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1.
Nat Commun ; 11(1): 1222, 2020 03 06.
Artigo em Inglês | MEDLINE | ID: mdl-32144273

RESUMO

Stable inheritance of DNA methylation is critical for maintaining differentiated phenotypes in multicellular organisms. We have recently identified dual mono-ubiquitylation of histone H3 (H3Ub2) by UHRF1 as an essential mechanism to recruit DNMT1 to chromatin. Here, we show that PCNA-associated factor 15 (PAF15) undergoes UHRF1-dependent dual mono-ubiquitylation (PAF15Ub2) on chromatin in a DNA replication-coupled manner. This event will, in turn, recruit DNMT1. During early S-phase, UHRF1 preferentially ubiquitylates PAF15, whereas H3Ub2 predominates during late S-phase. H3Ub2 is enhanced under PAF15 compromised conditions, suggesting that H3Ub2 serves as a backup for PAF15Ub2. In mouse ES cells, loss of PAF15Ub2 results in DNA hypomethylation at early replicating domains. Together, our results suggest that there are two distinct mechanisms underlying replication timing-dependent recruitment of DNMT1 through PAF15Ub2 and H3Ub2, both of which are prerequisite for high fidelity DNA methylation inheritance.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Ubiquitinação , Animais , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Cromatina/metabolismo , Humanos , Masculino , Camundongos , Células-Tronco Embrionárias Murinas/metabolismo , Ligação Proteica , Espermatozoides/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Xenopus laevis
2.
Biochim Biophys Acta Gene Regul Mech ; 1863(2): 194481, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31923609

RESUMO

DNA methylation and histone acetylation, the most studied epigenetic changes, drive and maintain cancer phenotypes. DNA methyltransferase (DNMT) dysregulation promoted localized hypermethylation in CpG rich regions while upregulated histone deacetylases (HDAC) deacetylated histone tails. Both changes led to close chromatin conformation, suppressing transcription and silencing tumor suppressor genes. Consequently, HDAC and DNMT inhibitors appeared to reprogram the transcriptional circuit and potentiate anti-tumoral activity. Here, we report that eicosapentaenoic acid (EPA), a fatty acid with anti-cancer properties, inhibited HDAC1 and DNMT expression and activity, thus promoting tumor suppressor gene expression. In hepatocarcinoma cells (HCC) EPA bound and activated PPARγ thus downregulating HDAC1 which sequentially reduced expression of DNMT1, 3A and 3B. At the same time, activated PPARγ physically interacted with DNMT1 and HDAC1 in a CpG island on the Hic-1 gene to assemble PPARγ/DNMT1 and PPARγ/HDAC1 protein complexes, which exited from DNA. When EPA and PPARγ were no longer bound, the protein complexes separated into individual proteins. Consequently, DNMT1 and HDAC1 down-regulation and release from DNA inhibited their activities. Overall, EPA-bound PPARγ induced re-expression of the tumor suppressor gene Hic-1. In the present study PPARγ emerged as a master regulator acting synergistically through diverse targets and ways to reveal the epigenetic action of EPA as an HDAC1 and DNMT1 inhibitor.


Assuntos
Antineoplásicos/farmacologia , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Ácido Eicosapentaenoico/farmacologia , Epigênese Genética/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Histona Desacetilase 1/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Ilhas de CpG , DNA/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Genes Supressores de Tumor , Histona Desacetilase 1/metabolismo , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Neoplasias Hepáticas Experimentais/genética , Neoplasias Hepáticas Experimentais/metabolismo , PPAR gama/metabolismo , Ratos
3.
Arch Insect Biochem Physiol ; 103(4): e21651, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31943343

RESUMO

DNA methylation refers to the addition of cytosine residues in a CpG context (5'-cytosine-phosphate-guanine-3'). As one of the most common mechanisms of epigenetic modification, it plays a crucial role in regulating gene expression and in a diverse range of biological processes across all multicellular organisms. The relationship between temperature and DNA methylation and how it acts on the adaptability of migratory insects remain unknown. In the present work, a 5,496 bp full-length complementary DNA encoding 1,436 amino acids (named MsDnmt1) was cloned from the devastating migratory pest oriental armyworm, Mythimna separata Walker. The protein shares 36.8-84.4% identity with other insect Dnmt1 isoforms. Spatial and temporal expression analysis revealed that MsDnmt1 was highly expressed in adult stages and head tissue. The changing temperature decreased the expression of MsDnmt1 in both high and low temperature condition. Besides, we found that M. separata exhibited the shortest duration time from the last instar to pupae under 36°C environment when injected with DNA methylation inhibitor. Therefore, our data highlight a potential role for DNA methylation in thermal resistance, which help us to understand the biological role adaptability and colonization of migratory pest in various environments.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Proteínas de Insetos/genética , Mariposas/fisiologia , Sequência de Aminoácidos , Animais , Temperatura Corporal , DNA (Citosina-5-)-Metiltransferase 1/química , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Insetos/química , Proteínas de Insetos/metabolismo , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Mariposas/genética , Mariposas/crescimento & desenvolvimento , Óvulo/crescimento & desenvolvimento , Óvulo/fisiologia , Filogenia , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Alinhamento de Sequência
4.
Chem Biodivers ; 17(2): e1900659, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31995280

