ABSTRACT
Metabolic switch is critical for cell fate determination through metabolic functions, epigenetic modifications, and gene expression. However, the mechanisms underlying these alterations and their functional roles remain unclear. Here, we show that Plin2-mediated moderate lipid hydrolysis is critical for pluripotency of embryonic stem cells (ESCs). Upon exit from pluripotency, lipid droplet (LD)-associated protein Plin2 is recognized by Hsc70 and degraded via chaperone-mediated autophagy to facilitate LD mobilization. Enhancing lipid hydrolysis by Plin2 knockout promotes pluripotency exit, which is recovered by ATGL inhibition. Mechanistically, excessive lipid hydrolysis induces a dramatic lipidomic remodeling characterized by decreased cardiolipin and phosphatidylethanolamine, which triggers defects in mitochondrial cristae and fatty acid oxidation, resulting in reduced acetyl-CoA and histone acetylation. Our results reveal how LD mobilization is regulated and its critical role in ESC pluripotency, and indicate the mechanism linking LD homeostasis to mitochondrial remodeling and epigenetic regulation, which might shed light on development and diseases.
Subject(s)
Histones , Lipid Droplets , Lipid Droplets/metabolism , Acetylation , Histones/metabolism , Epigenesis, Genetic , Lipidomics , Perilipin-2/genetics , Perilipin-2/metabolism , LipidsABSTRACT
Somatic cell reprogramming is an ideal model for studying epigenetic regulation as it undergoes dramatic chromatin remodeling. However, a role for phosphorylation signaling in chromatin protein modifications for reprogramming remains unclear. Here, we identified mitogen-activated protein kinase kinase 6 (Mkk6) as a chromatin relaxer and found that it could significantly enhance reprogramming. The function of Mkk6 in heterochromatin loosening and reprogramming requires its kinase activity but does not depend on its best-known target, P38. We identified Gatad2b as a novel target of Mkk6 phosphorylation that acts downstream to elevate histone acetylation levels and loosen heterochromatin. As a result, Mkk6 over-expression facilitates binding of Sox2 and Klf4 to their targets and promotes pluripotency gene expression during reprogramming. Our studies not only reveal an Mkk phosphorylation mediated modulation of chromatin status in reprogramming, but also provide new rationales to further investigate and improve the cell fate determination processes.
Subject(s)
Chromatin , Heterochromatin , Cellular Reprogramming , Epigenesis, Genetic , MAP Kinase Kinase 6/genetics , MAP Kinase Kinase 6/metabolism , PhosphorylationABSTRACT
Cell fate determination as a fundamental question in cell biology has been extensively studied at different regulatory levels for many years. However, the mechanisms of multilevel regulation of cell fate determination remain unclear. Recently, we have proposed an Epigenome-Metabolome-Epigenome (E-M-E) signaling cascade model to describe the cross-over cooperation during mouse somatic cell reprogramming. In this review, we summarize the broad roles of E-M-E signaling cascade in different cell biological processes, including cell differentiation and dedifferentiation, cell specialization, cell proliferation, and cell pathologic processes. Precise E-M-E signaling cascades are critical in these cell biological processes, and it is of worth to explore each step of E-M-E signaling cascade. E-M-E signaling cascade model sheds light on and may open a window to explore the mechanisms of multilevel regulation of cell biological processes.
Subject(s)
Biological Phenomena , Epigenome , Animals , Cell Differentiation , Epigenome/genetics , Metabolome , Mice , Signal Transduction/geneticsABSTRACT
We describe a fluorescence recovery after photobleaching (FRAP) protocol for assessing the dynamics of heterochromatin/euchromatin and identifying chromatin relaxers for cell fate transition. Here, we developed a system to track heterochromatin foci with HP1α-cherry and performed FRAP assay of H1-GFP to analyze the dynamics of heterochromatin and euchromatin during somatic cell reprogramming. This protocol is used to screen factors that impact chromatin structure, which could also be used to identify chromatin relaxers and repressors in various cell fate transitions. For complete details on the use and execution of this protocol, please refer to Chen et al. (2016) and Chen et al. (2020).
