Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mous...Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.展开更多
The TET family is well known for active DNA demethylation and plays important roles in regulating transcription,the epigenome and development.Nevertheless,previous studies using knockdown(KD)or knockout(KO)models to i...The TET family is well known for active DNA demethylation and plays important roles in regulating transcription,the epigenome and development.Nevertheless,previous studies using knockdown(KD)or knockout(KO)models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles,as well as compensatory effects among TET family members,which has made the understanding of the enzymatic role of TET not accurate enough.To solve this problem,we successfully generated mice catalytically inactive for specific Tet members(Tetm/m).We observed that,compared with the reported KO mice,mutant mice exhibited distinct developmental defects,including growth retardation,sex imbalance,infertility,and perinatal lethality.Notably,Tetm/mmouse embryonic stem cells(mESCs)were successfully established but entered an impaired developmental program,demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation.Intriguingly,Tet3,traditionally considered less critical for m ESCs due to its lower expression level,had a significant impact on the global hydroxymethylation,gene expression,and differentiation potential of mESCs.Notably,there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation.In summary,our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.展开更多
With advancements in deep space exploration missions,long-term spaceflights pose potential hazards to the reproductive and developmental functions of astronauts.Embryonic stem cells(ESCs),which are crucial to the deve...With advancements in deep space exploration missions,long-term spaceflights pose potential hazards to the reproductive and developmental functions of astronauts.Embryonic stem cells(ESCs),which are crucial to the development and growth of individual organisms,are observably altered by a microgravity environment.However,the role and mechanisms of microgravity in other activities of ESCs are still unclear.Here,mouse embryonic stem cells(mESCs)were used to investigate and understand the effect of microgravity on their activities.Combined with the SJ-10 satellite and random position machine,which were utilized for spaceflight and microgravity simulation,respectively,the bioinformatic tools were also used to assess the effect that microgravity might have on mESC activities.Based on differentially expressed genes(DEGs)analysis,114 DEGs were significantly up-regulated and 859 DEGs were significantly down-regulated in mESCs after being subjected to spaceflight.The activities,such as cell proliferation,senescence,and apoptosis,were selected and confirmed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses.It demonstrated a reduced proliferation capability of mESCs but increased the number of senescent and apoptotic cells after being subjected to simulated microgravity.Real-time polymerase chain reaction analysis of the screened activity-related DEGs demonstrated approximately consistent trends of these gene expressions in both spaceflight and simulated microgravity,as was predicted by bioinformatics analysis.Overall,these findings suggest that microgravity inhibits the proliferation of mESCs and induces senescence and apoptosis,shedding light on the impact of microgravity on the fundamental functions of mESCs in reproductive and embryonic development.展开更多
METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m^(6)A methyltransferase complex(MTC)that installs m^(6)A.Surprisingly,depletion of METTL3 or METTL14 displayed distinct effects on ...METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m^(6)A methyltransferase complex(MTC)that installs m^(6)A.Surprisingly,depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell(mESC).While comparable global hypo-methylation in RNA m^(6)A was observed in Mettl3 or Mettl14 knockout mESCs,respectively.Mettl14 knockout led to a globally decreased nascent RNA synthesis,whereas Mettl3 depletion resulted in transcription upregulation,suggesting that METTL14 might possess an mA-independent role in gene regulation.We found that METTL14 colocalizes with the repressive H3K27me3 modification.Mechanistically,METTL14,but not METTL3,binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3.Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression.The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs.This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m^(6)A,and critically impacts transcriptional regulation,stemness maintenance,and differentiation ofmESCs.展开更多