RESUMO

Breast Cancer (BCa) is the most often diagnosed cancer among women who were in the late 1940's. Breast cancer growth is largely dependent on the expression of estrogen and progesterone receptor. Breast cancer cells may have one, both, or none of these receptors. The treatment for breast cancer may involve surgery, hormonal therapy (Tamoxifen, an aromatase inhibitor, etc.) and oral chemotherapeutic drugs. The molecular docking technique reported the findings on the potential binding modes of the 2-(2-bromo-3-nitrophenyl)-5-phenyl-1,3,4-oxadiazole derivatives with the estrogen receptor (PDB ID: 3ERT). The 1,3,4-oxadiazole derivatives 4a-4j have been synthesized and described by spectroscopic method. 2-(2-Bromo-6-nitrophenyl)-5-(4-bromophenyl)-1,3,4-oxadiazole (4c) was reconfirmed by single-crystal XRD. All the compounds have been tested in combination with generic Imatinib pharmaceutical drug against breast cancer cell lines isolated from Caucasian woman MCF-7, MDA-MB-453 and MCF-10A non-cancer cell lines. The compounds with the methoxy (in 4c) and methyl (in 4j) substitution were shown to have significant cytotoxicity, with 4c showing dose-dependent activation and decreased cell viability. The mechanism of action was reported by induced apoptosis and tested by a DNA enzyme inhibitor experiment (ELISA) for Methyl Transferase. Molecular dynamics simulations were made for hit molecule 4c to study the stability and interaction of the protein-ligand complex. The toxicity properties of ADME were calculated for all the compounds. All these results provide essential information for further clinical trials.


Assuntos
Antineoplásicos/síntese química , Desenho de Fármacos , Oxidiazóis/química , Antineoplásicos/metabolismo , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Sítios de Ligação , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Ensaios de Seleção de Medicamentos Antitumorais , Inibidores Enzimáticos/química , Inibidores Enzimáticos/metabolismo , Inibidores Enzimáticos/farmacologia , Receptor alfa de Estrogênio/química , Receptor alfa de Estrogênio/metabolismo , Feminino , Humanos , Mesilato de Imatinib/farmacologia , Conformação Molecular , Simulação de Acoplamento Molecular , Oxidiazóis/metabolismo , Oxidiazóis/farmacologia , Relação Estrutura-Atividade
5.
Artif Cells Nanomed Biotechnol ; 47(1): 3687-3696, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31842626

RESUMO

Rheumatoid arthritis (RA) is characterized by tumor-like expansion of the synovium and the subsequent destruction of adjacent articular cartilage and bone. The latest studies proved phosphatase and tension homolog deleted on chromosome 10 (PTEN) might contribute to the surviving, proliferation and pro-inflammatory cytokines in RA. The purpose of this study was to explore the function and underlying mechanisms of PTEN in RA pro-inflammatory cytokines and chemokines of fibroblast-like synoviocytes (FLSs). Increased level of PTEN was observed in adjuvant-induced arthritis (AIA) FLSs in comparison to normal rats. Increased concentrations of pro-inflammatory cytokines (TNF-α, IL-6 and IL-1ß), chemokines (CCL-2 and CCL-3), VCAM-1 and VEGF-α expression were observed in FLSs with PTEN inhibitor bpv or PTEN-RNAi. Moreover, co-incubation FLSs with overexpression vector with PTEN-GV141 reduced the expression of pro-inflammatory cytokines, chemokines, VCAM-1 and VEGF-α in AIA. Interestingly, we also found DNA methylation could regulate PTEN expression and activation of AKT signaling was with a change of PTEN. Altogether, our findings in the present study suggested that PTEN might play a pivotal role during pro-inflammatory cytokines and chemokines of FLSs through activation of AKT signaling pathway. In addition, PTEN expression may be regulated by DNA methylation in the pathogenesis of AIA.


Assuntos
Artrite Experimental/patologia , Quimiocinas/metabolismo , Fibroblastos/patologia , Regulação da Expressão Gênica , PTEN Fosfo-Hidrolase/metabolismo , Sinoviócitos/metabolismo , Animais , Artrite Experimental/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Regulação para Baixo , Ativação Enzimática , Proteínas Proto-Oncogênicas c-akt/metabolismo , Ratos , Ratos Sprague-Dawley , Transdução de Sinais , Sinoviócitos/patologia
6.
Fertil Steril ; 112(6): 1180-1189, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31843095

