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1.
Cell ; 175(5): 1228-1243.e20, 2018 11 15.
Artículo en Inglés | MEDLINE | ID: mdl-30392959

RESUMEN

Genetic drivers of cancer can be dysregulated through epigenetic modifications of DNA. Although the critical role of DNA 5-methylcytosine (5mC) in the regulation of transcription is recognized, the functions of other non-canonical DNA modifications remain obscure. Here, we report the identification of novel N6-methyladenine (N6-mA) DNA modifications in human tissues and implicate this epigenetic mark in human disease, specifically the highly malignant brain cancer glioblastoma. Glioblastoma markedly upregulated N6-mA levels, which co-localized with heterochromatic histone modifications, predominantly H3K9me3. N6-mA levels were dynamically regulated by the DNA demethylase ALKBH1, depletion of which led to transcriptional silencing of oncogenic pathways through decreasing chromatin accessibility. Targeting the N6-mA regulator ALKBH1 in patient-derived human glioblastoma models inhibited tumor cell proliferation and extended the survival of tumor-bearing mice, supporting this novel DNA modification as a potential therapeutic target for glioblastoma. Collectively, our results uncover a novel epigenetic node in cancer through the DNA modification N6-mA.


Asunto(s)
Adenina/análogos & derivados , Neoplasias Encefálicas/patología , Metilación de ADN , Glioblastoma/patología , Adenina/análisis , Adenina/química , Adulto , Anciano , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/antagonistas & inhibidores , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/genética , Histona H2a Dioxigenasa, Homólogo 1 de AlkB/metabolismo , Animales , Astrocitos/citología , Astrocitos/metabolismo , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/mortalidad , Hipoxia de la Célula , Niño , Epigenómica , Femenino , Glioblastoma/metabolismo , Glioblastoma/mortalidad , Heterocromatina/metabolismo , Histonas/metabolismo , Humanos , Estimación de Kaplan-Meier , Masculino , Ratones , Persona de Mediana Edad , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/metabolismo , Proteína p53 Supresora de Tumor/metabolismo
2.
Cell ; 171(7): 1495-1507.e15, 2017 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-29224783

RESUMEN

Current genome-editing systems generally rely on inducing DNA double-strand breaks (DSBs). This may limit their utility in clinical therapies, as unwanted mutations caused by DSBs can have deleterious effects. CRISPR/Cas9 system has recently been repurposed to enable target gene activation, allowing regulation of endogenous gene expression without creating DSBs. However, in vivo implementation of this gain-of-function system has proven difficult. Here, we report a robust system for in vivo activation of endogenous target genes through trans-epigenetic remodeling. The system relies on recruitment of Cas9 and transcriptional activation complexes to target loci by modified single guide RNAs. As proof-of-concept, we used this technology to treat mouse models of diabetes, muscular dystrophy, and acute kidney disease. Results demonstrate that CRISPR/Cas9-mediated target gene activation can be achieved in vivo, leading to measurable phenotypes and amelioration of disease symptoms. This establishes new avenues for developing targeted epigenetic therapies against human diseases. VIDEO ABSTRACT.


Asunto(s)
Sistemas CRISPR-Cas , Epigénesis Genética , Marcación de Gen/métodos , Terapia Genética/métodos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/terapia , Utrofina/genética , Animales , Secuencia de Bases , Modelos Animales de Enfermedad , Distrofina/genética , Interleucina-10/genética , Proteínas Klotho , Proteínas de la Membrana/genética , Ratones , Ratones Endogámicos C57BL , Activación Transcripcional
3.
Cell Death Dis ; 14(7): 399, 2023 07 05.
Artículo en Inglés | MEDLINE | ID: mdl-37407581

