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1.
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
2.
Cancer Sci ; 111(1): 186-199, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31746077

RESUMO

Activity of transcriptional co-activator with PDZ binding domain (TAZ) protein is strongly implicated in the pathogenesis of human cancer and is influenced by tumor metabolism. High levels of lactate concentration in the tumor microenvironment as a result of metabolic reprogramming are inversely correlated with patient overall survival. Herein, we investigated the role of lactate in the regulation of the activity of TAZ and showed that glycolysis-derived lactate efficiently increased TAZ expression and activity in lung cancer cells. We showed that the reactive oxygen species (ROS) generated by lactate-fueled oxidative phosphorylation (OXPHOS) in mitochondria activated AKT and thereby inhibited glycogen synthase kinase 3 beta/beta-transducin repeat-containing proteins (GSK-3ß/ß-TrCP)-mediated ubiquitination and degradation of DNA methyltransferase 1 (DNMT1). Upregulation of DNMT1 by lactate caused hypermethylation of TAZ negative regulator of the LATS2 gene promoter, leading to TAZ activation. Moreover, TAZ binds to the promoter of DNMT1 and is necessary for DNMT1 transcription. Our study showed a molecular mechanism of DNMT1 in linking tumor metabolic reprogramming to the Hippo-TAZ pathway and functional significance of the DNMT1-TAZ feedback loop in the migratory and invasive potential of lung cancer cells.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Ácido Láctico/metabolismo , Estresse Oxidativo/genética , Transativadores/genética , Transcrição Genética/genética , Ativação Transcricional/genética , Linhagem Celular Tumoral , Glicogênio Sintase Quinase 3 beta/genética , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/genética , Espécies Reativas de Oxigênio/metabolismo
3.
Int J Mol Sci ; 20(20)2019 Oct 14.
Artigo em Inglês | MEDLINE | ID: mdl-31615086

RESUMO

MiRNAs, a small family of non-coding RNA, are now emerging as regulators of stem cell pluripotency, differentiation, and autophagy, thus controlling stem cell behavior. Stem cells are undifferentiated elements capable to acquire specific phenotype under different kind of stimuli, being a main tool for regenerative medicine. Within this context, we have previously shown that stem cells isolated from Wharton jelly multipotent stem cells (WJ-MSCs) exhibit gender differences in the expression of the stemness related gene OCT4 and the epigenetic modulator gene DNA-Methyltransferase (DNMT1). Here, we further analyze this gender difference, evaluating adipogenic and osteogenic differentiation potential, autophagic process, and expression of miR-145, miR-148a, and miR-185 in WJ-MSCs derived from males and females. These miRNAs were selected since they are involved in OCT4 and DNMT1 gene expression, and in stem cell differentiation. Our results indicate a difference in the regulatory circuit involving miR-148a/DNMT1/OCT4 autophagy in male WJ-MSCs as compared to female cells. Moreover, no difference was detected in the expression of the two-differentiation regulating miRNA (miR-145 and miR-185). Taken together, our results highlight a different behavior of WJ-MSCs from males and females, disclosing the chance to better understand cellular processes as autophagy and stemness, usable for future clinical applications.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , MicroRNAs/genética , Fator 3 de Transcrição de Octâmero/genética , Células-Tronco Pluripotentes/metabolismo , Adipogenia/genética , Autofagia/genética , Diferenciação Celular/genética , Epigênese Genética , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Humanos , Masculino , Células-Tronco Mesenquimais/metabolismo , Osteogênese/genética
4.
Nat Methods ; 16(11): 1087-1093, 2019 11.
Artigo em Inglês | MEDLINE | ID: mdl-31659326

RESUMO

Gene knock outs (KOs) are efficiently engineered through CRISPR-Cas9-induced frameshift mutations. While the efficiency of DNA editing is readily verified by DNA sequencing, a systematic understanding of the efficiency of protein elimination has been lacking. Here we devised an experimental strategy combining RNA sequencing and triple-stage mass spectrometry to characterize 193 genetically verified deletions targeting 136 distinct genes generated by CRISPR-induced frameshifts in HAP1 cells. We observed residual protein expression for about one third of the quantified targets, at variable levels from low to original, and identified two causal mechanisms, translation reinitiation leading to N-terminally truncated target proteins or skipping of the edited exon leading to protein isoforms with internal sequence deletions. Detailed analysis of three truncated targets, BRD4, DNMT1 and NGLY1, revealed partial preservation of protein function. Our results imply that systematic characterization of residual protein expression or function in CRISPR-Cas9-generated KO lines is necessary for phenotype interpretation.


