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1.
Epigenetics Chromatin ; 14(1): 56, 2021 12 15.
Artículo en Inglés | MEDLINE | ID: mdl-34906184

RESUMEN

BACKGROUND: DNA methylation plays an important role in regulating gene expression in mammals. The covalent DNMT1 inhibitors 5-azacytidine and decitabine are widely used in research to reduce DNA methylation levels, but they impart severe cytotoxicity which limits their demethylation capability and confounds interpretation of experiments. Recently, a non-covalent inhibitor of DNMT1 called GSK-3484862 was developed by GlaxoSmithKline. We sought to determine whether GSK-3484862 can induce demethylation more effectively than 5-azanucleosides. Murine embryonic stem cells (mESCs) are an ideal cell type in which to conduct such experiments, as they have a high degree of DNA methylation but tolerate dramatic methylation loss. RESULTS: We determined the cytotoxicity and optimal concentration of GSK-3484862 by treating wild-type (WT) or Dnmt1/3a/3b triple knockout (TKO) mESC with different concentrations of the compound, which was obtained from two commercial sources. Concentrations of 10 µM or below were readily tolerated for 14 days of culture. Known DNA methylation targets such as germline genes and GLN-family transposons were upregulated within 2 days of the start of GSK-3484862 treatment. By contrast, 5-azacytidine and decitabine induced weaker upregulation of methylated genes and extensive cell death. Whole-genome bisulfite sequencing showed that treatment with GSK-3484862 induced dramatic DNA methylation loss, with global CpG methylation levels falling from near 70% in WT mESC to less than 18% after 6 days of treatment with GSK-3484862. The treated cells showed a methylation level and pattern similar to that observed in Dnmt1-deficient mESCs. CONCLUSIONS: GSK-3484862 mediates striking demethylation in mESCs with minimal non-specific toxicity.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas , Células Madre Embrionarias , Animales , Azacitidina/toxicidad , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , Desmetilación , Células Madre Embrionarias/metabolismo , Ratones
2.
Nat Commun ; 12(1): 5716, 2021 09 29.
Artículo en Inglés | MEDLINE | ID: mdl-34588438

RESUMEN

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.


Asunto(s)
Metilación de ADN/genética , Antígenos de Histocompatibilidad/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Proteínas Nucleares/genética , Neoplasias de la Próstata/genética , Proteínas Represoras/genética , Animales , Protocolos de Quimioterapia Combinada Antineoplásica/farmacología , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapéutico , Azacitidina/farmacología , Azacitidina/uso terapéutico , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/antagonistas & inhibidores , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Docetaxel/farmacología , Docetaxel/uso terapéutico , Regulación hacia Abajo/efectos de los fármacos , Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Sinergismo Farmacológico , Epigénesis Genética/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Genes Supresores de Tumor , Humanos , Masculino , Ratones , Mutación , Proteínas Nucleares/metabolismo , Neoplasias de la Próstata/tratamiento farmacológico , Neoplasias de la Próstata/patología , Estabilidad Proteica/efectos de los fármacos , Proteolisis/efectos de los fármacos , Proteínas Represoras/metabolismo , Ensayos Antitumor por Modelo de Xenoinjerto
3.
Cells ; 10(9)2021 08 26.
Artículo en Inglés | MEDLINE | ID: mdl-34571854

RESUMEN

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.


Asunto(s)
Desaminasas APOBEC/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteínas Musculares/metabolismo , Factor de Transcripción PAX7/metabolismo , Desaminasas APOBEC/genética , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/genética , Proliferación Celular/genética , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Femenino , Expresión Génica/genética , Regulación del Desarrollo de la Expresión Génica/genética , Masculino , Ratones , Ratones Endogámicos C57BL , Desarrollo de Músculos/genética , Proteínas Musculares/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiología , Factor de Transcripción PAX7/genética , Factor de Transcripción PAX7/fisiología , Células Madre Pluripotentes/metabolismo , Células Madre Pluripotentes/fisiología , Células Satélite del Músculo Esquelético/metabolismo
4.
Nat Commun ; 12(1): 5541, 2021 09 20.
Artículo en Inglés | MEDLINE | ID: mdl-34545097

RESUMEN

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.