RESUMEN

Insufficient insulin secretion is a hallmark of type 2 diabetes and has been attributed to beta cell identity loss characterized by decreased expression of several key beta cell genes. The pro-inflammatory factor BMP-2 is upregulated in islets of Langerhans from individuals with diabetes and acts as an inhibitor of beta cell function and proliferation. Exposure to BMP-2 induces expression of Id1-4, Hes-1, and Hey-1 which are transcriptional regulators associated with loss of differentiation. The aim of this study was to investigate the mechanism by which BMP-2 induces beta cell dysfunction and loss of cell maturity. Mouse islets exposed to BMP-2 for 10 days showed impaired glucose-stimulated insulin secretion and beta cell proliferation. BMP-2-induced beta cell dysfunction was associated with decreased expression of cell maturity and proliferation markers specific to the beta cell such as Ins1, Ucn3, and Ki67 and increased expression of Id1-4, Hes-1, and Hey-1. The top 30 most regulated proteins significantly correlated with corresponding mRNA expression. BMP-2-induced gene expression changes were associated with a predominant reduction in acetylation of H3K27 and a decrease in NeuroD1 chromatin binding activity. These results show that BMP-2 induces loss of beta cell maturity and suggest that remodeling of H3K27ac and decreased NeuroD1 DNA binding activity participate in the effect of BMP-2 on beta cell dysfunction.


Asunto(s)
Diabetes Mellitus Tipo 2 , Código de Histonas , Animales , Ratones , Proteína Morfogenética Ósea 2/metabolismo , Cromatina , Diabetes Mellitus Tipo 2/metabolismo , Procesamiento Proteico-Postraduccional , Transducción de Señal
4.
J Clin Invest ; 133(8)2023 04 17.
Artículo en Inglés | MEDLINE | ID: mdl-36821378

RESUMEN

Adaptation of the islet ß cell insulin-secretory response to changing insulin demand is critical for blood glucose homeostasis, yet the mechanisms underlying this adaptation are unknown. Here, we have shown that nutrient-stimulated histone acetylation plays a key role in adapting insulin secretion through regulation of genes involved in ß cell nutrient sensing and metabolism. Nutrient regulation of the epigenome occurred at sites occupied by the chromatin-modifying enzyme lysine-specific demethylase 1 (Lsd1) in islets. ß Cell-specific deletion of Lsd1 led to insulin hypersecretion, aberrant expression of nutrient-response genes, and histone hyperacetylation. Islets from mice adapted to chronically increased insulin demand exhibited shared epigenetic and transcriptional changes. Moreover, we found that genetic variants associated with type 2 diabetes were enriched at LSD1-bound sites in human islets, suggesting that interpretation of nutrient signals is genetically determined and clinically relevant. Overall, these studies revealed that adaptive insulin secretion involves Lsd1-mediated coupling of nutrient state to regulation of the islet epigenome.


Asunto(s)
Diabetes Mellitus Tipo 2 , Células Secretoras de Insulina , Islotes Pancreáticos , Ratones , Humanos , Animales , Secreción de Insulina/genética , Diabetes Mellitus Tipo 2/genética , Diabetes Mellitus Tipo 2/metabolismo , Histonas/genética , Histonas/metabolismo , Epigenoma , Islotes Pancreáticos/metabolismo , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Glucosa/metabolismo
5.
Nat Genet ; 53(4): 455-466, 2021 04.
Artículo en Inglés | MEDLINE | ID: mdl-33795864

RESUMEN

Single-nucleus assay for transposase-accessible chromatin using sequencing (snATAC-seq) creates new opportunities to dissect cell type-specific mechanisms of complex diseases. Since pancreatic islets are central to type 2 diabetes (T2D), we profiled 15,298 islet cells by using combinatorial barcoding snATAC-seq and identified 12 clusters, including multiple alpha, beta and delta cell states. We cataloged 228,873 accessible chromatin sites and identified transcription factors underlying lineage- and state-specific regulation. We observed state-specific enrichment of fasting glucose and T2D genome-wide association studies for beta cells and enrichment for other endocrine cell types. At T2D signals localized to islet-accessible chromatin, we prioritized variants with predicted regulatory function and co-accessibility with target genes. A causal T2D variant rs231361 at the KCNQ1 locus had predicted effects on a beta cell enhancer co-accessible with INS and genome editing in embryonic stem cell-derived beta cells affected INS levels. Together our findings demonstrate the power of single-cell epigenomics for interpreting complex disease genetics.


