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1.
Nat Commun ; 12(1): 5541, 2021 09 20.
Artigo em Inglês | MEDLINE | ID: mdl-34545097

RESUMO

Human Immunodeficiency Virus (HIV-1) produces a persistent latent infection. Control of HIV-1 using combination antiretroviral therapy (cART) comes at the cost of life-shortening side effects and development of drug-resistant HIV-1. An ideal and safer therapy should be deliverable in vivo and target the stable epigenetic repression of the virus, inducing a stable "block and lock" of virus expression. Towards this goal, we developed an HIV-1 promoter-targeting Zinc Finger Protein (ZFP-362) fused to active domains of DNA methyltransferase 3 A to induce long-term stable epigenetic repression of HIV-1. Cells were engineered to produce exosomes packaged with RNAs encoding this HIV-1 repressor protein. We find here that the repressor loaded anti-HIV-1 exosomes suppress virus expression and that this suppression is mechanistically driven by DNA methylation of HIV-1 in humanized NSG mouse models. The observations presented here pave the way for an exosome-mediated systemic delivery platform of therapeutic cargo to epigenetically repress HIV-1 infection.


Assuntos
Repressão Epigenética/genética , Exossomos/metabolismo , HIV-1/genética , Animais , Encéfalo/patologia , Encéfalo/virologia , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Exossomos/ultraestrutura , Regulação Viral da Expressão Gênica , Vetores Genéticos/metabolismo , Células HEK293 , Infecções por HIV/virologia , Humanos , Lentivirus/metabolismo , Leucócitos Mononucleares/metabolismo , Camundongos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sequências Repetidas Terminais/genética , Carga Viral , Dedos de Zinco
2.
Cells ; 10(9)2021 08 26.
Artigo em Inglês | MEDLINE | ID: mdl-34571854

RESUMO

PAX7 transcription factor plays a crucial role in embryonic myogenesis and in adult muscles in which it secures proper function of satellite cells, including regulation of their self renewal. PAX7 downregulation is necessary for the myogenic differentiation of satellite cells induced after muscle damage, what is prerequisite step for regeneration. Using differentiating pluripotent stem cells we documented that the absence of functional PAX7 facilitates proliferation. Such action is executed by the modulation of the expression of two proteins involved in the DNA methylation, i.e., Dnmt3b and Apobec2. Increase in Dnmt3b expression led to the downregulation of the CDK inhibitors and facilitated cell cycle progression. Changes in Apobec2 expression, on the other hand, differently impacted proliferation/differentiation balance, depending on the experimental model used.


Assuntos
Desaminases APOBEC/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteínas Musculares/metabolismo , Fator de Transcrição PAX7/metabolismo , Desaminases APOBEC/genética , Animais , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciação Celular/genética , Proliferação de Células/genética , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Feminino , Expressão Gênica/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Desenvolvimento Muscular/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Fator de Transcrição PAX7/genética , Fator de Transcrição PAX7/fisiologia , Células-Tronco Pluripotentes/metabolismo , Células-Tronco Pluripotentes/fisiologia , Células Satélites de Músculo Esquelético/metabolismo
3.
Nat Commun ; 12(1): 5716, 2021 09 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588438

RESUMO

Mutations in SPOP E3 ligase gene are reportedly associated with genome-wide DNA hypermethylation in prostate cancer (PCa) although the underlying mechanisms remain elusive. Here, we demonstrate that SPOP binds and promotes polyubiquitination and degradation of histone methyltransferase and DNMT interactor GLP. SPOP mutation induces stabilization of GLP and its partner protein G9a and aberrant upregulation of global DNA hypermethylation in cultured PCa cells and primary PCa specimens. Genome-wide DNA methylome analysis shows that a subset of tumor suppressor genes (TSGs) including FOXO3, GATA5, and NDRG1, are hypermethylated and downregulated in SPOP-mutated PCa cells. DNA methylation inhibitor 5-azacytidine effectively reverses expression of the TSGs examined, inhibits SPOP-mutated PCa cell growth in vitro and in mice, and enhances docetaxel anti-cancer efficacy. Our findings reveal the GLP/G9a-DNMT module as a mediator of DNA hypermethylation in SPOP-mutated PCa. They suggest that SPOP mutation could be a biomarker for effective treatment of PCa with DNA methylation inhibitor alone or in combination with taxane chemotherapeutics.


