histone modifications. In this study, we investigated the role of KDM6A in the regulation of Xist expression during the onset of X inactivation. We found that KDM6A binds to the Xist gene to remove repressive histone marks and facilitate its expression in early development. Indeed, depletion of KDM6A prevents upregulation of Xist due to abnormal persistence of repressive histone modifications. In turn, this results in aberrant overexpression of genes from the inactive X chromosome. Our findings point to a novel mechanism of Xist regulation during the initiation of X inactivation, which may lead to new avenues of treatment to alleviate congenital disorders such as Kabuki syndrome and sex-biased immune disorders where X-linked gene dosage is perturbed., Competing Interests: Declarations. Ethics approval and consent to participate: Not applicable. Consent for publications: All authors have given their consent for publication of the manuscript in Biology of Sex Differences. Competing interests: The authors declare no competing interests, (© 2024. The Author(s).)

Gene expression is orchestrated by transcription factors, which function within the context of a three-dimensional genome. Zinc-finger protein 143 (ZNF143/ZFP143) is a transcription factor that has been implicated in both gene activation and chromatin looping. To study the direct consequences of ZNF143/ZFP143 loss, we generated a ZNF143/ZFP143 depletion system in mouse embryonic stem cells. Our results show that ZNF143/ZFP143 degradation has no effect on chromatin looping. Systematic analysis of ZNF143/ZFP143 occupancy data revealed that a commonly used antibody cross-reacts with CTCF, leading to its incorrect association with chromatin loops. Nevertheless, ZNF143/ZFP143 specifically activates nuclear-encoded mitochondrial genes, and its loss leads to severe mitochondrial dysfunction. Using an in vitro embryo model, we find that ZNF143/ZFP143 is an essential regulator of organismal development. Our results establish ZNF143/ZFP143 as a conserved transcriptional regulator of cell proliferation and differentiation by safeguarding mitochondrial activity., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Polymer modeling has been playing an increasingly important role in complementing 3D genome experiments, both to aid their interpretation and to reveal the underlying molecular mechanisms. This chapter illustrates an application of Hi-C metainference, a Bayesian approach to explore the 3D organization of a target genomic region by integrating experimental contact frequencies into a prior model of chromatin. The method reconstructs the conformational ensemble of the target locus by combining molecular dynamics simulation and Monte Carlo sampling from the posterior probability distribution given the data. Using prior chromatin models at both 1 kb and nucleosome resolution, we apply this approach to a 30 kb locus of mouse embryonic stem cells consisting of two well-defined domains linking several gene promoters together. Retaining the advantages of both physics-based and data-driven strategies, Hi-C metainference can provide an experimentally consistent representation of the system while at the same time retaining molecular details necessary to derive physical insights., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

Identification of the occupancy of transcription factors (TFs) and nucleosomes across the genome yields insights into the regulation of gene expression patterns. While several independent techniques can be performed and then analyzed in composite to reveal this chromatin landscape, the use of micrococcal nuclease (MNase) digestion can resolve the footprints of nearly all chromatin proteins simultaneously. The protocol below describes the use of MNase to identify chromatin footprints of both TFs and nucleosomes in two vastly different cell types, Mouse embryonic stem cells (mESCs) and sperm, with differing levels of chromatin compaction., (© 2025. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

A longevity factor α-Klotho has been shown to be neuroprotective and enhance brain function. However, it has not been elucidated whether α-Klotho is involved in the embryonic brain development. To address this, we investigated whether α-Klotho regulates embryonic neural stem cell proliferation and differentiation. We found that α-Klotho is expressed in the mouse embryonic brain and neural stem cells isolated from the embryonic day 13.5 brains. When α-Klotho was knocked down in the embryonic neural stem cells, basal level of apoptosis increased while proliferation decreased. Overexpression of membrane-bound or secreted form of α-Klotho promoted proliferation of the mouse embryonic neural stem cells. Knockdown of α-Klotho suppressed the phosphorylation of Akt and ERK, suggesting that α-Klotho may promote neural stem cell survival and proliferation by activating signaling pathway involving Akt and ERK. In addition, knockdown of α-Klotho suppressed differentiation of embryonic neural stem cells into neurons or oligodendrocytes but not astrocytes. These results suggest that α-Klotho may have a role in neuronal development by promoting neural stem cell survival, proliferation and differentiation., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Organoids are multicellular structures formed in vitro from populations of individual cells allowing modeling of structural and functional aspects of organs and tissues in normal and diseased states. They offer unique opportunities to model and treat disease. Using a mouse embryonic stem cell line, we have cultured organoids that express markers of spinal cord motor neurons as well as motor neurons found within the peripheral nervous system. The morphology and select neurotransmitter content of the organoids and spinal cord explants were compared at different developmental time points. We found indications of maturation in the organoids over time, mirrored by similar trends in the spinal cord explants. Although the organoids contained the same neurotransmitters as the spinal cord explants, the developmental changes of these neurotransmitter levels were less marked in organoids. Given these differences, further work is required to optimize organoid growth conditions to better reproduce in vivo models when using organoids to study development.

Mitogen-activated extracellular signal-regulated kinase (MEK) inhibitors are widely applied to maintain pluripotency, while prolonged MEK inhibition compromises the developmental potential of mouse embryonic stem cells (ESCs). To understand the mechanism of MEK in pluripotency maintenance, we first demonstrated that MEK regulates gene expression at post-transcriptional steps. Consistently, many of the 66 MEK substrates identified by quantitative phosphoproteomics analysis are involved in RNA processing. We further confirmed that MEK1 phosphorylates S563 of DCP1A, an mRNA decapping cofactor and processing body (P body) component. DCP1A, as well as two other P body components, EDC4 and DCP2, are required for the self-renewal and differentiation of ESCs, indicating the role of P bodies in ESCs. Dephosphorylation of DCP1A S563 facilitates both self-renewal and differentiation of ESCs through promoting P body formation and RNA storage. In summary, our study identified 66 MEK substrates supporting the extracellular signal-regulated kinase (ERK)-independent function of MEK and revealed that DCP1A, phosphorylated by MEK, regulates ESC self-renewal and differentiation through modulating P body formation., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Three-dimensional (3D) tissue culture models provide in vivo-like conditions for studying cell physiology. This study aimed to examine the efficiency of pyramidal microwell geometries in microfluidic devices on spheroid formation, cell growth, viability, and differentiation in mouse embryonic stem cells (mESCs). The static culture using the hanging drop (HD) method served as a control. The microfluidic chips were fabricated to have varying pyramidal tip angles, including 66°, 90°, and 106°. From flow simulations, when the tip angle increased, streamline distortion decreased, resulting in more uniform flow and a lower velocity gradient near the spheroids. These findings demonstrate the significant influence of microwell geometry on fluid dynamics. The 90° microwells provide optimal conditions, including uniform flow and reduced shear stress, while maintaining the ability for waste removal, resulting in superior spheroid growth compared to the HD method and other microwell designs. From the experiments, by Day 3, spheroids in the 90° microwells reached approximately 400 µm in diameter which was significantly larger than those in the 66° microwells, 106° microwells, and HD cultures. Brachyury gene expression in the 90° microwells was four times higher than the HD method, indicating enhanced mesodermal differentiation essential for cardiac differentiation. Immunofluorescence staining confirmed cardiomyocyte differentiation. In conclusion, microwell geometry significantly influences 3D cell culture outcomes. The pyramidal microwells with a 90° tip angle proved most effective in promoting spheroid growth and cardiac differentiation of mESC differentiation, providing insights for optimizing microfluidic systems in tissue engineering and regenerative medicine.