Assuntos
Sistemas CRISPR-Cas/genética , Técnicas de Inativação de Genes , Proteínas de Ciclo Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Éxons , Humanos , Mutação , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/genética , Fatores de Transcrição/genética
5.
Mol Med Rep ; 20(4): 3597-3608, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31485618

RESUMO

Clofarabine (2­chloro­2'­fluoro­2'­deoxyarabinosyladenine, CIF), a second­generation 2'­deoxyadenosine analog, possesses a variety of anti­cancer activities, including the capacity to modulate DNA methylation marks. Bioactive nutrients, including resveratrol (RSV) and all­trans retinoic acid (ATRA) have been indicated to regulate epigenetic machinery in malignant cells. The purpose of the current study was to evaluate whether the tested phytochemicals, RSV or ATRA, can improve the therapeutic epigenetic effects of CIF in chronic myeloid leukemia (CML) cells. The present study investigates, to the best of our knowledge, for the first time, the influence of CIF in combination with RSV or ATRA on the expression of relevant modifiers of DNA methylation machinery, including DNA Methyltransferase 1 (DNMT1) and Cyclin dependent kinase inhibitor 1A (CDKN1A) in CML cells. Subsequently, the combinatorial effects on promoter methylation and transcript levels of methylation­silenced tumor suppressor genes (TSGs), including phosphatase and tensin homologue (PTEN) and retinoic acid receptor beta (RARB), were estimated using MSRA and qPCR, respectively. The tested TSGs were chosen according to bioinformatical analysis of publicly available clinical data of human DNA methylation and gene expression arrays in leukemia patients. The K562 cell line was used as an experimental CML in vitro model. Following a period of 72 h exposure of K562 cells, the tested combinations led to significant cell growth inhibition and induction of caspase­3­dependent apoptosis. These observations were accompanied by DNMT1 downregulation and CDKN1A upregulation, with a concomitant enhanced decrease in DNMT1 protein level, especially after ATRA treatment with CIF. Concurrent methylation­mediated RARB and PTEN reactivation was detected. The results of the current study demonstrated that CIF that was used in combination with the tested phytochemicals, RSV or ATRA, exhibited a greater ability to remodel DNA methylation marks and promote cell death in CML cells. These results may support the application of CIF combinations with natural bioactive agents in anti­leukemic epigenetic therapy.


Assuntos
Antineoplásicos/farmacologia , Clofarabina/farmacologia , Metilação de DNA/efeitos dos fármacos , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Resveratrol/farmacologia , Tretinoína/farmacologia , Protocolos de Quimioterapia Combinada Antineoplásica/farmacologia , Antioxidantes/farmacologia , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Sinergismo Farmacológico , Epigênese Genética/efeitos dos fármacos , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia
6.
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
7.
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
8.
Nat Commun ; 10(1): 3182, 2019 07 18.
Artigo em Inglês | MEDLINE | ID: mdl-31320637

RESUMO

DNA methylation contributes to the maintenance of genomic integrity in somatic cells, in part through the silencing of transposable elements. In this study, we use CRISPR-Cas9 technology to delete DNMT1, the DNA methyltransferase key for DNA methylation maintenance, in human neural progenitor cells (hNPCs). We observe that inactivation of DNMT1 in hNPCs results in viable, proliferating cells despite a global loss of DNA CpG-methylation. DNA demethylation leads to specific transcriptional activation and chromatin remodeling of evolutionarily young, hominoid-specific LINE-1 elements (L1s), while older L1s and other classes of transposable elements remain silent. The activated L1s act as alternative promoters for many protein-coding genes involved in neuronal functions, revealing a hominoid-specific L1-based transcriptional network controlled by DNA methylation that influences neuronal protein-coding genes. Our results provide mechanistic insight into the role of DNA methylation in silencing transposable elements in somatic human cells, as well as further implicating L1s in human brain development and disease.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/genética , Desmetilação do DNA , Metilação de DNA/genética , Elementos Nucleotídeos Longos e Dispersos/genética , Células-Tronco Neurais/citologia , Encéfalo/embriologia , Sistemas CRISPR-Cas/genética , Montagem e Desmontagem da Cromatina/genética , Ilhas de CpG/genética , Inativação Gênica/fisiologia , Humanos , Células-Tronco Neurais/metabolismo , Ativação Transcricional/genética
9.
Int J Med Sci ; 16(4): 576-582, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31171909

