RESUMO
In eukaryotes, DNA compacts into chromatin through nucleosomes1,2. Replication of the eukaryotic genome must be coupled to the transmission of the epigenome encoded in the chromatin3,4. Here we report cryo-electron microscopy structures of yeast (Saccharomyces cerevisiae) replisomes associated with the FACT (facilitates chromatin transactions) complex (comprising Spt16 and Pob3) and an evicted histone hexamer. In these structures, FACT is positioned at the front end of the replisome by engaging with the parental DNA duplex to capture the histones through the middle domain and the acidic carboxyl-terminal domain of Spt16. The H2A-H2B dimer chaperoned by the carboxyl-terminal domain of Spt16 is stably tethered to the H3-H4 tetramer, while the vacant H2A-H2B site is occupied by the histone-binding domain of Mcm2. The Mcm2 histone-binding domain wraps around the DNA-binding surface of one H3-H4 dimer and extends across the tetramerization interface of the H3-H4 tetramer to the binding site of Spt16 middle domain before becoming disordered. This arrangement leaves the remaining DNA-binding surface of the other H3-H4 dimer exposed to additional interactions for further processing. The Mcm2 histone-binding domain and its downstream linker region are nested on top of Tof1, relocating the parental histones to the replisome front for transfer to the newly synthesized lagging-strand DNA. Our findings offer crucial structural insights into the mechanism of replication-coupled histone recycling for maintaining epigenetic inheritance.
Assuntos
Cromatina , Replicação do DNA , Epistasia Genética , Histonas , Saccharomyces cerevisiae , Sítios de Ligação , Cromatina/química , Cromatina/genética , Cromatina/metabolismo , Cromatina/ultraestrutura , Microscopia Crioeletrônica , Replicação do DNA/genética , DNA Fúngico/biossíntese , DNA Fúngico/química , DNA Fúngico/metabolismo , DNA Fúngico/ultraestrutura , Epistasia Genética/genética , Histonas/química , Histonas/metabolismo , Histonas/ultraestrutura , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/ultraestrutura , Nucleossomos/química , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Ligação Proteica , Domínios Proteicos , Multimerização Proteica , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/ultraestruturaRESUMO
Parental histone recycling is vital for maintaining chromatin-based epigenetic information during replication, yet its underlying mechanisms remain unclear. Here, we uncover an unexpected role of histone chaperone FACT and its N-terminus of the Spt16 subunit during parental histone recycling and transfer in budding yeast. Depletion of Spt16 and mutations at its middle domain that impair histone binding compromise parental histone recycling on both the leading and lagging strands of DNA replication forks. Intriguingly, deletion of the Spt16-N domain impairs parental histone recycling, with a more pronounced defect observed on the lagging strand. Mechanistically, the Spt16-N domain interacts with the replicative helicase MCM2-7 and facilitates the formation of a ternary complex involving FACT, histone H3/H4 and Mcm2 histone binding domain, critical for the recycling and transfer of parental histones to lagging strands. Lack of the Spt16-N domain weakens the FACT-MCM interaction and reduces parental histone recycling. We propose that the Spt16-N domain acts as a protein-protein interaction module, enabling FACT to function as a shuttle chaperone in collaboration with Mcm2 and potentially other replisome components for efficient local parental histone recycling and inheritance.
Assuntos
Histonas , Proteínas de Saccharomyces cerevisiae , Fatores de Elongação da Transcrição , Cromatina/genética , DNA Helicases/genética , Chaperonas de Histonas/genética , Chaperonas de Histonas/metabolismo , Histonas/metabolismo , Chaperonas Moleculares/genética , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Fatores de Elongação da Transcrição/metabolismo , Complexos Multiproteicos/metabolismoRESUMO
In eukaryotes, the packaging of genomic DNA into chromatin plays a critical role in gene regulation. However, the dynamic organization of chromatin fibers and its regulatory mechanisms remain poorly understood. Using single-molecule force spectroscopy, we reveal that the tetranucleosomes-on-a-string appears as a stable secondary structure during hierarchical organization of chromatin fibers. The stability of the tetranucleosomal unit is attenuated by histone chaperone FACT (facilitates chromatin transcription) in vitro. Consistent with in vitro observations, our genome-wide analysis further shows that FACT facilitates gene transcription by destabilizing the tetranucleosomal unit of chromatin fibers in yeast. Additionally, we found that the linker histone H1 not only enhances the stability but also facilitates the folding and unfolding kinetics of the outer nucleosomal wrap. Our study demonstrates that the tetranucleosome is a regulatory structural unit of chromatin fibers beyond the nucleosome and provides crucial mechanistic insights into the structure and dynamics of chromatin fibers during gene transcription.