RESUMO

OBJECTIVE: To determine the expression of miR-29c and its target gene transforming growth factor-ß3 (TGF-ß3) in leiomyoma and the mechanisms of their reciprocal regulation. DESIGN: Experimental study. SETTING: Academic research laboratory. PATIENT(S): Women undergoing hysterectomy for leiomyoma. INTERVENTION(S): Overexpression and underexpression of miR-29c; blockade of DNA methyltransferase 1 (DNMT1). MAIN OUTCOME MEASURE(S): The miR-29c and its target gene TGF-ß3 in leiomyoma and the effects of TGF-ß3 and blockade of DNMT1 on miR-29c expression. RESULT(S): Leiomyoma expressed significantly lower levels of miR-29c, but higher expression of TGF-ß3 compared with matched myometrium. The expression of TGF-ß3 and miR-29c were independent of race/ethnicity. Using 3' untranslated region luciferase reporter assay we confirmed that TGF-ß3 is a direct target of miR-29c in leiomyoma smooth muscle cells (LSMCs). Gain-of-function of miR-29c in LSMCs inhibited the expression of TGF-ß3 at protein and messenger RNA levels, whereas loss-of-function of miR-29c had the opposite effect. Treatment of LSMCs with TGF-ß3 inhibited the expression of miR-29c, whereas it stimulated DNMT1 expression. Knockdown of DNMT1 through transfection with small interfering RNA significantly decreased the expression of TGF-ß3, and induced miR-29c expression. Knockdown of DNMT1 also attenuated the inhibitory effect of TGF-ß3 on miR-29c expression. Furthermore, we demonstrated that TGF-ß3 increased the methylation level of miR-29c promoter in LSMCs. CONCLUSION(S): There is an inverse relationship in the expression of TGF-ß3 and miR-29c in leiomyoma. The TGF-ß3 is a direct target of miR-29c and inhibits the expression of miR-29c through an epigenetic mechanism. The cross-talk between miR-29c and TGF-ß3 provides a feed forward mechanism of fibrosis in leiomyoma.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Epigênese Genética , Leiomioma/enzimologia , MicroRNAs/metabolismo , Fator de Crescimento Transformador beta3/metabolismo , Neoplasias Uterinas/enzimologia , Adulto , DNA (Citosina-5-)-Metiltransferase 1/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Leiomioma/genética , Leiomioma/patologia , Leiomioma/cirurgia , MicroRNAs/genética , Pessoa de Meia-Idade , Transdução de Sinais , Fator de Crescimento Transformador beta3/genética , Células Tumorais Cultivadas , Neoplasias Uterinas/genética , Neoplasias Uterinas/patologia , Neoplasias Uterinas/cirurgia
7.
Acta Biochim Pol ; 66(4): 409-414, 2019 Dec 01.
Artigo em Inglês | MEDLINE | ID: mdl-31880901

RESUMO

OBJECTIVE: Breast cancer is known as one of very important cancers among females, given that a variety of external (i.e., environmental risk factors) and internal factors (i.e., genetics, and epigenetics) are related to the emergence and progression of breast cancer. Among genetic and epigenetic factors, DNA methyltransferase and EMT related genes have critical roles in breast cancer pathogenesis. In the study presented here, we investigated expression of DNA methyltransferases (e.g., DNMT1, DNMT3A and DNMT3B) and EMT related genes (e.g., E-cadherin, Snail, ZEB-1). METHODS AND MATERIALS: Tissue samples were collected from 18 cancer and 24 normal breast tissues. We evaluated the expression levels of DNA methyltransferases and EMT related genes using Quantitative real-time PCR (qRT-PCR). RESULTS: Our results indicated that the expression levels of ZEB-1, Snail, and DNMT3B were increased in breast cancer subjects in comparison to the control group. On the other hand, there was a significant decrease in E-cadherin expression in breast cancer tissues in comparison to the normal tissues. Moreover, there were no significant changes for DNMT1 and DNMT3A expression in breast cancer tissues when compared to the normal tissues. CONCLUSION: Taken together, our finding show that up regulation of ZEB-1 and Snail could be associated with down regulation of E-cadherin and results in promotion of cancer cell invasion. Moreover, down regulation of E-cadherin may be related to deterioration of DNMT3B inpatients with breast cancer.


Assuntos
Neoplasias da Mama/genética , Metilases de Modificação do DNA/genética , Regulação Neoplásica da Expressão Gênica , Adulto , Idoso , Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Estudos de Casos e Controles , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Feminino , Expressão Gênica , Humanos , Pessoa de Meia-Idade , Fatores de Transcrição da Família Snail/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo
8.
Proc Natl Acad Sci U S A ; 116(49): 24852-24860, 2019 12 03.
Artigo em Inglês | MEDLINE | ID: mdl-31748268

RESUMO

ErbB3-binding protein 1 (EBP1) is implicated in diverse cellular functions, including apoptosis, cell proliferation, and differentiation. Here, by generating genetic inactivation of Ebp1 mice, we identified the physiological roles of EBP1 in vivo. Loss of Ebp1 in mice caused aberrant organogenesis, including brain malformation, and death between E13.5 and 15.5 owing to severe hemorrhages, with massive apoptosis and cessation of cell proliferation. Specific ablation of Ebp1 in neurons caused structural abnormalities of brain with neuron loss in [Nestin-Cre; Ebp1 flox/flox ] mice. Notably, global methylation increased with high levels of the gene-silencing unit Suv39H1/DNMT1 in Ebp1-deficient mice. EBP1 repressed the transcription of Dnmt1 by binding to its promoter region and interrupted DNMT1-mediated methylation at its target gene, Survivin promoter region. Reinstatement of EBP1 into embryo brain relived gene repression and rescued neuron death. Our findings uncover an essential role for EBP1 in embryonic development and implicate its function in transcriptional regulation.


Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Desenvolvimento Embrionário/genética , Desenvolvimento Embrionário/fisiologia , Inativação Gênica/fisiologia , Proteínas de Ligação a RNA/genética , Proteínas de Ligação a RNA/metabolismo , Animais , Apoptose , Ciclo Celular , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Modelos Animais , Transcrição Genética
9.
Nat Commun ; 10(1): 4986, 2019 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-31676777

RESUMO

Chemotherapy-resistant cancer recurrence is a major cause of mortality. In acute myeloid leukemia (AML), chemorefractory relapses result from the complex interplay between altered genetic, epigenetic and transcriptional states in leukemic cells. Here, we develop an experimental model system using in vitro lineage tracing coupled with exome, transcriptome and in vivo functional readouts to assess the AML population dynamics and associated molecular determinants underpinning chemoresistance development. We find that combining standard chemotherapeutic regimens with low doses of DNA methyltransferase inhibitors (DNMTi, hypomethylating drugs) prevents chemoresistant relapses. Mechanistically, DNMTi suppresses the outgrowth of a pre-determined set of chemoresistant AML clones with stemness properties, instead favoring the expansion of rarer and unfit chemosensitive clones. Importantly, we confirm the capacity of DNMTi combination to suppress stemness-dependent chemoresistance development in xenotransplantation models and primary AML patient samples. Together, these results support the potential of DNMTi combination treatment to circumvent the development of chemorefractory AML relapses.


Assuntos
Metilação de DNA , Resistencia a Medicamentos Antineoplásicos/genética , Leucemia Mieloide/genética , Transcriptoma/genética , Doença Aguda , Antibióticos Antineoplásicos/uso terapêutico , Linhagem Celular Tumoral , Linhagem da Célula/genética , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Decitabina/uso terapêutico , Doxorrubicina/uso terapêutico , Inibidores Enzimáticos/uso terapêutico , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia
10.
Curr Drug Metab ; 20(10): 799-803, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31608839

RESUMO

BACKGROUND: To investigate MiRNA-126 amounts in serum exosomes from allergic asthma patients as well as lung tissues of asthmatic mice, evaluating the expression of its target gene DNMT1 in mouse specimens. METHODS: MiRNA-126 amounts in serum exosomes from asthmatic patients were detected by real-time PCR. The mouse model of allergic asthma was established by OVA-sensitization, and allergic symptoms were recorded; serum IL-4 and sIgE level evaluation (ELISA), broncho alveolar lavage fluid (BALF) cell count and H&E staining were performed to assess airway inflammation. MiRNA-126 and DNMT1 levels in the lung of asthmatic and control mice were detected by real-time PCR; DNMT1 protein levels were detected by immunoblot. RESULTS: MiRNA-126 amounts in peripheral blood exosomes from patients with allergic asthma were significantly higher than that of healthy volunteers (P<0.05). The frequencies of scratching of both sides of the nose and sneezing were elevated within 10 min of excitation in asthmatic rats compared with controls. Meanwhile, OVA-sIgE and IL-4 levels were significantly higher in asthmatic animals than controls (P<0.05). In the asthma group, narrowed bronchial lumen and thickened wall were observed, and bronchial and peripheral vessels showed overt inflammatory cell infiltration. Eosinophil, neutrophil and mast cell amounts in the BALF of asthmatic mice were significantly higher than control values. Furthermore, lung miRNA-126 expression in asthmatic mice was significantly higher than that of controls. Finally, DNMT1 mRNA and protein levels were significantly lower in asthmatic animals compared with controls (P < 0.01). CONCLUSION: MiRNA-126 is highly expressed in serum exosomes from allergic asthma patients and lung tissues of asthmatic mice, suggesting that it may be involved in the pathogenesis of bronchial asthma.


Assuntos
Asma/sangue , Exossomos/genética , MicroRNAs/sangue , Animais , Asma/genética , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar/citologia , Contagem de Células , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Feminino , Humanos , Pulmão/metabolismo , Pulmão/patologia , Camundongos , Camundongos Endogâmicos BALB C
11.
ACS Chem Biol ; 14(10): 2315-2326, 2019 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-31479228