RESUMO

Background: Kawasaki disease (KD) is the most common acute coronary vasculitis to occur in children. Although we have uncovered global DNA hypomethylation in KD, its underlying cause remains uncertain. In this study, we performed a survey of transcript levels of DNA methyltransferases and demethylases in KD patients. Materials and Methods: We recruited 145 participants for this study. The chip studies consisted of 18 KD patients that were analyzed before undergoing intravenous immunoglobulin (IVIG) treatment and at least 3 weeks after IVIG treatment, as well as 36 control subjects, using Affymetrix GeneChip® Human Transcriptome Array 2.0. An additional study of 91 subjects was performed in order to validate real-time quantitative PCR. Results: In our microarray study, the mRNA levels of DNMT1 and DNMT3A were significantly lower while TET2 was higher in acute-stage KD patients compared to the healthy controls. Through PCR validation, we observed that the expression of DNMT1 and TET2 are consistent with the Transcriptome Array 2.0 results. Furthermore, we observed significantly lower DMNT1 mRNA levels following IVIG treatment between those who developed CAL and those who did not. Conclusion: Our findings provide an evidence of DNA methyltransferases and demethylases changes and are among the first report that transient DNA hypomethylation is induced during acute inflammatory phase of Kawasaki disease.


Assuntos
Doença da Artéria Coronariana/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Ligação a DNA/genética , Síndrome de Linfonodos Mucocutâneos/genética , Proteínas Proto-Oncogênicas/genética , Adolescente , Criança , Pré-Escolar , Doença da Artéria Coronariana/complicações , Doença da Artéria Coronariana/patologia , Vasos Coronários/metabolismo , Vasos Coronários/patologia , Metilação de DNA/genética , Feminino , Humanos , Imunoglobulinas Intravenosas/administração & dosagem , Lactente , Recém-Nascido , Inflamação/genética , Inflamação/patologia , Masculino , Síndrome de Linfonodos Mucocutâneos/complicações , Síndrome de Linfonodos Mucocutâneos/patologia , RNA Mensageiro/genética , Transcriptoma/genética
10.
BMC Cancer ; 19(1): 609, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31226958

RESUMO

BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is the major subtype of esophageal cancer with high aggressiveness and poor prognosis. There is an urgent need for understanding the molecular mechanism underlying the development and progression of ESCC. METHODS: ESCC tissues and corresponding non-neoplastic tissues were collected. The expression and function of miR-124-3p and BCAT1 in two cell lines KYSE-150 and Eca109 were determined. RESULTS: We show downregulation of miR-124-3p expression in ESCC tissues, which is highly correlated with proliferation and migration of ESCC cell lines KYSE-150 and Eca109. miR-124-3p show high correlation with TNM stage and differentiation grade. Furthermore, miR-124-3p directly targets mRNA 3'UTR region of BCAT1, which results in upregulation of BCAT1 expression as observed in ESCC tissues and cell lines. Also, our data indicates that BCAT1 high expression is strongly linked to the disease-free survival, tumor size, pathologic stage, T classification and differentiation grade. On the other hand, we clarified the upstream mechanism regulating miR-124-3p expression in ESCC, which involves in the hypermethylation-silencing regulation mediated by DNA methyltransferase 1(DNMT1), which is of high expression in ESCC tissues and cell lines in the present study. In addition, DNMT1 knockdown or inhibition of DNMT1 function contributes to downregulation of miR-124-3p and BCAT1 expression. CONCLUSIONS: Our study thus clarifies a new mechanism that DNMT1/miR-124/BCAT1 axis regulates the development and progression of ESCC.


