The role of Netrin-1 is identified as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naïve pluripotency.
Abstract
We investigated the role of Netrin-1 (NTN1) in human naïve pluripotency using complementary loss- and gain-of-function approaches. In primate embryos and human embryonic stem cells (hESCs), Netrin-1 expression is associated with the naïve pluripotent state. Disruption of NTN1 had no detectable effect on hESCs maintained on murine embryonic fibroblasts. However, under sub-optimal culture conditions, NTN1-knockout cells exhibited compromised naïve pluripotency, which was rescued with feeder cells overexpressing Netrin-1. Netrin-1 overexpression in hESCs accelerated acquisition of the naïve state and markedly increased resistance to differentiation. These effects were accompanied by extensive epigenetic remodeling, including H3K27ac and H2K27me3. Proteomic and phospho-proteomic analyses further revealed rapid Netrin-1-dependent alterations in pathways controlling cell adhesion, signaling, and chromatin regulation. Together, these findings extend the role of Netrin-1 beyond its established functions and identify it as a coordinator of extracellular cues, intracellular signaling, and nuclear regulatory mechanisms that support human naïve pluripotency.
Embryonic stem cells (ESCs) are refractory to inflammatory stimuli but acquire inflammatory competence upon differentiation, yet the underlying mechanisms remain unclear. Here, we report that H2BK5ac regulates the acquisition of inflammatory potential in mouse ESCs during differentiation. H2BK5ac levels increased at the regulatory regions of inflammatory genes upon differentiation, and this modification was required for their transcriptional upregulation. An acetylation-deficient H2BK5A mutant impaired the upregulation of inflammatory genes, whereas an acetylation-mimicking H2BK5Q mutant had the opposite effect. We identified p300 and HDAC3 as the acetyltransferase and deacetylase, respectively, that control H2BK5ac: p300 protein levels increased upon differentiation and its depletion reduced H2BK5ac levels, whereas loss of HDAC3 increased H2BK5ac levels despite its unchanged protein levels. H2BK5Q overexpression in mouse embryos increased embryonic mortality and inflammatory cytokine secretion, consistent with its pro-inflammatory effect. Thus, our study reveals H2BK5ac as a regulator that establishes inflammatory potential in mouse ESCs during differentiation.
Yan Xi, Shuai Pei, Qian-Qian Lou et al.· Cell Reports· 0 citations
It is demonstrated that coordinated regulation of both cytosolic and mitochondrial translation during totipotent-like reprogramming is offered, offering a new perspective for understanding cell fate determination.
Lingci Huang, Jun Zhang, Xin-Wei Wu et al.· Journal of Biological Chemis...· 0 citations
Mouse embryonic stem cell (ESC) cultures contain a rare subpopulation of two-cell-like cells (2CLCs) that transiently reactivate a two-cell embryo-like transcriptional program characteristic of zygotic genome activation (ZGA), including the endogenous retrovirus MERVL and Zscan4, and thereby regain a totipotent-like state. Polycomb repressive complex 1 (PRC1)-mediated H2AK119ub1 has been implicated in restraining entry into the 2C-like state through Pcgf6, yet the factors governing exit from this state and loss of totipotency remain poorly defined. Here, we show that among the six Pcgf paralogs, Pcgf5, which is most prominently upregulated in 2CLCs and forms an MERVL-driven chimeric transcript (Pcgf5MT2C_Mm) during ZGA in 2-cell embryos, controls exit from the 2C-like state in mouse ESCs. Using a reporter ESC line carrying MERVL-tdTomato and Zscan4c-EGFP (MtZG), we manipulated Pcgf5 dosage bidirectionally. Doxycycline (Dox)-inducible overexpression (OE) of Pcgf5 reduced the double-positive (DP) 2C-like population. Conversely, CRISPR-mediated knockout (KO) of Pcgf5 by targeting a common exon shared by all Pcgf5 variants (hereafter, total Pcgf5) increased the DP population. Time-lapse imaging directly confirmed that these changes reflected genuine differences in duration of the 2C-like state: OE shortened, whereas KO prolonged, the time cells spent in this state. These findings reveal that a Polycomb group factor controls not only entry into but also exit from the 2C-like state.
Satoshi Mashiko, Shinnosuke Honda, Shunta Ikeda et al.· Biochemical and Biophysical...· 0 citations
It is demonstrated that TUG1, which is highly expressed in tumors, regulates early embryonic development in mice and is transformed from a cancer-specific effector to a critical regulator of the ZGA, raising the possibility that related regulatory principles may operate in other biological contexts.
Jian-Wu Wang, Guang Yang, Qingbo Yang et al.· Cellular and Molecular Life...· 0 citations
Neural progenitor cell differentiation is a complex process requiring the proper integration of instructive and permissive factors. Instructive cues including signaling molecules and transcription factor networks have been well studied in this context, but permissive factors such as cell homeostasis have not. Cell homeostasis is critical to support the health and stability of a cell and enable the cell to act on instructive differentiation cues. Our study investigates a homeostasis protein, FAF2, and its function in neural progenitor cells. FAF2 is an adaptor protein involved in endoplasmic reticulum (ER) associated degradation to remove misfolded proteins and restore ER homeostasis. Here we show that knocking out Faf2 in neural progenitor cells results in increased ER stress signature at the protein and transcription level, indicating a conserved functional role in neural progenitor cells. Induced neural differentiation of FAF2 deletion cells shows a failure of neurite development but RNA-seq indicates genes that support neural differentiation are induced. Reducing ER stress in FAF2 knockout cells with a small molecule inhibitor can rescue neural differentiation, providing evidence that excess ER stress contributes to the inhibited differentiation. Taken together, these results reveal that FAF2 is a critical protein in neural progenitor cells for the maintenance of ER homeostasis and execution of neural differentiation.
A. Kakebeen, Leo F. Dunphy, Heather K. Hazen et al.· Frontiers in Cell and Develo...· 0 citations
Summary Deciphering the mechanisms that govern lineage commitment of human pluripotent stem cells (hPSCs) is essential for optimizing differentiation strategies. Here, we uncover the role of the mitochondrial protein OCIAD2 in hPSC fate decisions. We show that OCIAD2 is transiently upregulated during mesendoderm differentiation in response to TGFβ/Activin A signaling. OCIAD2 depletion disrupted TGFβ and WNT signaling, impaired mesendoderm specification, and resulted in an incomplete epithelial-to-mesenchymal transition (EMT). Furthermore, OCIAD2 loss reduced mitochondrial dynamics and fatty acid oxidation, indicating dysregulated lipid metabolism. Transcriptome and biochemical analysis of OCIAD2-knockout (KO) and -overexpressing (OV) hESCs identified OCIAD2 as a positive regulator of TGFβ signaling. Supplementation with acetate alongside Wnt3a increased SMAD2/3 acetylation and restored mesendoderm differentiation in KO. We propose that OCIAD2 integrates metabolic cues with TGFβ signaling to regulate EMT. This study provides new insight into how mitochondrial proteins influence lineage commitment, highlighting broader implications for developmental biology and tumor progression.
Kajal Kamat, M. Inamdar· Stem Cell Reports· 0 citations
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