Fate Determination Research Articles

Elongator is a microtubule polymerase selective for polyglutamylated tubulin

Abstract Elongator is a tRNA-modifying complex that regulates protein translation. Recently, a moonlighting function of Elongator has been identified in regulating the polarization of the microtubule cytoskeleton during asymmetric cell division. Elongator induces symmetry breaking of the anaphase midzone by selectively stabilizing microtubules on one side of the spindle, contributing to the downstream polarized segregation of cell-fate determinants, and therefore to cell fate determination. Here, we investigate how Elongator controls microtubule dynamics. Elongator binds both to the tip of microtubules and to free GTP-tubulin heterodimers using two different subcomplexes, Elp123 and Elp456, respectively. We show that these activities must be coupled for Elongator to decrease the tubulin critical concentration for microtubule elongation. As a consequence, Elongator increases the growth speed and decreases the catastrophe rate of microtubules. Surprisingly, the Elp456 subcomplex binds to tubulin tails and has strong selectivity towards polyglutamylated tubulin. Hence, microtubules assembled by Elongator become selectively enriched with polyglutamylated tubulin, as observed in vitro, in mouse and Drosophila cell lines, as well as in vivo in Drosophila Sensory Organ Precursor cells. Therefore, Elongator rewrites the tubulin code of growing microtubules, placing it at the core of cytoskeletal dynamics and polarization during asymmetric cell division.

MicroRNA analysis reveals two modules that antagonistically regulate xylem tracheary element development in Arabidopsis.

Tracheary elements (TEs) are vital in the transport of various substances and contribute to plant growth. The differentiation of TEs is complex and regulated by a variety of microRNAs (miRNAs). However, the dynamic changes in miRNAs during each stage of TE differentiation remain unclear, and the miRNA regulatory network is not yet complete. This study employed Vascular cell Induction culture System Using Arabidopsis Leaves (VISUAL) to profile the miRNome during TE differentiation in Arabidopsis (Arabidopsis thaliana) and established comprehensive miRNA co-expression networks functioning at the different stages of TE differentiation. Two negatively correlated modules exist in the miRNA networks, each exhibiting strong intra-module positive correlation and strong inter-module negative correlation. Thus, the two modules may play opposite roles in TE differentiation and vascular development. Indeed, we found that miR408 promotes cambium formation and TE differentiation, consistent with miR408 as a key node in the networks of fate determination and the initiation of TE differentiation. Additionally, we found that miR163 inhibits secondary cell wall formation and TE differentiation, corresponding to miR163 as a key node in the TE maturation network. Moreover, we discovered that the miRNA co-expression network in poplar (Populus tomentosa) xylem development is also composed of two negatively correlated modules that contain miRNAs orthologous to those in Arabidopsis. Therefore, the two negatively correlated modules of the miRNA co-expression network are likely conserved and fundamental to xylem TE differentiation. These results provide insights into microRNA regulation in plant development.

Irisin reshapes bone metabolic homeostasis to delay age-related osteoporosis by regulating the multipotent differentiation of BMSCs via Wnt pathway

IntroductionBone aging is linked to changes in the lineage differentiation of bone marrow stem cells (BMSCs), which show a heightened tendency to differentiate into adipocytes instead of osteoblasts. The therapeutic potential of irisin in addressing age-related diseases has garnered significant attention. More significantly, irisin has the capacity to enhance bone mass recovery and sustain overall bone health. Its mechanism of action in preventing osteoporosis has generated considerable interest within the research community. Nonetheless, the targeting effect of irisin on age-related osteoporosis and its underlying molecular biological mechanisms remain unclear.MethodsThe specific role of irisin in osteogenic-adipogenic differentiation in young or aging BMSCs was evaluated by multiple cells staining and quantitative real-time PCR (RT-qPCR) analysis. RNA-seq and protein Western blotting excavated and validated the key pathway by which irisin influences the fate determination of aging BMSCs. The macroscopic and microscopic changes of bone tissue in aging mice were examined using Micro-computed tomography (Micro-CT) and morphological staining.ResultsIt was noted that irisin affected the multilineage differentiation of BMSCs in a manner dependent on the dosage. Simultaneously, the Wnt signaling pathway might be a crucial mechanism through which irisin sustains the bone-fat balance in aging BMSCs and mitigates the decline in pluripotency. In vivo, irisin reduced bone marrow fat deposition in aging mice and effectively alleviating the occurrence of bone loss.ConclusionIrisin mediates the Wnt signaling pathway, thereby influencing the fate determination of BMSCs. In addition, it is essential for preserving metabolic equilibrium in the bone marrow microenvironment and significantly contributes to overall bone health. The findings provide new evidence for the use of iris extract in the treatment of age-related osteoporosis.