Assuntos
DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Neoplasias Esofágicas/metabolismo , Neoplasias Esofágicas/patologia , Carcinoma de Células Escamosas do Esôfago/metabolismo , Carcinoma de Células Escamosas do Esôfago/patologia , Transaminases/metabolismo , Regiões 3' não Traduzidas , Linhagem Celular Tumoral , Movimento Celular , Proliferação de Células , Sobrevivência Celular , DNA (Citosina-5-)-Metiltransferase 1/genética , Metilação de DNA , Progressão da Doença , Intervalo Livre de Doença , Feminino , Humanos , Masculino , MicroRNAs/genética , MicroRNAs/metabolismo , Pessoa de Meia-Idade , Invasividade Neoplásica/fisiopatologia , Estadiamento de Neoplasias , Transaminases/genética , Transfecção
11.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 35(4): 351-356, 2019 Apr.
Artigo em Chinês | MEDLINE | ID: mdl-31167695

RESUMO

Objective To explore the genome-wide DNA methylation level and the expression of DNA methylation-related enzymes in the villi tissue of patients with unexplained recurrent spontaneous abortion (URSA). Methods The villi samples were obtained from thirty-one URSA patients (URSA group) and thirty pregnancy women who underwent induced abortion (control group). Total DNA methylation was determined by ELISA. Real-time quantitative PCR was used to detect mRNA levels of DNMT1, DNMT3a, DNMT3b, TET1, TET2 and TET3, and Western blot analysis was used to detect protein levels of DNMT1, DNMT2, DNMT3, TET1, TET2 and TET3. Immunohistochemistry was performed to detect the expression and distribution of DNMT1, DNMT3a, DNMT3b, TET1, TET2 and TET3 in villi tissues. Results Methylation level of total DNA in the URSA group was significantly lower than that of the control group. Compared with the control group, mRNA and protein expression levels of DNMT1 and DNMT3b significantly decreased, while TET1 and TET2 significantly increased in the villi of the URSA group. Conclusion The methylation level is reduced in the villi of URSA women, which may be correlated with up-regulated expression of DNMT1 and DNMT3b and down-regulated expression of TET1 and TET2.


Assuntos
Aborto Habitual/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA , Proteínas de Ligação a DNA/genética , Oxigenases de Função Mista/genética , Proteínas Proto-Oncogênicas/genética , Feminino , Humanos , Imuno-Histoquímica , Gravidez , RNA Mensageiro
12.
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
13.
Nat Cell Biol ; 21(7): 835-844, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31209294

RESUMO

Totipotency refers to the ability of a cell to generate all of the cell types of an organism. Unlike pluripotency, the establishment of totipotency is poorly understood. In mouse embryonic stem cells, Dux drives a small percentage of cells into a totipotent state by expressing 2-cell-embryo-specific transcripts. To understand how this transition takes place, we performed single-cell RNA-seq, which revealed a two-step transcriptional reprogramming process characterized by downregulation of pluripotent genes in the first step and upregulation of the 2-cell-embryo-specific elements in the second step. To identify factors controlling the transition, we performed a CRISPR-Cas9-mediated screen, which revealed Myc and Dnmt1 as two factors preventing the transition. Mechanistic studies demonstrate that Myc prevents downregulation of pluripotent genes in the first step, while Dnmt1 impedes 2-cell-embryo-specific gene activation in the second step. Collectively, the findings of our study reveal insights into the establishment and regulation of the totipotent state in mouse embryonic stem cells.


Assuntos
Reprogramação Celular/genética , DNA (Citosina-5-)-Metiltransferase 1/genética , Células-Tronco Embrionárias/metabolismo , Genes myc/genética , Animais , Diferenciação Celular/genética , Epigênese Genética/genética , Camundongos , Células-Tronco Embrionárias Murinas , Células-Tronco Pluripotentes/citologia , Células-Tronco Totipotentes/citologia
14.
Nutrients ; 11(6)2019 Jun 22.
Artigo em Inglês | MEDLINE | ID: mdl-31234503