Asunto(s)
Cromatina/química , Diabetes Mellitus Tipo 2/genética , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Canal de Potasio KCNQ1/genética , Células Secretoras de Polipéptido Pancreático/metabolismo , Células Secretoras de Somatostatina/metabolismo , Glucemia/metabolismo , Diferenciación Celular , Cromatina/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Epigenómica , Ayuno , Perfilación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Células Secretoras de Glucagón/patología , Secuenciación de Nucleótidos de Alto Rendimiento , Células Madre Embrionarias Humanas/citología , Humanos , Células Secretoras de Insulina/patología , Canal de Potasio KCNQ1/metabolismo , Familia de Multigenes , Células Secretoras de Polipéptido Pancreático/patología , Polimorfismo Genético , Análisis de la Célula Individual , Células Secretoras de Somatostatina/patología , Factores de Transcripción/clasificación , Factores de Transcripción/genética , Factores de Transcripción/metabolismo
6.
Mol Cell Endocrinol ; 496: 110524, 2019 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-31362031

RESUMEN

Decreased insulin secretory capacity in Type 2 diabetes mellitus is associated with beta-cell dedifferentiation and inflammation. We hypothesize that prolonged exposure of beta-cells to low concentrations of IL-1ß induce beta-cell dedifferentiation characterized by impaired glucose-stimulated insulin secretion, reduced expression of key beta-cell genes and changes in histone modifications at gene loci known to affect beta-cell function. Ten days exposure to IL-1ß at non-cytotoxic concentrations reduced insulin secretion and beta-cell proliferation and decreased expression of key beta-cell identity genes, including MafA and Ucn3 and decreased H3K27ac at the gene loci, suggesting that inflammatory cytokines directly affects the epigenome. Following removal of IL-1ß, beta-cell function was normalized and mRNA expression of beta-cell identity genes, such as insulin and Ucn3 returned to pre-stimulation levels. Our findings indicate that prolonged exposure to low concentrations of IL-1ß induces epigenetic changes associated with loss of beta-cell identity as observed in Type 2 diabetes.


Asunto(s)
Diabetes Mellitus Tipo 2/metabolismo , Regulación de la Expresión Génica , Histonas/metabolismo , Células Secretoras de Insulina/metabolismo , Interleucina-1beta/metabolismo , Procesamiento Proteico-Postraduccional , Animales , Diabetes Mellitus Tipo 2/patología , Epigénesis Genética , Células Secretoras de Insulina/patología , Factores de Transcripción Maf de Gran Tamaño/biosíntesis , Masculino , Ratones , Urocortinas/biosíntesis
7.
iScience ; 21: 681-694, 2019 Nov 22.
Artículo en Inglés | MEDLINE | ID: mdl-31733514

RESUMEN

Pancreatic endocrine cell differentiation is orchestrated by the action of transcription factors that operate in a gene regulatory network to activate endocrine lineage genes and repress lineage-inappropriate genes. MicroRNAs (miRNAs) are important modulators of gene expression, yet their role in endocrine cell differentiation has not been systematically explored. Here we characterize miRNA-regulatory networks active in human endocrine cell differentiation by combining small RNA sequencing, miRNA over-expression, and network modeling approaches. Our analysis identified Let-7g, Let-7a, miR-200a, miR-127, and miR-375 as endocrine-enriched miRNAs that drive endocrine cell differentiation-associated gene expression changes. These miRNAs are predicted to target different transcription factors, which converge on genes involved in cell cycle regulation. When expressed in human embryonic stem cell-derived pancreatic progenitors, these miRNAs induce cell cycle exit and promote endocrine cell differentiation. Our study delineates the role of miRNAs in human endocrine cell differentiation and identifies miRNAs that could facilitate endocrine cell reprogramming.