Posttranscriptional Control of Neural Progenitors Temporal Dynamics During Neocortical Development by Syncrip.

The development of the mammalian neocortex is precisely regulated by temporal gene expression, yet the temporal regulatory mechanisms of cortical neurogenesis, particularly how radial glial cells (RGCs) sequentially generate deep to superficial neurons, remain unclear. Here, the hnRNP family member Syncrip (hnRNP Q) is identified as a key modulator of superficial neuronal differentiation in neocortical neurogenesis. Syncrip knockout in RGCs disrupts differentiation and abnormal neuronal localization, ultimately resulting in superficial cortical layer defects as well as learning and memory impairments in mice. Single-cell RNA sequencing analysis demonstrated that the knockout of Syncrip disrupts the late-stage neurogenesis, stalling transcriptional progression in RGCs. Mechanistically, Syncrip maintains the transcription of temporal process-related transcription factors by recruiting stabilization complexes through phase separation, crucially regulating the Notch signaling pathway that determines the fate of RGCs. Furthermore, pathogenic human mutations in Syncrip weaken its phase-separation capability, failing to form stable complexes normally. Thus, Syncrip acts as a mediator of posttranscriptional regulatory mechanisms, governing the fate progression of RGCs and the advancement of intrinsic temporal programs. This study establishes an intracellular mechanism for posttranscriptional regulation of progressive fate determination in cortical neurogenesis.

RNA N4‐acetylcytidine modification and its role in health and diseases

AbstractN4‐acetylcytidine (ac4C) modification is a crucial RNA modification widely present in eukaryotic RNA. Previous studies have demonstrated that ac4C plays a pivotal role in viral infections. Despite numerous studies highlighting the strong correlation between ac4C modification and cancer progression, its detailed roles and molecular mechanisms in normal physiological processes and cancer progression remain incompletely understood. This review first outlines the key regulatory enzyme mediating ac4C modification, N‐acetyltransferase 10 (NAT10), including its critical roles in regulating RNA stability, transcriptional efficiency, and translational fidelity. Additionally, it systematically summarizes the essential functions and mechanisms of ac4C modification in normal biological processes, including stem cell fate determination, spermatogenesis and oogenesis, embryonic development, cellular senescence, and bone remodeling. Furthermore, this review delves into the central roles and molecular mechanisms of ac4C modification in regulating malignant proliferation, cell cycle arrest, EMT, drug resistance, cell death, cancer metabolism, and tumor immunotherapy. It also emphasizes the potential of NAT10 as a prognostic biomarker and its therapeutic potential as a target for disease treatment. In summary, this review clarifies the multifaceted roles of ac4C modification in both health and disease and explores NAT10‐targeted therapies with the aim of advancing cancer research and improving patient outcomes.

Organ injury accelerates stem cell differentiation by modulating a fate-trans-ducing lateral inhibition circuit.

Injured epithelial organs must rapidly replace damaged cells to restore barrier integrity and physiological function. In response, injury-born stem cell progeny differentiate faster compared to healthy-born counterparts, yet the mechanisms that pace differentia-tion are unclear. Using the adult Drosophila intestine, we find that injury speeds cell differentiation by altering the lateral inhibition circuit that transduces a fate-determin-ing Notch signal. During healthy intestinal turnover, a balanced ratio of terminal (Notch-active) and stem (Notch-inactive) fates arises through canonical lateral inhibi-tion feedback, in which mutual Notch-Delta signaling between two stem cell daughters evolves to activate Notch and extinguish Delta in exactly one cell. When we damage in-testines by feeding flies toxin, mutual signaling persists, but a cytokine relay from dam-aged cells to differentiating daughters prevents the Notch co-repressor Groucho from extinguishing Delta. Despite Delta persistence, injured organs preserve the Notch-inac-tive stem cell pool; thus, fate balance does not hinge on an intact circuit. Mathematical modeling predicts that increased Delta prompts faster Notch signaling; indeed, in vivo live imaging reveals that the real-time speed of Notch signal transduction doubles in in-jured guts. These results show that in tissue homeostasis, lateral inhibition feedback be-tween stem cell daughters throttles the speed of Notch-mediated fate determination by constraining Delta. Tissue-level damage signals relax this constraint to accelerate cell differentiation for expedited organ repair.

