RESUMO
The RNA modification N6-methyladenosine (m6A) has critical roles in many biological processes1,2. However, the function of m6A in the early phase of mammalian development remains poorly understood. Here we show that the m6A reader YT521-B homology-domain-containing protein 1 (YTHDC1) is required for the maintenance of mouse embryonic stem (ES) cells in an m6A-dependent manner, and that its deletion initiates cellular reprogramming to a 2C-like state. Mechanistically, YTHDC1 binds to the transcripts of retrotransposons (such as intracisternal A particles, ERVK and LINE1) in mouse ES cells and its depletion results in the reactivation of these silenced retrotransposons, accompanied by a global decrease in SETDB1-mediated trimethylation at lysine 9 of histone H3 (H3K9me3). We further demonstrate that YTHDC1 and its target m6A RNAs act upstream of SETDB1 to repress retrotransposons and Dux, the master inducer of the two-cell stage (2C)-like program. This study reveals an essential role for m6A RNA and YTHDC1 in chromatin modification and retrotransposon repression.
Assuntos
Adenosina/análogos & derivados , Inativação Gênica , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , RNA/genética , Retroelementos/genética , Adenosina/metabolismo , Animais , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Histonas/química , Histonas/metabolismo , Masculino , Camundongos , RNA/química , RNA/metabolismo , Proteínas Repressoras/metabolismoRESUMO
Somatic cells can be reprogrammed into induced pluripotent stem cells (iPSCs), which is a highly heterogeneous process. Here we report the cell fate continuum during somatic cell reprogramming at single-cell resolution. We first develop SOT to analyze cell fate continuum from Oct4/Sox2/Klf4- or OSK-mediated reprogramming and show that cells bifurcate into two categories, reprogramming potential (RP) or non-reprogramming (NR). We further show that Klf4 contributes to Cd34+/Fxyd5+/Psca+ keratinocyte-like NR fate and that IFN-γ impedes the final transition to chimera-competent pluripotency along the RP cells. We analyze more than 150,000 single cells from both OSK and chemical reprograming and identify additional NR/RP bifurcation points. Our work reveals a generic bifurcation model for cell fate decisions during somatic cell reprogramming that may be applicable to other systems and inspire further improvements for reprogramming.
Assuntos
Diferenciação Celular/genética , Linhagem da Célula/genética , Técnicas de Reprogramação Celular , Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Células-Tronco Embrionárias Murinas/fisiologia , Análise de Sequência de RNA , Análise de Célula Única , Animais , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Pluripotentes Induzidas/metabolismo , Interferon gama/genética , Interferon gama/metabolismo , Fator 4 Semelhante a Kruppel , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Fenótipo , Transdução de Sinais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
Sensing of environmental challenges, such as mechanical injury, by a single plant tissue results in the activation of systemic signaling, which attunes the plant's physiology and morphology for better survival and reproduction. As key signals, both calcium ions (Ca2+ ) and hydrogen peroxide (H2 O2 ) interplay with each other to mediate plant systemic signaling. However, the mechanisms underlying Ca2+ -H2 O2 crosstalk are not fully revealed. Our previous study showed that the interaction between glycolate oxidase and catalase, key enzymes of photorespiration, serves as a molecular switch (GC switch) to dynamically modulate photorespiratory H2 O2 fluctuations via metabolic channeling. In this study, we further demonstrate that local wounding induces a rapid shift of the GC switch to a more interactive state in systemic leaves, resulting in a sharp decrease in peroxisomal H2 O2 levels, in contrast to a simultaneous outburst of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-derived apoplastic H2 O2 . Moreover, the systemic response of the two processes depends on the transmission of Ca2+ signaling, mediated by glutamate-receptor-like Ca2+ channels 3.3 and 3.6. Mechanistically, by direct binding and/or indirect mediation by some potential biochemical sensors, peroxisomal Ca2+ regulates the GC switch states in situ, leading to changes in H2 O2 levels. Our findings provide new insights into the functions of photorespiratory H2 O2 in plant systemic acclimation and an optimized systemic H2 O2 signaling via spatiotemporal interplay between the GC switch and NADPH oxidases.