Understanding the molecular signatures of individual cells within complex biological systems is crucial for deciphering cellular heterogeneity and uncovering regulatory mechanisms. Here, we present a protocol for simultaneous multiplexed detection of selected mRNAs and (phospho-)proteins in mouse embryonic stem cells using spatial single-cell profiling. We describe steps for employing single-stranded DNA (ssDNA)-labeled antibo'dies, padlock probes, and rolling circle amplification to achieve simultaneous visualization of mRNAs and (phospho-)proteins at subcellular resolution. This protocol has potential application in identifying cells in heterogeneous biological microenvironments. For complete details on the use and execution of this protocol, please refer to Hu et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The proneural transcription factor ASCL1 regulates neurogenesis and drives somatic cell reprogramming into neurons. However, not all cell types can be reprogrammed by ASCL1, raising the questions of what provides competence and how we can overcome barriers to enable directed differentiation. Here, we investigate how levels of ASCL1 and its phosphorylation modulate its activity over progressive lineage restriction of mouse embryonic stem cells. We find that inhibition of ASCL1 phosphorylation enhances reprogramming of both mesodermal and neuroectodermal cells, while pluripotent cells remain refractory to ASCL1-directed neuronal differentiation. By performing RNA-seq and ATAC-seq in neuroectoderm, we find that un(der)phosphorylated ASCL1 causes increased chromatin accessibility at sites proximal to neuronal genes, accompanied by their increased expression. Combined analysis of protein stability and proneural function of phosphomutant and phosphomimetic ASCL1 reveals that protein stability plays only a marginal role in regulating activity, while changes in amino acid charge cannot fully explain enhanced activity of the serine-proline mutant variants of ASCL1. Our work provides new insights into proneural factor activity and regulation, and suggests ways to optimize reprogramming protocols in cancer and regenerative medicine., Competing Interests: Competing interests S.G. is now employed at bit.bio. The other authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Enhancers play a critical role in regulating precise gene expression patterns essential for development and cellular identity; however, how gene-enhancer specificity is encoded within the genome is not clearly defined. To investigate how this specificity arises within topologically associated domains (TAD), we performed allele-specific genome editing of sequences surrounding the Lefty1 and Lefty2 paralogs in mouse embryonic stem cells. The Lefty genes arose from a tandem duplication event and these genes interact with each other in chromosome conformation capture assays which place these genes within the same TAD. Despite their physical proximity, we demonstrate that these genes are primarily regulated by separate enhancer elements. Through CRISPR-Cas9 mediated deletions to remove the intervening chromatin between the Lefty genes, we reveal a distance-dependent dosage effect of the Lefty2 enhancer on Lefty1 expression. These findings indicate a role for chromatin distance in insulating gene expression domains in the Lefty locus in the absence of architectural insulation., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Taylor et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

Endogenous condensates with transient constituents are notoriously difficult to study with common biological assays like mass spectrometry and other proteomics profiling. Here, we report a method for light-induced targeting of endogenous condensates (LiTEC) in living cells. LiTEC combines the identification of molecular zip codes that target the endogenous condensates with optogenetics to enable controlled and reversible partitioning of an arbitrary cargo, such as enzymes commonly used in proteomics, into the condensate in a blue light-dependent manner. We demonstrate a proof of concept by combining LiTEC with proximity-based biotinylation (BioID) and uncover putative components of transcriptional condensates in mouse embryonic stem cells. Our approach opens the road to genome-wide functional studies of endogenous condensates., Competing Interests: Declaration of interests I.I.C. is a member of the editorial advisory board at Cell. I.I.C. and C.L. are co-inventors on a US and EU patent application (US patent application no. 63387982 and International Publication number WO 2024/133013 A1, titled: Means and methods to target endogenous condensates)., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Cells need to overcome both intrinsic and extrinsic threats. Although pluripotency is associated with damage responses, how stem cells respond to DNA damage remains controversial. Here, we elucidate that DNA damage activates Chk2, leading to the phosphorylation of serine 164 on C-terminal binding protein 2 (Ctbp2). The phosphorylation of Ctbp2 induces the disruption of Ctbp2 tetramer, weakening interactions with zinc finger proteins, leading to the dissociation of phosphorylated Ctbp2 from chromatin. This transition to a monomeric state results in the separation of histone deacetylase 1 from Ctbp2, consequently slowing the rate of H3K27 deacetylation. In contrast to the nucleosome remodeling and deacetylase complex, phosphorylated Ctbp2 increased binding affinity to polycomb repressive complex (PRC)2, interacting through the N-terminal domain of Suz12. Through this domain, Ctbp2 competes with Jarid2, inhibiting the function of PRC2. Thus, the phosphorylation of Ctbp2 under stress conditions represents a precise mechanism aimed at preserving stemness traits by inhibiting permanent transcriptional shutdown., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Many RNAs associate with chromatin, either directly or indirectly. Several technologies for mapping regions where RNAs interact across the genome have been developed to investigate the function of these RNAs. Obtaining information on the proteins involved in these RNA-chromatin interactions is critical for further analysis. Here, we developed RADIP [RNA and DNA interacting complexes ligated and sequenced (RADICL-seq) with immunoprecipitation], a novel technology that combines RADICL-seq technology with chromatin immunoprecipitation to characterize RNA-chromatin interactions mediated by individual proteins. Building upon the foundational principles of RADICL-seq, RADIP extends its advantages by increasing genomic coverage and unique mapping rate efficiency compared to existing methods. To demonstrate its effectiveness, we applied an anti-H3K27me3 antibody to the RADIP technology and generated libraries from mouse embryonic stem cells (mESCs). We identified a multitude of RNAs, including RNAs from protein-coding genes and non-coding RNAs, that are associated with chromatin via H3K27me3 and that likely facilitate the spread of Polycomb repressive complexes over broad regions of the mammalian genome, thereby affecting gene expression, chromatin structures and pluripotency of mESCs. Our study demonstrates the applicability of RADIP to investigations of the functions of chromatin-associated RNAs., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The orchestration of our genes heavily relies on coordinated communication between enhancers and promoters, yet the mechanisms behind this dynamic interplay during active transcription remain unclear. Here, we investigated enhancer-promoter (E-P) interactions in relation to transcriptional bursting in mouse embryonic stem cells using sequential DNA/RNA/immunofluorescence-fluorescence in situ hybridization analyses. Our data reveal that the active state of specific genes is characterized by specific proximities between different genomic regions and the accumulation of transcriptional regulatory factors. Mathematical simulations suggest that an increase in local viscosity could potentially contribute to stabilizing the duration of these E-P proximities. Our study provides insights into the association among E-P proximity, protein accumulation, and transcriptional dynamics, paving the way for a more nuanced understanding of gene-specific regulatory mechanisms.