Asunto(s)
Represión Epigenética/genética , Exosomas/metabolismo , VIH-1/genética , Animales , Encéfalo/patología , Encéfalo/virología , Línea Celular , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Exosomas/ultraestructura , Regulación Viral de la Expresión Génica , Vectores Genéticos/metabolismo , Células HEK293 , Infecciones por VIH/virología , Humanos , Lentivirus/metabolismo , Leucocitos Mononucleares/metabolismo , Ratones , ARN Mensajero/genética , ARN Mensajero/metabolismo , Secuencias Repetidas Terminales/genética , Carga Viral , Dedos de Zinc
5.
BMC Cancer ; 21(1): 914, 2021 Aug 12.
Artículo en Inglés | MEDLINE | ID: mdl-34380460

RESUMEN

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.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN , Epistasis Genética , Regulación Neoplásica de la Expresión Génica , Cadenas Pesadas de Miosina/genética , Neoplasias Gástricas/genética , Neoplasias Gástricas/patología , Adulto , Anciano , Animales , Línea Celular Tumoral , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Modelos Animales de Enfermedad , Progresión de la Enfermedad , Femenino , Xenoinjertos , Humanos , Inmunohistoquímica , Masculino , Ratones , Persona de Mediana Edad , Cadenas Pesadas de Miosina/metabolismo , Estadificación de Neoplasias , Regiones Promotoras Genéticas , Unión Proteica , Neoplasias Gástricas/metabolismo , Carga Tumoral
6.
FASEB J ; 35(9): e21847, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34405464

RESUMEN

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.


Asunto(s)
Composición Corporal , Dieta Rica en Proteínas , Epigénesis Genética , Padre , Microbioma Gastrointestinal , Insulina/metabolismo , Exposición Paterna , Tejido Adiposo/metabolismo , Adiposidad , Envejecimiento , Animales , Peso Corporal , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Dieta Alta en Grasa , Sacarosa en la Dieta , Ingestión de Energía , Metabolismo Energético , Ácidos Grasos/metabolismo , Femenino , Fertilidad , Prueba de Tolerancia a la Glucosa , Hormonas/metabolismo , Resistencia a la Insulina , Hígado/metabolismo , Masculino , Tamaño de los Órganos , Embarazo , ARN Pequeño no Traducido/metabolismo , Ratas , Ratas Sprague-Dawley , Respuesta de Saciedad , Destete
7.
Biomolecules ; 11(8)2021 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-34439754

RESUMEN

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.


Asunto(s)
Tejido Adiposo Pardo/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Factor 5 Regulador Miogénico/metabolismo , Obesidad/metabolismo , Adipocitos Marrones/metabolismo , Tejido Adiposo Blanco/metabolismo , Alelos , Animales , Peso Corporal , Cruzamientos Genéticos , ADN (Citosina-5-)-Metiltransferasas/genética , Modelos Animales de Enfermedad , Metabolismo Energético , Epigénesis Genética , Femenino , Resistencia a la Insulina , Ratones , Ratones Noqueados , Células Musculares/metabolismo , RNA-Seq , Termogénesis
8.
J Mol Biol ; 433(19): 167186, 2021 09 17.
Artículo en Inglés | MEDLINE | ID: mdl-34375615

RESUMEN

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.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , ADN/genética , ADN/metabolismo , Animales , Islas de CpG , ADN (Citosina-5-)-Metiltransferasas/química , Humanos , Ratones , Modelos Moleculares , Conformación Proteica
9.
Psychopharmacology (Berl) ; 238(11): 3107-3118, 2021 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-34328516

RESUMEN

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.


Asunto(s)
Cannabinoides , Cocaína , Animales , Cannabinoides/farmacología , Cocaína/farmacología , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Ratones , Corteza Prefrontal/metabolismo , Recompensa
10.
Int J Mol Sci ; 22(14)2021 Jul 15.
Artículo en Inglés | MEDLINE | ID: mdl-34299205

RESUMEN

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.


Asunto(s)
Metilación de ADN , Impresión Genómica , Factor II del Crecimiento Similar a la Insulina/metabolismo , Región de Control de Posición , ARN Largo no Codificante/genética , Factores de Transcripción/metabolismo , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Fibroblastos/metabolismo , Fibroblastos/patología , Edición Génica , Células HEK293 , Humanos , Factor II del Crecimiento Similar a la Insulina/genética , Regiones Promotoras Genéticas , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética , Proteínas de Motivos Tripartitos/genética , Proteínas de Motivos Tripartitos/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo
11.
Nat Protoc ; 16(8): 4004-4030, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34244697

RESUMEN

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.