Assuntos
Oxirredutases do Álcool , Plantas , Catalase/metabolismo , Plantas/metabolismo , Oxirredutases do Álcool/metabolismo , Receptores de Glutamato , Peróxido de Hidrogênio/metabolismoRESUMO
The homeostasis of hydrogen peroxide (H2 O2 ), a key regulator of basic biological processes, is a result of the cooperation between its generation and scavenging. However, the mechanistic basis of this balance is not fully understood. We previously proposed that the interaction between glycolate oxidase (GLO) and catalase (CAT) may serve as a molecular switch that modulates H2 O2 levels in plants. In this study, we demonstrate that the GLO-CAT complex in plants exists in different states, which are dynamically interchangeable in response to various stimuli, typically salicylic acid (SA), at the whole-plant level. More crucially, changes in the state of the complex were found to be closely linked to peroxisomal H2 O2 fluctuations, which were independent of the membrane-associated NADPH oxidase. Furthermore, evidence suggested that H2 O2 channeling occurred even in vitro when GLO and CAT worked cooperatively. These results demonstrate that dynamic changes in H2 O2 levels are physically created via photorespiratory metabolic channeling in plants, and that such H2 O2 fluctuations may serve as signals that are mechanistically involved in the known functions of photorespiratory H2 O2 . In addition, our study also revealed a new way for SA to communicate with H2 O2 in plants.
Assuntos
Peróxido de Hidrogênio , Plantas , Peróxido de Hidrogênio/metabolismo , Plantas/metabolismo , HomeostaseRESUMO
BMI-1, a polycomb ring finger oncogene, is highly expressed in multiple cancer cells and is involved in cancer cell proliferation, invasion, and apoptosis. BMI-1 represents a cancer stemness marker that is associated with the regulation of stem cell self-renewal. In this study, pharmacological inhibition (PTC596) or knockdown (siRNA) of BMI-1 reduced cancer stem-like cells and enhanced cancer cell death. Mechanistically, the inhibition of BMI-1 induced the downregulation of Mcl-1 protein, but not Mcl-1 mRNA. PTC596 downregulated Mcl-1 protein expression at the post-translational level through the proteasome-ubiquitin system. PTC596 and BMI-1 siRNA induced downregulation of DUB3 deubiquitinase, which was strongly linked to Mcl-1 destabilization. Furthermore, overexpression of Mcl-1 or DUB3 inhibited apoptosis by PTC596. Taken together, our findings reveal that the inhibition of BMI-1 induces Mcl-1 destabilization through downregulation of DUB3, resulting in the induction of cancer cell death.
Assuntos
Apoptose , Benzimidazóis/farmacologia , Regulação para Baixo , Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Complexo Repressor Polycomb 1/antagonistas & inibidores , Pirazinas/farmacologia , Células A549 , Índice de Massa Corporal , Caspase 3/metabolismo , Morte Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Fragmentação do DNA , Ativação Enzimática , Células HeLa , Humanos , Células-Tronco Neoplásicas/metabolismo , RNA Interferente Pequeno/metabolismo , Ubiquitina/químicaRESUMO
BACKGROUND: Fungicides are often applied to pears before they are kept in storage facilities. The scientific application of pesticides can reduce unnecessary exposure, which in turn could benefit both humans and the environment. RESULTS: We investigated dissipation behavior and residue distribution, and conducted risk assessments for prochloraz, pyraclostrobin, and tebuconazole in pears stored under different conditions using ultra-performance liquid chromatography (UPLC). The recoveries of the three fungicides ranged from 76.5% to 114.5%, and the coefficients of variation were 1.0%-8.5%. The half-life (t1/2 ) ranges for degradation of the three fungicides in Dangshan Su pear peel were 8.8-13.9 days after storage at 25 °C and 99.0-346.6 days after storage at 2 °C. Among the three fungicides, tebuconazole had the lowest residue concentration in pear pulp (maximum of 0.226 mg·kg-1 ) and the longest half-life (≥ 231.0 days). Accordingly, among these fungicides, tebuconazole is the most suitable for the preservation of Dangshan Su pears during storage. Finally, we analyzed samples of six pear varieties from markets in China and found that the residue concentrations of the three fungicides in pear pulp and fruit met Chinese standards. CONCLUSION: The results provide a scientific basis for rationalizing the use of prochloraz, pyraclostrobin, and tebuconazole, and improving the safety of pears for eating. © 2019 Society of Chemical Industry.