Assuntos
Metilação de DNA/genética , Antígenos de Histocompatibilidade/metabolismo , Histona-Lisina N-Metiltransferase/metabolismo , Proteínas Nucleares/genética , Neoplasias da Próstata/genética , Proteínas Repressoras/genética , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Azacitidina/farmacologia , Azacitidina/uso terapêutico , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/efeitos dos fármacos , Docetaxel/farmacologia , Docetaxel/uso terapêutico , Regulação para Baixo/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Supressores de Tumor , Humanos , Masculino , Camundongos , Mutação , Proteínas Nucleares/metabolismo , Neoplasias da Próstata/tratamento farmacológico , Neoplasias da Próstata/patologia , Estabilidade Proteica/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Proteínas Repressoras/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
4.
FASEB J ; 35(9): e21847, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34405464

RESUMO

Mounting evidence demonstrates that paternal diet programs offspring metabolism. However, the contribution of a pre-conception paternal high protein (HP) diet to offspring metabolism, gut microbiota, and epigenetic changes remains unclear. Here we show that paternal HP intake in Sprague Dawley rats programs protective metabolic outcomes in offspring. Compared to paternal high fat/sucrose (HF/S), HP diet improved body composition and insulin sensitivity and improved circulating satiety hormones and cecal short-chain fatty acids compared to HF/S and control diet (P < .05). Further, using 16S rRNA gene sequencing to assess gut microbial composition, we observed increased alpha diversity, distinct bacterial clustering, and increased abundance of Bifidobacterium, Akkermansia, Bacteroides, and Marvinbryantia in HP fathers and/or male and female adult offspring. At the epigenetic level, DNMT1and 3b expression was altered intergenerationally. Our study identifies paternal HP diet as a modulator of gut microbial composition, epigenetic markers, and metabolic function intergenerationally.


Assuntos
Composição Corporal , Dieta Rica em Proteínas , Epigênese Genética , Pai , Microbioma Gastrointestinal , Insulina/metabolismo , Exposição Paterna , Tecido Adiposo/metabolismo , Adiposidade , Envelhecimento , Animais , Peso Corporal , DNA (Citosina-5-)-Metiltransferases/metabolismo , Dieta Hiperlipídica , Sacarose na Dieta , Ingestão de Energia , Metabolismo Energético , Ácidos Graxos/metabolismo , Feminino , Fertilidade , Teste de Tolerância a Glucose , Hormônios/metabolismo , Resistência à Insulina , Fígado/metabolismo , Masculino , Tamanho do Órgão , Gravidez , Pequeno RNA não Traduzido/metabolismo , Ratos , Ratos Sprague-Dawley , Resposta de Saciedade , Desmame
5.
BMC Cancer ; 21(1): 914, 2021 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-34380460

RESUMO

BACKGROUND: Gastric cancer (GC) has an unwelcoming prognosis when diagnosed at an advanced stage. The purpose of this study was to examine the expression of myosin heavy chain 11 (MYH11) in GC and mechanisms related. METHODS: The MYH11 expression in GC was investigated via the SangerBox platform. MYH11 expression in GC tissues and cell lines was examined by immunohistochemistry, RT-qPCR, and western blot. The relationship between MYH11 expression and patients' prognosis was analyzed. The effects of MYH11 on the biological behaviors of GC cells were investigated by gain-of-function experiments. Bioinformatics analysis was used to find genes with relevance to MYH11 expression in GC. The relationship was verified by luciferase and ChIP-qPCR assays, followed by rescue assay validation. The causes of MYH11 downregulation in GC were verified by quantitative methylation-specific PCR. Finally, the effect of MYH11 on tumor growth was examined. RESULTS: MYH11 was downregulated in GC and predicted poor prognoses. MYH11 reverted the malignant phenotype of GC cells. MYH11 repressed the TNFRSF14 expression by binding to the TNFRSF14 promoter. TNFRSF14 reversed the inhibitory effect of MYH11 on the malignant phenotype of GC cells. The methylation of the MYH11 promoter was elevated in GC, which was correlated with the elevated DNMT3B in GC. Overexpression of DNMT3B repressed transcription of MYH11 by promoting its methylation. Also, MYH11 upregulation inhibited tumor growth. CONCLUSION: DNMT3B inhibits MYH11 expression by promoting its DNA methylation, thereby attenuating the repressive effect of MYH11 on the transcriptional of TNFRSF14 and promoting the progression of GC.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Epistasia Genética , Regulação Neoplásica da Expressão Gênica , Cadeias Pesadas de Miosina/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patologia , Adulto , Idoso , Animais , Linhagem Celular Tumoral , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/metabolismo , Modelos Animais de Doenças , Progressão da Doença , Feminino , Xenoenxertos , Humanos , Imuno-Histoquímica , Masculino , Camundongos , Pessoa de Meia-Idade , Cadeias Pesadas de Miosina/metabolismo , Estadiamento de Neoplasias , Regiões Promotoras Genéticas , Ligação Proteica , Neoplasias Gástricas/metabolismo , Carga Tumoral
6.
Biomolecules ; 11(8)2021 07 23.
Artigo em Inglês | MEDLINE | ID: mdl-34439754