Asunto(s)
ADN/metabolismo , Análisis de la Célula Individual , Secuencia de Aminoácidos , Antibacterianos/farmacología , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Doxiciclina/farmacología , Epigenoma , Regulación de la Expresión Génica/efectos de los fármacos , Regulación de la Expresión Génica/fisiología , Humanos , Péptidos y Proteínas de Señalización Intracelular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Transcriptoma
12.
Cell Death Dis ; 12(7): 667, 2021 07 02.
Artículo en Inglés | MEDLINE | ID: mdl-34215719

RESUMEN

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.


Asunto(s)
Relojes Biológicos , Diferenciación Celular , Metilación de ADN , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/genética , Células Madre Embrionarias de Ratones/metabolismo , Miocitos Cardíacos/metabolismo , Regiones Promotoras Genéticas , ARN Largo no Codificante/genética , Nodo Sinoatrial/metabolismo , Animales , Células Cultivadas , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación hacia Abajo , Regulación del Desarrollo de la Expresión Génica , Canales Regulados por Nucleótidos Cíclicos Activados por Hiperpolarización/metabolismo , Ratones , ARN Largo no Codificante/metabolismo , Nodo Sinoatrial/citología
13.
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi ; 37(7): 608-615, 2021 Jul.
Artículo en Chino | MEDLINE | ID: mdl-34140072

RESUMEN

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.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas , Células Epiteliales , Vía de Señalización Wnt , Animales , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Células Epiteliales/metabolismo , Fibrosis , Glucosa , Glucógeno Sintasa Quinasa 3 beta/genética , Ratones , beta Catenina/genética , beta Catenina/metabolismo
14.
Aging (Albany NY) ; 13(12): 16267-16286, 2021 06 18.
Artículo en Inglés | MEDLINE | ID: mdl-34148029

RESUMEN

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.


Asunto(s)
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patología , ADN (Citosina-5-)-Metiltransferasas/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patología , MicroARNs/metabolismo , ARN Largo no Codificante/metabolismo , Regulación hacia Arriba/genética , Anciano , Apoptosis/genética , Secuencia de Bases , Línea Celular Tumoral , Movimiento Celular/genética , Proliferación Celular/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Progresión de la Enfermedad , Transición Epitelial-Mesenquimal/genética , Femenino , Regulación Neoplásica de la Expresión Génica , Humanos , Masculino , MicroARNs/genética , Persona de Mediana Edad , Invasividad Neoplásica , Metástasis de la Neoplasia , Fosforilación , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Largo no Codificante/genética
15.
Front Immunol ; 12: 653030, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-34093541

RESUMEN

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.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/genética , Metilación de ADN/inmunología , Regulación Leucémica de la Expresión Génica/inmunología , Leucemia Mieloide Aguda/genética , Escape del Tumor/genética , Animales , Técnicas de Cultivo de Célula , Técnicas de Cocultivo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Técnicas de Silenciamiento del Gen , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/inmunología , Macrófagos/inmunología , Ratones , Mutación , RNA-Seq , Resistina/farmacología , Resistina/uso terapéutico , Células THP-1 , Escape del Tumor/efectos de los fármacos , Microambiente Tumoral/efectos de los fármacos , Microambiente Tumoral/genética , Microambiente Tumoral/inmunología , Ensayos Antitumor por Modelo de Xenoinjerto
16.
Aging (Albany NY) ; 13(11): 15193-15213, 2021 06 08.
Artículo en Inglés | MEDLINE | ID: mdl-34100772

RESUMEN

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.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Daño del ADN , Reparación del ADN , Células de la Granulosa/patología , Insuficiencia Ovárica Primaria/metabolismo , Insuficiencia Ovárica Primaria/patología , 5-Metilcitosina/metabolismo , Animales , Apoptosis , Línea Celular Tumoral , Metilación de ADN/genética , Femenino , Modelos Biológicos , Estrés Oxidativo , Ratas Sprague-Dawley
17.
Cell Death Dis ; 12(7): 642, 2021 06 23.
Artículo en Inglés | MEDLINE | ID: mdl-34162834

RESUMEN

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.


