Cell fate plasticity - the capacity of cells to transition between distinct identities - is particularly evident in pluripotent and totipotent cells, including embryonic cells and their in vitro stem cell counterparts. Accumulating evidence indicates that these cell states are accompanied by extensive remodeling of three-dimensional (3D) chromatin organization, including changes in compartments, topologically associating domains, and enhancer-promoter interactions. Engineering chromatin architecture to modulate pluripotency- or totipotency-associated genes has emerged as a promising strategy to reprogram cell identity and acquire pluripotent or totipotent features. Realizing this potential, however, requires a comprehensive understanding of both the universal principles of 3D chromatin regulation and the mechanisms specific to pluripotency or totipotency. Here, we review recent advances linking 3D chromatin structure to pluripotency and totipotency, and outline future directions for elucidating their regulatory logic. We also highlight key open questions that must be addressed to harness 3D genome engineering for controlling cell pluripotency or totipotency.

Selective gene expression is pivotal in orchestrating human development. Specifically, trithorax group (TrxG) and polycomb group (PcG) components play crucial roles in transcriptional activation and repression of state-specific stem cell expression programs, yet the mechanisms underlying their selective genomic binding remain poorly understood. In this study, we report that the polycomb repressive complex 1 (PRC1) subunit RYBP co-localizes with TrxG component WDR5 and selectively enriches PcG component RING1B in condensates in murine embryonic stem cells (ESCs). RYBP deficiency impairs the genomic binding of WDR5 and RING1B. Further, STAT3 excludes RING1B binding at RYBP-associated transcriptionally active loci. Additionally, RYBP depletion attenuates WDR5-dependent activation of DNA repair gene expression and facilitates the transition of ESCs to 2-cell-like cells. Finally, RYBP depletion disrupts RING1B deposition at lineage-specific genes, promoting ESC differentiation towards mesendoderm fate. These findings uncover RYBP as a regulator of selective genomic binding of TrxG and PcG components, providing insights into their roles in cell fate determination during development.

In this issue of Cell Genomics, Tortora and Fudenberg develop a first-principles framework in which loop extrusion is quantitatively regulated by multiple cohesin-associated factors, giving rise to “bursty extrusion.” This model predicts regulator-dependent changes in motor kinetics, chromatin contact patterns, and chromosome-scale morphology across spatial scales, providing a mechanistically grounded basis for quantitative modeling of 3D genome architecture.

Introduction: Microglial activation is a key contributor to neuroinflammation in the pathogenesis of major depressive disorder (MDD). Isoliquiritigenin (ILG), a compound derived from medicinal plants such as Glycyrrhiza glabra, has been widely reported to inhibit microglial activation and reduce neuroinflammation; however, its antidepressant effects and underlying mechanisms remain unclear. Objectives: Here, we show that ILG exerts antidepressant effects that are dependent on its suppression of hippocampal microglia. Methods: This study first evaluated the antidepressant efficacy of ILG using multiple established depression models. We then employed pathological, molecular biological, and targeted inhibition approaches to establish the necessity of microglial involvement in ILG's antidepressant action. Furthermore, single-cell RNA sequencing was utilized to characterize the microglial landscape in depression and to identify the key microglial subset associated with ILG's therapeutic effect. Finally, we elucidated the regulatory mechanism of ILG on this specific microglial subset through molecular docking, microscale thermophoresis, and site-directed mutagenesis. Results: Specifically, single‑cell RNA sequencing revealed that ILG inhibits a subset of highly phagocytic microglia (HP‑MG), a population characterized by high expression of phagocytosis‑associated genes and markedly activated during depression. Mechanistically, ILG binds to the Thr446 phosphorylation site of PKR, blocking its activation and the downstream NF‑κB/ATF4 signaling cascade, thereby downregulating transcription of the HP‑MG marker gene Saa3 and ultimately suppressing HP‑MG. Conclusion: Taken together, these findings indicate that ILG possesses considerable antidepressant potential, which may be attributable to its inhibition of PKR‑mediated highly phagocytic microglia.

The acquisition of totipotency requires transcriptional activation of endogenous retroviruses (MERVL/HERVL) and zygotic genome activation (ZGA) related genes, yet the molecular mechanisms linking chromatin architecture to this process remain elusive. Here, we demonstrate that mouse Dux and human DUX4, double homeobox transcription factors essential for totipotency, form liquid-liquid phase-separated (LLPS) condensates through conserved arginine residues within intrinsically disordered regions (IDRs) in the Homeobox domain. These condensates recruit CBP/p300 and CTCF to establish super-enhancers (SEs) at MERVL/MT2 loci, enabling H3K27ac deposition and chromatin accessibility. Hi-C analysis revealed that DUX-driven phase separation facilitates 3D genome reorganization, including de novo formation of enhancer-promoter loops and TAD boundary shifts. Disruption of LLPS (DUXR70A) abolished SE assembly, transcriptional activation, and embryonic chimerism. Strikingly, human DUX4 required phase separation for both myotoxic gene activation and cytotoxicity in facioscapulohumeral muscular dystrophy (FSHD) models. Our study establishes a paradigm wherein phase separation integrates transcriptional control with 3D genome remodeling to license totipotency, with direct implications for developmental biology and disease therapy.