Overexpression of miR-192 Inhibits In Vitro Porcine Embryo Development by Inducing Oxidative Stress Damage and Impairing Mitochondrial Function.

Early embryonic development relies on intricately regulated gene expression, and miRNAs influence zygotic genome activation (ZGA), cleavage, and cell fate determination through post-transcriptional regulatory mechanisms. miR-192 is expressed in early pig embryos and participates in various reproductive processes. However, its role in pre-implantation pig embryo development remains poorly understood. In this study, we microinjected the miR-192 agonist (miR-192 agomir) into parthenogenetically activated pig embryos to evaluate its effects on early pig embryo development. Our findings indicate that compared to the control group (agomir NC), miR-192 agomir impairs the developmental capacity of parthenogenetic pig embryos to reach the 2-cell, 4-cell, and blastocyst stages. This impairment leads to imbalances in the oxidative-reductive system and abnormalities in mitochondrial function during the 4-cell stage, resulting in the significant accumulation of ROS, notable decreases in the expression of antioxidant enzymes CAT and SOD1 mRNA, reduction in mitochondrial membrane potential, and induction of apoptosis in pig blastocysts. Additionally, the overexpression of miR-192 inhibits the expression of its target genes YY1 and the pluripotency factor NANOG mRNA. In conclusion, this study reveals that the overexpression of miR-192 adversely affects early pig embryo development, providing new evidence for understanding the role miR-192 plays in reproduction.

Retinoic acid drives surface epithelium fate determination through the TCF7-MSX2 axis

Understanding how embryonic progenitors decode extrinsic signals and transform into lineage-specific regulatory networks to drive cell fate specification is a fundamental, yet challenging question. Here, we develop a new model of surface epithelium (SE) differentiation induced by human embryonic stem cells (hESCs) using retinoic acid (RA), and identify BMP4 as an essential downstream signal in this process. We show that the retinoid X receptors, RXRA and RXRB, orchestrate SE commitment by shaping lineage-specific epigenetic and transcriptomic landscapes. Moreover, we find that TCF7, as a RA effector, regulates the transition from pluripotency to SE initiation by directly silencing pluripotency genes and activating SE genes. MSX2, a downstream activator of TCF7, primes the SE chromatin accessibility landscape and activates SE genes. Our work reveals the regulatory hierarchy between key morphogens RA and BMP4 in SE development, and demonstrates how the TCF7-MSX2 axis governs SE fate, providing novel insights into RA-mediated regulatory principles.

Mitonuclear Communication in Stem Cell Function.

Mitochondria perform multiple functions within the cell, including the production of ATP and a great deal of metabolic intermediates, while also contributing to the cellular stress response. The majority of mitochondrial proteins are encoded by nuclear genomes, highlighting the importance of mitonuclear communication for sustaining mitochondrial homeostasis and functional. As a crucial part of the intracellular signalling network, mitochondria can impact stem cell fate determinations. Considering the essential function of stem cells in tissue maintenance, regeneration and aging, it is important to understand how mitochondria influence stem cell fate. This review explores the significant roles of mitonuclear communication and mitochondrial proteostasis, highlighting their influence on stem cells. We also examine how mitonuclear interactions contribute to cellular homeostasis, stem cell therapies, and the potential for extending lifespan.

Dual-nuclease single-cell lineage tracing by Cas9 and Cas12a.