Subject(s)
Chromatin Assembly and Disassembly/drug effects , Drug Evaluation, Preclinical/methods , Fluorescence Recovery After Photobleaching/methods , Animals , Cell Line , Chromatin , Chromatin Assembly and Disassembly/physiology , Chromosomal Proteins, Non-Histone/metabolism , Euchromatin , Fibroblasts/metabolism , Heterochromatin , Histones/genetics , Mice , NIH 3T3 CellsABSTRACT
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
ABSTRACT
Somatic cell reprogramming provides insight into basic principles of cell fate determination, which remain poorly understood. Here we show that the transcription factor Glis1 induces multi-level epigenetic and metabolic remodelling in stem cells that facilitates the induction of pluripotency. We find that Glis1 enables reprogramming of senescent cells into pluripotent cells and improves genome stability. During early phases of reprogramming, Glis1 directly binds to and opens chromatin at glycolytic genes, whereas it closes chromatin at somatic genes to upregulate glycolysis. Subsequently, higher glycolytic flux enhances cellular acetyl-CoA and lactate levels, thereby enhancing acetylation (H3K27Ac) and lactylation (H3K18la) at so-called 'second-wave' and pluripotency gene loci, opening them up to facilitate cellular reprogramming. Our work highlights Glis1 as a powerful reprogramming factor, and reveals an epigenome-metabolome-epigenome signalling cascade that involves the glycolysis-driven coordination of histone acetylation and lactylation in the context of cell fate determination.
Subject(s)
DNA-Binding Proteins/metabolism , Epigenome , Induced Pluripotent Stem Cells , Metabolome , Signal Transduction/genetics , Signal Transduction/physiology , Transcription Factors/metabolism , Acetyl Coenzyme A/metabolism , Animals , Cellular Reprogramming , Cellular Senescence , Chromatin Immunoprecipitation , Glucose/metabolism , Lactic Acid/metabolism , Male , Mice , Plasmids/geneticsABSTRACT
Selective elimination of mitochondria by autophagy is a critical strategy for a variety of physiological processes, including development, cell-fate determination and stress response. Although several mechanisms have been identified as responsible for selective degradation of mitochondria, such as the PINK1-PRKN/PARKIN- and receptor-dependent pathways, aspects of the mechanisms and particularly the principles underlying the selection process of mitochondria remain obscure. Here, we addressed a new selection strategy in which the selective elimination of mitochondria is dependent on organellar topology. We found that populations of mitochondria undergo different topological transformations under serum starvation, either swelling or forming donut shapes. Swollen mitochondria are associated with mitochondrial membrane potential dissipation and PRKN recruitment, which promote their selective elimination, while the donut topology maintains mitochondrial membrane potential and helps mitochondria resist autophagy. Mechanistic studies show that donuts resist autophagy even after depolarization through preventing recruitment of autophagosome receptors CALCOCO2/NDP52 and OPTN even after PRKN recruitment. Our results demonstrate topology-dependent, bifurcated mitochondrial recycling under starvation, that is swollen mitochondria undergo removal by autophagy, while donut mitochondria undergo fission and fusion cycles for reintegration. This study reveals a novel morphological selection for control of mitochondrial quality and quantity under starvation.
Subject(s)
Mitochondria/metabolism , Animals , Autophagy/drug effects , Autophagy-Related Protein 5/metabolism , Carbonyl Cyanide p-Trifluoromethoxyphenylhydrazone/pharmacology , Cell Cycle Proteins/metabolism , Cell Line, Tumor , Culture Media, Serum-Free , Humans , Membrane Potential, Mitochondrial/drug effects , Membrane Transport Proteins/metabolism , Mice , Mitochondria/ultrastructure , Mitophagy/drug effects , Ubiquitin-Protein Ligases/metabolism , Ubiquitination/drug effectsABSTRACT
The success of Yamanaka factor reprogramming of somatic cells into induced pluripotent stem cells suggests that some factor(s) must remodel the nuclei from a condensed state to a relaxed state. How factor-dependent chromatin opening occurs remains unclear. Using FRAP and ATAC-seq, we found that Oct4 acts as a pioneer factor that loosens heterochromatin and facilitates the binding of Klf4 and the expression of epithelial genes in early reprogramming, leading to enhanced mesenchymal-to-epithelial transition. A mutation in the Oct4 linker, L80A, which shows impaired interaction with the BAF complex component Brg1, is inactive in heterochromatin loosening. Oct4-L80A also blocks the binding of Klf4 and retards MET. Finally, vitamin C or Gadd45a could rescue the reprogramming deficiency of Oct4-L80A by enhancing chromatin opening and Klf4 binding. These studies reveal a cooperation between Oct4 and Klf4 at the chromatin level that facilitates MET at the cellular level and shed light into the research of multiple factors in cell fate determination.