RESUMO

Increasing energy expenditure through activation of brown fat thermogenesis is a promising therapeutic strategy for the treatment of obesity. Epigenetic regulation has emerged as a key player in regulating brown fat development and thermogenic program. Here, we aimed to study the role of DNA methyltransferase 3b (Dnmt3b), a DNA methyltransferase involved in de novo DNA methylation, in the regulation of brown fat function and energy homeostasis. We generated a genetic model with Dnmt3b deletion in brown fat-skeletal lineage precursor cells (3bKO mice) by crossing Dnmt3b-floxed (fl/fl) mice with Myf5-Cre mice. Female 3bKO mice are prone to diet-induced obesity, which is associated with decreased energy expenditure. Dnmt3b deficiency also impairs cold-induced thermogenic program in brown fat. Surprisingly, further RNA-seq analysis reveals a profound up-regulation of myogenic markers in brown fat of 3bKO mice, suggesting a myocyte-like remodeling in brown fat. Further motif enrichment and pyrosequencing analysis suggests myocyte enhancer factor 2C (Mef2c) as a mediator for the myogenic alteration in Dnmt3b-deficient brown fat, as indicated by decreased methylation at its promoter. Our data demonstrate that brown fat Dnmt3b is a key regulator of brown fat development, energy metabolism and obesity in female mice.


Assuntos
Tecido Adiposo Marrom/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fator Regulador Miogênico 5/metabolismo , Obesidade/metabolismo , Adipócitos Marrons/metabolismo , Tecido Adiposo Branco/metabolismo , Alelos , Animais , Peso Corporal , Cruzamentos Genéticos , DNA (Citosina-5-)-Metiltransferases/genética , Modelos Animais de Doenças , Metabolismo Energético , Epigênese Genética , Feminino , Resistência à Insulina , Camundongos , Camundongos Knockout , Células Musculares/metabolismo , RNA-Seq , Termogênese
7.
J Mol Biol ; 433(19): 167186, 2021 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-34375615

RESUMO

DNA interacting enzymes recognize their target sequences embedded in variable flanking sequence context. The influence of flanking sequences on enzymatic activities of DNA methyltransferases (DNMTs) can be systematically studied with "deep enzymology" approaches using pools of double-stranded DNA substrates, which contain target sites in random flanking sequence context. After incubation with DNMTs and bisulfite conversion, the methylation states and flanking sequences of individual DNA molecules are determined by NGS. Deep enzymology studies with different human and mouse DNMTs revealed strong influences of flanking sequences on their CpG and non-CpG methylation activity and the structures of DNMT-DNA complexes. Differences in flanking sequence preferences of DNMT3A and DNMT3B were shown to be related to the prominent role of DNMT3B in the methylation of human SATII repeat elements. Mutational studies in DNMT3B discovered alternative interaction networks between the enzyme and the DNA leading to a partial equalization of the effects of different flanking sequences. Structural studies in DNMT1 revealed striking correlations between enzymatic activities and flanking sequence dependent conformational changes upon DNA binding. Correlation of the biochemical data with cellular methylation patterns demonstrated that flanking sequence preferences are an important parameter that influences genomic DNA methylation patterns together with other mechanisms targeting DNMTs to genomic sites.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , DNA/genética , DNA/metabolismo , Animais , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferases/química , Humanos , Camundongos , Modelos Moleculares , Conformação Proteica
8.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-34299205

RESUMO

Gain and loss of DNA methylation in cells is a dynamic process that tends to achieve an equilibrium. Many factors are involved in maintaining the balance between DNA methylation and demethylation. Previously, it was shown that methyl-DNA protein Kaiso may attract NCoR, SMRT repressive complexes affecting histone modifications. On the other hand, the deficiency of Kaiso resulted in reduced methylation of ICR in H19/Igf2 locus and Oct4 promoter in mouse embryonic fibroblasts. However, nothing is known about how Kaiso influences DNA methylation at the genome level. Here we show that deficiency of Kaiso led to whole-genome hypermethylation, using Kaiso deficient human renal cancer cell line obtained via CRISPR/CAS9 genome editing. However, Kaiso serves to protect genic regions, enhancers, and regions with a low level of histone modifications from demethylation. We detected hypomethylation of binding sites for Oct4 and Nanog in Kaiso deficient cells. Kaiso immunoprecipitated with de novo DNA methyltransferases DNMT3a/3b, but not with maintenance methyltransferase DNMT1. Thus, Kaiso may attract methyltransferases to surrounding regions and modulate genome methylation in renal cancer cells apart from being methyl DNA binding protein.