Assuntos
Fungicidas Industriais/química , Resíduos de Praguicidas/química , Pyrus/química , China , Qualidade de Produtos para o Consumidor , Contaminação de Alimentos/análise , Frutas/química , Humanos , Imidazóis/química , Cinética , Estrobilurinas/química , Espectrometria de Massas em Tandem , Triazóis/químicaRESUMO
Quinoxaline-1,4-dioxides (QdNOs) are a class of quinoxaline derivatives that are widely used in humans or animals as drugs or feed additives. However, the metabolic mechanism, especially the involved enzymes, has not been reported in detail. In this study, the N-oxide reduction enzyme, porcine aldehyde oxidase SsAOX1 was identified and characterized. The SsAOX1 gene was cloned from pig liver through reverse-transcription polymerase chain reaction using degenerate primers, which encode a 147-kDa protein with typical aldehyde oxidase motifs, two [2Fe-2S] centers, a flavin adenine dinucleotide (FAD) binding domain, and a molybdenum cofactor domain. After heterologous expression in a prokaryote, purified SsAOX1 formed a functional homodimer under native conditions. Importantly, the SsAOX1 catalyzed the N-oxide reduction at the N1 position of three representative QdNOs (quinocetone, mequindox, and cyadox), which are commonly used as animal feed additives. SsAOX1 has the highest activity toward quinocetone, followed by mequindox and cyadox, with kcat/K(m) values of 1.94 ± 0.04, 1.27 ± 0.15, and 0.43 ± 0.09 minute(-1) µM(-1), respectively. However, SsAOX1 has the lowest substrate affinity for quinocetone, followed by the cyadox and mequindox, with K(m) values of 4.36 ± 0.56, 3.16 ± 0.48, and 2.96 ± 0.51 µM, respectively. In addition, using site-directed mutagenesis, we found that substitution of glycine 1019 with threonine endows SsAOX1 with N-oxide reductive activity at the N4 position. The goal of this study was to identify and characterize the N-oxide reduction enzyme for a class of veterinary drugs, QdNOs, which will aid in the elucidation of the metabolic pathways of QdNOs and will provide a theoretical basis for their administration and new veterinary drug design.
Assuntos
Aldeído Oxidase/metabolismo , Fígado/enzimologia , Quinoxalinas/metabolismo , Aldeído Oxidase/química , Aldeído Oxidase/genética , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Catálise , Dicroísmo Circular , Clonagem Molecular , Feminino , Glicina/genética , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oxirredução , Conformação Proteica , Quinoxalinas/química , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Alinhamento de Sequência , Sus scrofa , Treonina/genéticaRESUMO
Based on the innovation-driven theory and the ability-motivation-opportunity (AMO) perspective, we explore the role of intellectual property protection (IPP) in enhancing the radical technological innovation (RTI) of national research project teams (NRPT). Survey data from 336 national research project team members from universities and enterprises were used to analyze the theoretical model of this study, bringing in the chain-mediated effects of innovation milieu (IM) and group potential (GP) for analysis, as well as two-stage hybrid partial least squares structural equation modeling (PLS-SEM) and artificial neural network techniques (ANN) to evaluate the hypotheses. The empirical findings of this paper show that the strength of IPP has a positive relationship with RTI of NRPT, and that IPP is the most important predictor of this. These new findings expand the scope of innovation-driven theory and AMO theory, and provide a constructive model for NRPT to provide suggestions for the improvement of IPP system as a way to improve the realization of RTI. The results of this study can guide policymakers in strengthening IPP systems to encourage research teams to explore innovation more proactively and to facilitate the reasonable sharing and transfer of innovative outcomes. By creating a supportive innovation environment and maximizing the potential of research teams, technological breakthroughs can be achieved more effectively.