Asunto(s)
Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/metabolismo , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Neuropatías Diabéticas/enzimología , Fosfatidilinositol 3-Quinasa/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Células de Schwann/enzimología , Nervio Ciático/enzimología , Tiorredoxinas/metabolismo , Animales , Apoptosis , Proteínas Reguladoras de la Apoptosis/metabolismo , Autofagia , Proteínas Relacionadas con la Autofagia/metabolismo , Glucemia/metabolismo , Proteínas Portadoras/genética , Proteínas de Ciclo Celular/genética , Línea Celular , ADN (Citosina-5-)-Metiltransferasa 1/genética , ADN (Citosina-5-)-Metiltransferasas/genética , Neuropatías Diabéticas/genética , Neuropatías Diabéticas/patología , Modelos Animales de Enfermedad , Humanos , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Ratas , Células de Schwann/patología , Nervio Ciático/patología , Transducción de Señal , Tiorredoxinas/genética
18.
Adv Healthc Mater ; 10(16): e2100821, 2021 08.
Artículo en Inglés | MEDLINE | ID: mdl-34174172

RESUMEN

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.


Asunto(s)
ADN (Citosina-5-)-Metiltransferasas , Metilación de ADN , Proteína Quinasa C-alfa/metabolismo , Transporte Activo de Núcleo Celular , Animales , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Matriz Extracelular/metabolismo , Ratones , Proteína Quinasa C-alfa/genética
19.
Biomolecules ; 11(6)2021 05 22.
Artículo en Inglés | MEDLINE | ID: mdl-34067359

RESUMEN

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.


Asunto(s)
Proteína Quinasa CDC2/biosíntesis , Carcinogénesis/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Regulación Enzimológica de la Expresión Génica , Regulación Leucémica de la Expresión Génica , Leucemia Mieloide Aguda/metabolismo , Mutación , Proteínas de Neoplasias/metabolismo , Animales , Proteína Quinasa CDC2/genética , Carcinogénesis/genética , Línea Celular Tumoral , ADN (Citosina-5-)-Metiltransferasas/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Humanos , Leucemia Mieloide Aguda/genética , Ratones , Células 3T3 NIH , Proteínas de Neoplasias/genética
20.
Neurochem Res ; 46(8): 2181-2191, 2021 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-34032956

RESUMEN

Application of chemotherapeutic oxaliplatin represses gene transcription through induction of DNA methylation, which may contribute to oxaliplatin-induced chronic pain. Here, Ddr1, which showed an increased methylation in the promoter, was screened from the SRA methylation database (PRJNA587622) after oxaliplatin treatment. qPCR and MeDIP assays verified that oxaliplatin treatment increased the methylation in Ddr1 promoter region and decreased the expression of DDR1 in the neurons of spinal dorsal horn. In addition, overexpression of DDR1 by intraspinal injection of AAV-hSyn-Ddr1 significantly alleviated the mechanical allodynia induced by oxaliplatin. Furthermore, we found that oxaliplatin treatment increased the expression of DNMT3b and ZEB1 in dorsal horn neurons, and promoted the interaction between DNMT3b and ZEB1. Intrathecal injection of ZEB1 siRNA inhibited the enhanced recruitment of DNMT3b and the hypermethylation in Ddr1 promoter induced by oxaliplatin. Finally, ZEB1 siRNA rescued the DDR1 downregulation and mechanical allodynia induced by oxaliplatin. In conclusion, these results suggested that the ZEB1 recruited DNMT3b to the Ddr1 promoter, which induced the DDR1 downregulation and contributed to the oxaliplatin-induced chronic pain.


Asunto(s)
Dolor Crónico/metabolismo , Metilación de ADN/fisiología , Receptor con Dominio Discoidina 1/genética , Oxaliplatino/efectos adversos , Asta Dorsal de la Médula Espinal/metabolismo , Homeobox 1 de Unión a la E-Box con Dedos de Zinc/metabolismo , Animales , Dolor Crónico/inducido químicamente , ADN/metabolismo , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Hiperalgesia/inducido químicamente , Hiperalgesia/metabolismo , Masculino , Neuralgia/inducido químicamente , Neuralgia/metabolismo , Regiones Promotoras Genéticas/fisiología , ARN Interferente Pequeño/farmacología , Ratas Sprague-Dawley , Asta Dorsal de la Médula Espinal/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos
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