Hair pigmentation is regulated by melanocyte stem cells(MesCs)within the hair follicle.Mitochondrial dysfunction is associated with hair depigmentation,primarily due to defects in melanogenesis.However,the mechanisms ...Hair pigmentation is regulated by melanocyte stem cells(MesCs)within the hair follicle.Mitochondrial dysfunction is associated with hair depigmentation,primarily due to defects in melanogenesis.However,the mechanisms by which mitochondria support MeSCs during hair pigmentation remain obscure.In this study,we investigated the role of mitochondrial deoxyguanosine kinase(DGUOK),which provides guanosine and adenosine nucleotides for mitochondrial DNA(mtDNA)replication,in hair pigmentation and MeSCs maintenance.Dguok depleted and con-ditional knockout mice exhibit premature hair greying.This phenotype was not due to impaired melanin production by melanocytes but was associated with a significant loss of MesCs and mature melanocytes.Notably,Dguok defi-ciency decreased the expression of 13 mtDNA-encoded genes,increased the levels of reactive oxygen species(ROS)and apoptosis in MeSCs.Treatment with N-acetylcysteine(NAC),an ROS inhibitor,effectively mitigated the depig-mentation and rejuvenated the MeSCs population.These findings underscore the critical role of DGUOK in regulat-ing mtDNA integrity,which is vital for sustaining MeSCs and ensuring hair pigmentation,providing valuable insights that may inform therapeutic strategies for combating hair greying.展开更多
基金supported by the Hong Kong Research Grants Council(RGC)General Research Fund awards(662113,16101714,16100115)the ANR/RGC joint research scheme award(AHKUST601/13)+1 种基金the Hong Kong Theme-based Research Scheme award(T13-706/11-1)the Hong Kong Innovation and Technology Commission(ITCPD/17-9)
文摘Transient receptor potential canonical subfamily member 3(TRPC3) is known to be important for neural development and the formation of neuronal networks. Here, we investigated the role of TRPC3 in undifferentiated mouse embryonic stem cells(mESCs) and during the differentiation of mESCs into neurons. CRISPR/Cas9-mediated knockout(KO) of TRPC3 induced apoptosis and the disruption of mitochondrial membrane potential both in undifferentiated mESCs and in those undergoing neural differentiation. In addition, TRPC3 KO impaired the pluripotency of mESCs. TRPC3 KO also dramatically repressed the neural differentiation of mESCs by inhibiting the expression of markers for neural progenitors, neurons, astrocytes and oligodendrocytes.Taken together, our new data demonstrate an important function of TRPC3 with regards to the survival, pluripotency and neural differentiation of mESCs.
基金supported by the National Key Research and Development Program of China(2020YFA0112500,2021YFA1100300,2021YFC2700300 and 2022YFC2702200)supported by the Fundamental Research Funds for the Central Universities+2 种基金National Natural Science Foundation of China(32070857 and 32270856,and 32270858)the Science and Technology Commission of Shanghai Municipality(23JC1403700)Peak Disciplines(TypeⅣ)of Institutions of Higher Learning in Shanghai。
文摘The TET family is well known for active DNA demethylation and plays important roles in regulating transcription,the epigenome and development.Nevertheless,previous studies using knockdown(KD)or knockout(KO)models to investigate the function of TET have faced challenges in distinguishing its enzymatic and nonenzymatic roles,as well as compensatory effects among TET family members,which has made the understanding of the enzymatic role of TET not accurate enough.To solve this problem,we successfully generated mice catalytically inactive for specific Tet members(Tetm/m).We observed that,compared with the reported KO mice,mutant mice exhibited distinct developmental defects,including growth retardation,sex imbalance,infertility,and perinatal lethality.Notably,Tetm/mmouse embryonic stem cells(mESCs)were successfully established but entered an impaired developmental program,demonstrating extended pluripotency and defects in ectodermal differentiation caused by abnormal DNA methylation.Intriguingly,Tet3,traditionally considered less critical for m ESCs due to its lower expression level,had a significant impact on the global hydroxymethylation,gene expression,and differentiation potential of mESCs.Notably,there were common regulatory regions between Tet1 and Tet3 in pluripotency regulation.In summary,our study provides a more accurate reference for the functional mechanism of Tet hydroxymethylase activity in mouse development and ESC pluripotency regulation.
基金supported by the Natural Science Foundation of China(32101055,82072106,and 81901917)the Shaanxi Provincial Key R&D Program(2021GXLH-01-02,2022SF-295,and 2021SF-242)+1 种基金the Strategic Priority Research Program of the Chinese Academy of Sciences(grant numbers XDA04020202-13 and XDA04020413)the Master’s Graduate Practice and Innovation Capacity Cultivation Fund Project of the School of Life Sciences,Northwestern Polytechnical University(PF2023121 and PF2023124).