Assuntos
DNA Fúngico/química , Proteínas de Ligação a DNA/química , Regulação Fúngica da Expressão Gênica , Proteínas de Grupo de Alta Mobilidade/química , Histonas/química , Nucleossomos/genética , Proteínas de Saccharomyces cerevisiae/química , Transcrição Gênica , Fatores de Elongação da Transcrição/química , Sequência de Aminoácidos , DNA Fúngico/genética , DNA Fúngico/metabolismo , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Grupo de Alta Mobilidade/genética , Proteínas de Grupo de Alta Mobilidade/metabolismo , Histonas/genética , Histonas/metabolismo , Modelos Moleculares , Nucleossomos/metabolismo , Nucleossomos/ultraestrutura , Dobramento de Proteína , Multimerização Proteica , Estabilidade Proteica , Estrutura Secundária de Proteína , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/ultraestrutura , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Imagem Individual de Molécula , Fatores de Elongação da Transcrição/genética , Fatores de Elongação da Transcrição/metabolismoRESUMO
The histone chaperone facilitates chromatin transactions (FACT) functions in various DNA transactions. How FACT performs these multiple functions remains largely unknown. Here, we found, for the first time, that the N-terminal domain of its Spt16 subunit interacts with the Set3 histone deacetylase complex (Set3C) and that FACT and Set3C function in the same pathway to regulate gene expression in some settings. We observed that Spt16-G132D mutant proteins show defects in binding to Set3C but not other reported FACT interactors. At the permissive temperature, induction of the GAL1 and GAL10 genes is reduced in both spt16-G132D and set3Δ cells, whereas transient upregulation of GAL10 noncoding RNA (ncRNA), which is transcribed from the 3' end of the GAL10 gene, is elevated. Mutations that inhibit GAL10 ncRNA transcription reverse the GAL1 and GAL10 induction defects in spt16-G132D and set3Δ mutant cells. Mechanistically, set3Δ and FACT (spt16-G132D) mutants show reduced histone acetylation and increased nucleosome occupancy at the GAL1 promoter under inducing conditions and inhibition of GAL10 ncRNA transcription also partially reverses these chromatin changes. These results indicate that FACT interacts with Set3C, which in turn prevents uncontrolled GAL10 ncRNA expression and fine-tunes the expression of GAL genes upon a change in carbon source.
Assuntos
Cromatina/metabolismo , Galactoquinase/metabolismo , Regulação Fúngica da Expressão Gênica , Histona Desacetilases/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , Transcrição Gênica , RNA não Traduzido/metabolismo , Transativadores , Ativação TranscricionalRESUMO
Generation of single-stranded DNA (ssDNA) is required for the template strand formation during DNA replication. Replication Protein A (RPA) is an ssDNA-binding protein essential for protecting ssDNA at replication forks in eukaryotic cells. While significant progress has been made in characterizing the role of the RPA-ssDNA complex, how RPA is loaded at replication forks remains poorly explored. Here, we show that the Saccharomyces cerevisiae protein regulator of Ty1 transposition 105 (Rtt105) binds RPA and helps load it at replication forks. Cells lacking Rtt105 exhibit a dramatic reduction in RPA loading at replication forks, compromised DNA synthesis under replication stress, and increased genome instability. Mechanistically, we show that Rtt105 mediates the RPA-importin interaction and also promotes RPA binding to ssDNA directly in vitro, but is not present in the final RPA-ssDNA complex. Single-molecule studies reveal that Rtt105 affects the binding mode of RPA to ssDNA These results support a model in which Rtt105 functions as an RPA chaperone that escorts RPA to the nucleus and facilitates its loading onto ssDNA at replication forks.