Transcriptional induction coincides with the formation of various chromatin topologies. Strong evidence supports that gene activation is accompanied by a general increase in promoter-enhancer interactions. However, it remains unclear how these topological changes are coordinated across time and space during transcriptional activation. Here, we combine chromatin conformation capture with transcription and chromatin profiling during an embryonic stem cell (ESC) differentiation time course to determine how 3D genome restructuring is related to transcriptional transitions. This approach allows us to identify distinct topological alterations that are associated with the magnitude of transcriptional induction. We detect transiently formed interactions and demonstrate by genetic deletions that associated distal regulatory elements (DREs), as well as appropriate formation and disruption of these interactions, can contribute to the transcriptional induction of linked genes. Together, our study links topological dynamics to the magnitude of transcriptional induction and detects an uncharacterized type of transcriptionally important DREs., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Intricate microenvironment signals orchestrate to affect cell behavior and fate during tissue morphogenesis. However, the underlying mechanisms on how specific local niche signals influence cell behavior and fate are not fully understood, owing to the lack of in vitro platform able to precisely, quantitatively, spatially, and independently manipulate individual niche signals. Here, microarrays of protein-based 3D single cell micro-niche (3D-SCμN), with precisely engineered biophysical and biochemical niche signals, are micro-printed by a multiphoton microfabrication and micropatterning technology. Mouse embryonic stem cell (mESC) is used as the model cell to study how local niche signals affect stem cell behavior and fate. By precisely engineering the internal microstructures of the 3D SCμNs, we demonstrate that the cell division direction can be controlled by the biophysical niche signals, in a cell shape-independent manner. After confining the cell division direction to a dominating axis, single mESCs are exposed to asymmetric biochemical niche signals, specifically, cell-cell adhesion molecule on one side and extracellular matrix on the other side. We demonstrate that, symmetry-breaking (asymmetric) niche signals successfully trigger cell polarity formation and bias the orientation of asymmetric cell division, the mitosis process resulting in two daughter cells with differential fates, in mESCs., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: N.H. and B.P.C. are inventors of a patent associated with this work., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

The widespread application of nanoparticles (NPs) in various fields has raised health concerns, especially in reproductive health. Our research has shown zinc oxide nanoparticles (ZnONPs) exhibit the most significant toxicity to pre-implantation embryos in mice compared to other common NPs. In patients undergoing assisted reproduction technology (ART), a significant negative correlation was observed between Zn concentration and clinical outcomes. Therefore, this study explores the impact of ZnONPs exposure on pre-implantation embryonic development and its underlying mechanisms. We revealed that both in vivo and in vitro exposure to ZnONPs impairs pre-implantation embryonic development. Moreover, ZnONPs were found to reduce the pluripotency of mouse embryonic stem cells (mESCs), as evidenced by teratoma and diploid chimera assays. Employing multi-omics approaches, including RNA-Seq, CUT&Tag, and ATAC-seq, the embryotoxicity mechanisms of ZnONPs were elucidated. The findings indicate that ZnONPs elevate H3K9me3 levels, leading to increased heterochromatin and consequent inhibition of gene expression related to development and pluripotency. Notably, Chaetocin, a H3K9me3 inhibitor, sucessfully reversed the embryotoxicity effects induced by ZnONPs. Additionally, the direct interaction between ZnONPs and H3K9me3 was verified through pull-down and immunoprecipitation assays. Collectively, these findings offer new insights into the epigenetic mechanisms of ZnONPs toxicity, enhancing our understanding of their impact on human reproductive health., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Eukaryotic mRNAs are transcribed, processed, translated, and degraded in different subcellular compartments. Here, we measured mRNA flow rates between subcellular compartments in mouse embryonic stem cells. By combining metabolic RNA labeling, biochemical fractionation, mRNA sequencing, and mathematical modeling, we determined the half-lives of nuclear pre-, nuclear mature, cytosolic, and membrane-associated mRNAs from over 9000 genes. In addition, we estimated transcript elongation rates. Many matured mRNAs have long nuclear half-lives, indicating nuclear retention as the rate-limiting step in the flow of mRNAs. In contrast, mRNA transcripts coding for transcription factors show fast kinetic rates, and in particular short nuclear half-lives. Differentially localized mRNAs have distinct rate constant combinations, implying modular regulation. Membrane stability is high for membrane-localized mRNA and cytosolic stability is high for cytosol-localized mRNA. mRNAs encoding target signals for membranes have low cytosolic and high membrane half-lives with minor differences between signals. Transcripts of nuclear-encoded mitochondrial proteins have long nuclear retention and cytoplasmic kinetics that do not reflect co-translational targeting. Our data and analyses provide a useful resource to study spatiotemporal gene expression regulation., Competing Interests: Disclosure and competing interests statement. The authors declare no competing interests., (© 2024. The Author(s).)