RESUMO

Nutrition in the postnatal period is associated with metabolic programming. One of the presumed underlying mechanisms involves epigenetic modifications (e.g., DNA methylation). Breastfeeding has an unknown impact on DNA methylation at a young age. Within the Maternal Nutrition and Offspring's Epigenome (MANOE) study, we assessed the effect of breastfeeding duration on infant growth and buccal methylation in obesity-related genes (n = 101). A significant difference was found between infant growth and buccal RXRA and LEP methylation at 12 months of breastfeeding. For RXRA CpG2 methylation, a positive association was found with duration of breastfeeding (slope = 0.217; 95% confidence interval (CI) 1.03, 0.330; p < 0.001). For RXRA CpG3 and CpG, mean methylation levels were significantly lower when children were breastfed for 4-6 months compared to non-breastfed children (only CpG3), and those breastfed for 7-9 months, 10-12 months, or 1-3 months. On the other hand, higher LEP CpG3 methylation was observed when mothers breastfed 7-9 months (6.1%) as compared to breastfeeding for 1-3 months (4.3%; p = 0.007) and 10-12 months (4.6%; p = 0.04). In addition, we observed that infant weight was significantly lower when children were breastfed for 10-12 months. Breastfeeding duration was associated with epigenetic variations in RXRA and LEP at 12 months and with infant biometry/growth. Our results support the hypothesis that breastfeeding could induce epigenetic changes in infants.


Assuntos
Aleitamento Materno , Metilação de DNA , Leptina/genética , Metaboloma/genética , Obesidade Pediátrica/genética , Receptor X Retinoide alfa/genética , Adulto , Fatores Etários , Desenvolvimento Infantil , Ilhas de CpG , DNA (Citosina-5-)-Metiltransferase 1/genética , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Lactente , Fator de Crescimento Insulin-Like II/genética , Obesidade Pediátrica/metabolismo , Obesidade Pediátrica/fisiopatologia , Estudos Prospectivos , Fatores de Risco , Fatores de Tempo , Ganho de Peso/genética , Adulto Jovem
15.
BMB Rep ; 52(5): 342-347, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31068247

RESUMO

Methylation is a primary epigenetic mechanism regulating gene expression. 5-aza-2'-deoxycytidine is an FDA-approved drug prescribed for treatment of cancer by inhibiting DNA-Methyl-Transferase 1 (DNMT1). Results of this study suggest that prolonged treatment with 5-aza-2'-deoxycytidine could induce centrosome abnormalities in cancer cells and that CEP131, a centrosome protein, is regulated by DNMT1. Interestingly, cancer cell growth was attenuated in vitro and in vivo by inhibiting the expression of Cep131. Finally, Cep131-deficient cells were more sensitive to treatment with DNMT1 inhibitors. These findings suggest that Cep131 is a potential novel anti-cancer target. Agents that can inhibit this protein may be useful alone or in combination with DNMT1 inhibitors to treat cancer. [BMB Reports 2019; 52(5): 342-347].


Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , DNA (Citosina-5-)-Metiltransferase 1/antagonistas & inibidores , Decitabina/farmacologia , Proteínas dos Microtúbulos/antagonistas & inibidores , Neoplasias do Colo do Útero/tratamento farmacológico , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Metilação de DNA/efeitos dos fármacos , DNA de Neoplasias/genética , DNA de Neoplasias/metabolismo , Epigênese Genética , Feminino , Células HEK293 , Células HeLa , Humanos , Proteínas dos Microtúbulos/genética , Proteínas dos Microtúbulos/metabolismo , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/metabolismo
16.
Genes (Basel) ; 10(5)2019 05 14.
Artigo em Inglês | MEDLINE | ID: mdl-31091831

RESUMO

Despite a large body of evidence supporting the role of aberrant DNA methylation in etiology of several human diseases, the fundamental mechanisms that regulate the activity of mammalian DNA methyltransferases (DNMTs) are not fully understood. Recent advances in whole genome association studies have helped identify mutations and genetic alterations of DNMTs in various diseases that have a potential to affect the biological function and activity of these enzymes. Several of these mutations are germline-transmitted and associated with a number of hereditary disorders, which are potentially caused by aberrant DNA methylation patterns in the regulatory compartments of the genome. These hereditary disorders usually cause neurological dysfunction, growth defects, and inherited cancers. Biochemical and biological characterization of DNMT variants can reveal the molecular mechanism of these enzymes and give insights on their specific functions. In this review, we introduce roles and regulation of DNA methylation and DNMTs. We discuss DNMT mutations that are associated with rare diseases, the characterized effects of these mutations on enzyme activity and provide insights on their potential effects based on the known crystal structure of these proteins.