RESUMO
KMT2D, a H3K4me1 methyltransferase primarily regulating enhancers, is a leading cause of KABUKI syndrome. This multisystem disorder leads to craniofacial and cognitive abnormalities, possibly through neural crest and neuronal lineages. However, the impacted cell-of-origin and molecular mechanism of KMT2D during the development of KABUKI disease remains unknown. Here we have optimized a brain organoid model to investigate neural crest and neuronal differentiation. To pinpoint KMT2D's enhancer target, we developed a genome-wide cis-regulatory element explorer (GREE) based on single-cell multiomic integration. Single cell RNA-seq revealed that KMT2D-knockout (KO) and patient-derived organoids exhibited neural crest deformities and GABAergic overproduction. Mechanistically, GREE identified that KMT2D targets a roof-plate-like niche cell and activates the niche cell-specific WNT3A enhancer, providing the microenvironment for neural crest and neuronal development. Interestingly, KMT2D-mutated mice displayed decreased WNT3A expression in the diencephalon roof plate, indicating impaired niche cell function. Deleting the WNT3A enhancer in the organoids presented phenotypic similarities to KMT2D-depletion, emphasizing the WNT3A enhancer as the predominant target of KMT2D. Conversely, reactivating WNT signaling in KMT2D-KO rescued the lineage defects by restoring the microenvironment. Overall, our discovery of KMT2D's primary target provides insights for reconciling complex phenotypes of KABUKI syndrome and establishes a new paradigm for dissecting the mechanisms of genetic disorders from genotype to phenotype.
RESUMO
T-2 toxin is a mycotoxin that is toxic to plants, animals, and humans. However, its molecular mechanism remains unclear, especially in chickens. In this study, using 2D electrophoresis with MALDI-TOF/TOF-MS, 53 proteins were identified as up- or downregulated by T-2 toxin in chicken primary hepatocytes. Functional network analysis by ingenuity pathway analysis showed that the top network altered by T-2 toxin is associated with neurological disease, cancer, organismal injury, and abnormalities. Most of the identified proteins were associated with one of eight functional classes, including cell redox homeostasis, transcriptional or translational regulation, cell cycle or cell proliferation, stress response, lipid metabolism, transport, carbohydrate metabolism, and protein degradation. Subcellular location categorization showed that the identified proteins were predominantly located in the mitochondrion (34%) and interestingly, the expression of all the identified mitochondrial proteins was increased. Further cellular analysis showed that T-2 toxin was able to induce the ROS accumulation and could lead to an increase in mitochondrial mass and adenosine 5'-triphosphate content, which indicated that oxidative stress and mitochondrial enhancement occurred in T-2 toxin-treated cells. Overall, these results characterize the global proteomic response of chicken primary hepatocytes to T-2 toxin, which may lead to a better understanding of the molecular mechanisms underlying its toxicity.