Single-cell lineage tracing based on CRISPR-Cas9 gene editing enables the simultaneous linkage of cell states and lineage history at a high resolution. Despite its immense potential in resolving the cell fate determination and genealogy within an organism, existing implementations of this technology suffer from limitations in recording capabilities and considerable barcode dropout. Here, we introduce DuTracer, a versatile tool that utilizes two orthogonal gene editing systems to record cell lineage history at single-cell resolution in an inducible manner. DuTracer shows the ability to enhance lineage recording with minimized target dropouts and potentially deeper tree depths. Applying DuTracer in mouse embryoid bodies and neuromesodermal organoids illustrates the lineage relationship of different cell types and proposes potential lineage-biased molecular drivers, showcased by identifying transcription factor Foxb1 as a modulator in the cell fate determination of neuromesodermal progenitors. Collectively, DuTracer facilitates the precise and regulatory interrogation of cellular lineages of complex biological processes.

Inclusive, exclusive and hierarchical atlas of NFATc1+/PDGFR-α+ cells in dental and periodontal mesenchyme.

Platelet-derived growth factor receptor alpha (PDGFR-α) activity is crucial in the process of dental and periodontal mesenchyme regeneration facilitated by autologous platelet concentrates (APCs), such as platelet-rich fibrin (PRF), platelet-rich plasma (PRP) and concentrated growth factors (CGF), as well as by recombinant PDGF drugs. However, it is largely unclear about the physiological patterns and cellular fate determinations of PDGFR-α+ cells in the homeostasis maintaining of adult dental and periodontal mesenchyme. We previously identified NFATc1 expressing PDGFR-α+ cells as a subtype of skeletal stem cells (SSCs) in limb bone in mice, but their roles in dental and periodontal remain unexplored. To this end, in the present study we investigated the spatiotemporal atlas of NFATc1+ and PDGFR-α+ cells residing in dental and periodontal mesenchyme in mice, their capacity for progeny cell generation, and their inclusive, exclusive and hierarchical relations in homeostasis. We utilized CRISPR/Cas9-mediated gene editing to generate two dual recombination systems, which were Cre-loxP and Dre-rox combined intersectional and exclusive reporters respectively, to concurrently demonstrate the inclusive, exclusive, and hierarchical distributions of NFATc1+ and PDGFR-α+ cells and their lineage commitment. By employing the state-of-the-art transgenic lineage tracing techniques in cooperating with tissue clearing-based advanced imaging and three-dimensional slices reconstruction, we systematically mapped the distribution atlas of NFATc1+ and PDGFR-α+ cells in dental and periodontal mesenchyme and tracked their in vivo fate trajectories in mice. Our findings extend current understanding of NFATc1+ and PDGFR-α+ cells in dental and periodontal mesenchyme homeostasis, and furthermore enhance our comprehension of their sustained therapeutic impact for future clinical investigations.

3D cryo-printed hierarchical porous scaffolds provide immobilization of surface-functionalized sleep-inspired small extracellular vesicles: synergistic therapeutic strategies for vascularized bone regeneration based on macrophage phenotype modulation and angiogenesis-osteogenesis coupling

Bone defect healing is a multi-factorial process involving the inflammatory microenvironment, bone regeneration and the formation of blood vessels, and remains a great challenge in clinical practice. Combined use of three-dimensional (3D)-printed scaffolds and bioactive factors is an emerging strategy for the treatment of bone defects. Scaffolds can be printed using 3D cryogenic printing technology to create a microarchitecture similar to trabecular bone. Melatonin (MT) has attracted attention in recent years as an excellent factor for promoting cell viability and tissue repair. In this study, porous scaffolds were prepared by cryogenic printing with poly(lactic-co-glycolic acid) and ultralong hydroxyapatite nanowires. The hierarchical pore size distribution of the scaffolds was evaluated by scanning electron microscopy (SEM) and micro-computed tomography (micro-CT). Sleep-inspired small extracellular vesicles (MT-sEVs) were then obtained from MT-stimulated cells and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-poly(ethylene glycol)-inorganic pyrophosphate (DSPE-PEG-PPi) was used to modify the membrane of MT-sEVs to obtain PPi-MT-sEVs. RNA sequencing was performed to explore the potential mechanisms. The results demonstrated that PPi-MT-sEVs not only enhanced cell proliferation, migration and angiogenesis, but also regulated the osteogenic/adipogenic fate determination and M1/M2 macrophage polarization switch in vitro. PPi-MT-sEVs were used to coat scaffolds, enabled by the capacity of PPi to bind to hydroxyapatite, and computational simulations were used to analyze the interfacial bonding of PPi and hydroxyapatite. The macrophage phenotype-modulating and osteogenesis–angiogenesis coupling effects were evaluated in vivo. In summary, this study suggests that the combination of hierarchical porous scaffolds and PPi-MT-sEVs could be a promising candidate for the clinical treatment of bone defects.Graphical abstract