Assuntos
Genoma Fúngico , Instabilidade Genômica , Modelos Biológicos , Chaperonas Moleculares/metabolismo , Proteína de Replicação A/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Fúngico/genética , DNA Fúngico/metabolismo , DNA de Cadeia Simples/genética , DNA de Cadeia Simples/metabolismo , Carioferinas/genética , Carioferinas/metabolismo , Chaperonas Moleculares/genética , Proteína de Replicação A/genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
Diabetic nephropathy (DN) is the primary cause of end-stage renal disease, and renal tubular cell dysfunction contributes to the pathogenesis of many kidney diseases. Our previous study demonstrated that dual-specificity protein phosphatase 1 (DUSP1) reduced hyperglycemia-mediated mitochondrial damage; however, its role in hyperglycemia-driven dysfunction of tubular cells is still not fully understood. In this study, we found that DUSP1 is reduced in human proximal tubular epithelial (HK-2) cells under high-glucose conditions. DUSP1 overexpression in HK-2 cells partially restored autophagic flux, improved mitochondrial function, and reduced reactive oxygen species generation and cell apoptosis under high-glucose conditions. Surprisingly, overexpressing DUSP1 abolished the decrease in mitochondrial parkin expression caused by high-glucose stimulation. In addition, knockdown of parkin in HK-2 cells reversed the effects of DUSP1 overexpression on mitophagy and apoptosis under high-glucose conditions. Overall, these data indicate that DUSP1 plays a defensive role in the pathogenesis of DN by restoring parkin-mediated mitophagy, suggesting that it may be considered a prospective therapeutic strategy for the amelioration of DN.
Assuntos
Nefropatias Diabéticas/patologia , Fosfatase 1 de Especificidade Dupla/metabolismo , Túbulos Renais/patologia , Mitocôndrias/patologia , Mitofagia , Ubiquitina-Proteína Ligases/metabolismo , Linhagem Celular , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Fosfatase 1 de Especificidade Dupla/genética , Humanos , Túbulos Renais/metabolismo , Mitocôndrias/metabolismo , Regulação para CimaRESUMO
Excessive mitochondrial fission has been identified as the pathogenesis of diabetic nephropathy (DN), although the upstream regulatory signal for mitochondrial fission activation in the setting of DN remains unknown. In the current study, we found that dual-specificity protein phosphatase-1 (DUSP1) was actually downregulated by chronic hyperglycemia stimulus. Lower DUSP1 expression was associated with glucose metabolism disorder, renal dysfunction, kidney hypertrophy, renal fibrosis, and glomerular apoptosis. At the molecular level, defective DUSP1 expression activated JNK pathway, and the latter selectively opened mitochondrial fission by modulating mitochondrial fission factor (Mff) phosphorylation. Excessive Mff-related mitochondrial fission evoked mitochondrial oxidative stress, promoted mPTP opening, exacerbated proapoptotic protein leakage into the cytoplasm, and finally initiated mitochondria-dependent cellular apoptosis in the setting of diabetes. However, overexpression of DUSP1 interrupted Mff-related mitochondrial fission, reducing hyperglycemia-mediated mitochondrial damage and thus improving renal function. Overall, we have shown that DUSP1 functions as a novel malefactor in diabetic renal damage that mediates via modifying Mff-related mitochondrial fission. Thus, finding strategies to regulate the balance of the DUSP1-JNK-Mff signaling pathway and mitochondrial homeostasis may be a therapeutic target for treating diabetic nephropathy in clinical practice.