Subject(s)
Cellular Reprogramming , Epithelial Cells/metabolism , Heterochromatin/metabolism , Histones/metabolism , Induced Pluripotent Stem Cells/cytology , Kruppel-Like Transcription Factors/metabolism , Octamer Transcription Factor-3/metabolism , Animals , Antioxidants/pharmacology , Ascorbic Acid/pharmacology , Cell Cycle Proteins/genetics , Cell Cycle Proteins/metabolism , Cell Differentiation , Cells, Cultured , DNA Helicases/genetics , DNA Helicases/metabolism , Epithelial Cells/cytology , Epithelial-Mesenchymal Transition , Fibroblasts/cytology , Fibroblasts/metabolism , Heterochromatin/genetics , Histones/genetics , Humans , Induced Pluripotent Stem Cells/metabolism , Kruppel-Like Factor 4 , Kruppel-Like Transcription Factors/genetics , Mice , Nuclear Proteins/genetics , Nuclear Proteins/metabolism , Octamer Transcription Factor-3/genetics , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
Metabolic reprogramming has emerged as a key regulator of cell fate decisions. Roles of glucose and amino acid metabolism have been extensively documented, whereas lipid metabolism in pluripotency remains largely unexplored. Using a high-coverage lipidomics approach, we reveal dynamic changes in phospholipids occurring during reprogramming and show that the CDP-ethanolamine (CDP-Etn) pathway for phosphatidylethanolamine (PE) synthesis is required at the early stage of reprogramming. Mechanistically, the CDP-Etn pathway inhibits NF-κB signaling and mesenchymal genes in a Pebp1-dependent manner, without affecting autophagy, resulting in accelerated mesenchymal-to-epithelial transition (MET) and enhanced reprogramming. Furthermore, PE binding to Pebp1 enhances the interaction of Pebp1 with IKKα/ß and reduces the phosphorylation of IKKα/ß. The CDP-Etn-Pebp1 axis is associated with EMT/MET in hepatocyte differentiation, indicating that Etn/PE is a broad-spectrum MET/EMT-regulating metabolite. Collectively, our study reveals an unforeseen connection between phospholipids, cell migration, and pluripotency and highlights the importance of phospholipids in cell fate transitions.
Subject(s)
Cell Differentiation , Epithelial-Mesenchymal Transition , Hepatocytes/metabolism , Phosphatidylethanolamines/metabolism , Pluripotent Stem Cells/metabolism , Signal Transduction , Animals , Cell Line , Cell Movement , Cytidine Diphosphate/analogs & derivatives , Cytidine Diphosphate/metabolism , Ethanolamines/metabolism , Hepatocytes/cytology , I-kappa B Kinase/metabolism , Mice , NF-kappa B/metabolism , Phosphatidylethanolamine Binding Protein/metabolism , Pluripotent Stem Cells/cytologyABSTRACT
Hexadimethrine bromide (Polybrene) was once used clinically as a heparin neutralizer and has recently found use as a promoter in virus-mediated gene therapy trials and gene transfer in research. However, the potential for tissue-specific toxicity of polybrene at low doses has been ignored so far. Here, we found that after intracerebroventricular (ICV) polybrene injection, mice showed disability of movement accompanied neural death and gliosis in brain, and in human neurons, polybrene induces concentration-dependent neuritic beading and fragmentation. Mechanistically, polybrene induces a rapid voltage-dependent calcium channel (VDCC)-mediated influx of extracellular Ca2+. The elevated cytoplasmic Ca2+ activates DRP1, which leads to mitochondrial fragmentation and metabolic dysfunction. At the same time, Ca2+ influx induces endoplasmic reticulum (ER) fragmentation and tightened associations between ER and mitochondria, which makes mitochondria prone to Ca2+ overloading and ensuing permeability transition. These results reveal an unexpected neuronal toxicity of polybrene, wherein Ca2+ influx serves as a regulator for both mitochondrial dynamics and ER-mitochondrial remodeling.