Glutamine-αKG axis affects dentin regeneration and regulates osteo/odontogenic differentiation of mesenchymal adult stem cells via IGF2 m6A modification

BackgroundMulti-lineage differentiation of mesenchymal adult stem cells (m-ASCs) is crucial for tissue regeneration and accompanied with metabolism reprogramming, among which dental-pulp-derived m-ASCs has obvious advantage of easy accessibility. Stem cell fate determination and differentiation are closely related to metabolism status in cell microenvironment, which could actively interact with epigenetic modification. In recent years, glutamine-α-ketoglutarate (αKG) axis was proved to be related to aging, tumorigenesis, osteogenesis etc., while its role in m-ASCs still lack adequate research evidence.MethodsWe employed metabolomic analysis to explore the change pattern of metabolites during dental-pulp-derived m-ASCs differentiation. A murine incisor clipping model was established to investigate the influence of αKG on dental tissue repairment. shRNA technique was used to knockdown the expression of related key enzyme-dehydrogenase 1(GLUD1). RNA-seq, m6A evaluation and MeRIP-qPCR were used to dig into the underlying epigenetic mechanism.ResultsHere we found that the glutamine-αKG axis displayed an increased tendency along with the osteo/odontogenic differentiation of dental-pulp-derived m-ASCs, same as expression pattern of GLUD1. Further, the key metabolite αKG was found able to accelerate the repairment of clipped mice incisor and promote dentin formation. Exogenous DM-αKG was proved able to promote osteo/odontogenic differentiation of dental-pulp-derived m-ASCs, while the inhibition of glutamine-derived αKG level via GLUD1 knockdown had the opposite effect. Under the circumstance of GLUD1 knockdown, extracellular matrix (ECM) function and PI3k-Akt signaling pathway was screened out to be widely involved in the process with insulin-like growth factor 2 (IGF2) participation via RNA-seq. Inhibition of glutamine-αKG axis may affect IGF2 translation efficiency via m6A methylation and can be significantly rescued by αKG supplementation.ConclusionOur findings indicate that glutamine-αKG axis may epigenetically promote osteo/odontogenic differentiation of dental-pulp-derived m-ASCs and dentin regeneration, which provide a new research vision of potential dental tissue repairment therapy method or metabolite-based drug research.

H3K4me3 Genome-Wide Distribution and Transcriptional Regulation of Transposable Elements by RNA Pol2 Deposition.

Zygotic genome activation (ZGA) is critical for early embryo development and is meticulously regulated by epigenetic modifications. H3K4me3 is a transcription-permissive histone mark preferentially found at promoters, but its distribution across genome features remains incompletely understood. In this study, we investigated the genome-wide enrichment of H3K4me3 during early embryo development and embryonic stem cells (ESCs) in both sheep and mice. We discovered that broad H3K4me3 domains were present in MII stage oocytes and were progressively diminished, while promoter H3K4me3 enrichment was increased and correlated with gene upregulation during ZGA in sheep. Additionally, we reported the dynamic distribution of H3K4me3 at the transposable elements (TEs) during early embryo development in both sheep and mice. Specifically, the H3K4me3 distribution of LINE1 and ERVL, two subsets of TEs, was associated with their expression during early embryo development in sheep. Furthermore, H3K4me3 enrichment in TEs was greatly increased during ZGA following Kdm5b knockdown, and the distribution of RNA polymerase II (Pol2) in TEs was also markedly increased in Kdm5b knockout ESCs in mice. These findings suggest that H3K4me3 plays important roles in regulating TE expression through interaction with RNA Pol2, providing valuable insights into the regulation of ZGA initiation and cell fate determination by H3K4me3.