Assuntos
Nefropatias Diabéticas/genética , Fosfatase 1 de Especificidade Dupla/genética , Proteínas de Membrana/genética , Dinâmica Mitocondrial/genética , Proteínas Mitocondriais/genética , Animais , Apoptose/genética , Nefropatias Diabéticas/patologia , Regulação da Expressão Gênica/genética , Humanos , Rim/metabolismo , Rim/patologia , MAP Quinase Quinase 4/genética , Sistema de Sinalização das MAP Quinases/genética , Camundongos , Mitocôndrias/genética , Mitofagia/genética , Fosforilação , Transdução de Sinais/genéticaRESUMO
Single-stranded DNA (ssDNA) is widely generated during DNA metabolisms including DNA replication, repair and recombination and is susceptible to digestion by nucleases and secondary structure formation. It is vital for DNA metabolism and genome stability that ssDNA is protected and stabilized, which are performed by the major ssDNA-binding protein, and replication protein A (RPA) in these processes. In addition, RPA-coated ssDNA also serves as a protein-protein-binding platform for coordinating multiple events during DNA metabolisms. However, little is known about whether and how the formation of RPA-ssDNA platform is regulated. Here we highlight our recent study of a novel RPA-binding protein, Regulator of Ty1 transposition 105 (Rtt105) in Saccharomyces cerevisiae, which regulates the RPA-ssDNA platform assembly at replication forks. We propose that Rtt105 functions as an "RPA chaperone" during DNA replication, likely also promoting the assembly of RPA-ssDNA platform in other processes in which RPA plays a critical role.
Assuntos
DNA de Cadeia Simples/genética , DNA/metabolismo , Proteína de Replicação A/genética , DNA/genética , Reparo do DNA/genética , Replicação do DNA/genética , Instabilidade Genômica/genética , Humanos , Chaperonas Moleculares/genética , Proteínas de Ligação a RNA/genética , Recombinação Genética , Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/genéticaRESUMO
We propose a novel fiber chemical sensing system based on reflective Mach-Zehnder interference with temperature calibration for the measurement of Cd2+ in aqueous solution. The sensing system has high measurement sensitivity of Cd2+ with an estimated minimum detection limit of 4×10-7 mol/L at a spectral resolution of 0.02 nm and with long-term stability. The fiber sensing head is prepared by coating a sensing membrane on a fused tapering single-mode fiber. The thiourea group of the sensing membrane has an effective combination effect on Cd2+. Disturbance from ambient temperature fluctuation on the measurement of Cd2+ concentration can be eliminated with the fiber Bragg grating.
RESUMO
BACKGROUND/AIMS: Amniotic fluid stem cells (AFSCs) transplantation is a promising therapeutic strategy for diabetic nephropathy. Sirtuin3 (SIRT3) is a novel mitochondrial protective factor. In the present study, we aimed to investigate whether SIRT3 protects against hyperglycemia-induced AFSCs damage and enhances the therapeutic efficiency of AFSCs in diabetic nephropathy. METHODS: To establish the diabetic nephropathy model, db/ db mice were used. AFSCs were obtained and transplanted into the kidney tissue of db/ db mice. Gain-of-function assay with SIRT3 overexpression was performed in AFSCs via adenoviral transfections (Ad/SIRT3). Cellular viability and apoptosis were measured via MTT, TUNEL assay and western blotting. Mitochondrial function was assessed via JC1 staining, mPTP opening assay, mitochondrial respiratory function analysis, and immunofluorescence analysis of cyt-c. Mitophagy was assessed via western blotting and immunofluorescence analysis. Renal histopathology and morphometric analysis were conducted via H&E, Masson and PASM staining. Kidney function was detected via ELISA assay, western blotting and qPCR. RESULTS: SIRT3 was downregulated in AFSCs under high glucose stimulation, where its expression was positively correlated with AFSCs survival and proliferation. Regaining SIRT3 activated mitophagy protecting AFSCs against high glucose-induced apoptosis via preserving mitochondrial function. Transplanting SIRT3-overexpressing AFSCs in db/db mice improved the abnormalities in glucose metabolic parameters, including the levels of glucose, insulin, C-peptide, HbA1c and inflammatory markers. In addition, the engraftment of SIRT3-modified AFSCs also reversed renal function, decreased renal hypertrophy, and ameliorated renal histological changes in db/db mice. Functional studies confirmed that SIRT3-modified AFSCs promoted glomerulus survival and reduced renal fibrosis. CONCLUSION: Collectively, our results demonstrate that AFSCs may be a promising therapeutic treatment for ameliorating diabetes and the development of diabetic nephropathy and that the overexpression of SIRT3 in AFSCs may further increase the efficiency of stem cell-based therapy.