Subject(s)
Calcium/metabolism , Endoplasmic Reticulum/metabolism , Hexadimethrine Bromide/toxicity , Mitochondria/metabolism , Nerve Degeneration/chemically induced , Neurons/cytology , Neurons/drug effects , Animals , Cells, Cultured , Humans , Mice , Mice, Inbred BALB C , Mitochondrial Dynamics , Reactive Oxygen Species/metabolismABSTRACT
Reprogramming of somatic cells to induced pluripotent stem cells reconfigures chromatin modifications. Whether and how this process is regulated by signals originating in the mitochondria remain unknown. Here we show that the mitochondrial permeability transition pore (mPTP), a key regulator of mitochondrial homeostasis, undergoes short-term opening during the early phase of reprogramming and that this transient activation enhances reprogramming. In mouse embryonic fibroblasts, greater mPTP opening correlates with higher reprogramming efficiency. The reprogramming-promoting function of mPTP opening is mediated by plant homeodomain finger protein 8 (PHF8) demethylation of H3K9me2 and H3K27me3, leading to reduction in their occupancies at the promoter regions of pluripotency genes. mPTP opening increases PHF8 protein levels downstream of mitochondrial reactive oxygen species production and miR-101c and simultaneously elevates levels of PHF8's cofactor, α-ketoglutarate. Our findings represent a novel mitochondria-to-nucleus pathway in cell fate determination by mPTP-mediated epigenetic regulation.
Subject(s)
Cellular Reprogramming , Fibroblasts/metabolism , Histone Demethylases/metabolism , Histones/metabolism , Lysine/metabolism , Mitochondria/metabolism , Mitochondrial Membrane Transport Proteins/physiology , Mitochondrial Membranes/metabolism , Mouse Embryonic Stem Cells/metabolism , Transcription Factors/metabolism , Animals , HEK293 Cells , Humans , Ketoglutaric Acids/metabolism , Methylation , Mice , Mice, Inbred C57BL , MicroRNAs/metabolism , Mitochondrial Permeability Transition Pore , Reactive Oxygen Species/metabolismABSTRACT
MicroRNAs (miRNAs) play crucial roles in the establishment of pluripotent state by controlling pluripotent network. However, the molecular mechanisms controlling miRNAs during somatic cell reprogramming remain obscure. In this study, we show Gadd45a (growth arrest and DNA-damage-inducible protein 45a) enhances reprogramming by activating miR-295. Furthermore, we show that Gadd45a binds the promoter regions of miR-295. Nuclease accessibility assay indicates that Gadd45a opens the promoter regions of miR-295. Levels of H3K9Ac and H3K27Ac on the promoter regions of miR-295 were also increased. In conclusion, our results indicate that Gadd45a relaxes the promoter regions of miR-295 and promotes the expression of miR-295 during reprogramming, implying a concise mechanism of Gadd45a and miR-290 cluster cooperation in cell-fate determination.
