RESUMO
DNA methylation is a fundamental epigenetic modification regulating gene expression. Aberrant DNA methylation is the most common molecular lesion in cancer cells. However, medical intervention has been limited to the use of broadly acting, small molecule-based demethylating drugs with significant side-effects and toxicities. To allow for targeted DNA demethylation, we integrated two nucleic acid-based approaches: DNMT1 interacting RNA (DiR) and RNA aptamer strategy. By combining the RNA inherent capabilities of inhibiting DNMT1 with an aptamer platform, we generated a first-in-class DNMT1-targeted approach - aptaDiR. Molecular modelling of RNA-DNMT1 complexes coupled with biochemical and cellular assays enabled the identification and characterization of aptaDiR. This RNA bio-drug is able to block DNA methylation, impair cancer cell viability and inhibit tumour growth in vivo. Collectively, we present an innovative RNA-based approach to modulate DNMT1 activity in cancer or diseases characterized by aberrant DNA methylation and suggest the first alternative strategy to overcome the limitations of currently approved non-specific hypomethylating protocols, which will greatly improve clinical intervention on DNA methylation.
Assuntos
Metilação de DNA , RNA , RNA/genética , RNA/metabolismo , DNA (Citosina-5-)-Metiltransferases/metabolismo , Regulação Neoplásica da Expressão Gênica , DNA (Citosina-5-)-Metiltransferase 1/genética , DNA (Citosina-5-)-Metiltransferase 1/metabolismo , Epigênese GenéticaRESUMO
Nutritional intake impacts the human epigenome by directing epigenetic pathways in normal cell development via as yet unknown molecular mechanisms. Consequently, imbalance in the nutritional intake is able to dysregulate the epigenetic profile and drive cells towards malignant transformation. Here we present a novel epigenetic effect of the essential nutrient, NAD. We demonstrate that impairment of DNMT1 enzymatic activity by NAD-promoted ADP-ribosylation leads to demethylation and transcriptional activation of the CEBPA gene, suggesting the existence of an unknown NAD-controlled region within the locus. In addition to the molecular events, NAD- treated cells exhibit significant morphological and phenotypical changes that correspond to myeloid differentiation. Collectively, these results delineate a novel role for NAD in cell differentiation, and indicate novel nutri-epigenetic strategies to regulate and control gene expression in human cells.
Assuntos
Diferenciação Celular , Metilação de DNA/genética , NAD/farmacologia , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Diferenciação Celular/efeitos dos fármacos , Linhagem Celular , Desmetilação do DNA/efeitos dos fármacos , Humanos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Células Mieloides/citologia , Células Mieloides/efeitos dos fármacos , Neoplasias/genética , Neoplasias/patologia , Fosforilação Oxidativa/efeitos dos fármacos , Poli Adenosina Difosfato Ribose/metabolismo , Poli(ADP-Ribose) Polimerases/metabolismo , Regiões Promotoras Genéticas/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Gênica/efeitos dos fármacosRESUMO
E-cadherin is a calcium-dependent cell-cell adhesion molecule extensively studied for its involvement in tissue formation, epithelial cell behavior, and suppression of cancer. However, E-cadherin expression in the hematopoietic system has not been fully elucidated. Combining single-cell RNA-sequencing analyses and immunophenotyping, we revealed that progenitors expressing high levels of E-cadherin and contained within the granulocyte-monocyte progenitors (GMPs) fraction have an enriched capacity to differentiate into basophils and mast cells. We detected E-cadherin expression on committed progenitors before the expression of other reported markers of these lineages. We named such progenitors pro-BMPs (pro-basophil and mast cell progenitors). Using RNA sequencing, we observed transcriptional priming of pro-BMPs to the basophil and mast cell lineages. We also showed that GATA-2 directly regulates E-cadherin expression in the basophil and mast cell lineages, thus providing a mechanistic connection between the expression of this cell surface marker and the basophil and mast cell fate specification.
Assuntos
Caderinas/genética , Fator de Transcrição GATA2/metabolismo , Células-Tronco Hematopoéticas/fisiologia , Animais , Basófilos/fisiologia , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Linhagem da Célula/genética , Linhagem da Célula/imunologia , Células Cultivadas , Mastócitos/fisiologia , Camundongos , Cultura Primária de Células , RNA-Seq , Análise de Célula ÚnicaRESUMO
DNA methylation controls several cellular processes, from early development to old age, including biological responses to endogenous or exogenous stimuli contributing to disease transition. As a result, minimal DNA methylation changes during developmental stages drive severe phenotypes, as observed in germ-line imprinting disorders, while genome-wide alterations occurring in somatic cells are linked to cancer onset and progression. By summarizing the molecular events governing DNA methylation, we focus on the methods that have facilitated mapping and understanding of this epigenetic mark in healthy conditions and diseases. Overall, we review the bright (health-related) and dark (disease-related) side of DNA methylation changes, outlining how bulk and single-cell genomic analyses are moving toward the identification of new molecular targets and driving the development of more specific and less toxic demethylating agents.
Assuntos
Metilação de DNA/fisiologia , Animais , Epigênese Genética , Epigenômica/métodos , Genoma , Impressão Genômica , Humanos , Neoplasias/metabolismoRESUMO
Dementia contributes substantially to the burden of disability experienced at old age, and mitochondrial dysfunction (MD) was identified as common final pathway in brain aging and Alzheimer's disease. Due to its early appearance, MD is a promising target for nutritional prevention strategies and polyphenols as potential neurohormetic inducers may be strong neuroprotective candidates. This study aimed to investigate the effects of a polyphenol-rich grape skin extract (PGE) on age-related dysfunctions of brain mitochondria, memory, life span and potential hormetic pathways in C57BL/6J mice. PGE was administered at a dose of 200 mg/kg body weight/d in a 3-week short-term, 6-month long-term and life-long study. MD in the brains of aged mice (19-22 months old) compared to young mice (3 months old) was demonstrated by lower ATP levels and by impaired mitochondrial respiratory complex activity (except for mice treated with antioxidant-depleted food pellets). Long-term PGE feeding partly enhanced brain mitochondrial respiration with only minor beneficial effect on brain ATP levels and memory of aged mice. Life-long PGE feeding led to a transient but significant shift of survival curve toward higher survival rates but without effect on the overall survival. The moderate effects of PGE were associated with elevated SIRT1 but not SIRT3 mRNA expressions in brain and liver tissue. The beneficial effects of the grape extract may have been influenced by the profile of bioavailable polyphenols and the starting point of interventions.