Assuntos
Nefropatias Diabéticas/patologia , Mitocôndrias/metabolismo , Mitofagia/fisiologia , Sirtuína 3/metabolismo , Trifosfato de Adenosina/metabolismo , Líquido Amniótico/citologia , Animais , Apoptose/efeitos dos fármacos , Nitrogênio da Ureia Sanguínea , Peptídeo C/metabolismo , Células Cultivadas , Nefropatias Diabéticas/terapia , Nefropatias Diabéticas/veterinária , Regulação para Baixo/efeitos dos fármacos , Vetores Genéticos/genética , Vetores Genéticos/metabolismo , Glucose/farmacologia , Hemoglobinas Glicadas/metabolismo , Insulina/metabolismo , Rim/metabolismo , Rim/patologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Obesos , Espécies Reativas de Oxigênio/metabolismo , Sirtuína 3/genética , Transplante de Células-Tronco , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
BACKGROUND/AIMS: Disrupted mitochondrial dynamics, including excessive mitochondrial fission and mitophagy arrest, has been identified as a pathogenic factor in diabetic nephropathy (DN), although the upstream regulatory signal for mitochondrial fission activation and mitophagy arrest in the setting of DN remains unknown. METHODS: Wild-type (WT) mice and NR4A1 knockout (NR4A1-KO) mice were used to establish a DN model. Mitochondrial fission and mitophagy were evaluated by western blotting and immunofluorescence. Mitochondrial function was assessed by JC-1 staining, the mPTP opening assay, immunofluorescence and western blotting. Renal histopathology and morphometric analyses were conducted via H&E, Masson and PASM staining. Kidney function was evaluated via ELISA, western blotting and qPCR. RESULTS: In the present study, we found that nuclear receptor subfamily 4 group A member 1 (NR4A1) was actually activated by a chronic hyperglycemic stimulus. Higher NR4A1 expression was associated with glucose metabolism disorder, renal dysfunction, kidney hypertrophy, renal fibrosis, and glomerular apoptosis. At the molecular level, increased NR4A1 expression activated p53, and the latter selectively stimulated mitochondrial fission and inhibited mitophagy by modulating Mff and Parkin transcription. Excessive Mff-related mitochondrial fission caused mitochondrial oxidative stress, promoted mPTP opening, exacerbated proapoptotic protein leakage into the cytoplasm, and finally initiated mitochondria-dependent cellular apoptosis in the setting of diabetes. In addition, defective Parkin-mediated mitophagy repressed cellular ATP production and failed to correct the uncontrolled mitochondrial fission. However, NR4A1 knockdown interrupted the Mff-related mitochondrial fission and recused Parkin-mediated mitophagy, reducing the hyperglycemia-mediated mitochondrial damage and thus improving renal function. CONCLUSION: Overall, we have shown that NR4A1 functions as a novel malefactor in diabetic renal damage and operates by synchronously enhancing Mff-related mitochondrial fission and repressing Parkin-mediated mitophagy. Thus, finding strategies to regulate the balance of the NR4A1-p53 signaling pathway and mitochondrial homeostasis may be a therapeutic option for treating diabetic nephropathy in clinical practice.
Assuntos
Proteínas de Membrana/metabolismo , Dinâmica Mitocondrial/fisiologia , Proteínas Mitocondriais/metabolismo , Mitofagia/fisiologia , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/genética , Ubiquitina-Proteína Ligases/metabolismo , Animais , Diabetes Mellitus Experimental/induzido quimicamente , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/patologia , Nefropatias Diabéticas/etiologia , Nefropatias Diabéticas/patologia , Nefropatias Diabéticas/veterinária , Humanos , Rim/patologia , Potencial da Membrana Mitocondrial , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Poro de Transição de Permeabilidade Mitocondrial , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/antagonistas & inibidores , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases/antagonistas & inibidores , Ubiquitina-Proteína Ligases/genéticaRESUMO
During lagging strand chromatin replication, multiple Okazaki fragments (OFs) require processing and nucleosome assembly, but the mechanisms linking these processes remain unclear. Here, using transmission electron microscopy and rapid degradation of DNA ligase Cdc9, we observed flap structures accumulated on lagging strands, controlled by both Pol δ's strand displacement activity and Fen1's nuclease digestion. The distance between neighboring flap structures exhibits a regular pattern, indicative of matured OF length. While fen1Δ or enhanced strand displacement activities by polymerase δ (Pol δ; pol3exo-) minimally affect inter-flap distance, mutants affecting replication-coupled nucleosome assembly, such as cac1Δ and mcm2-3A, do significantly alter it. Deletion of Pol32, a subunit of DNA Pol δ, significantly increases this distance. Mechanistically, Pol32 binds to histone H3-H4 and is critical for nucleosome assembly on the lagging strand. Together, we propose that Pol32 establishes a connection between nucleosome assembly and the processing of OFs on lagging strands.