Assuntos
Metilases de Modificação do DNA/genética , Metilases de Modificação do DNA/metabolismo , Mutação em Linhagem Germinativa/genética , Animais , Ataxia Cerebelar/genética , DNA , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Genoma , Mutação em Linhagem Germinativa/fisiologia , Perda Auditiva Neurossensorial/genética , Neuropatias Hereditárias Sensoriais e Autônomas/genética , Humanos , Mutação , Narcolepsia/genética , Relação Estrutura-Atividade
17.
FEBS Open Bio ; 9(6): 1063-1070, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31090214

RESUMO

DNA methylation of promoter regions is often associated with epigenetic silencing of gene expression, and DNA methyltransferase (DNMTs) has been used to suppress gene expression. In order to explore the synergistic roles of two methyltransferase members Dnmt3a and Dnmt1, we constructed expression plasmid that could express a recombinant DNMTs consisting of the C-terminal domains of both Dnmt3a and Dnmt1 fused to a zinc finger domain which binds to the PD-L1 promoter of human prostate cancer cells (DU145). Programmed death ligand 1 (PD-L1, B7-H1, CD-274) is a transmembrane protein widely expressed on antigen-presenting and other immune cells. The interaction of PD-L1 with its receptor PD-1 is considered an 'immune checkpoint' for possible cancer therapy. DU145 cells treated with the Dnmt3aC-1C plasmid showed significantly reduced expression of PD-L1 as compared to Dnmt3aC or Dnmt1C alone. Our results show that the fusion of Dnmt1 improves the methylation activity of Dnmt3a and enhances its biological functions. This combinatorial strategy can be used to better control PD-L1 expression to support cytotoxic T lymphocytes (CTL) response against tumors.


Assuntos
Antígeno B7-H1/genética , Metilação de DNA/genética , Regiões Promotoras Genéticas , Neoplasias da Próstata/genética , Neoplasias da Próstata/patologia , Dedos de Zinco/genética , Antígeno B7-H1/antagonistas & inibidores , Antígeno B7-H1/metabolismo , Sítios de Ligação , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , Inativação Gênica , Humanos , Ligação de Hidrogênio , Masculino , Plasmídeos , Ligação Proteica , Domínios Proteicos , Proteínas Recombinantes/farmacologia , Transdução Genética
18.
Food Chem Toxicol ; 130: 161-173, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31112703

RESUMO

Aberrant epigenetic modifications are responsible for tumor development and cancer progression; however, readily reversible. Bioactive molecules from diets are promising to cure cancer by modulating epigenetic marks and changing immune response. These compounds specifically target the activity of DNMTs and HDACs to cure various human cancers. In view of this, we investigated the anticancer and epigenetic regulatory activities of an edible-plant Paederia foetida. The efficacy of methanolic extract of P. foetida leaves (MEPL) was tested for the modulation of epigenetic factors in gene silencing, i.e. DNMT and HDAC and expression pattern of certain tumor-suppressor genes. After treatment of prostate cancer cells (PC-3 and DU-145) with MEPL, lupeol and ß-sitosterol; induction of apoptosis, decrease in cellular-viability and inhibition of cellular-migration were noticed. Simultaneously there was inhibition of DNMT1, HDACs and pro-inflammatory, IL-6, IL1-ß, TNF-α and anti-inflammatory, IL-10 genes in cancer and THP1 cell lines. The DNMT1 protein content, enzyme activity and Bcl2 expression decreased significantly; however, expression of E-cadherin (CDH1) and pro-apoptotic gene Bax increased significantly after the treatment of cells with drugs. We conclude plant-derived compounds can be considered to target epigenetic machineries involved with malignant transformation and can open new avenues for cancer therapeutics provoking immune response.