Cell fate decisions in the early embryo rely on reciprocal transcriptional networks that balance pluripotency with lineage commitment. NANOG is essential for directing the epiblast-primitive endoderm (PrE) fate choice, but the molecular mechanisms underlying its repressive activity remain incompletely understood. Here we show that NANOG partners with TBX3 and the PRC2 complex to maintain embryonic stem cell (ESC) identity by silencing PrE genes through newly identified distal enhancers. Loss of Nanog reduces PRC2-mediated repression of Gata6, initiating its expression independently of TBX3. Subsequent TBX3 upregulation enables its association with GATA6, driving a feed-forward programme that activates Gata6, Gata4 and Sox17 and promotes PrE differentiation. Thus, NANOG suppresses PrE fate not only by direct repression but also by preventing TBX3 from switching partners. These findings define a Nanog-Tbx3-Gata6 regulatory axis that integrates enhancer control, chromatin regulation and transcription factor redeployment to couple ESC maintenance with lineage commitment.

Polysaccharides are known to be synthesized by enzymes in the endoplasmic reticulum and Golgi apparatus and transported through the secretory pathway to the cell surface or extracellular space, where they mediate essential biological processes. While classical localization and functions of polysaccharides are well established, their presence and potential roles in the nucleus remain unclear. Here we demonstrate that N-glycans, a type of polysaccharides, modify inner nuclear membrane (INM) proteins and are present in the cell nucleus across diverse cell types-a modification referred to as N-linked glycosylation (N-glycosylation). N-glycosylation is enriched in chromatin regions marked by H3K9me3 and long interspersed nuclear element-1 (LINE-1) retrotransposons. N-glycosylation inhibition and INM protein N-glycosylation site mutation both downregulate H3K9me3 within lamina-associated domains and lead to genomic instability. Mechanistically, N-glycosylation regulates the interaction between the histone H3K9 methyltransferase SETDB1 and INM proteins, promotes the association of SETDB1 with the INM, and maintains H3K9me3. Moreover, we reveal that canonical N-glycan biosynthetic machinery in the endoplasmic reticulum contributes to the N-glycosylation of INM proteins. These findings uncover a previously unrecognized nuclear role for polysaccharides, broadening our understanding beyond their traditional subcellular distributions and functional profiles.

Rationale: Endothelial cell senescence leads to endothelial dysfunction, thereby promoting the progression of atherosclerosis. Super-enhancers are crucial epigenetic cis-regulatory elements whose extensive reprogramming drives aberrant transcription in human diseases. However, the underlying mechanisms by which super-enhancers regulate endothelial cell senescence remain unclear. This study reveals the effect of liquid-liquid phase separation (LLPS) mediated by super-enhancer-driven core transcription factor FOXP1 on endothelial cell senescence. Methods: The landscape of super-enhancers, chromatin accessibility, and transcriptome profiling were characterized during endothelial cell senescence by conducting CUT&Tag-seq with antibodies against H3K27ac, H3K4me1, and H3K4me3, along with assays for ATAC-seq and RNA-seq. The Coltron algorithm was used to identify core transcription factors in the process of endothelial cell senescence. Fluorescence recovery after photobleaching (FRAP), dCas9-KRAB CRISPRi, and the Optodroplet assay were utilized to confirm the phase separation properties of FOXP1. Functional experiments were employed to elucidate the effect of FOXP1 on endothelial cell senescence through LLPS. Results: Senescent endothelial cells undergo significant changes in their epigenome. FOXP1 is identified as a core transcription factor, driven by super-enhancers, which delays endothelial cell senescence and inhibits atherosclerosis. Moreover, FOXP1 undergoes LLPS, which the 19 phase-forming amino acids within the intrinsically disordered region of FOXP1 are capable of maintaining its ability to delay endothelial cell senescence. Mechanistically, FOXP1 activates the target gene SESN3 and inhibits the mTORC1 signaling pathway through phase separation, a key event in delaying endothelial cell senescence. The clinical evidences support the potential role of FOXP1 and SESN3 as protective factors against atherosclerosis. Conclusion: FOXP1 undergoes phase separation at its super-enhancer, recruiting transcription coactivators to form condensates. These condensates, in turn, facilitate binding with the SESN3 promoter and inhibit the mTORC1 signaling pathway, thereby delaying endothelial cell senescence.