The basic helix-loop-helix transcription factor HAND2 has multiple roles during vertebrate organogenesis, including cardiogenesis. However, much remains to be uncovered about its mechanism of action. Here, we show the generation of several hand2 mutant alleles in zebrafish and demonstrate that dimerization-deficient mutants display the null phenotype but DNA-binding-deficient mutants do not. Rescue experiments with Hand2 variants using a newly identified hand2 enhancer confirmed these observations. To identify Hand2 effectors critical for cardiogenesis, we analyzed the transcriptomes of hand2 loss- and gain-of-function embryonic cardiomyocytes and tested the function of eight candidate genes in vivo; pdgfra was most effective in rescuing myocardial migration in hand2 mutants. Accordingly, we identified a putative Hand2-binding region in the zebrafish pdgfra locus that is important for its expression. In addition, Hand2 loss- and gain-of-function experiments in mouse embryonic stem cell-derived cardiac cells decreased and increased Pdgfra expression, respectively. Altogether, these results further our mechanistic understanding of HAND2 function during early cardiogenesis., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

H3K9me3 heterochromatin, established by lysine methyltransferases (KMTs) and compacted by heterochromatin protein 1 (HP1) isoforms, represses alternative lineage genes and DNA repeats. Our understanding of H3K9me3 heterochromatin stability is presently limited to individual domains and DNA repeats. Here we engineered Suv39h2-knockout mouse embryonic stem cells to degrade remaining two H3K9me3 KMTs within 1 hour and found that both passive dilution and active removal contribute to H3K9me3 decay within 12-24 hours. We discovered four different H3K9me3 decay rates across the genome and chromatin features and transcription factor binding patterns that predict the stability classes. A 'binary switch' governs heterochromatin compaction, with HP1 rapidly dissociating from heterochromatin upon KMT depletion and a particular threshold level of HP1 limiting pioneer factor binding, chromatin opening and exit from pluripotency within 12 h. Unexpectedly, receding H3K9me3 domains unearth residual HP1β peaks enriched with heterochromatin-inducing proteins. Our findings reveal distinct H3K9me3 heterochromatin maintenance dynamics governing gene networks and repeats that together safeguard pluripotency., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

LIF maintains self-renewal growth in mouse embryonic stem cells (mESC) by activating STAT3, which translocates into nucleus for pluripotent gene induction. However, the ERK signaling pathway activated by LIF at large counteract with pluripotent gene induction during self-renewal growth. Here, it is reported that in mESC STAT3 undergoes multivesicular endosomes (MVEs) translocation and subsequent secretion, LIF-activated STAT3 is acetylated on K177/180 and phosphorylated on Y293 residues within the N-terminal coiled-coil domain, which is responsible for the interaction between STAT3 and Secl5b, an exocyst complex component 6B (EXOC6B). STAT3 translocation into MVEs resulted in the downregulation of T202/Y204-ERK1/2 phosphorylation and up-regulation of S9-GSK3β phosphorylation for maintaining mESC self-renewal growth. STAT3 with K177R/K180R or Y293F substitution fails to execute MVEs translocation and Secl5b-dependent secretion. Mice expressing K177RK180R substitution (STAT3 mut/mut ) are partially embryonic lethal. In STAT3 mut/mut embryos, gene expressions related to hematological system function changed significantly and those living ones carry a series of abnormalities in the hematopoietic system. Furthermore, mice with Secl5b knockout exhibit embryonic lethality. Thus, Secl5b mediated STAT3 MVEs translocation regulates the balance of ERK and GSK3β signaling pathways and maintain mESC self-renewal growth, which is involved in regulating the stability of hematopoietic system., (© 2024 The Author(s). Advanced Science published by Wiley‐VCH GmbH.)

Mouse embryonic stem cells (mESCs) exist in two distinct pluripotent states: the naive and the primed. Mainly by inducing differentiation of mESCs in vitro, conducting RNA sequencing analyses, and specifying expression of the regulatory genes, we explored the regulatory mechanisms underlying the transition between the naive and primed states. We found that, under the defined differentiation-inducing conditions, the naive state of mESCs shifted to the primed state within 2 days of differentiation induction, during which the cell cycle- and differentiation-related proteins changes significantly. Specifically, we uncovered that the expression of STAG2, a subunit of the Cohesin complex, was upregulated. We further revealed that knockout of STAG2 resulted in upregulation of the naive gene sets and downregulation of the primed gene sets, indicating importance of STAG2 in regulating the naive-primed transition. More importantly, STAG2 knockout led to a reduction in number of the bivalent genes, a decrease in Lin28a transcription, and a reduced cytoplasmic localization of Lin28a. Overexpressing Lin28a or a Lin28a variant lacking the nucleolar localization signal (Lin28aΔNoLS) in STAG2 knockout cells rescued the downregulation of the primed marker genes Dnmt3a/3b. Collectively, we conclude that STAG2 facilitates the naive-primed transition of mESCs by activating Lin28a transcription and that this work may offer a new insight into the regulation of pluripotency in mESCs., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The mammalian p57 protein is a member of the CIP/KIP family of cyclin-dependent kinase inhibitors and plays an essential role in the development of multiple tissues during embryogenesis as well as in the maintenance of tissue stem cells in adults. Although several transcription factors have been implicated in regulating the p57 gene, cis-elements such as enhancers that regulate its expression have remained ill-defined. Here we identify a candidate enhancer for the mouse p57 gene (Cdkn1c) within an intron of the Kcnq1 locus by 4C-seq analysis in mouse embryonic stem cells (mESCs). Deletion of this putative enhancer region with the CRISPR-Cas9 system or its suppression by CRISPR interference resulted in a marked attenuation of Cdkn1c expression in differentiating mESCs. Our results thus suggest that this region may serve as an enhancer for the p57 gene during early mouse embryogenesis., (© 2024 The Author(s). Genes to Cells published by Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Repair of double-stranded breaks generated by CRISPR/Cas9 is highly dependent on the flanking DNA sequence. To learn about interactions between DNA repair and target sequence, we measure frequencies of over 236,000 distinct Cas9-generated mutational outcomes at over 2800 synthetic target sequences in 18 DNA repair deficient mouse embryonic stem cells lines. We classify the outcomes in an unbiased way, finding a specialised role for Prkdc (DNA-PKcs protein) and Polm in creating 1 bp insertions matching the nucleotide on the protospacer-adjacent motif side of the break, a variable involvement of Nbn and Polq in the creation of different deletion outcomes, and uni-directional deletions dependent on both end-protection and end-resection. Using our dataset, we build predictive models of the mutagenic outcomes of Cas9 scission that outperform the current standards. This work improves our understanding of DNA repair gene function, and provides avenues for more precise modulation of Cas9-generated mutations., Competing Interests: Competing interests: B.M. is an employee of Merck Healthcare, Darmstadt, Germany. O.S. is an employee of BioMed X Institute (GmbH), Heidelberg, Germany, which receives research grants from Merck KGaA. L.P. Receives remuneration and stock options from ExpressionEdits. The remaining authors declare no competing interests., (© 2024. The Author(s).)