RESUMO

The proliferating cell nuclear antigen (PCNA)-associated factor p15PAF is a nuclear protein that acts as a regulator of DNA repair during DNA replication. The p15PAF gene is overexpressed in several types of human cancer, and its function is regulated by monoubiquitination of two lysines (K15 and K24) at the protein N-terminal region. We have previously shown that p15PAF is an intrinsically disordered protein which partially folds upon binding to PCNA and independently contacts DNA through its N-terminal tail. Here we present an NMR conformational characterization of p15PAF monoubiquitinated at both K15 and K24 via a disulfide bridge mimicking the isopeptide bond. We show that doubly monoubiquitinated p15PAF is monomeric, intrinsically disordered, and binds to PCNA as nonubiquitinated p15PAF does but interacts with DNA with reduced affinity. Our SAXS-derived conformational ensemble of doubly monoubiquitinated p15PAF shows that the ubiquitin moieties, separated by eight disordered residues, form transient dimers because of the high local effective ubiquitin concentration. This observation and the sequence similarity with histone H3 N-terminal tail suggest that doubly monoubiquitinated p15PAF is a binding target of DNA methyl transferase Dnmt1, as confirmed by calorimetry. Therefore, doubly monoubiquitinated p15PAF directly interacts with PCNA and recruits Dnmt1 for maintenance of DNA methylation during replication.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Ubiquitinação , DNA/metabolismo , Proteínas de Ligação a DNA/química , Escherichia coli , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/metabolismo , Antígeno Nuclear de Célula em Proliferação/química , Antígeno Nuclear de Célula em Proliferação/metabolismo , Ligação Proteica , Conformação Proteica , Domínios Proteicos , Processamento de Proteína Pós-Traducional
12.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(7): 625-630, 2019 Jul.
Artigo em Chinês | MEDLINE | ID: mdl-31537248

RESUMO

Objective To investigate the effect of homocysteine (Hcy) on the cardiomyocytes cultured in vitro, and to analyze the role of folic acid in DNA methylation to explore the protective effect and mechanism of folic acid during Hcy exposure of H9C2 cardiomyocytes. Methods H9C2 cells were treated with Hcy at different concentrations (0, 0.5, 1, 2) mmol/L for 24 hours. Cell viability was tested by CCK-8 assay. The apoptosis was detected by flow cytometry. H9C2 cells were divided into 2 mmol/L Hcy group, 0.1 mmol/L folic acid combined with 2 mmol/L Hcy group, 0.1 mmol/L folic acid group and DMSO control group. The above corresponding treatment lasted 24 hours. Then we detected the cell viability and apoptosis. The total DNA methylation level was determined by MethylFlash ELISA kit. DNMT1, DNMT3a, DNMT3b mRNA and protein expression were detected by real-time quantitative PCR and Western blot analysis. Results The number of H9C2 cells treated with different concentrations of Hcy for 24 hours decreased with the increase of Hcy concentration. Compared with the control group, the activity and apoptosis of the cells in the 2 mmol/L Hcy treatment group were reduced, and the number of cells in the folic acid combined with Hcy treatment group was significantly higher than that in the Hcy treatment group. Compared with the other groups, the total apoptosis rate of Hcy treatment group increased, methylation level decreased significantly, and the level of DNA methylation increased in the folic acid combined with Hcy treatment group. The level of DNMT1 mRNA significantly increased only in the folic acid treatment group, and the levels of DNMT1, DNMT3a and DNMT3b were not significantly changed. Conclusion Folic acid can relieve the damage of Hcy to myocardial cells by DNA methylation.


Assuntos
Apoptose , Metilação de DNA , Ácido Fólico/farmacologia , Miócitos Cardíacos/efeitos dos fármacos , Animais , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Homocisteína , Ratos
13.
Nat Commun ; 10(1): 3536, 2019 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-31387996

RESUMO

Tonicity-responsive enhancer binding protein (TonEBP or NFAT5) is a regulator of cellular adaptation to hypertonicity, macrophage activation and T-cell development. Here we report that TonEBP is an epigenetic regulator of thermogenesis and obesity. In mouse subcutaneous adipocytes, TonEBP expression increases > 50-fold in response to high-fat diet (HFD) feeding. Mice with TonEBP haplo-deficiency or adipocyte-specific TonEBP deficiency are resistant to HFD-induced obesity and metabolic defects (hyperglycemia, hyperlipidemia, and hyperinsulinemia). They also display increased oxygen consumption, resistance to hypothermia, and beiging of subcutaneous fat tissues. TonEBP suppresses the promoter of ß3-adrenoreceptor gene, a critical regulator of lipolysis and thermogenesis, in ex vivo and cultured adipocytes. This involves recruitment of DNMT1 DNA methylase and methylation of the promoter. In human subcutaneous adipocytes TonEBP expression displays a correlation with body mass index but an inverse correlation with ß3-adrenoreceptor expression. Thus, TonEBP is an attractive therapeutic target for obesity, insulin resistance, and hyperlipidemia.