Assuntos
Metilação de DNA , Impressão Genômica , Fator de Crescimento Insulin-Like II/metabolismo , Região de Controle de Locus Gênico , RNA Longo não Codificante/genética , Fatores de Transcrição/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patologia , Edição de Genes , Células HEK293 , Humanos , Fator de Crescimento Insulin-Like II/genética , Regiões Promotoras Genéticas , Fatores de Transcrição/antagonistas & inibidores , Fatores de Transcrição/genética , Proteínas com Motivo Tripartido/genética , Proteínas com Motivo Tripartido/metabolismo , Ubiquitina-Proteína Ligases/genética , Ubiquitina-Proteína Ligases/metabolismo
9.
Cell Death Dis ; 12(7): 667, 2021 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-34215719

RESUMO

Long non-coding RNA (lncRNA) is receiving increasing attention in embryonic stem cells (ESCs) research. However, the roles of lncRNA in the differentiation of ESCs into pacemaker-like cells are still unclear. Therefore, the present study aims to explore the roles and mechanisms of lncRNA in the differentiation of ESCs into pacemaker-like cells. ESCs were cultured and induced differentiation to pacemaker-like cells. RNA sequencing was used to identify the differential expression lncRNAs during the differentiation of ESCs into pacemaker-like cells. Cell morphology observation, flow cytometry, quantitative real-time polymerase chain reaction, western blot, and immunofluorescence were used to detect the differentiation of ESCs into pacemaker-like cells. LncRNA and genes overexpression or knockdown through transfected adenovirus in the differentiation process. The fluorescence in situ hybridization (FISH) detected the lncRNA location in the differentiated ESCs. Luciferase reporter gene assay, methylation-specific PCR, chromatin immunoprecipitation assay, and RNA immunoprecipitation assay were performed to reveal the mechanism of lncRNA-regulating HCN4 expression. Rescue experiments were used to confirm that lncRNA regulates the differentiation of ESCs into pacemaker-like cells through HCN4. We cultured the ESCs and induced the differentiation of ESCs into pacemaker-like cells successfully. The expression of lncRNA RCPCD was significantly decreased in the differentiation of ESCs into pacemaker-like cells. Overexpression of RCPCD inhibited the differentiation of ESCs into pacemaker-like cells. RCPCD inhibited the expression of HCN4 by increasing HCN4 methylation at the promoter region through DNMT1, DNMT2, and DNMT3. RCPCD inhibited the differentiation of ESCs into pacemaker-like cells by inhibiting the expression of HCN4. Our results confirm the roles and mechanism of lncRNA RCPCD in the differentiation of ESCs into pacemaker-like cells, which could pave the path for the development of a cell-based biological pacemaker.


Assuntos
Relógios Biológicos , Diferenciação Celular , Metilação de DNA , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Embrionárias Murinas/metabolismo , Miócitos Cardíacos/metabolismo , Regiões Promotoras Genéticas , RNA Longo não Codificante/genética , Nó Sinoatrial/metabolismo , Animais , Células Cultivadas , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação para Baixo , Regulação da Expressão Gênica no Desenvolvimento , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/metabolismo , Camundongos , RNA Longo não Codificante/metabolismo , Nó Sinoatrial/citologia
10.
Nat Protoc ; 16(8): 4004-4030, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34244697

RESUMO

The integration of DNA methylation and transcriptional state within single cells is of broad interest. Several single-cell dual- and multi-omics approaches have been reported that enable further investigation into cellular heterogeneity, including the discovery and in-depth study of rare cell populations. Such analyses will continue to provide important mechanistic insights into the regulatory consequences of epigenetic modifications. We recently reported a new method for profiling the DNA methylome and transcriptome from the same single cells in a cancer research study. Here, we present details of the protocol and provide guidance on its utility. Our Smart-RRBS (reduced representation bisulfite sequencing) protocol combines Smart-seq2 and RRBS and entails physically separating mRNA from the genomic DNA. It generates paired epigenetic promoter and RNA-expression measurements for ~24% of protein-coding genes in a typical single cell. It also works for micro-dissected tissue samples comprising hundreds of cells. The protocol, excluding flow sorting of cells and sequencing, takes ~3 d to process up to 192 samples manually. It requires basic molecular biology expertise and laboratory equipment, including a PCR workstation with UV sterilization, a DNA fluorometer and a microfluidic electrophoresis system.


Assuntos
DNA/metabolismo , Análise de Célula Única , Sequência de Aminoácidos , Antibacterianos/farmacologia , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Doxiciclina/farmacologia , Epigenoma , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma
11.
Psychopharmacology (Berl) ; 238(11): 3107-3118, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34328516

RESUMO

RATIONALE: Cannabis sativa is the most widely used drug by adolescents globally. The recreational use of synthetic cannabinoids by teenagers has also grown in recent years. Despite the wrong perception that exposure to these drugs does not cause harm, repeated exposure to cannabinoids at early stages of life compromises important maturation processes and brain development. Chronic early cannabinoid use has been related to a higher risk of psychiatric outcomes, including cocaine addiction. Evidence suggests that exposure to natural and synthetic cannabinoids during adolescence modifies molecular and behavioral effects of cocaine in adulthood. Responses to cocaine are regulated by epigenetic mechanisms, such as DNA methylation, in the brain's reward regions. However, the involvement of these processes in modulation of the vulnerability to the effects of cocaine induced by prior exposure to cannabinoids remains poorly understood. OBJECTIVES: Investigate whether exposure to the synthetic cannabinoid WIN55,212-2 during adolescence modulates anxiety- and depression-like behavior, memory, and cocaine reward in adult mice. We also evaluated whether exposure to cannabinoids during adolescence modulates the expression of enzymes that are involved in DNA methylation. RESULTS: Exposure to WIN55,212-2 during adolescence did not alter anxiety- or depressive-like behavior. However, prior exposure to cannabinoids inhibited cocaine-induced conditioned place preference without modulating cocaine-induced hyperlocomotion, accompanied by an increase in expression of the enzyme DNA methyltransferase 3a (DNMT3a) in the prefrontal cortex. CONCLUSIONS: Our findings suggest that exposure to WIN55,212-2 during adolescence leads to changes in DNMT3a expression, and this pathway appears to be relevant to modulating the rewarding effects of cocaine.