Assuntos
DNA Polimerase III , DNA , Histonas , Nucleossomos , Proteínas de Saccharomyces cerevisiae , Saccharomyces cerevisiae , Nucleossomos/metabolismo , Histonas/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , DNA Polimerase III/metabolismo , DNA Polimerase III/genética , Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/genética , DNA/metabolismo , Replicação do DNA , Ligação Proteica , DNA Polimerase Dirigida por DNARESUMO
The differentiation of embryonic stem cells (ESCs) begins with the transition from the naive to the primed state. The formative state was recently established as a critical intermediate between the two states. Here, we demonstrate the role of the histone chaperone FACT in regulating the naive-to-formative transition. We found that the Q265K mutation in the FACT subunit SSRP1 increased the binding of FACT to histone H3-H4, impaired nucleosome disassembly in vitro, and reduced the turnover of FACT on chromatin in vivo. Strikingly, mouse ESCs harboring this mutation showed elevated naive-to-formative transition. Mechanistically, the SSRP1-Q265K mutation enriched FACT at the enhancers of formative-specific genes to increase targeted gene expression. Together, these findings suggest that the turnover of FACT on chromatin is crucial for regulating the enhancers of formative-specific genes, thereby mediating the naive-to-formative transition. This study highlights the significance of FACT in fine-tuning cell fate transition during early development.
RESUMO
Transcriptional control of marginal zone (MZ) and follicular (FO) B cell development remains incompletely understood. The transcription factor, IFN regulatory factor (IRF)8, is known to play important roles in the differentiation of early B cells. In this article, we demonstrate that IRF8 is also required for normal development of MZ and FO B cells. Mice with a conventional knockout of Irf8 (IRF8(-/-)) or a point mutation in the IRF association domain of IRF8 had increased numbers of MZ B cells. To determine the B cell-intrinsic effects of IRF8 deficiency, we generated mice with a conditional allele of Irf8 crossed with CD19-Cre mice (designated IRF8-conditional knockout [CKO]). These mice had enlarged MZ and increased numbers of MZ and FO B cells compared with controls. The FO B cells of CKO mice exhibited reduced expression of CD23 and moderately increased expression of CD21. Gene-expression profiling showed that increased B cell production in IRF8-CKO mice was associated with changes in expression of genes involved in regulation of transcription, signaling, and inflammation. Functional studies showed that IRF8-CKO mice generated normal Ab responses to T-independent and T-dependent Ags. Thus, IRF8 controls the expansion and maturation of MZ and FO B cells but has little effect on B cell function.
Assuntos
Subpopulações de Linfócitos B/imunologia , Subpopulações de Linfócitos B/metabolismo , Fatores Reguladores de Interferon/fisiologia , Baço/citologia , Baço/imunologia , Animais , Subpopulações de Linfócitos B/patologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Feminino , Deleção de Genes , Fatores Reguladores de Interferon/deficiência , Fatores Reguladores de Interferon/genética , Contagem de Linfócitos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Mutagênese Insercional , Mutação Puntual , Estrutura Terciária de Proteína/genética , Baço/patologiaRESUMO
Diabetic nephropathy (DN) is the direct cause of end-stage renal disease, and nephritic inflammation plays a role in its growth and advancement. Aberrant expression of long non-coding RNAs (lncRNAs) correlates with many diseases, including DN. In this study, we investigated whether lncRNA small nucleolar RNA host gene 1 (Snhg1) was mechanistically involved in inflammation and mesangial cell (MC) proliferation in DN. We found that Snhg1 was significantly upregulated in DN renal tissues and high glucose (HG)-treated MCs. Overexpression of Snhg1 promoted inflammatory cytokine expression in MCs and MC proliferation under low-glucose conditions; meanwhile, Snhg1 knockdown suppressed inflammatory cytokine production and MC proliferation under HG conditions. Mechanistically, Snhg1 was found to directly bind miR-27b, thereby preventing the miRNA from binding its target KDM6B mRNA. Furthermore, miR-27b overexpression recapitulated the inhibitory effects of Snhg1 knockdown, whereas restoration of Snhg1 expression attenuated the function of miR-27b in MCs under HG conditions. Taken together, these results indicate that suppression of Snhg1 inhibited HG-induced inflammation and proliferation of MCs by regulating the miR-27b/KDM6B axis.