Assuntos
Epigênese Genética , Resistência à Insulina/genética , Obesidade/metabolismo , Fatores de Transcrição/metabolismo , Células 3T3 , Adipócitos/metabolismo , Tecido Adiposo Bege/citologia , Tecido Adiposo Bege/metabolismo , Animais , Índice de Massa Corporal , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/genética , Dieta Hiperlipídica/efeitos adversos , Modelos Animais de Doenças , Metabolismo Energético/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Transgênicos , MicroRNAs/genética , MicroRNAs/metabolismo , Obesidade/etiologia , Cultura Primária de Células , Receptores Adrenérgicos beta 3/metabolismo , Gordura Subcutânea/citologia , Gordura Subcutânea/metabolismo , Termogênese/genética , Fatores de Transcrição/genética
14.
Phytomedicine ; 63: 153058, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31394414

RESUMO

BACKGROUND: Sulforaphane (SFN), a natural compound present in cruciferous vegetable, has been shown to possess anti-cancer activities. Cancer stem cell (CSC) in bulk tumor is generally considered as treatment resistant cell and involved in cancer recurrence. The effects of SFN on nasopharyngeal carcinoma (NPC) CSCs have not yet been explored. PURPOSE: The present study aims to examine the anti-tumor activities of SFN on NPC cells with CSC-like properties and the underlying mechanisms. METHODS: NPC cells growing in monolayer culture, CSCs-enriched NPC tumor spheres, and also the NPC nude mice xenograft were used to study the anti-tumor activities of SFN on NPC. The population of cells expressing CSC-associated markers was evaluated using flow cytometry and aldehyde dehydrogenase (ALDH) activity assay. The effect of DNA methyltransferase 1 (DNMT1) on the growth of NPC cells was analyzed by using small interfering RNA (siRNA)-mediated silencing method. RESULTS: SFN was found to inhibit the formation of CSC-enriched NPC tumor spheres and reduce the population of cells with CSC-associated properties (SRY (Sex determining Region Y)-box 2 (SOX2) and ALDH). In the functional study, SFN was found to restore the expression of Wnt inhibitory factor 1 (WIF1) and the effect was accompanied with the downregulation of DNMT1. The functional activities of WIF1 and DNMT1 were confirmed using exogenously added recombinant WIF1 and siRNA knockdown of DNMT1. Moreover, SFN was found to inhibit the in vivo growth of C666-1 cells and enhance the anti-tumor effects of cisplatin. CONCLUSION: Taken together, we demonstrated that SFN could suppress the growth of NPC cells via the DNMT1/WIF1 axis.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Antineoplásicos Fitogênicos/farmacologia , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Isotiocianatos/farmacologia , Carcinoma Nasofaríngeo/tratamento farmacológico , Neoplasias Nasofaríngeas/tratamento farmacológico , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Antineoplásicos Fitogênicos/administração & dosagem , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Brassicaceae/química , Linhagem Celular Tumoral , Cisplatino/administração & dosagem , DNA (Citosina-5-)-Metiltransferase 1/genética , Humanos , Isotiocianatos/administração & dosagem , Masculino , Camundongos Endogâmicos BALB C , Camundongos Nus , Carcinoma Nasofaríngeo/metabolismo , Carcinoma Nasofaríngeo/patologia , Neoplasias Nasofaríngeas/metabolismo , Neoplasias Nasofaríngeas/patologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Células-Tronco Neoplásicas/patologia , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Ensaios Antitumorais Modelo de Xenoenxerto
15.
Nucleic Acids Res ; 47(17): 9053-9068, 2019 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-31400111

RESUMO

Faithful inheritance of DNA methylation across cell division requires DNMT1 and its accessory factor UHRF1. However, how this axis is regulated to ensure DNA methylation homeostasis remains poorly understood. Here we show that SET8, a cell-cycle-regulated protein methyltransferase, controls protein stability of both UHRF1 and DNMT1 through methylation-mediated, ubiquitin-dependent degradation and consequently prevents excessive DNA methylation. SET8 methylates UHRF1 at lysine 385 and this modification leads to ubiquitination and degradation of UHRF1. In contrast, LSD1 stabilizes both UHRF1 and DNMT1 by demethylation. Importantly, SET8 and LSD1 oppositely regulate global DNA methylation and do so most likely through regulating the level of UHRF1 than DNMT1. Finally, we show that UHRF1 downregulation in G2/M by SET8 has a role in suppressing DNMT1-mediated methylation on post-replicated DNA. Altogether, our study reveals a novel role of SET8 in promoting DNA methylation homeostasis and identifies UHRF1 as the hub for tuning DNA methylation through dynamic protein methylation.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Histona-Lisina N-Metiltransferase/metabolismo , Ubiquitinação , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Ciclo Celular , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Replicação do DNA , Células HEK293 , Células HeLa , Histona Desmetilases/genética , Histona-Lisina N-Metiltransferase/genética , Humanos , Metilação , Camundongos , Células NIH 3T3 , Processamento de Proteína Pós-Traducional , Estabilidade Proteica
16.
J Pept Sci ; 25(9): e3200, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31309659

RESUMO

Posttranslational modifications of histone proteins, which form nucleosome cores, play an important role in gene regulation. Ubiquitination is one such modification. We previously reported on the synthesis of ubiquitinated histone H3 with an isopeptide mimetic structure. In this report, we describe the preparation of ubiquitinated histone H3 peptides with a native isopeptide structure, which showed a slightly weaker effect on the enzymatic activity of DNA methyltransferase 1 than the previous ubiquitinated H3 peptide analogs. These findings show that a native structure is important for determining the mechanism of the function, although ubiquitinated H3 peptide analogs can mimic the role of the original ubiquitinated H3. We also report on the successful preparation of the ubiquitinated full length histone H3.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Histonas/química , Peptídeos/farmacologia , Humanos , Estrutura Molecular , Peptídeos/síntese química , Peptídeos/química , Processamento de Proteína Pós-Traducional , Ubiquitinação
17.
Am J Respir Cell Mol Biol ; 61(4): 417-428, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31264905