Assuntos
Canabinoides , Cocaína , Animais , Canabinoides/farmacologia , Cocaína/farmacologia , DNA (Citosina-5-)-Metiltransferases/metabolismo , Camundongos , Córtex Pré-Frontal/metabolismo , Recompensa
12.
Aging (Albany NY) ; 13(12): 16267-16286, 2021 06 18.
Artigo em Inglês | MEDLINE | ID: mdl-34148029

RESUMO

Multiple studies have revealed that long non-coding RNA (lncRNAs) served as regulatory factors in modulating tumorigenesis of hepatocellular carcinoma (HCC). In the present study, we demonstrated that lncRNA HCP5 was overexpressed in HCC tissues and cell lines, and these findings were obvious even in metastatic and recurrent cases. Knockdown of HCP5 significantly alleviated cell growth, metastasis, and invasion both in vitro and in vivo through promoting apoptosis and by inactivating the epithelial-mesenchymal transition (EMT) progress. Moreover, miR-29b-3p has been identified as a negatively regulatory target gene of HCP5, and served as a tumor suppressor of HCC to prevent cell proliferation, migration, and invasion. Subsequently, DNMT3A was identified as a downstream regulatory factor of miR-29b-3p, and acted as a participated element of HCC progression by activating AKT phosphorylation. Taken together, our study elucidated for the first time that HCP5 plays a crucial role in HCC via the HCP5/miR-29b-3p/DNMT3A/AKT axis and our findings demonstrated a novel diagnostic and therapeutic strategy with potentiality to treat HCC.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , DNA (Citosina-5-)-Metiltransferases/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , MicroRNAs/metabolismo , RNA Longo não Codificante/metabolismo , Regulação para Cima/genética , Idoso , Apoptose/genética , Sequência de Bases , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Progressão da Doença , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , MicroRNAs/genética , Pessoa de Meia-Idade , Invasividade Neoplásica , Metástase Neoplásica , Fosforilação , Proteínas Proto-Oncogênicas c-akt/metabolismo , RNA Longo não Codificante/genética
13.
Aging (Albany NY) ; 13(11): 15193-15213, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34100772

RESUMO

The molecular mechanisms underlying premature ovarian failure, which seriously impacts the physical and psychological health of patients, are not fully understood. Here, we present the role of TRDMT1 in reactive oxygen species-induced granulosa cells death, which is considered an important cause of premature ovarian failure. We found that reactive oxygen species were increased in a H2O2 dose-dependent manner and accompanied by the nuclear shuttling of TRDMT1, increased DNA damage and increased apoptosis of granulosa cells. In addition, reactive oxygen species-induced granulosa cells apoptosis could be prevented by the antioxidant N-acetylcysteine or overexpression of TRDMT1. Furthermore, DNA repair following reactive oxygen species induction was severely impaired/enhanced in TRDMT1 mutants, which exhibited reduced/increased RNA m5C methylation activity. Altogether, our results reveal a novel role of TRDMT1 in the regulation of premature ovarian failure through the repair of reactive oxygen species-triggered DNA damage in granulosa cells and provide an improved understanding of the mechanisms underlying granulosa cells apoptosis, which could potentially be useful for future clinical treatments of premature ovarian failure.


Assuntos
DNA (Citosina-5-)-Metiltransferases/metabolismo , Dano ao DNA , Reparo do DNA , Células da Granulosa/patologia , Insuficiência Ovariana Primária/metabolismo , Insuficiência Ovariana Primária/patologia , 5-Metilcitosina/metabolismo , Animais , Apoptose , Linhagem Celular Tumoral , Metilação de DNA/genética , Feminino , Modelos Biológicos , Estresse Oxidativo , Ratos Sprague-Dawley
14.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(7): 608-615, 2021 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-34140072