Mitochondrial dynamics govern whole-body regeneration through stem cell pluripotency and mitonuclear balance

Tissue regeneration is a complex process involving large changes in cell proliferation, fate determination, and differentiation. Mitochondrial dynamics and metabolism play a crucial role in development and wound repair, but their function in large-scale regeneration remains poorly understood. Planarians offer an excellent model to investigate this process due to their remarkable regenerative abilities. In this study, we examine mitochondrial dynamics during planarian regeneration. We find that knockdown of the mitochondrial fusion gene, opa1, impairs both tissue regeneration and stem cell pluripotency. Interestingly, the regeneration defects caused by opa1 knockdown are rescued by simultaneous knockdown of the mitochondrial fission gene, drp1, which partially restores mitochondrial dynamics. Furthermore, we discover that Mitolow stem cells exhibit an enrichment of pluripotency due to their fate choices at earlier stages. Transcriptomic analysis reveals the delicate mitonuclear balance in metabolism and mitochondrial proteins in regeneration, controlled by mitochondrial dynamics. These findings highlight the importance of maintaining mitochondrial dynamics in large-scale tissue regeneration and suggest the potential for manipulating these dynamics to enhance stem cell functionality and regenerative processes.

Caspar specifies primordial germ cell count and identity in Drosophila melanogaster.

Repurposing of pleiotropic factors during execution of diverse cellular processes has emerged as a regulatory paradigm. Embryonic development in metazoans is controlled by maternal factors deposited in the egg during oogenesis. Here, we explore maternal role(s) of Caspar (Casp), the Drosophila orthologue of human Fas-associated factor-1 (FAF1) originally implicated in host-defense as a negative regulator of NF-κB signaling. Maternal loss of either Casp or it's protein partner, transitional endoplasmic reticulum 94 (TER94) leads to partial embryonic lethality correlated with aberrant centrosome behavior, cytoskeletal abnormalities, and defective gastrulation. Although ubiquitously distributed, both proteins are enriched in the primordial germ cells (PGCs), and in keeping with the centrosome problems, mutant embryos display a significant reduction in the PGC count. Moreover, the total number of pole buds is directly proportional to the level of Casp. Consistently, it's 'loss' and 'gain' results in respective reduction and increase in the Oskar protein levels, the master determinant of PGC fate. To elucidate this regulatory loop, we analyzed several known components of mid-blastula transition and identify the translational repressor Smaug, a zygotic regulator of germ cell specification, as a potential critical target. We present a detailed structure-function analysis of Casp aimed at understanding its novel involvement during PGC development.

The Drosophila toothrin Gene Related to the d4 Family Genes: An Evolutionary View on Origin and Function.

D. melanogaster has two paralogs, tth and dd4, related to the evolutionarily conserved d4 family genes. In mammals, the family consists of Dpf1-3, encoding transcription co-factors involved in the regulation of development and cell fate determination. The function of tth and dd4 in Drosophila remains unclear. The typical domain structure of the proteins encoded by the d4 family consists of an N-terminal 2/3 domain (Requiem_N), a central Kruppel-type zinc finger, and a C-terminal D4 domain of paired PHD zinc fingers (DPFs). In Drosophila, both paralogs lack the Kruppel-type ZF, and tth encodes a protein that contains only Requiem_N. In contrast, vertebrate Dpf1-3 paralogs encode all the domains, but some paralogs have specific splice isoforms. For example, the DPF3a isoform lacks the D4 domain necessary for histone reading. The occurrence of proteins without the D4 domain in mammals and flies implies functional significance and analogous roles across animal taxa. In this study, we reconstructed the evolutionary events that led to the emergence of Drosophila tth by analyzing the divergence of d4 paralogs across different evolutionary lineages. Our genomic and transcriptomic data analysis revealed duplications and gene copy loss events. Among insects, gene duplication was only observed in Diptera. In other lineages, we found the specialization of paralogs for producing isoforms and further specialization for coding proteins with specific domain organizations. We hypothesize that this pathway is a common mechanism for the emergence of paralogues lacking the D4 domain across different evolutionary lineages. We, thus, postulate that TTH may function as a splice isoform of the ancestral single-copy gene, possibly a DPF3a-like isoform characteristic of related insect species. Our analysis provides insights into the possible impact of paralogue divergence, emphasizing the functional significance of the 2/3 domain and the potential roles of isoforms lacking the D4 domain.

Exosomes released from immature neurons regulate adult neural stem cell differentiation through microRNA-7a-5p.