RESUMO

DNA methylation represents a fundamental epigenetic mark that is associated with transcriptional repression during development, maintenance of homeostasis, and disease. In addition to methylation-sensitive PCR and targeted deep-amplicon bisulfite sequencing to measure DNA methylation at defined genomic loci, numerous unsupervised techniques exist to quantify DNA methylation on a genome-wide scale, including affinity enrichment strategies and methods involving bisulfite conversion. Both affinity-enriched and bisulfite-converted DNA can serve as input material for array hybridization or sequencing using next-generation technologies. In this practical guide to the measurement and analysis of DNA methylation, the goal is to convey basic concepts in DNA methylation biology and explore genome-scale bisulfite sequencing as the current gold standard for assessment of DNA methylation. Bisulfite conversion chemistry and library preparation are discussed in addition to a bioinformatics approach to quality assessment, trimming, alignment, and methylation calling of individual cytosine residues. Bisulfite-converted DNA presents challenges for standard next-generation sequencing library preparation protocols and data-processing pipelines, but these challenges can be met with elegant solutions that leverage the power of high-performance computing systems. Quantification of DNA methylation, data visualization, statistical approaches to compare DNA methylation between sample groups, and examples of integrating DNA methylation data with other -omics data sets are also discussed. The reader is encouraged to use this article as a foundation to pursue advanced topics in DNA methylation measurement and data analysis, particularly the application of bioinformatics and computational biology principles to generate a deeper understanding of mechanisms linking DNA methylation to cellular function.


Assuntos
5-Metilcitosina/análise , Metilação de DNA , 5-Metilcitosina/imunologia , 5-Metilcitosina/isolamento & purificação , Sequência de Bases , Biologia Computacional/métodos , Ilhas de CpG , DNA/química , DNA/efeitos dos fármacos , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Sequenciamento de Nucleotídeos em Larga Escala , Imunoprecipitação , Metilação , Estrutura Molecular , Hibridização de Ácido Nucleico , Controle de Qualidade , Alinhamento de Sequência , Sulfitos/farmacologia
18.
Gastroenterology ; 157(3): 744-759.e4, 2019 09.
Artigo em Inglês | MEDLINE | ID: mdl-31154022

RESUMO

BACKGROUND & AIMS: Many genetic and environmental factors, including family history, dietary fat, and inflammation, increase risk for colon cancer development. Peroxisome proliferator-activated receptor alpha (PPARα) is a nuclear receptor that regulates systemic lipid homeostasis. We explored the role of intestinal PPARα in colon carcinogenesis. METHODS: Colon cancer was induced in mice with intestine-specific disruption of Ppara (PparaΔIE), Pparafl/fl (control), and mice with disruption of Ppara that express human PPARA (human PPARA transgenic mice), by administration of azoxymethane with or without dextran sulfate sodium (DSS). Colons were collected from mice and analyzed by immunoblots, quantitative polymerase chain reaction, and histopathology. Liquid chromatography coupled with mass spectrometry-based metabolomic analyses were performed on urine and colons. We used molecular biology and biochemical approaches to study mechanisms in mouse colons, primary intestinal epithelial cells, and colon cancer cell lines. Gene expression data and clinical features of patients with colorectal tumors were obtained from Oncomine, and human colorectal-tumor specimens and adjacent normal tissues were collected and analyzed by immunohistochemistry. RESULTS: Levels of Ppara messenger RNA were reduced in colon tumors from mice. PparaΔIE mice developed more and larger colon tumors than control mice following administration of azoxymethane, with or without DSS. Metabolomic analyses revealed increases in methylation-related metabolites in urine and colons from PparaΔIE mice, compared with control mice, following administration of azoxymethane, with or without DSS. Levels of DNA methyltransferase 1 (DNMT1) and protein arginine methyltransferase 6 (PRMT6) were increased in colon tumors from PparaΔIE mice, compared with colon tumors from control mice. Depletion of PPARα reduced the expression of retinoblastoma protein, resulting in increased expression of DNMT1 and PRMT6. DNMT1 and PRMT6 decreased expression of the tumor suppressor genes Cdkn1a (P21) and Cdkn1b (p27) via DNA methylation and histone H3R2 dimethylation-mediated repression of transcription, respectively. Fenofibrate protected human PPARA transgenic mice from azoxymethane and DSS-induced colon cancer. Human colon adenocarcinoma specimens had lower levels of PPARA and retinoblastoma protein and higher levels of DNMT1 and PRMT6 than normal colon tissues. CONCLUSIONS: Loss of PPARα from the intestine promotes colon carcinogenesis by increasing DNMT1-mediated methylation of P21 and PRMT6-mediated methylation of p27 in mice. Human colorectal tumors have lower levels of PPARA messenger RNA and protein than nontumor tissues. Agents that activate PPARα might be developed for chemoprevention or treatment of colon cancer.