Assuntos
Nefropatias Diabéticas , MicroRNAs , RNA Longo não Codificante , Camundongos , Animais , RNA Longo não Codificante/genética , Células Mesangiais/metabolismo , Proliferação de Células , MicroRNAs/genética , MicroRNAs/metabolismo , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/metabolismo , Inflamação/induzido quimicamente , Inflamação/metabolismo , Citocinas/metabolismo , Glucose/toxicidade , Glucose/metabolismoRESUMO
Epigenetic regulations play crucial roles in the pathogenesis of metabolic-associated fatty liver disease; therefore, elucidating the biological functions of differential miRNAs helps us to understand the pathogenesis. Herein, we discovered miR-337-3p was decreased in patients with NAFLD from Gene Expression Omnibus dataset, which was replicated in various cell and mouse models with lipid disorders. Subsequently, overexpression of miR-337-3p in vivo could ameliorate hepatic lipid accumulation, reduce fasting blood glucose, and improve insulin resistance. Meanwhile, we determined miR-337-3p might influence multiple genes involved in glycolipid metabolism through mass spectrometry detection, bioinformatics analysis, and experimental verification. Finally, we selected HMGCR as a representative example to investigate the molecular mechanism of miR-337-3p regulating these genes, where the seed region of miR-337-3p bound to 3'UTR of HMGCR to inhibit HMGCR translation. In conclusion, we discovered a new function of miR-337-3p in glycolipid metabolism and that might be a new therapeutic target of MAFLD.
RESUMO
The successful assembly of nucleosomes following DNA replication is critically important for both the inheritance of epigenetic information and the maintenance of genome integrity. This process, termed DNA replication-coupled (RC) nucleosome assembly, requires that DNA replication and nucleosome assembly function in a highly coordinated fashion to transmit both genetic and epigenetic information. In this chapter, we describe a genome-wide method for measuring nucleosome occupancy patterns on nascent strands, which we have termed Replication-Intermediate Nucleosome Mapping (ReIN-Map), to monitor the RC nucleosome assembly level genome-wide in vivo. This method takes advantage of next-generation sequencing and in vivo labeling of newly synthesized DNA using a thymidine analogue, 5-bromo-2'-deoxyuridine (BrdU), and involves parallel analyses of the nucleosome formation using micrococcal nuclease (MNase) digestion of chromatin (MNase-seq) and of the newly synthesized DNA levels using sonication shearing of chromatin s (Sonication-seq). Replicated chromatin was enriched by immunoprecipitation using antibodies against BrdU (BrdU-IP), which is incorporated into DNA during DNA synthesis; the DNA is then subjected to strand-specific sequencing.
Assuntos
Mapeamento Cromossômico , Replicação do DNA , Genoma , Genômica , Nucleossomos/metabolismo , Ciclo Celular/genética , Cromatina/genética , Mapeamento Cromossômico/métodos , Fragmentação do DNA , DNA de Cadeia Simples , Análise de Dados , Biblioteca Gênica , Genômica/métodos , Sequenciamento de Nucleotídeos em Larga Escala , Hidrolases/química , Hidrolases/metabolismo , Desnaturação de Ácido Nucleico , Fosforilação , SonicaçãoRESUMO
PU.1, IKAROS, E2A, EBF, and PAX5 comprise a transcriptional network that orchestrates B-cell lineage specification, commitment, and differentiation. Here we identify interferon regulatory factor 8 (IRF8) as another component of this complex, and show that it also modulates lineage choice by hematopoietic stem cells (HSCs). IRF8 binds directly to an IRF8/Ets consensus sequence located in promoter regions of Sfpi1 and Ebf1, which encode PU.1 and EBF, respectively, and is associated with transcriptional repression of Sfpi1 and transcriptional activation of Ebf1. Bone marrows of IRF8 knockout mice (IRF8(-/-)) had significantly reduced numbers of pre-pro-B cells and increased numbers of myeloid cells. Although HSCs of IRF8(-/-) mice failed to differentiate to B220(+) B-lineage cells in vitro, the defect could be rescued by transfecting HSCs with wild-type but not with a signaling-deficient IRF8 mutant. In contrast, overexpression of IRF8 in HSC-differentiated progenitor cells resulted in growth inhibition and apoptosis. We also found that IRF8 was expressed at higher levels in pre-pro-B cells than more mature B cells in wild-type mice. Together, these results indicate that IRF8 modulates lineage choice by HSCs and is part of the transcriptional network governing B-cell lineage specification, commitment, and differentiation.