Assuntos
Hepatócitos/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Proteoma/análise , Proteoma/efeitos dos fármacos , Toxina T-2/toxicidade , Animais , Células Cultivadas , Embrião de Galinha , Galinhas , Hepatócitos/química , Hepatócitos/metabolismo , Espaço Intracelular , Mitocôndrias/metabolismo , Proteínas/análise , Proteínas/química , Proteínas/classificação , Proteoma/química , Proteômica/métodosRESUMO
Deficiency of the histone H3K9 methyltransferase SETDB1 induces RIPK3-dependent necroptosis in mouse embryonic stem cells (mESCs). However, how necroptosis pathway is activated in this process remains elusive. Here we report that the reactivation of transposable elements (TEs) upon SETDB1 knockout is responsible for the RIPK3 regulation through both cis and trans mechanisms. IAPLTR2_Mm and MMERVK10c-int, both of which are suppressed by SETDB1-dependent H3K9me3, act as enhancer-like cis-regulatory elements and their RIPK3 nearby members enhance RIPK3 expression when SETDB1 is knockout. Moreover, reactivated endogenous retroviruses generate excessive viral mimicry, which promotes necroptosis mainly through Z-DNA-binding protein 1 (ZBP1). These results indicate TEs play an important role in regulating necroptosis.
Assuntos
Elementos de DNA Transponíveis , Células-Tronco Embrionárias Murinas , Animais , Camundongos , Elementos de DNA Transponíveis/genética , Necroptose/genética , Histona Metiltransferases , Proteínas de Ligação a RNARESUMO
Lysine-specific histone demethylase 1 (LSD1), also known as KDM1A, can remove the methyl group from lysine 4 and 9 at histone H3, which regulates transcriptional suppression and activation. Recently, high expression of LSD1 in tumors has been shown to be involved in cancer cell proliferation, metastasis, and poor prognosis. We found that SP2509, a potent and reversible inhibitor of LSD1, induced apoptosis in human renal carcinoma (Caki and ACHN) and glioma (U87MG) cells. Pharmacological inhibition and siRNA-mediated silencing of LSD1 expression effectively downregulated anti-apoptotic proteins such as Bcl-2 and Mcl-1. Ectopic expression of these proteins markedly attenuated SP2509-induced apoptosis. At a mechanistic level, we found that inhibition of LSD1 downregulated Bcl-2 at a transcriptional level. Interestingly, protein expression of Mcl-1 was modulated at a post-translation level. Our results reveal that LSD1 could induce apoptotic cell death in renal carcinoma cells through downregulation of Bcl-2 and Mcl-1.
RESUMO
N6-methyladenosine (m6A), the most abundant reversible modification on eukaryote messenger RNA, is recognized by a series of readers, including the YT521-B homology domain family (YTHDF) proteins, which are coupled to perform physiological functions. Here, we report that YTHDF2 and YTHDF3, but not YTHDF1, are required for reprogramming of somatic cells into induced pluripotent stem cells (iPSCs). Mechanistically, we found that YTHDF3 recruits the PAN2-PAN3 deadenylase complex and conduces to reprogramming by promoting mRNA clearance of somatic genes, including Tead2 and Tgfb1, which parallels the activity of the YTHDF2-CCR4-NOT deadenylase complex. Ythdf2/3 deficiency represses mesenchymal-to-epithelial transition (MET) and chromatin silencing at loci containing the TEAD motif, contributing to decreased reprogramming efficiency. Moreover, RNA interference of Tgfb1 or the Hippo signaling effectors Yap1, Taz, and Tead2 rescues Ythdf2/3-defective reprogramming. Overall, YTHDF2/3 couples RNA deadenylation and regulation with the clearance of somatic genes and provides insights into iPSC reprogramming at the posttranscriptional level.