Assuntos
Adenocarcinoma/prevenção & controle , Colo/enzimologia , Neoplasias do Colo/prevenção & controle , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA , Proteínas Nucleares/metabolismo , PPAR alfa/metabolismo , Proteína-Arginina N-Metiltransferases/metabolismo , Adenocarcinoma/enzimologia , Adenocarcinoma/genética , Adenocarcinoma/patologia , Animais , Anticarcinógenos/farmacologia , Estudos de Casos e Controles , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/metabolismo , Transformação Celular Neoplásica/patologia , Colo/patologia , Neoplasias do Colo/enzimologia , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA/efeitos dos fármacos , Bases de Dados Genéticas , Modelos Animais de Doenças , Fenofibrato/farmacologia , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Nucleares/genética , PPAR alfa/agonistas , PPAR alfa/deficiência , PPAR alfa/genética , Proteína-Arginina N-Metiltransferases/genética , Transdução de Sinais
19.
Toxicology ; 424: 152225, 2019 08 01.
Artigo em Inglês | MEDLINE | ID: mdl-31150806

RESUMO

Growing evidences indicate that epigenetic modification involves in the mechanisms of atherosclerosis, which intersects with oxidative stress pathway. Arsenic is an important environmental contaminant and has been linked to atherosclerosis. However, the exact mechanism is not well understood. In the present study, we analyzed the effect of arsenic on oxidative stress, ABCA1 promoter methylation and cholesterol efflux of THP-1 macrophages. Results showed that arsenic could induce ROS-mediated DNA methyltransferase 1 (DNMT1) transcription and activity up-regulation, causing ABCA1 promoter to be hypermethylated with repressed expression. In addition, arsenic depleted the methyl donor S-adenosylmethionine (SAM) and induced global DNA hypomethylation. Arsenic inhibited cholesterol efflux of THP-1 macrophages, which could be attenuated after pretreatment with NAC or DNMT inhibitor 5-Aza-2'-deoxycytidine, but not with SAM. All of the findings suggest that arsenic inhibit cholesterol efflux of THP-1 macrophages via ROS-mediated ABCA1 hypermethylation.


Assuntos
Transportador 1 de Cassete de Ligação de ATP/efeitos dos fármacos , Transportador 1 de Cassete de Ligação de ATP/metabolismo , Arsênico/farmacologia , Colesterol/metabolismo , Macrófagos/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Acetilcisteína/farmacologia , Antioxidantes/farmacologia , Linhagem Celular , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , Humanos , Macrófagos/efeitos dos fármacos , Metilação/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , S-Adenosilmetionina/farmacologia
20.
Ann Rheum Dis ; 78(10): 1420-1429, 2019 10.
Artigo em Inglês | MEDLINE | ID: mdl-31239244

RESUMO

OBJECTIVES: Osteoarthritis (OA) is the most common degenerative joint disease in aged population and its development is significantly influenced by aberrant epigenetic modifications of numerous OA susceptible genes; however, the precise mechanisms that DNA methylation alterations affect OA pathogenesis remain undefined. This study investigates the critical role of epigenetic PPARγ (peroxisome proliferator-activated receptor-gamma) suppression in OA development. METHODS: Articular cartilage expressions of PPARγ and bioactive DNA methyltransferases (DNMTs) from OA patients and mice incurred by DMM (destabilisation of medial meniscus) were examined. DNA methylation status of both human and mouse PPARγ promoters were assessed by methylated specific PCR and/or bisulfite-sequencing PCR. OA protections by a pharmacological DNA demethylating agent 5Aza (5-Aza-2'-deoxycytidine) were compared between wild type and PPARγ knockout mice. RESULTS: Articular cartilages from both OA patients and DMM mice display substantial PPARγ suppressions likely due to aberrant elevations of DNMT1 and DNMT3a and consequential PPARγ promoter hypermethylation. 5Aza known to inhibit both DNMT1 and DNMT3a reversed the PPARγ promoter hypermethylation, recovered the PPARγ loss and effectively attenuated the cartilage damage in OA mice. 5Aza also inhibited the OA-associated excessive inflammatory cytokines and deficit anti-oxidant enzymes, which were blocked by a specific PPARγ inhibitor in cultured chondrocytes. Further, 5Aza-confered protections against the cartilage damage and the associated abnormalities of OA-susceptible factors were significantly abrogated in PPARγ knockout mice. CONCLUSION: Epigenetic PPARγ suppression plays a key role in OA development and PPARγ preservation via promoter demethylation possesses promising therapeutic potentials in clinical treatment of OA and the related joint diseases.


Assuntos
Metilação de DNA/genética , Osteoartrite/genética , PPAR gama/fisiologia , Animais , Cartilagem Articular/metabolismo , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Modelos Animais de Doenças , Epigênese Genética , Humanos , Meniscos Tibiais/metabolismo , Camundongos , Camundongos Knockout , Regiões Promotoras Genéticas
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