Assuntos
Sobrevivência Celular/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Extratos Vegetais/farmacologia , Neoplasias da Próstata , Rubiaceae/química , Linhagem Celular Tumoral , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Histona Desacetilase 1/genética , Histona Desacetilase 1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Inflamação/genética , Masculino , Triterpenos Pentacíclicos , Compostos Fitoquímicos , Extratos Vegetais/química , Folhas de Planta/química , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Sitosteroides
19.
Future Oncol ; 15(15): 1729-1744, 2019 May.
Artigo em Inglês | MEDLINE | ID: mdl-31038361

RESUMO

Aims: To investigate roles of miR-29a-DNMT1-SOCS1 axis in cervical cancer invasion and migration. Materials & methods: The methylation level of SOCS1 was determined by methylation specific PCR. The cell apoptosis, proliferation, migration and invasion were examined by Annexin-V/PI staining, MTT and colony formation assays, plus scratch and transwell assays respectively. The expressions of epithelial-mesenchymal transition and NF-κB related proteins were determined by western blotting. Results: MiR-29a was downregulated, accompanied with DNMT1 upregulation and SOCS1 downregulation in cervical cancer tissues. MiR-29a suppressed DNMT1, inhibited SOCS1 promoter methylation and upregulated its expression. Moreover, miR-29a promoted cell apoptosis, suppressed proliferation, inhibited migration and invasion via inactivation of NF-κB signaling pathway in cervical cancer cells. Conclusion: MiR-29a-DNMT1-SOCS1 axis plays an important role on invasion and metastasis in cervical cancer via NF-κB signaling pathway.


Assuntos
Metilação de DNA , Regulação Neoplásica da Expressão Gênica , MicroRNAs/genética , Interferência de RNA , Proteína 1 Supressora da Sinalização de Citocina/genética , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Adulto , Idoso , Apoptose/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Proliferação de Células , DNA (Citosina-5-)-Metiltransferase 1/genética , Decitabina/farmacologia , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes Reporter , Humanos , Pessoa de Meia-Idade , NF-kappa B/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Estadiamento de Neoplasias
20.
FEBS Open Bio ; 9(6): 1137-1143, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31001930

RESUMO

Abdominal arterial aneurysm (AAA) shares many features with autoimmune diseases and appears to be a T-cell-mediated process. In addition, certain epigenetic changes, including DNA methylation, are associated with AAA. In this study, we investigated epigenetic modifications in regulatory T cells (Tregs) from AAA patients. We used flow cytometry to sort FOXP3+ CD4+ CD25+ Tregs from the peripheral blood of AAA patients and from healthy controls (HC), and then detected DNA methylation and histone modifications by ELISA. The DNA methylation rate of Tregs was significantly higher in AAA patients than in the HC group (0.159 ± 0.08% vs 0.098 ± 0.03%, P < 0.05), while the acetylation rates of H3 and H3K9 histones were lower in the AAA than in the HC group. We also examined the expression of mRNA encoding enzymes that catalyze making and removing epigenetic modifications by real-time PCR: we found that mRNA levels of DNA methyltransferase (DNMT) 1 and DNMT3A were higher in the AAA than in the HC group, mRNA levels of methyl-CpG-binding domain protein (MBD) 2 and MBD4 were higher in the AAA than in the HC group (MBD2: 6.21 ± 2.57 vs 3.04 ± 1.45; MBD4: 7.76 ± 3.48 vs 4.97 ± 3.10; both P < 0.05), and mRNA levels of histone deacetylase (HDAC) 1 and HDAC5 were significantly up-regulated in the AAA compared with the HC group (HDAC1: 2.17 ± 1.18 vs 1.51 ± 0.99; HDAC5: 1.35 ± 0.49 vs 0.94 ± 0.76; both P < 0.05). Together, our results reveal that rates of DNA methylation and histone modifications of Tregs are significantly altered in AAA patients.


Assuntos
Aneurisma da Aorta Abdominal/sangue , Aneurisma da Aorta Abdominal/genética , Epigênese Genética/genética , Linfócitos T Reguladores/fisiologia , Acetilação , Idoso , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferases/genética , Metilação de DNA/genética , Proteínas de Ligação a DNA/genética , Endodesoxirribonucleases/genética , Feminino , Histona Desacetilase 1/genética , Histona Desacetilases/genética , Histonas/genética , Humanos , Masculino , Pessoa de Meia-Idade , RNA Mensageiro/genética , Regulação para Cima/genética
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