Ribosome biogenesis plays a pivotal role in maintaining stem cell homeostasis, yet the precise regulatory mechanisms governing this process in mouse embryonic stem cells (mESCs) remain largely unknown. In this investigation, we ascertain that DEAD-box RNA helicase 10 (DDX10) is indispensable for upholding cellular homeostasis and the viability of mESCs. Positioned predominantly at the nucleolar dense fibrillar component (DFC) and granular component (GC), DDX10 predominantly binds to 45S ribosomal RNA (rRNA) and orchestrates ribosome biogenesis. Degradation of DDX10 prevents the release of U3 snoRNA from pre-rRNA, leading to perturbed pre-rRNA processing and compromised maturation of the 18S rRNA, thereby disrupting the biogenesis of the small ribosomal subunit. Moreover, DDX10 participates in the process of liquid-liquid phase separation (LLPS), which is necessary for efficient ribosome biogenesis. Notably, the NUP98-DDX10 fusion associated with acute myelocytic leukemia (AML) alters the cellular localization of DDX10 and results in loss of ability to regulate pre-rRNA processing. Collectively, this study reveals the critical role of DDX10 as a pivotal regulator of ribosome biogenesis in mESCs., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Cardiopharyngeal mesoderm contributes to the formation of the heart and head muscles. However, the mechanisms governing cardiopharyngeal mesoderm specification remain unclear. Here, we reproduce cardiopharyngeal mesoderm specification towards cardiac and skeletal muscle lineages with gastruloids from mouse embryonic stem cells. By conducting a comprehensive temporal analysis of cardiopharyngeal mesoderm development and differentiation in gastruloids compared to mouse embryos, we present the evidence for skeletal myogenesis in gastruloids. We identify different subpopulations of cardiomyocytes and skeletal muscles, the latter of which most likely correspond to different states of myogenesis with "head-like" and "trunk-like" skeletal myoblasts. In this work, we unveil the potential of gastruloids to undergo specification into both cardiac and skeletal muscle lineages, allowing the investigation of the mechanisms of cardiopharyngeal mesoderm differentiation in development and how this could be affected in congenital diseases., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

The formation of transcription regulatory complexes by the association of Smad4 with Smad2 and Smad3 (Smad2/3) is crucial in the canonical TGFβ pathway. Although the central requirement of Smad4 as a common mediator is emphasized in regulating TGFβ signaling, it is not obligatory for all responses. The role of Smad2/3 independently of Smad4 remains understudied. Here, we introduce a stepwise paradigm in which Smad2/3 regulate the lineage priming and differentiation of mouse embryonic stem cells (mESCs) by collaboration with different effectors. During the naïve-to-primed transition, Smad2/3 upregulate DNA methyltransferase 3b (Dnmt3b), which establishes the proper DNA methylation patterns and, in turn, enables Smad2/3 binding to the hypomethylated centers of promoters and enhancers of epiblast marker genes. Consequently, in the absence of Smad2/3, Smad4 alone cannot initiate epiblast-specific gene transcription. When primed epiblast cells begin to differentiate, Dnmt3b becomes less actively engaged in global genome methylation, and Smad4 takes over the baton in this relay race, forming a complex with Smad2/3 to support mesendoderm induction. Thus, mESCs lacking Smad4 can undergo the priming process but struggle with the downstream differentiation. This work sheds light on the intricate mechanisms underlying TGFβ signaling and its role in cellular processes., Competing Interests: Competing interests: The authors declare no competing interests., (© 2024. The Author(s).)

Lysine-specific histone demethylase 1 (LSD1) is a histone demethylase that plays a critical role in epigenetic regulation by removing the methyl group from mono- and di-methylated lysine 4 on histone H3 (H3K4me1/2), acting as a repressor of gene expression. Recently, catalytically independent functions of LSD1, serving as a scaffold for assembling chromatin-regulator and transcription factor complexes, have been identified. Herein, we show for the first time that LSD1 interacts with chromodomain-helicase-DNA-binding protein 7 (CHD7) in mouse embryonic stem cells (ESCs). To further investigate the CHD7-LSD1 crosstalk, we engineered Chd7 and Chd7/Lsd1 knockout (KO) mouse ESCs. We show that CHD7 is dispensable for ESC self-renewal and survival, while Chd7 KO ESCs can differentiate towards embryoid bodies (EBs) with defective expression of ectodermal markers. Intriguingly, Chd7/Lsd1 double KO mouse ESCs exhibit proliferation defects similar to Lsd1 KO ESCs and have lost the capacity to differentiate properly. Furthermore, the increased co-occupancy of H3K4me1 and CHD7 on chromatin following Lsd1 deletion suggests that LSD1 is required for facilitating the proper binding of CHD7 to chromatin and regulating differentiation. Collectively, our results suggest that LSD1 and CHD7 work in concert to modulate gene expression and influence proper cell fate determination., Competing Interests: Declarations Competing interests The authors declare no competing interests., (© 2024. The Author(s).)

Minimal in vitro systems composed of embryonic stem cells (ESCs) have been shown to recapitulate the establishment of the anteroposterior (AP) axis. In contrast to the native embryo, ESC aggregates - such as gastruloids - can break symmetry, which is demarcated by polarization of the mesodermal marker T, autonomously without any localized external cues. However, associated earliest patterning events, such as the spatial restriction of cell fates and concomitant transcriptional changes, remain poorly understood. Here, we dissect the dynamics of AP axis establishment in mouse gastruloids, particularly before external Wnt stimulation. Through single-cell RNA sequencing, we identify key cell state transitions and the molecular signatures of T+ and T- populations underpinning AP polarization. We also show that this process is robust to modifications of aggregate size. Finally, transcriptomic comparison with the mouse embryo indicates that gastruloids develop similar mesendodermal cell types, despite initial differences in their primed pluripotent populations, which adopt a more mesenchymal state in lieu of an epiblast-like transcriptome. Hence, our findings suggest the possibility of alternate ESC states in vivo and in vitro that can converge onto similar cell fates., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Background: Cellular metabolism regulates the pluripotency of embryonic stem cells (ESCs). Yet, how metabolism regulates the transition among different pluripotent states remains elusive. It has been shown that protein lactylation, which uses lactate, a metabolic product of glycolysis, as a substrate, plays a critical role in various biological events. Here we focused on that glycolysis regulates the conversion between ESCs and 2-cell-like cells (2CLCs) through protein lactylation., Methods: RNA-seq revealed the activation of 2-cell (2C) genes by suppression of Ldh. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with lactylated peptide enrichment and quantitative mass spectrometric analysis was carried out to investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition. And we focused on Hdac1. Lactylation of Hdac1 required for silencing 2C genes was proved by quantitative reverse-transcription PCR (qRT-PCR), immunofluorescence (IF), Western blot and chimeric embryos. Chromatin immunoprecipitation coupled with sequencing (ChIP-seq) and in vitro deacetylation assay confirmed lactylation of Hdac1 promoting its binding at 2C genes and enhancing its deacetylase activity, thereby facilitating the removal of H3K27ac and the silencing of 2C genes., Results: We found that inhibition or depletion of Ldha, the enzyme converting pyruvate to lactate, leads to the activation of 2C genes, as well as reduced global lactylation in ESCs. To investigate the mechanism how protein lactylation regulates the pluripotent-to-2C transition, quantitative lactylome analysis was performed, and 1716 lactylated proteins were identified. We then focused on Hdac1, a histone deacetylase involved in the silencing of 2C genes. Lactylation of Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes., Conclusions: In summary, our study reveals a mechanistic link between cellular metabolism and pluripotency regulation through protein lactylation. Our research is the first time to reveal that quantitative lactylome analysis in mouse ESCs. We found that lactylated Hdac1 promotes its binding at 2C genes and enhances its deacetylase activity, thus facilitating the removal of H3K27ac and the silencing of 2C genes., Competing Interests: Declarations Ethics approval and consent to participate All procedures and protocols have been reviewed and approved by the Nankai University Animal Care and Use Committee. Ethics has been reviewed under the title “Application Form for Ethical Review of the Use of Experimental Animals” on December 20,2021 (Approval Number: 2021-SYDWLL-000469). Consent for publication All authors approved the publication for this manuscript. Competing interests The authors declare no competing interests. Declaration of Generative AI and AI-assisted Technologies in the Writing Process The authors declare that they have not use AI-generated work in this manuscript., (© 2024. The Author(s).)