Assuntos
RNA Mensageiro/metabolismo , Proteínas de Ligação a RNA/metabolismo , Adenosina/análogos & derivados , Adenosina/metabolismo , Animais , Reprogramação Celular/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , RNA Mensageiro/genética , Proteínas de Ligação a RNA/genéticaRESUMO
Known as a histone H3K9 methyltransferase, SETDB1 is essential for embryonic development and pluripotent inner cell mass (ICM) establishment. However, its function in pluripotency regulation remains elusive. In this study, we find that under the "ground state" of pluripotency with two inhibitors (2i) of the MEK and GSK3 pathways, Setdb1-knockout fails to induce trophectoderm (TE) differentiation as in serum/LIF (SL), indicating that TE fate restriction is not the direct target of SETDB1. In both conditions, Setdb1-knockout activates a group of genes targeted by SETDB1-mediated H3K9 methylation, including Dux. Notably, Dux is indispensable for the reactivation of 2C-like state genes upon Setdb1 deficiency, delineating the mechanistic role of SETDB1 in totipotency restriction. Furthermore, Setdb1-null ESCs maintain pluripotent marker (e.g., Nanog) expression in the 2i condition. This "ground state" Setdb1-null population undergoes rapid cell death by activating Ripk3 and, subsequently, RIPK1/RIPK3-dependent necroptosis. These results reveal the essential role of Setdb1 between totipotency and pluripotency transition.
Assuntos
Linhagem da Célula , Histona-Lisina N-Metiltransferase/metabolismo , Células-Tronco Pluripotentes/metabolismo , Trofoblastos/metabolismo , Animais , Diferenciação Celular , Células Cultivadas , Ectoderma/metabolismo , Técnicas de Inativação de Genes , Camundongos , Camundongos Endogâmicos C57BL , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteína Homeobox Nanog/metabolismo , Necroptose , Células-Tronco Pluripotentes/citologia , Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Células-Tronco Totipotentes/metabolismoRESUMO
BMP4 regulates a plethora of developmental processes, including the dorsal-ventral axis and neural patterning. Here, we report that BMP4 reconfigures the nuclear architecture during the primed-to-naive transition (PNT). We first established a BMP4-driven PNT and show that BMP4 orchestrates the chromatin accessibility dynamics during PNT. Among the loci opened early by BMP4, we identified Zbtb7a and Zbtb7b (Zbtb7a/b) as targets that drive PNT. ZBTB7A/B in turn facilitate the opening of naive pluripotent chromatin loci and the activation of nearby genes. Mechanistically, ZBTB7A not only binds to chromatin loci near to the genes that are activated, but also strategically occupies those that are silenced, consistent with a role of BMP4 in both activating and suppressing gene expression during PNT at the chromatin level. Our results reveal a previously unknown function of BMP4 in regulating nuclear architecture and link its targets ZBTB7A/B to chromatin remodelling and pluripotent fate control.
Assuntos
Proteína Morfogenética Óssea 4/metabolismo , Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/citologia , Camadas Germinativas/citologia , Células-Tronco Pluripotentes/citologia , Fatores de Transcrição/metabolismo , Animais , Blastocisto/citologia , Blastocisto/metabolismo , Proteína Morfogenética Óssea 4/genética , Diferenciação Celular , Células Cultivadas , Cromatina/genética , Proteínas de Ligação a DNA/genética , Células-Tronco Embrionárias/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos CBA , Células-Tronco Pluripotentes/metabolismo , Transdução de Sinais , Fatores de Transcrição/genéticaRESUMO
The majority of mammalian genomes are devoted to transposable elements (TEs). Whilst TEs are increasingly recognized for their important biological functions, they are a potential danger to genomic stability and are carefully regulated by the epigenetic system. However, the full complexity of this regulatory system is not understood. Here, using mouse embryonic stem cells, we show that TEs are suppressed by heterochromatic marks like H3K9me3, and are also labelled by all major types of chromatin modification in complex patterns, including bivalent activatory and repressive marks. We identified 29 epigenetic modifiers that significantly deregulated at least one type of TE. The loss of Setdb1, Ncor2, Rnf2, Kat5, Prmt5, Uhrf1, and Rrp8 caused widespread changes in TE expression and chromatin accessibility. These effects were context-specific, with different chromatin modifiers regulating the expression and chromatin accessibility of specific subsets of TEs. Our work reveals the complex patterns of epigenetic regulation of TEs.