RESUMO

Objective To observe the effects of DNA methyltransferase 3B (DNMT3B) on the expression of secreted frizzled-related protein 1 (SFRP1) and regulation of Wnt/ß-catenin signaling pathway in renal tubular epithelial cells (RTECs) of mice under high glucose conditions. Methods in vitro cultured mouse RTECs were divided into normal glucose (NG) group and high glucose (HG) group. After DNMT3B short-hairclip RNA (sh-DNMT3B) and DNMT3B over-expression (DNMT3B-OE) plasmids were transfected separately into RTECs, mRNA expression of DNMT3B, SFRP1, collagen IV (Col4) and fibronectin (FN) were detected by reverse-transcription PCR. Protein expression of DNMT3B, SFRP1, glycogen synthase kinase 3ß (GSK3ß), phosphorylated glycogen synthase kinase 3ß (p-GSK3ß), ß-catenin, Col4 and FN were detected by Western blotting. The localization of DNMT3B and SFRP1 in RTECs was observed by immunofluorescence cytochemistry combined with confocal microscopy. Results Compared with the NG group, the protein expression of DNMT3B, ß-catenin, p-GSK3ß, Col4 and FN increased in the HG group, while SFRP1 protein expression was reduced in the HG group. Compared with the sh-vector group, SFRP1 mRNA and protein expression increased in the sh-DNMT3B group, while the expression of ß-catenin, p-GSK3ß and Col4 proteins decreased. FN mRNA and protein expression dropped in the sh-DNMT3B group, however, the expression of ß-catenin mRNA did not change significantly. Visually, DNMT3B over-expression reversed the above changes. Both DNMT3B and SFRP1 were expressed in the nucleus and cytoplasm of RTECs, and DNMT3B was aggregated in the nuclei of the cells in the HG group and the co-localization between DNMT3B and SFRP1 was also promoted in the HG group. Conclusion The expression of DNMT3B increases and the expression of SFRP1 decreases when the mouse RTECs were stimulated by HG. This subsequently leads to the activation of the Wnt/ß-catenin signaling pathway and promotes the formation of extracellular matrix.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Células Epiteliais , Via de Sinalização Wnt , Animais , DNA (Citosina-5-)-Metiltransferases/metabolismo , Células Epiteliais/metabolismo , Fibrose , Glucose , Glicogênio Sintase Quinase 3 beta/genética , Camundongos , beta Catenina/genética , beta Catenina/metabolismo
15.
Adv Healthc Mater ; 10(16): e2100821, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34174172

RESUMO

Extracellular matrix (ECM) stiffness has profound effects on the regulation of cell functions. DNA methylation is an important epigenetic modification governing gene expression. However, the effects of ECM stiffness on DNA methylation remain elusive. Here, it is reported that DNA methylation is sensitive to ECM stiffness, with a global hypermethylation under stiff ECM condition in mouse embryonic stem cells (mESCs) and embryonic fibroblasts compared with soft ECM. Stiff ECM enhances DNA methylation of both promoters and gene bodies, especially the 5' promoter regions of pluripotent genes. The enhanced DNA methylation is functionally required for the loss of pluripotent gene expression in mESCs grown on stiff ECM. Further experiments reveal that the nuclear transport of DNA methyltransferase 3-like (DNMT3L) is promoted by stiff ECM in a protein kinase C α (PKCα)-dependent manner and DNMT3L can be binding to Nanog promoter regions during cell-ECM interactions. These findings unveil DNA methylation as a novel target for the mechanical sensing mechanism of ECM stiffness, which provides a conserved mechanism for gene expression regulation during cell-ECM interactions.


Assuntos
DNA (Citosina-5-)-Metiltransferases , Metilação de DNA , Proteína Quinase C-alfa/metabolismo , Transporte Ativo do Núcleo Celular , Animais , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Matriz Extracelular/metabolismo , Camundongos , Proteína Quinase C-alfa/genética
16.
Front Immunol ; 12: 653030, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34093541

RESUMO

DNA (cytosine-5)-methyltransferase 3A (DNMT3A)-mutated acute myeloid leukemia (AML) has a poor prognosis, but the exact mechanism is still unclear. Here, we aimed to explore the mechanism of immune escape in AML with DNMT3A mutation. We constructed a DNMT3A knockout clone and DNMT3A-R882H-mutated clones. RNA-seq results showed that transcription factors and macrophage inflammatory proteins were significantly downregulated in the DNMT3A mutant clones. KEGG enrichment and gene set enrichment analysis (GSEA) showed that a large number of genes were enriched in inflammatory immune-related pathways, such as the toll-like receptor signaling pathway. Therefore, we co-cultured AML cells with macrophages. The DNMT3A-mutated AML cells attenuated M1 macrophage polarization and resisted its killing effect in vitro and in vivo. In xenografts, the tumor volumes in the experimental group were significantly larger than those in the control group, and the proportion of M2 macrophages was significantly higher. After the co-culture, the increase in pro-inflammatory cytokine expression in the mutant cells was significantly lower than that in the control group, while that in immunosuppressive factors was not significantly different. In co-cultivated supernatants, the concentration of inflammatory factors in the experimental group was significantly lower than that in the control group, while that of immunosuppressive factors was significantly higher. Resistin significantly promoted the expression of inflammatory proteins in AML cells. It relieved the inhibitory effect of DNMT3A mutation, promoted the phenotypic recovery of the co-cultured macrophages, eliminated resistance, and regulated the immune microenvironment. Thus, resistin may serve as an ancillary drug for patients with DNMT3A-mutated AML.


