These findings establish IRAK4 as a regulator that constrains cellular plasticity potentially by coordinating chromatin accessibility and cell cycle dynamics, which may facilitate the establishment of multi-lineage states.
Abstract
Chemical reprogramming holds transformative potential for regenerative medicine. However, the regulatory mechanisms governing cell fate transitions are not well understood. Here, we identify Interleukin-1 Receptor-Associated Kinase 4 (IRAK4) as a barrier to multi-lineage reprogramming. Pharmacological inhibition of IRAK4 enhances the reprogramming of mouse embryonic fibroblasts (MEFs) through a chemically activated multi-lineage priming (CaMP) state and extraembryonic endoderm (XEN)-like intermediates, increasing colony formation, and the expression of core XEN regulators (Sox17, Gata4, Sall4, and Foxa2). Genetic knockdown of Irak4 similarly accelerates reprogramming, whereas its overexpression blocks cell fate transitions. IRAK4 inhibition enhances chromatin accessibility and reshapes cell cycle dynamics, characterized by G0/G1 shortening and G2/M lengthening, potentially contributing to multi-lineage state establishment. Furthermore, IRAK4 suppression enhances the direct conversion of MEFs to neuron-like and hepatocyte-like cells, which exhibit enhanced functional maturity, including increased glycogen storage and improved detoxification capacity. Our findings establish IRAK4 as a regulator that constrains cellular plasticity potentially by coordinating chromatin accessibility and cell cycle dynamics. IRAK4 is a barrier to chemical reprogramming and lineage conversion. Pharmacological or genetic inhibition of IRAK4 enhances multi-lineage priming, accelerates reprogramming, and improves the functional maturity of directly converted neuron-like and hepatocyte-like cells. IRAK4 inhibition promotes the chemical reprogramming of mouse embryonic fibroblasts through a CaMP state and XEN-like intermediates, increasing colony formation and the expression of core XEN regulators (Sox17, Gata4, Sall4, Foxa2). IRAK4 suppression enhances chromatin accessibility and reshapes cell cycle dynamics, which may facilitate the establishment of multi-lineage states. Inhibition of IRAK4 boosts direct conversion to neuron-like and hepatocyte-like cells. IRAK4 inhibition promotes the chemical reprogramming of mouse embryonic fibroblasts through a CaMP state and XEN-like intermediates, increasing colony formation and the expression of core XEN regulators (Sox17, Gata4, Sall4, Foxa2). IRAK4 suppression enhances chromatin accessibility and reshapes cell cycle dynamics, which may facilitate the establishment of multi-lineage states. Inhibition of IRAK4 boosts direct conversion to neuron-like and hepatocyte-like cells. IRAK4 is a barrier to chemical reprogramming and lineage conversion. Pharmacological or genetic inhibition of IRAK4 enhances multi-lineage priming, accelerates reprogramming, and improves the functional maturity of directly converted neuron-like and hepatocyte-like cells.
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, Xinwei Wu et al.· Journal of Biological Chemis...· 0 citations
Differentiated cells maintain their identity through active mechanisms that suppress alternative cell fates, but disrupting these barriers can enhance direct reprogramming for organ repair. Among the regulators of cell fate stability, glycosylation-associated genes have emerged as barriers to cardiac reprogramming. Here we show that carbohydrate sulfotransferases are central fate-stabilizing regulators, with CHST7 acting through CD44 to control nuclear JUNB levels, chromatin binding, and downstream transcriptional activity. Integrated RNA-seq and ATAC-seq analyses reveal that CHST7 maintains open chromatin at JUNB- and CTCF-enriched loci while restricting accessibility at MEF2C-enriched regions, collectively reinforcing fibroblast identity and suppressing cardiac fate acquisition. We further identify PIP4K2C as a downstream effector whose inhibition enhances cardiac reprogramming efficiency and improves myocardial repair in vivo. These findings define a sulfotransferase-dependent barrier to cell fate conversion with therapeutic implications for heart regeneration. Cells maintain stable identities that resist conversion into other cell types. Here, the authors show that the sulfotransferase CHST7 stabilizes fibroblast fate via CD44/JUNB signalling, and that its inhibition enhances cardiac reprogramming and improves heart repair.