8.
Cell Rep ; 25(10): 2904-2918.e8, 2018 12 04.
Artículo en Inglés | MEDLINE | ID: mdl-30517875

RESUMEN

Pancreatic ß cell physiology changes substantially throughout life, yet the mechanisms that drive these changes are poorly understood. Here, we performed comprehensive in vivo quantitative proteomic profiling of pancreatic islets from juvenile and 1-year-old mice. The analysis revealed striking differences in abundance of enzymes controlling glucose metabolism. We show that these changes in protein abundance are associated with higher activities of glucose metabolic enzymes involved in coupling factor generation as well as increased activity of the coupling factor-dependent amplifying pathway of insulin secretion. Nutrient tracing and targeted metabolomics demonstrated accelerated accumulation of glucose-derived metabolites and coupling factors in islets from 1-year-old mice, indicating that age-related changes in glucose metabolism contribute to improved glucose-stimulated insulin secretion with age. Together, our study provides an in-depth characterization of age-related changes in the islet proteome and establishes metabolic rewiring as an important mechanism for age-associated changes in ß cell function.


Asunto(s)
Senescencia Celular , Células Secretoras de Insulina/metabolismo , Metabolómica/métodos , Proteómica/métodos , Envejecimiento , Animales , Carbono/metabolismo , Respiración de la Célula/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Glucosa/metabolismo , Glucosa/farmacología , Secreción de Insulina , Masculino , Ratones Endogámicos C57BL , Proteoma/metabolismo
9.
Cell Metab ; 25(5): 1160-1175.e11, 2017 May 02.
Artículo en Inglés | MEDLINE | ID: mdl-28467932

RESUMEN

Pancreatic ß cell mass for appropriate blood glucose control is established during early postnatal life. ß cell proliferative capacity declines postnatally, but the extrinsic cues and intracellular signals that cause this decline remain unknown. To obtain a high-resolution map of ß cell transcriptome dynamics after birth, we generated single-cell RNA-seq data of ß cells from multiple postnatal time points and ordered cells based on transcriptional similarity using a new analytical tool. This analysis captured signatures of immature, proliferative ß cells and established high expression of amino acid metabolic, mitochondrial, and Srf/Jun/Fos transcription factor genes as their hallmark feature. Experimental validation revealed high metabolic activity in immature ß cells and a role for reactive oxygen species and Srf/Jun/Fos transcription factors in driving postnatal ß cell proliferation and mass expansion. Our work provides the first high-resolution molecular characterization of state changes in postnatal ß cells and paves the way for the identification of novel therapeutic targets to stimulate ß cell regeneration.


Asunto(s)
Proliferación Celular , Células Secretoras de Insulina/citología , Redes y Vías Metabólicas , Transcriptoma , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Células Cultivadas , Femenino , Regulación del Desarrollo de la Expresión Génica , Células HEK293 , Humanos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones Endogámicos C57BL , Mitocondrias/genética , Mitocondrias/metabolismo , Especies Reactivas de Oxígeno/metabolismo
10.
Cell Stem Cell ; 19(4): 516-529, 2016 10 06.
Artículo en Inglés | MEDLINE | ID: mdl-27570066

RESUMEN

Transit-amplifying nephron progenitor cells (NPCs) generate all of the nephrons of the mammalian kidney during development. Their limited numbers, poor in vitro expansion, and difficult accessibility in humans have slowed basic and translational research into renal development and diseases. Here, we show that with appropriate 3D culture conditions, it is possible to support long-term expansion of primary mouse and human fetal NPCs as well as NPCs derived from human induced pluripotent stem cells (iPSCs). Expanded NPCs maintain genomic stability, molecular homogeneity, and nephrogenic potential in vitro, ex vivo, and in vivo. Cultured NPCs are amenable to gene targeting and can form nephron organoids that engraft in vivo, functionally couple to the host's circulatory system, and produce urine-like metabolites via filtration. Together, these findings provide a technological platform for studying human nephrogenesis, modeling and diagnosing renal diseases, and drug discovery.


Asunto(s)
Técnicas de Cultivo de Célula/métodos , Nefronas/citología , Organogénesis , Células Madre/citología , Lesión Renal Aguda/patología , Lesión Renal Aguda/fisiopatología , Animales , Proliferación Celular , Células Cultivadas , Modelos Animales de Enfermedad , Edición Génica , Humanos , Pruebas de Función Renal , Ratones , Organoides/citología , Comunicación Paracrina , Células Madre/metabolismo , Factores de Tiempo
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