Assuntos
DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/imunologia , Regulação Leucêmica da Expressão Gênica/imunologia , Leucemia Mieloide Aguda/genética , Evasão Tumoral/genética , Animais , Técnicas de Cultura de Células , Técnicas de Cocultura , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Humanos , Leucemia Mieloide Aguda/tratamento farmacológico , Leucemia Mieloide Aguda/imunologia , Macrófagos/imunologia , Camundongos , Mutação , RNA-Seq , Resistina/farmacologia , Resistina/uso terapêutico , Células THP-1 , Evasão Tumoral/efeitos dos fármacos , Microambiente Tumoral/efeitos dos fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/imunologia , Ensaios Antitumorais Modelo de Xenoenxerto
17.
Biomolecules ; 11(6)2021 05 22.
Artigo em Inglês | MEDLINE | ID: mdl-34067359

RESUMO

DNMT3A mutations are frequently identified in acute myeloid leukemia (AML) and indicate poor prognosis. Previously, we found that the hotspot mutation DNMT3A R882H could upregulate CDK1 and induce AML in conditional knock-in mice. However, the mechanism by which CDK1 is involved in leukemogenesis of DNMT3A mutation-related AML, and whether CDK1 could be a therapeutic target, remains unclear. In this study, using fluorescence resonance energy transfer and immunoprecipitation analysis, we discovered that increased CDK1 could compete with EZH2 to bind to the PHD-like motif of DNMT3A, which may disturb the protein interaction between EZH2 and DNMT3A. Knockdown of CDK1 in OCI-AML3 cells with DNMT3A mutation markedly inhibited proliferation and induced apoptosis. CDK1 selective inhibitor CGP74514A (CGP) and the pan-CDK inhibitor flavopiridol (FLA) arrested OCI-AML3 cells in the G2/M phase, and induced cell apoptosis. CGP significantly increased CD163-positive cells. Moreover, the combined application of CDK1 inhibitor and traditional chemotherapy drugs synergistically inhibited proliferation and induced apoptosis of OCI-AML3 cells. In conclusion, this study highlights CDK1 overexpression as a pathogenic factor and a potential therapeutic target for DNMT3A mutation-related AML.


Assuntos
Proteína Quinase CDC2/biossíntese , Carcinogênese/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Regulação Enzimológica da Expressão Gênica , Regulação Leucêmica da Expressão Gênica , Leucemia Mieloide Aguda/metabolismo , Mutação , Proteínas de Neoplasias/metabolismo , Animais , Proteína Quinase CDC2/genética , Carcinogênese/genética , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferases/genética , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Humanos , Leucemia Mieloide Aguda/genética , Camundongos , Células NIH 3T3 , Proteínas de Neoplasias/genética
18.
Cell Death Dis ; 12(7): 642, 2021 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-34162834

RESUMO

Diabetic peripheral neuropathy (DPN) is the most common complication of diabetes mellitus (DM) and the dysfunction of Schwann cells plays an important role in the pathogenesis of DPN. Thioredoxin-interacting protein (TXNIP) is known as an inhibitor of thioredoxin and associated with oxidative stress and inflammation. However, whether TXNIP is involved in dysfunction of Schwann cells of DPN and the exact mechanism is still not known. In this study, we first reported that TXNIP expression was significantly increased in the sciatic nerves of diabetic mice, accompanied by abnormal electrophysiological indexes and myelin sheath structure. Similarly, in vitro cultured Schwann cells TXNIP was evidently enhanced by high glucose stimulation. Again, the function experiment found that knockdown of TXNIP in high glucose-treated RSC96 cells led to a 4.12 times increase of LC3-II/LC3-I ratio and a 25.94% decrease of cleaved caspase 3/total caspase 3 ratio. Then, DNA methyltransferase (DNMT) inhibitor 5-Aza has been reported to benefit Schwann cell in DPN, and here 5-Aza treatment reduced TXNIP protein expression, improved autophagy and inhibited apoptosis in high glucose-treated RSC96 cells and the sciatic nerves of diabetic mice. Furthermore, DNMT1 and DNMT3a upregulation were found to be involved in TXNIP overexpression in high glucose-stimulated RSC96 cells. Silencing of DNMT1 and DNMT3a effectively reversed high glucose-enhanced TXNIP. Moreover, high glucose-inhibited PI3K/Akt pathway led to DNMT1, DNMT3a, and TXNIP upregulation in RSC96 cells. Knockdown of DNMT1 and DNMT3a prevented PI3K/Akt pathway inhibition-caused TXNIP upregulation in RSC96 cells. Finally, in vivo knockout of TXNIP improved nerve conduction function, increased autophagosome and LC3 expression, and decreased cleaved Caspase 3 and Bax expression in diabetic mice. Taken together, PI3K/Akt pathway inhibition mediated high glucose-induced DNMT1 and DNMT3a overexpression, leading to cell autophagy inhibition and apoptosis via TXNIP protein upregulation in Schwann cells of DPN.