Michaela R. Romero, S. Murphy, Yuzhu Chang et al.· Nature Communications· 0 citations
Objectives The transition between naive and primed pluripotency is governed by dynamic signaling networks and transcriptional circuits. While the janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathway is the master driver of naive pluripotency, the intrinsic negative feedback mechanisms that restrict its activation in primed epiblast stem cells (EpiSCs) remain incompletely defined. This study aimed to characterize the functional role of Suppressor of Cytokine Signaling 3 (SOCS3) in the primed-to-naive pluripotency transition. Methods CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated protein 9) -mediated Socs3 knockout (KO) was generated in mouse EpiSCs, followed by primed-to-naive reprogramming induction in 2i/LIF [LIF (leukemia inhibitory factor), PD0325901 and CHIR99021) culture system. Molecular and phenotypic changes were evaluated via quantative real time PCR (qRT-PCR), Western blot, flow cytometry and immunofluorescence. Multilineage differentiation as-says were performed to verify pluripotency, and the STAT3-specific inhibitor Stattic was used to confirm the pathway dependence of the reprogramming phenotype. Results Socs3 was highly expressed in naive embryonic stem cells (ESCs) but minimally detected in EpiSCs. Socs3 deletion uncoupled the JAK/STAT3 negative feedback loop, causing sustained STAT3 Tyr705 phosphorylation that drove rapid and successful primed-to-naive conversion. The resulting reprogrammed naive ESCs (rnESCs) reactivated the core naive transcriptional network and acquired multilineage differentiation potential. Socs3 deficiency also delayed exit from naive pluripotency, and Stattic treatment completely abrogated Socs3 KO-mediated reprogramming. Conclusion SOCS3 acts as a pivotal inducible barrier to the primed-to-naive pluripotency transition. Eliminating SOCS3-mediated negative regulation to sustain STAT3 activation is an effective strategy to overcome stem cell reprogramming barriers, providing a key target for the precise manipulation of pluripotent stem cell (PSC) fate.
Renhong Lu, Suoni Huang, Mingyang Du et al.· Frontiers in Genetics· 0 citations
Summary Direct reprogramming of human fibroblasts into hematopoietic stem cells (HSCs) offers a promising strategy for generating autologous cells to treat blood and immune disorders. Current protocols are limited by low efficiency and insufficient tools for evaluating reprogramming outcomes. Although functional assays are the standard for confirming cell identity, they require fully reprogrammed cells, limiting their utility during protocol development. To address this, we assembled a single-cell transcriptomic reference atlas of hematopoietic reprogramming and tested an algorithmically predicted transcription factor recipe for HSC induction. Long-read single-cell RNA sequencing of CD34+ reprogrammed cells revealed progressive loss of fibroblast identity alongside induction of early hematopoietic and endothelial programs, with reference-atlas benchmarking placing reprogrammed cells in an intermediate transcriptomic state between fibroblasts, endothelial cells, and HSCs. Isoform-level analysis further revealed transcriptional remodeling not captured by gene-level analyses. This experimental-computational framework offers a generalizable strategy for characterizing partially reprogrammed states and guiding optimization of reprogramming protocols.
Neuroblast cells play a pivotal role in adult neurogenesis. However, the detailed mechanisms underlying the acquisition of pluripotency or the process of differentiation remain unknown. Herein, the role of the AT-hook protein AKNA in regulating pluripotency and stemness in neuroblastoma cells is demonstrated through gene knockdown, immunofluorescence, chromatin immunoprecipitation (ChIP), localization of AKNA, signaling interactions, and transcriptional activity. AKNA was detected primarily in the nucleus during the induction of pluripotency and was retained in the cytosol by FAK signaling during differentiation. Loss of AKNA disrupts both the stemness and differentiation potential of neuroblastoma cells. In the nucleus, colocalization and physical association between AKNA and KDM6B promote H3K27me3 demethylation and subsequently H3K27ac deposition on the promoters of stemness genes, triggering their transcription. These findings establish AKNA as a critical regulator of neuroblast cell fate determinants in association with epigenetic modifiers and signaling pathways, offering potential targets for neuroblastoma therapies and regenerative medicine for neurodegenerative diseases.
Soumen Manna, R. Kirtana, Tirthankar Baral et al.· Biochimica et biophysica act...· 0 citations
This review evaluating how the antagonistic interplay between Polycomb and Trithorax complexes, chromatin modifier activity, and enhancer reprogramming affects lineage identity and enables multi-lineage plasticity in cancer underscores epigenetic deregulation as a primary driver of lineage plasticity and intratumoral heterogeneity.
Ezgi Boyvatlı, Burcu Akman, E. Bağırsakçı et al.· FEBS Letters· 0 citations
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