RAF protein kinases are major RAS effectors that function by phosphorylating MEK. Although all three RAF isoforms share a conserved RAS binding domain and bind to GTP-loaded RAS, only ARAF uniquely enhances RAS activity. Here we uncovered the molecular basis of ARAF in regulating RAS activation. The disordered N-terminal sequence of ARAF drives self-assembly, forming ARAF–RAS condensates tethered to the plasma membrane. These structures concentrate active RAS locally, impeding NF1-mediated negative regulation of RAS, thereby fostering receptor tyrosine kinase (RTK)-triggered RAS activation. In RAS-mutant tumors, loss of the ARAF N terminus sensitizes tumor cells to pan-RAF inhibition. In hormone-sensitive cancers, increased ARAF condensates drive endocrine therapy resistance, whereas ARAF depletion reverses RTK-dependent resistance. Our findings delineate ARAF–RAS protein condensates as distinct subcellular structures sustaining RAS activity and facilitating oncogenic RAS signaling. Targeting ARAF–RAS condensation may offer a strategy to overcome drug resistance in both wild-type and mutant ARAF-mediated scenarios. ARAF uniquely enhances RAS activity via N-terminal disordered sequence, forming membrane ARAF–RAS condensates that shield RAS from negative regulation and drive cancer drug resistance.

In migratory fibroblasts, front-rear polarity is defined by the centrosome positioned anterior to a rearward nucleus. To achieve this polarity, actin cables couple to nuclear membrane proteins nesprin-2G and SUN2 and drive the nucleus backward. Aging disrupts this polarity by increasing SUN1, a SUN2 homolog. Here, we investigated the molecular mechanisms behind this disruption and found that the dominant-negative effect of SUN1 and progerin, a lamin A variant, required direct SUN1-lamin A interaction. Microtubule interaction and force transmission through a nesprin, identified as nesprin-2, are crucial for SUN1's effect. We further discovered that stable microtubules are both necessary and sufficient to inhibit cell polarity. Using SUN1-SUN2 chimeric proteins, we demonstrated that the SUN domains determine their roles in cell polarization. Our findings reveal how elevated SUN1 disrupts cell polarity through coupling microtubules and nuclear lamina, emphasizing the impact of altered microtubule stability and nuclear mechanotransduction in polarity defects.

Chromatin domain boundaries are crucial for development by spatially constraining enhancer activity to ensure precise spatiotemporal gene regulation. In this issue of Developmental Cell, Chakraborty et al. demonstrate that the loss of a chromatin domain boundary caused by deletion of a single CTCF motif can lead to severe developmental defects.

In migratory fibroblasts, the front-rear polarity required for cell migration is defined by an anterior centrosome relative to the nucleus. To achieve this polarity, actin cables drive the nucleus backward by coupling to nuclear membrane proteins nesprin-2G and SUN2. Aging disrupts this cell polarity by increasing the protein levels of SUN1, a SUN2 homolog. Here, we investigated the molecular mechanisms behind this disruption and found that the dominant negative effect of SUN1 and progerin, an aging-related lamin A variant, required direct SUN1-lamin A interaction. Microtubule interaction and force transmission through a nesprin, identified as nesprin-2, are crucial for SUN1's effect. We further discovered that stable microtubules are both necessary and sufficient to inhibit cell polarity. Using SUN1-SUN2 chimeric proteins, we demonstrated that the SUN domains determine their roles in cell polarization. Our findings reveal how elevated SUN1 disrupts cell polarity through coupling microtubule and nuclear lamina, emphasizing the impact of altered microtubule stability and nuclear mechanotransduction in aging. ### Competing Interest Statement The authors have declared no competing interest.

Enhancer-promoter (E-P) interactions regulate transcription during cell fate determination. However, the regulatory mechanisms underlying E-P interactions have remained elusive. Here we present a chromatin-interaction-based proteomic approach, LoopID, to profile proteins (termed the looposome) at certain E-P anchors. We find that histone demethylase JMJD2, a key looposome component, can regulate E-P interactions and the looposome in a catalytic-independent manner through formation of biomolecular condensates. Furthermore, we introduce a system to engineer E-P interactions by assembling JMJD2 condensates at certain genomic loci, enabling construction of cell-type-specific E-P interactions to promote cellular reprogramming into pluripotent or two-cell-like cells. Our findings reveal a noncanonical function of a histone demethylase in regulation of chromatin organization and provide a strategy to regulate cell fate transitions through E-P interactions.