Copper oxide nanoparticles (CuONPs) have been widely applied, posing potential risks to human health. Although the toxicity of CuONPs on the liver and spleen has been reported, their effects on reproductive health remain unexplored. In this study, we investigate the effects of CuONPs on embryonic development and their potential mechanisms. Our results demonstrate that CuONPs exposure impairs mouse preimplantation embryonic development, particularly affecting the morula-to-blastocyst transition. Additionally, CuONPs were found to reduce the pluripotency of the inner cell mass (ICM) and mouse embryonic stem cells (mESCs). Mechanistically, CuONPs block autophagic flux and impair mitophagy, leading to the accumulation of damaged mitochondria. This mitochondrial dysfunction leads to reduced tricarboxylic acid (TCA) cycle activity and decreased α-ketoglutarate (α-KG) production. Insufficient α-KG induces the failure of DNA demethylation, reducing corresponding chromatin accessibility and consequently inhibiting ICM-specific genes expressions. Similar reduced development and inhibitions of pluripotency gene expression were observed in CuONPs-treated human blastocysts. Moreover, in women undergoing assisted reproductive technology (ART), a negative correlation was found between urinary Cu ion concentrations and clinical outcomes. Collectively, our study elucidates the mitophagy-mediated metabolic mechanisms of CuONPs embryotoxicity, improving our understanding of the potential reproductive toxicity associated with it.

Telomeres protect chromosome ends from DNA damage responses, and their dysfunction triggers genomic alterations like chromosome fusion and rearrangement, which can lead to cellular death. Certain cells, including specific cancer cells, adopt alternative lengthening of telomere (ALT) to counteract dysfunctional telomeres and proliferate indefinitely. While telomere instability and ALT activity are likely major sources of genomic alteration, the patterns and consequences of such changes at the nucleotide level in ALT cells remain unexplored. Here we generated haplotype-resolved genome assemblies for type I ALT mouse embryonic stem cells, facilitated by highly accurate or ultra-long reads and Hi-C reads. High-quality genome revealed ALT-specific complex chromosome end structures and various genomic alterations including over 1000 structural variants (SVs). The unique sequence (mTALT) used as a template for type I ALT telomeres showed traces of being recruited into the genome, with mTALT being replicated with remarkably high accuracy. Subtelomeric regions exhibited distinct characteristics: resistance to the accumulation of SVs and small variants. We genotyped SVs at allele resolution, identifying genes (Rgs6, Dpf3 and Tacc2) crucial for maintaining ALT telomere stability. Our genome assembly-based approach elucidated the unique characteristics of ALT genome, offering insights into the genome evolution of cells surviving telomere-derived crisis., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

In mammalian cells, primary miRNAs are cleaved at their hairpin structures by the Microprocessor complex, whose core is composed of DROSHA and DGCR8. Here, we show that 5' flanking regions, resulting from Microprocessor cleavage, are targeted by the RNA exosome in mouse embryonic stem cells (mESCs). This is facilitated by a physical link between DGCR8 and the nuclear exosome targeting (NEXT) component ZCCHC8. Surprisingly, however, both biochemical and mutagenesis studies demonstrate that a variant NEXT complex, containing the RNA helicase MTR4 but devoid of the RNA-binding protein RBM7, is the active entity. This Microprocessor-NEXT variant also targets stem-loop-containing RNAs expressed from other genomic regions, such as enhancers. By contrast, Microprocessor does not contribute to the turnover of less structured NEXT substrates. Our results therefore demonstrate that MTR4-ZCCHC8 can link to either RBM7 or DGCR8/DROSHA to target different RNA substrates depending on their structural context., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Teeth are comprised of epithelial and mesenchymal cells, and regenerative teeth rely on the regeneration of both cell types. Transcription factors play a pivotal role in cell fate determination. In this study, we establish fluorescence models based on transcription factors to monitor and analyze dental epithelial cells. Using Pitx2-P2A-copGFP mice, we observe that Pitx2+ epithelial cells, when combined with E14.5 dental mesenchymal cells, are sufficient for the reconstitution of teeth. Induced-Pitx2+ cells, directly isolated from the embryoid body that employs the Pitx2-GFP embryonic stem cell line, exhibit the capacity to differentiate into ameloblasts and develop into teeth when combined with dental mesenchymal cells. The regenerated teeth exhibit a complete structure, including dental pulp, dentin, enamel, and periodontal ligaments. Subsequent exploration via RNA-seq reveals that induced-Pitx2+ cells exhibit enrichment in genes associated with FGF receptors and WNT ligands compared with induced-Pitx2- cells. Our results indicate that both primary Pitx2+ and induced Pitx2+ cells possess the capability to differentiate into enamel-secreting ameloblasts and grow into teeth when combined with dental mesenchymal cells., (© The Author(s) 2024. Published by Oxford University Press. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