Subject(s)
Cell Cycle Proteins/metabolism , Cellular Reprogramming/genetics , MicroRNAs/genetics , Nuclear Proteins/metabolism , Pluripotent Stem Cells/cytology , Animals , Cell Differentiation/genetics , Cell Proliferation/genetics , DNA Damage/genetics , DNA Repair/genetics , DNA-Binding Proteins/metabolism , Mice , Pluripotent Stem Cells/virology , Promoter Regions, Genetic/geneticsABSTRACT
Induced pluripotent stem cells (iPSCs) have fewer and immature mitochondria than somatic cells and mainly rely on glycolysis for energy source. During somatic cell reprogramming, somatic mitochondria and other organelles get remodeled. However, events of organelle remodeling and interaction during somatic cell reprogramming have not been extensively explored. We show that both SKP/SKO (Sox2, Klf4, Pou5f1/Oct4) and SKPM/SKOM (SKP/SKO plus Myc/c-Myc) reprogramming lead to decreased mitochondrial mass but with different kinetics and by divergent pathways. Rapid, MYC/c-MYC-induced cell proliferation may function as the main driver of mitochondrial decrease in SKPM/SKOM reprogramming. In SKP/SKO reprogramming, however, mitochondrial mass initially increases and subsequently decreases via mitophagy. This mitophagy is dependent on the mitochondrial outer membrane receptor BNIP3L/NIX but not on mitochondrial membrane potential (ΔΨm) dissipation, and this SKP/SKO-induced mitophagy functions in an important role during the reprogramming process. Furthermore, endosome-related RAB5 is involved in mitophagosome formation in SKP/SKO reprogramming. These results reveal a novel role of mitophagy in reprogramming that entails the interaction between mitochondria, macroautophagy/autophagy and endosomes.
Subject(s)
Cellular Reprogramming , Membrane Proteins/metabolism , Mitochondria/metabolism , Mitochondrial Proteins/metabolism , Mitophagy , Animals , Embryo, Mammalian/cytology , Endosomes/metabolism , Endosomes/ultrastructure , Fibroblasts/metabolism , Kruppel-Like Factor 4 , Membrane Potential, Mitochondrial , Mice , Mitochondria/ultrastructure , Models, Biological , Transcription Factors/metabolism , rab5 GTP-Binding Proteins/metabolismABSTRACT
Reprogramming of somatic cells to induced pluripotent stem cells rewrites the code of cell fate at the chromatin level. Yet, little is known about this process physically. Here, we describe a fluorescence recovery after photobleaching method to assess the dynamics of heterochromatin/euchromatin and show significant heterochromatin loosening at the initial stage of reprogramming. We identify growth arrest and DNA damage-inducible protein a (Gadd45a) as a chromatin relaxer in mouse embryonic fibroblasts, which also enhances somatic cell reprogramming efficiency. We show that residue glycine 39 (G39) in Gadd45a is essential for interacting with core histones, opening chromatin and enhancing reprogramming. We further demonstrate that Gadd45a destabilizes histone-DNA interactions and facilitates the binding of Yamanaka factors to their targets for activation. Our study provides a method to screen factors that impact on chromatin structure in live cells, and identifies Gadd45a as a chromatin relaxer.
Subject(s)
Cell Cycle Proteins/genetics , Cellular Reprogramming , Heterochromatin/metabolism , Induced Pluripotent Stem Cells/physiology , Nuclear Proteins/genetics , Animals , Cell Cycle Proteins/metabolism , Cell Differentiation/genetics , Cellular Reprogramming/genetics , DNA/genetics , DNA/metabolism , Embryonic Stem Cells/metabolism , Fibroblasts/metabolism , Glycine/metabolism , Heterochromatin/genetics , Histones/genetics , Histones/metabolism , Induced Pluripotent Stem Cells/metabolism , Mice , Nuclear Proteins/metabolism , PhotobleachingABSTRACT
Somatic cell reprogramming is accompanied by changes in lipid metabolism. While attempting to dissect the molecular mechanisms of the lipid metabolic switch during reprogramming, we found that overexpression of sterol regulatory element binding protein-1 (Srebp-1), a transcriptional factor required for lipid homeostasis, enhances reprogramming efficiency, while knockdown or pharmaceutical inhibition of Srebp-1 is inhibitory. Srebp-1 overexpression blocks the formation of partially reprogrammed cells, and functions in the early phase of reprogramming. Furthermore, Srebp-1 functions in nucleus and depends on its transcriptional activity but not its ability to bind the E-box motif and regulation of canonical targets. Mechanistically, Srebp-1 interacts with c-Myc, facilitates its binding to downstream pluripotent targets, strengthens the function of c-Myc in enhancing other Yamanaka factors' binding, and thereby promotes the expression of pluripotent genes. These results elucidate a novel role for Srebp-1 in somatic cell reprogramming and provide insights into understanding the metabolic switch during reprogramming.