Exosomes in the hippocampal dentate gyrus (DG) are essential for modulating the cell signaling controlling the neural differentiation of hippocampal neural stem cells (NSCs), which may determine the level of hippocampal adult neurogenesis. In the present study, we found that exosomes secreted by immature neurons may promote the neuronal differentiation of mouse NSCs in vitro. By miRNA sequencing, we discovered that miR-7a-5p was significantly lower in exosomes from differentiated immature neurons than those from undifferentiated NSCs. By modulating the level of miR-7a-5p, the mimic and inhibitor of miR-7a-5p could either inhibit or promote the neuronal differentiation of NSCs, respectively. Moreover, we confirmed that miR-7a-5p affected neurogenesis by directly targeting Tcf12, a transcription factor responsible for the differentiation of NSCs. The siRNA of Tcf12 inhibited neuronal differentiation of NSCs, while overexpression of Tcf12 promoted NSC differentiation. Thus, we conclude that the miR-7a-5p content in neural exosomes is essential to the fate determination of adult hippocampal neurogenesis and that miR-7a-5p directly targets Tcf12 to regulate adult hippocampal neurogenesis.

Multiscale simulations reveal architecture of NOTCH protein and ligand specific features.

NOTCH, a single-pass transmembrane protein, plays a crucial role in cell fate determination through cell-to-cell communication. It interacts with two canonical ligands, Delta-like (DLL) and Jagged (JAG), located on neighboring cells to regulate diverse cellular processes. Despite extensive studies on the functional roles of NOTCH and its ligands in cellular growth, the structural details of full-length NOTCH and its ligands remain poorly understood. In this study, we employed fragment-based modeling and multiscale simulations to study the full-length structure of the human NOTCH ectodomain, comprising 1,756 amino acids. We performed coarse-grain dynamics simulations of NOTCH in both glycosylated and non-glycosylated forms to investigate the role of glycosylation in modulating its conformational dynamics. In apo form, coarse-grained simulations revealed that glycosylated NOTCH protein can transition from an elongated structure of ∼ 86 nm from the membrane surface to a semi-compact state (∼23.81 ± 9.98 nm), which aligns with cryo-EM data. To transition from the apo form to ligand-bound forms of NOTCH, we followed an atomistic and integrative modeling approach to model the interactions between NOTCH-DLL4 and NOTCH-JAG1. Atomistic simulations of the smaller bound fragment EGF8-13 patch revealed conformational plasticity critical for NOTCH binding, while integrative modeling of full-length complexes suggested a larger binding surface than previously reported. Simulations of pathogenic mutations revealed that E360K and R448Q disrupted the NOTCH-ligand interaction surfaces, causing dissociation. In contrast, C1133Y in the Abruptex domain compromised protein stability by disrupting the domain's interaction with the ligand-binding domain in the apo form of NOTCH-ECD. These findings provide a detailed molecular understanding of NOTCH and its ligands, offering insights that could enable the development of novel therapeutic approaches to selectively target pathogenic NOTCH signaling.

Uncovering the lung cancer mechanisms through the chromosome structural ensemble characteristics and nucleation seeds.

Lung cancer is one of the most common cancers in humans. However, there is still a need to understand the underlying mechanisms of a normal cell developing into a cancer cell. Here, we develop the chromosome dynamic structural model and quantify the important characteristics of the chromosome structural ensemble of the normal lung cell and the lung cancer A549 cell. Our results demonstrate the essential relationship among the chromosome ensemble, the epigenetic marks, and the gene expressions, which suggests the linkage between chromosome structure and function. The analysis reveals that the lung cancer cell may have a higher level of relative ensemble fluctuation (micro CFI) and a higher degree of phase separation between the two compartments than the normal lung cell. In addition, the significant conformational "switching off" events (from compartment A to B) are more than the significant conformational "switching on" events during the lung cancerization. We identify "nucleation seeds" or hot spots in chromosomes, which initiate the transitions and determine the mechanisms. The hot spots and interaction network results reveal that the lung cancerization process (from normal lung to A549) and the reversion process have different mechanisms. These investigations have revealed the cell fate determination mechanism of the lung cancer process, which will be helpful for the further prevention and control of cancers.