In eukaryotic organisms, the three-dimensional organization and epigenomic landscape of chromatin are fundamental to the regulation of gene expression. Previous studies have provided significant insights into CpG methylation, chromatin accessibility, and the dynamics of 3D architecture. However, a systematic delineation of how these epigenomic features regulate transcriptional activity remains limited. In this study, we develop nanoCAM-seq, a single-molecule sequencing technique designed to simultaneously profile higher-order chromatin interactions, chromatin accessibility, and endogenous CpG methylation. This approach provides an integrative view of chromatin features associated with cis-regulatory elements and reveals their coordinated dynamics during transitions of A/B compartments. Single-molecule analyses using nanoCAM-seq further reveal that promoters characterized by low CpG methylation and high chromatin accessibility more frequently interact with multiple enhancers. Collectively, our findings establish nanoCAM-seq as a powerful approach for resolving the coordinated dynamics of chromatin architecture and epigenetic modifications, offering critical insights into the regulatory mechanisms underlying gene expression.

Breast cancer is the most common malignancy in females and remains the leading cause of cancer-related deaths for women worldwide. The cellular and molecular basis of breast tumorigenesis is not completely understood partly due to the lack of human research models which simulate the development of breast cancer. Here, we developed a method for generating functional mammary-like cells (MCs) from human-induced pluripotent stem cells (iPSCs). The iPSC-MCs closely resemble human primary MCs at cellular, transcriptional, and functional levels. Using this method, a breast cancer model was generated using patient-derived iPSCs harboring germline BRCA1 mutation. The patient iPSC-MCs recapitulated the transcriptome, clinical genomic alteration, and tumorigenic ability of breast cancer cells. We also identified S100P as an oncogene downstream of mutated BRCA1 that promotes cancer cell stemness and tumorigenesis. Our study establishes a promising system of breast cancer for studying the mechanism of tumorigenesis and identifying potential therapeutic targets.

The genome is intricately folded into chromatin compartments, topologically associating domains (TADs) and loops unique to each cell type. How this higher-order genome organization regulates cell fate transition remains elusive. Here we show how a single non-neural progenitor transcription factor, PTF1A, reorchestrates the 3D genome during fibroblast transdifferentiation into neural stem cells (NSCs). Multiomics analyses integrating Hi-C data, PTF1A and CTCF DNA-binding profiles, H3K27ac modification, and gene expression, demonstrate that PTF1A binds to subTAD boundaries subsequently associated with elevated CTCF binding and enhanced boundary insulation, and reorganizes chromatin loops, leading to gene expression changes that drive transdifferentiation into NSCs. Moreover, PTF1A activates enhancers and super-enhancers near low-insulation boundaries and modulates H3K27ac deposition, promoting cell fate transitions. Together, our data implicate an involvement of 3D genome in transcriptional and cell fate alterations, and highlight an essential role for PTF1A in gene expression control and multiscale 3D genome remodeling during cell reprogramming. This study explores the role of the transcription factor PTF1A in remodeling 3D genome architecture during the transdifferentiation of fibroblasts to neural stem cells, highlighting changes in gene expression and TAD organization.

EDITORIAL article Front. Aging Neurosci., 07 March 2024Sec. Cellular and Molecular Mechanisms of Brain-aging Volume 16 - 2024 | https://doi.org/10.3389/fnagi.2024.1384586

CCCTC-binding factor (CTCF), a ubiquitously expressed and highly conserved protein, is known to play a critical role in chromatin structure. Post-translational modifications (PTMs) diversify the functions of protein to regulate numerous cellular processes. However, the effects of PTMs on the genome-wide binding of CTCF and the organization of three-dimensional (3D) chromatin structure have not been fully understood. In this study, we uncovered the PTM profiling of CTCF and demonstrated that CTCF can be O-GlcNAcylated and arginine methylated. Functionally, we demonstrated that O-GlcNAcylation inhibits CTCF binding to chromatin. Meanwhile, deficiency of CTCF O-GlcNAcylation results in the disruption of loop domains and the alteration of chromatin loops associated with cellular development. Furthermore, the deficiency of CTCF O-GlcNAcylation increases the expression of developmental genes and negatively regulates maintenance and establishment of stem cell pluripotency. In conclusion, these results provide key insights into the role of PTMs for the 3D chromatin structure. CTCF, which is known to play critical role in chromatin structure, undergoes post-translational modifications (PTMs). In this research, O-GlcNAcylation was found to inhibit CTCF binding, impacting 3D chromatin structure, gene expression and cellular development.