RNA-binding proteins (RBPs) regulate totipotency, pluripotency maintenance, and induction. The intricacies of how they modulate these processes through their interaction with RNAs remain to be elucidated. Here we employed Targets of RBPs Identified By Editing (TRIBE) with single-cell resolution (scTRIBE) to profile the mRNA targets of the key pluripotency regulator LIN28A in mouse embryonic stem cells (ESCs), 2-cell embryo-like cells (2CLCs), and somatic cell reprogramming. LIN28A is known to act by controlling the maturation of the let-7 microRNA, but, in addition, it binds to multiple mRNAs and influences their stability and translation efficiency. However, the mRNA targets of LIN28A in 2CLCs and reprogramming are unclear. Through quantitative single-cell analysis of the scTRIBE dataset, we observed a marked increase in the binding of LIN28A to mRNAs of ribosome biogenesis factors and a selected group of totipotency factors in 2CLCs within ESC cultures. Our results suggest that LIN28A extends the half-life of at least some of these mRNAs, providing new insights into its role in the totipotent state. We also uncovered the distinct trajectory-specific LIN28A-mRNA networks in reprogramming, helping explain how LIN28A facilitates the mesenchymal-to-epithelial transition and pluripotency acquisition. Our study not only clarifies the multifunctional role of LIN28A in these processes but also highlights the importance of decoding RNA-protein interactions at the single-cell level., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

The canonical BRG/BRM-associated factor (cBAF) complex is essential for chromatin opening at enhancers in mammalian cells. However, the nature of the open chromatin remains unclear. Here, we show that, in addition to producing histone-free DNA, cBAF generates stable hemisome-like subnucleosomal particles containing the four core histones associated with 50-80 bp of DNA. Our genome-wide analysis indicates that cBAF makes these particles by targeting and splitting fragile nucleosomes. In mouse embryonic stem cells, these subnucleosomes become an in vivo binding substrate for the master transcription factor OCT4 independently of the presence of OCT4 DNA motifs. At enhancers, the OCT4-subnucleosome interaction increases OCT4 occupancy and amplifies the genomic interval bound by OCT4 by up to one order of magnitude compared to the region occupied on histone-free DNA. We propose that cBAF-dependent subnucleosomes orchestrate a molecular mechanism that projects OCT4 function in chromatin opening beyond its DNA motifs., Competing Interests: Competing interests The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Canonical histone H3 and histone variant H3.3 are posttranslationally modified with the genomic distribution of these marks denoting different features and these modifications may influence transcription. While the majority of posttranslational modifications occur on histone tails, there are defined modifications within the globular domain, such as acetylation of H3K122/H3.3K122. To understand the function of the amino acid H3.3K122 in transcriptional regulation, we attempted to generate H3.3K122A mouse embryonic stem (mES) cells but were unsuccessful. Through multi-omic profiling of mutant cell lines harboring two or three of four H3.3 targeted alleles, we have uncovered that H3.3K122A is neomorphic and results in lethality. This is surprising as prior studies demonstrate H3.3-null mES cells are viable and pluripotent but exhibit a reduced differentiation capacity. Together, these studies have uncovered a novel dependence of a globular domain residue within H3.3 for viability and broadened our understanding of how histone variants contribute to transcription regulation and pluripotency in mES cells., (© 2024. The Author(s).)

Benzophenones (BPs) are widely used as photoinitiators (PIs) or printing inks in food packaging, which may migrate into foods. However, the toxicity information of some BP analogues, such as 4,4'-bis(diethylamino)-benzophenone (DEAB), 4-phenylbenzophenone (4-PBP), 4 (hydroxymethyl)benzophenone (4-HMBP), those are used as PIs is lacking. Developmental toxicity is a health concern associated with PIs exposure. Recently, alternative non-in vivo methods have been proposed to evaluate the concerned chemicals or better understand the modes of action of certain toxicological endpoints. In this study, using in silico methods, we predicted that BP, DEAB, 4-PBP and 4-HMBP might exhibit developmental toxicity. However, we found that only DEAB is strong embryotoxic and disturbs the early differentiation of mouse embryonic stem cells into three germ layers and cardiomyocytes. DEAB treatment also prevented cardiomyocyte differentiation in human induced pluripotent stem cells (hiPSCs) on day 10. However, BP, 4-PBP and 4-HMBP had no similar effects on cardiomyocyte differentiation on day 10. Transcriptomic analysis revealed that treatment with DEAB significantly decreased the mRNA levels of differentiation-related transcription factors SOX17 and FOXA1, in hiPSCs on day 4. Furthermore, DEAB treatment caused tail malformations and yolk sac edema in zebrafish embryos. To conclude, DEAB may be embryotoxic because it disturbs the early differentiation of stem cells. Further studies are warranted to better understand the health effects of DEAB exposure., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

Aims: Mammalian genomes encode 12 proteins that contain a CXXC zinc finger domain. Most members of this family are large multi-domain proteins that function in the control of DNA methylation and histone methylation patterns. CXXC5 is a smaller member of the family, along with its closest homologue CXXC4. These two proteins lack known catalytic domains. Here, we have characterized CXXC5 in mouse embryonic stem (ES) cells., Materials & Methods: We used gene knockouts, RNA sequencing, and DNA methylation analysis by whole-genome bisulfite sequencing., Results & Conclusions: We show that CXXC5 is a nuclear protein that interacts with 5-methylcytosine oxidases (TET proteins). Removal of CXXC5 from ES cells leads to very few changes in gene expression. CXXC5 extensively colocalizes with TET1 and TET2 at CpG islands. CXXC5 inactivation leads to a substantial reduction of DNA methylation levels that affects all genomic compartments including genic and intergenic regions and CpG island shores. We propose a model in which CXXC5 serves as an anchor for TET proteins at CpG islands. In the absence of CXXC5, the 5-methylcytosine oxidases become dislodged from CpG islands and are enabled to induce genome-scale DNA demethylation. Thus, CXXC5 serves as a stabilizer of DNA methylation patterns.

Erk signaling is indispensable for the self-renewal and differentiation of mouse embryonic stem cells (ESCs), as well as telomere homeostasis. But how Erk regulates these biological processes remains unclear. We identified 132 Erk2 interacting proteins by co-immunoprecipitation and mass spectrometric analysis, and focused on Ddx39 as a potential Erk2 substrate. We demonstrated that Erk2 phosphorylates Ddx39 on Y132 and Y138. Ddx39 knockout (KO) ESCs are defective in differentiation, due to reduced H3K27ac level upon differentiation. Phosphorylation of Ddx39 promotes the recruitment of Hat1 to acetylate H3K27 and activate differentiation genes. In addition, Ddx39 KO leads to telomere elongation in ESCs. Ddx39 is recruited to telomeres by the telomere-binding protein Trf1, consequently disrupting the DNA loop formed by Trf1 and suppressing the alternative lengthening of telomeres (ALT). Phosphorylation of Ddx39 weakens its interaction with Trf1, releasing it from telomeres. Thus, ALT activity is enhanced, and telomeres are elongated. Altogether, our studies reveal an essential role of Ddx39 in the differentiation and telomere homeostasis of ESCs., (© 2024. The Author(s), under exclusive licence to ADMC Associazione Differenziamento e Morte Cellulare.)