Assuntos
Proteínas de Transporte/metabolismo , Proteínas de Ciclo Celular/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Neuropatias Diabéticas/enzimologia , Fosfatidilinositol 3-Quinase/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Células de Schwann/enzimologia , Nervo Isquiático/enzimologia , Tiorredoxinas/metabolismo , Animais , Apoptose , Proteínas Reguladoras de Apoptose/metabolismo , Autofagia , Proteínas Relacionadas à Autofagia/metabolismo , Glicemia/metabolismo , Proteínas de Transporte/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Neuropatias Diabéticas/genética , Neuropatias Diabéticas/patologia , Modelos Animais de Doenças , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ratos , Células de Schwann/patologia , Nervo Isquiático/patologia , Transdução de Sinais , Tiorredoxinas/genética
19.
Nat Genet ; 53(6): 794-800, 2021 06.
Artigo em Inglês | MEDLINE | ID: mdl-33986537

RESUMO

Precise deposition of CpG methylation is critical for mammalian development and tissue homeostasis and is often dysregulated in human diseases. The localization of de novo DNA methyltransferase DNMT3A is facilitated by its PWWP domain recognizing histone H3 lysine 36 (H3K36) methylation1,2 and is normally depleted at CpG islands (CGIs)3. However, methylation of CGIs regulated by Polycomb repressive complexes (PRCs) has also been observed4-8. Here, we report that DNMT3A PWWP domain mutations identified in paragangliomas9 and microcephalic dwarfism10 promote aberrant localization of DNMT3A to CGIs in a PRC1-dependent manner. DNMT3A PWWP mutants accumulate at regions containing PRC1-mediated formation of monoubiquitylated histone H2A lysine 119 (H2AK119ub), irrespective of the amounts of PRC2-catalyzed formation of trimethylated histone H3 lysine 27 (H3K27me3). DNMT3A interacts with H2AK119ub-modified nucleosomes through a putative amino-terminal ubiquitin-dependent recruitment region, providing an alternative form of DNMT3A genomic targeting that is augmented by the loss of PWWP reader function. Ablation of PRC1 abrogates localization of DNMT3A PWWP mutants to CGIs and prevents aberrant DNA hypermethylation. Our study implies that a balance between DNMT3A recruitment by distinct reader domains guides de novo CpG methylation and may underlie the abnormal DNA methylation landscapes observed in select human cancer subtypes and developmental disorders.


Assuntos
Ilhas de CpG/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Proteínas do Grupo Polycomb/metabolismo , Animais , Catálise , Linhagem Celular , DNA (Citosina-5-)-Metiltransferases/química , Predisposição Genética para Doença , Genoma Humano , Histonas/metabolismo , Humanos , Lisina/metabolismo , Camundongos , Mutação/genética , Nucleossomos/metabolismo , Domínios Proteicos , Ubiquitinação
20.
Nat Commun ; 12(1): 3243, 2021 05 28.
Artigo em Inglês | MEDLINE | ID: mdl-34050148

RESUMO

Determining the effect of DNA methylation on chromatin structure and function in higher organisms is challenging due to the extreme complexity of epigenetic regulation. We studied a simpler model system, budding yeast, that lacks DNA methylation machinery making it a perfect model system to study the intrinsic role of DNA methylation in chromatin structure and function. We expressed the murine DNA methyltransferases in Saccharomyces cerevisiae and analyzed the correlation between DNA methylation, nucleosome positioning, gene expression and 3D genome organization. Despite lacking the machinery for positioning and reading methylation marks, induced DNA methylation follows a conserved pattern with low methylation levels at the 5' end of the gene increasing gradually toward the 3' end, with concentration of methylated DNA in linkers and nucleosome free regions, and with actively expressed genes showing low and high levels of methylation at transcription start and terminating sites respectively, mimicking the patterns seen in mammals. We also see that DNA methylation increases chromatin condensation in peri-centromeric regions, decreases overall DNA flexibility, and favors the heterochromatin state. Taken together, these results demonstrate that methylation intrinsically modulates chromatin structure and function even in the absence of cellular machinery evolved to recognize and process the methylation signal.


Assuntos
Montagem e Desmontagem da Cromatina , Metilação de DNA , Epigênese Genética , Nucleossomos/metabolismo , Saccharomyces cerevisiae/genética , Regiões 5' não Traduzidas/genética , Centrômero/metabolismo , Cromatina/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Genoma Fúngico , Histonas/genética , Histonas/metabolismo , Microscopia Intravital , Mutagênese Sítio-Dirigida , Mutação , Nucleossomos/genética , RNA-Seq , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/isolamento & purificação , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/isolamento & purificação , Proteínas de Saccharomyces cerevisiae/metabolismo , Sequenciamento Completo do Genoma
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