文摘With advancements in deep space exploration missions,long-term spaceflights pose potential hazards to the reproductive and developmental functions of astronauts.Embryonic stem cells(ESCs),which are crucial to the development and growth of individual organisms,are observably altered by a microgravity environment.However,the role and mechanisms of microgravity in other activities of ESCs are still unclear.Here,mouse embryonic stem cells(mESCs)were used to investigate and understand the effect of microgravity on their activities.Combined with the SJ-10 satellite and random position machine,which were utilized for spaceflight and microgravity simulation,respectively,the bioinformatic tools were also used to assess the effect that microgravity might have on mESC activities.Based on differentially expressed genes(DEGs)analysis,114 DEGs were significantly up-regulated and 859 DEGs were significantly down-regulated in mESCs after being subjected to spaceflight.The activities,such as cell proliferation,senescence,and apoptosis,were selected and confirmed by Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analyses.It demonstrated a reduced proliferation capability of mESCs but increased the number of senescent and apoptotic cells after being subjected to simulated microgravity.Real-time polymerase chain reaction analysis of the screened activity-related DEGs demonstrated approximately consistent trends of these gene expressions in both spaceflight and simulated microgravity,as was predicted by bioinformatics analysis.Overall,these findings suggest that microgravity inhibits the proliferation of mESCs and induces senescence and apoptosis,shedding light on the impact of microgravity on the fundamental functions of mESCs in reproductive and embryonic development.
基金supported by the National Institute of Health to C.H.(Nos.HG008935,ES030546,and R01ES030546)the National Natural Science Foundation of China to J.L.(No.32170595)+1 种基金the Beijing Nova Program to JL.(No.Z211100002121011)the Center for Life Sciences(CLS),the School of Life Sciences(SLS)of Peking University,the SLS-Qidong Innovation Fund,and the Li Ge Zhao Ning Life Science Junior Research Fellowship.C.H.is an investigator of the Howard Hughes Medical Institute.
文摘METTL3 and METTL14 are two components that form the core heterodimer of the main RNA m^(6)A methyltransferase complex(MTC)that installs m^(6)A.Surprisingly,depletion of METTL3 or METTL14 displayed distinct effects on stemness maintenance of mouse embryonic stem cell(mESC).While comparable global hypo-methylation in RNA m^(6)A was observed in Mettl3 or Mettl14 knockout mESCs,respectively.Mettl14 knockout led to a globally decreased nascent RNA synthesis,whereas Mettl3 depletion resulted in transcription upregulation,suggesting that METTL14 might possess an mA-independent role in gene regulation.We found that METTL14 colocalizes with the repressive H3K27me3 modification.Mechanistically,METTL14,but not METTL3,binds H3K27me3 and recruits KDM6B to induce H3K27me3 demethylation independent of METTL3.Depletion of METTL14 thus led to a global increase in H3K27me3 level along with a global gene suppression.The effects of METTL14 on regulation of H3K27me3 is essential for the transition from self-renewal to differentiation of mESCs.This work reveals a regulatory mechanism on heterochromatin by METTL14 in a manner distinct from METTL3 and independently of m^(6)A,and critically impacts transcriptional regulation,stemness maintenance,and differentiation ofmESCs.
基金supported by the National Natural Science Foundation of China No.32270846(W.N.)Applied Basic Research Foundation of Yunnan Province No.202401AT070443(H.L.)Applied Basic Research Foundation of Yunnan Province No.202501AT070205(W.N.)。
文摘Hair pigmentation is regulated by melanocyte stem cells(MesCs)within the hair follicle.Mitochondrial dysfunction is associated with hair depigmentation,primarily due to defects in melanogenesis.However,the mechanisms by which mitochondria support MeSCs during hair pigmentation remain obscure.In this study,we investigated the role of mitochondrial deoxyguanosine kinase(DGUOK),which provides guanosine and adenosine nucleotides for mitochondrial DNA(mtDNA)replication,in hair pigmentation and MeSCs maintenance.Dguok depleted and con-ditional knockout mice exhibit premature hair greying.This phenotype was not due to impaired melanin production by melanocytes but was associated with a significant loss of MesCs and mature melanocytes.Notably,Dguok defi-ciency decreased the expression of 13 mtDNA-encoded genes,increased the levels of reactive oxygen species(ROS)and apoptosis in MeSCs.Treatment with N-acetylcysteine(NAC),an ROS inhibitor,effectively mitigated the depig-mentation and rejuvenated the MeSCs population.These findings underscore the critical role of DGUOK in regulat-ing mtDNA integrity,which is vital for sustaining MeSCs and ensuring hair pigmentation,providing valuable insights that may inform therapeutic strategies for combating hair greying.