Assuntos
Linfócitos B/metabolismo , Diferenciação Celular/fisiologia , Células-Tronco Hematopoéticas/metabolismo , Fatores Reguladores de Interferon/metabolismo , Elementos de Resposta/fisiologia , Transdução de Sinais/fisiologia , Animais , Linfócitos B/citologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Células-Tronco Hematopoéticas/química , Fator de Transcrição Ikaros/genética , Fator de Transcrição Ikaros/metabolismo , Fatores Reguladores de Interferon/genética , Antígenos Comuns de Leucócito/genética , Antígenos Comuns de Leucócito/metabolismo , Camundongos , Camundongos Knockout , Fator de Transcrição PAX5/genética , Fator de Transcrição PAX5/metabolismo , Ligação Proteica/fisiologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Nucleosome assembly during DNA replication is tightly coupled to ongoing DNA synthesis. This process, termed DNA replication-coupled (RC) nucleosome assembly, is essential for chromatin replication and has a great impact on both genome stability maintenance and epigenetic inheritance. This review discusses a set of recent findings regarding the role of replisome components contributing to RC nucleosome assembly. Starting with a brief introduction to the factors involved in nucleosome assembly and some aspects of the architecture of the eukaryotic replisome, we discuss studies from yeast to mammalian cells and the interactions of replisome components with histones and histone chaperones. We describe the proposed functions of replisome components during RC nucleosome assembly and discuss their impacts on histone segregation and implications for epigenetic inheritance.
RESUMO
OBJECTIVE: To isolate mesenchymal stem cells (MSCs) from second-trimester amniotic fluid using an improved two-stage culture protocol, and to induce these MSCs into neuron-like cells. METHODS: An improved two-stage culture protocol for MSCs from amniotic fluid was developed. MSCs from amniotic fluid were induced to differentiate with beta-mercaptoethanol into neuron-like cells. Flow cytometry, reverse transcription-polymerase chain reaction (RT-PCR) and immunocytochemistry were employed for analysis of the phenotypic characteristics of the cultured MSCs from amniotic fluid. RESULTS: MSCs from amniotic fluid were successfully isolated, cultured and enriched without interfering with the routine process of fetal karyotyping. Flow cytometry analysis showed that they were positive for CD44, CD29, and CD105, but negative for CD45, CD34, or human leucocyte antigen-DR (HLA-DR). Importantly, a subpopulation of transcription factor Oct-4 positive cells could be detectable in the cultured MSCs from amniotic fluid. Moreover, MSCs from amniotic fluid obtained by two-stage culture protocol showed higher proliferation rate [(15.0+/-2.3)% vs. (10.0+/-1.8)%] and higher Oct-4 positive cell rate [(1.2+/-0.3)% vs. (0.9+/-0.2)%, both P<0.05]. After the induction, MSCs from amniotic fluid displayed processes that formed extensive networks. Positive cells for neurone specific enolase (NSE) constituted (54.76+/-3.65)%, and glial fibrillary acidic protein (GFAP) (36.28+/-4.27)%, respectively. CONCLUSION: We demonstrate that human pluripotent MSCs are present in second-trimester amniotic fluid. The two-stage culture protocol could be a kind of simple one with high performance and it does not interfere with the routine fetal karyotyping. MSCs from amniotic fluid can be induced to differentiate into neuron-like cells in vitro by beta-mercaptoethanol in optimal medium.