Genes are often regulated by multiple enhancers. It is poorly understood how the individual enhancer activities are combined to control promoter activity. Anecdotal evidence has shown that enhancers can combine sub-additively, additively, synergistically, or redundantly. However, it is not clear which of these modes are more frequent in mammalian genomes. Here, we systematically tested how pairs of enhancers activate promoters using a three-way combinatorial reporter assay in mouse embryonic stem cells. By assaying about 69,000 enhancer-enhancer-promoter combinations we found that enhancer pairs generally combine near-additively. This behaviour was conserved across seven developmental promoters tested. Surprisingly, these promoters scale the enhancer signals in a non-linear manner that depends on promoter strength. A housekeeping promoter showed an overall different response to enhancer pairs, and a smaller dynamic range. Thus, our data indicate that enhancers mostly act additively, but promoters transform their collective effect non-linearly., Competing Interests: MM, Bv No competing interests declared, FC co-founder of enGene Statistics GmbH, (© 2023, Martinez-Ara et al.)

The pluripotent mouse embryonic stem cell (mESCs) can transit into the totipotent-like state, and the transcription factor DUX is one of the master regulators of this transition. Intriguingly, this transition in mESCs is accompanied by massive cell death, which significantly impedes the establishment and maintenance of totipotent cells in vitro, yet the underlying mechanisms of this cell death remain largely elusive. In this study, we found that the totipotency transition in mESCs triggered cell death through the upregulation of DUX. Specifically, R-loops are accumulated upon DUX induction, which subsequently lead to DNA replication stress (RS) in mESCs. This RS further activates p53 and PMAIP1, ultimately leading to Caspase-9/7-dependent intrinsic apoptosis. Notably, inhibiting this intrinsic apoptosis not only mitigates cell death but also enhances the efficiency of the totipotency transition in mESCs. Our findings thus elucidate one of the mechanisms underlying cell apoptosis during the totipotency transition in mESCs and provide a strategy for optimizing the establishment and maintenance of totipotent cells in vitro., (© 2024. The Author(s).)

The recognition of core promoter sequences by TFIID is the first step in RNA polymerase II (Pol II) transcription initiation. Metazoan holo-TFIID is a trilobular complex, composed of the TATA binding protein (TBP) and 13 TBP-associated factors (TAFs). Why and how TAFs are necessary for the formation of TFIID domains and how they contribute to transcription initiation remain unclear. Inducible TAF7 or TAF10 depletion, followed by comprehensive analysis of TFIID subcomplex formation, chromatin binding, and nascent transcription in mouse embryonic stem cells, result in the formation of a TAF7-lacking TFIID or a minimal core-TFIID complex, respectively. These partial complexes support TBP recruitment at promoters and nascent Pol II transcription at most genes early after depletion, but importantly, TAF10 is necessary for efficient Pol II pausing. We show that partially assembled TFIID complexes can sustain Pol II transcription initiation but cannot replace holo-TFIID over several cell divisions and/or development., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Mammalian riboswitches that can regulate transgene expression via RNA-small molecule interaction have promising applications in medicine and biotechnology, as they involve no protein factors that can induce immunogenic reactions and are not dependent on specially engineered promoters. However, the lack of cell-permeable and low-toxicity small molecules and cognate aptamers that can be exploited as riboswitches and the modest switching performance of mammalian riboswitches have limited their applications. In this study, we systematically optimized the design of a riboswitch that regulates exon skipping via an RNA aptamer that binds ASP2905. We examined two design strategies to modulate the stability of the aptamer base stem that blocks the 5' splice site to fine-tune the riboswitch characteristics. Furthermore, an optimized riboswitch was used to generate a mouse embryonic stem cell line that can be chemically induced to differentiate into myogenic cells by activating Myod1 expression and a human embryonic kidney cell line that can be induced to trigger apoptosis by activating BAX expression. The results demonstrate the tight chemical regulation of transgenes in mammalian cells to control their phenotype without exogenous protein factors.

During mammalian embryogenesis, both the 5-cytosine DNA methylation (5meC) landscape and three dimensional (3D) chromatin architecture are profoundly remodeled during a process known as 'epigenetic reprogramming.' An understudied aspect of epigenetic reprogramming is how the 5meC flux, per se, affects the 3D genome. This is pertinent given the 5meC-sensitivity of DNA binding for a key regulator of chromosome folding: CTCF. We profiled the CTCF binding landscape using a mouse embryonic stem cell (ESC) differentiation protocol that models embryonic 5meC dynamics. Mouse ESCs lacking DNA methylation machinery are able to exit naive pluripotency, thus allowing for dissection of subtle effects of CTCF on gene expression. We performed CTCF HiChIP in both wild-type and mutant conditions to assess gained CTCF-CTCF contacts in the absence of 5meC. We performed H3K27ac HiChIP to determine the impact that ectopic CTCF binding has on cis-regulatory contacts. Using 5meC epigenome editing, we demonstrated that the methyl-mark is able to impair CTCF binding at select loci. Finally, a detailed dissection of the imprinted Zdbf2 locus showed how 5meC-antagonism of CTCF allows for proper gene regulation during differentiation. This work provides a comprehensive overview of how 5meC impacts the 3D genome in a relevant model for early embryonic events., (© The Author(s) 2024. Published by Oxford University Press on behalf of Nucleic Acids Research.)

The interplay between G4s and R-loops are emerging in regulating DNA repair, replication, and transcription. A comprehensive picture of native co-localized G4s and R-loops in living cells is currently lacking. Here, we describe the development of HepG4-seq and an optimized HBD-seq methods, which robustly capture native G4s and R-loops, respectively, in living cells. We successfully employed these methods to establish comprehensive maps of native co-localized G4s and R-loops in human HEK293 cells and mouse embryonic stem cells (mESCs). We discovered that co-localized G4s and R-loops are dynamically altered in a cell type-dependent manner and are largely localized at active promoters and enhancers of transcriptional active genes. We further demonstrated the helicase Dhx9 as a direct and major regulator that modulates the formation and resolution of co-localized G4s and R-loops. Depletion of Dhx9 impaired the self-renewal and differentiation capacities of mESCs by altering the transcription of co-localized G4s and R-loops -associated genes. Taken together, our work established that the endogenous co-localized G4s and R-loops are prevalently persisted in the regulatory regions of active genes and are involved in the transcriptional regulation of their linked genes, opening the door for exploring broader roles of co-localized G4s and R-loops in development and disease., Competing Interests: TL, XS, YR, HL, YL, CC, LY, ZX No competing interests declared, (© 2024, Liu, Shen, Ren et al.)