Subject(s)
Cellular Reprogramming , Proto-Oncogene Proteins c-myc/metabolism , Sterol Regulatory Element Binding Protein 1/metabolism , Animals , Cellular Reprogramming/genetics , Gene Expression Regulation , Mice , Pluripotent Stem Cells/metabolism , Protein BindingABSTRACT
The mechanisms of somatic cell reprogramming have been revealed at multiple levels. However, the lack of tools to monitor different reactive oxygen species (ROS) has left their distinct signals and roles in reprogramming unknown. We hypothesized that mitochondrial flashes (mitoflashes), recently identified spontaneous bursts of mitochondrial superoxide signaling, play a role in reprogramming. Here we show that the frequency of mitoflashes transiently increases, accompanied by flash amplitude reduction, during the early stages of reprogramming. This transient activation of mitoflashes at the early stage enhances reprogramming, whereas sustained activation impairs reprogramming. The reprogramming-promoting function of mitoflashes occurs via the upregulation of Nanog expression that is associated with decreases in the methylation status of the Nanog promoter through Tet2 occupancy. Together our findings provide a previously unknown role for superoxide signaling mediated epigenetic regulation in cell fate determination.
Subject(s)
Cellular Reprogramming , Homeodomain Proteins/metabolism , Mitochondria/physiology , Animals , Cells, Cultured , DNA Methylation , DNA-Binding Proteins/metabolism , Dioxygenases , Epigenesis, Genetic , Fibroblasts/physiology , Homeodomain Proteins/genetics , Humans , Mice , Nanog Homeobox Protein , Promoter Regions, Genetic , Protein Binding , Proto-Oncogene Proteins/metabolism , Signal Transduction , Superoxides/metabolism , Up-RegulationABSTRACT
UNLABELLED: Valproic acid (VPA) is widely used to treat epilepsy, migraine, chronic headache, bipolar disorder, and as adjuvant chemotherapy, but potentially causes idiosyncratic liver injury. Alpers-Huttenlocher syndrome (AHS), a neurometabolic disorder caused by mutations in mitochondrial DNA polymerase gamma (POLG), is associated with an increased risk of developing fatal VPA hepatotoxicity. However, the mechanistic link of this clinical mystery remains unknown. Here, fibroblasts from 2 AHS patients were reprogrammed to induced pluripotent stem cells (iPSCs) and then differentiated to hepatocyte-like cells (AHS iPSCs-Hep). Both AHS iPSCs-Hep are more sensitive to VPA-induced mitochondrial-dependent apoptosis than controls, showing more activated caspase-9 and cytochrome c release. Strikingly, levels of both soluble and oligomeric optic atrophy 1, which together keep cristae junctions tight, are reduced in AHS iPSCs-Hep. Furthermore, POLG mutation cells show reduced POLG expression, mitochondrial DNA (mtDNA) amount, mitochondrial adenosine triphosphate production, as well as abnormal mitochondrial ultrastructure after differentiation to hepatocyte-like cells. Superoxide flashes, spontaneous bursts of superoxide generation, caused by opening of the mitochondrial permeability transition pore (mPTP), occur more frequently in AHS iPSCs-Hep. Moreover, the mPTP inhibitor, cyclosporine A, rescues VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. This result suggests that targeting mPTP opening could be an effective method to prevent hepatotoxicity by VPA in AHS patients. In addition, carnitine or N-acetylcysteine, which has been used in the treatment of VPA-induced hepatotoxicity, is able to rescue VPA-induced apoptotic sensitivity in AHS iPSCs-Hep. CONCLUSION: AHS iPSCs-Hep are more sensitive to the VPA-induced mitochondrial-dependent apoptotic pathway, and this effect is mediated by mPTP opening. Toxicity models in genetic diseases using iPSCs enable the evaluation of drugs for therapeutic targets.