RESUMEN

Coactivator-associated arginine methyltransferase (CARM1/PRMT4)-mediated transcriptional coactivation and arginine methylation is known to regulate various tissue-specific differentiation events. Although CARM1 is expressed in the neural crest region in early development, coinciding with early neuronal progenitor specification, the role of CARM1 in any neuronal developmental pathways has been unexplored. Using a specific small-molecule inhibitor of CARM1-mediated H3R17 methylation in human embryonic stem cell line, we find that H3R17 methylation contributes to the maintenance of the astroglial cell population. A network of regulation was observed on the miR92a promoter by which H3R17-responsive Nanog bound to the miR92a promoter decreased upon inhibition, resulting in an abnormal gene expression program influencing the glial lineage. This was also true in zebrafish, in which, with the help of CARM1 inhibitor and CARM1 morpholinos, we show that inhibition of H3R17 methylation results in defective glial cell morphology and a sensory defect in a subpopulation. A gain-of-function strategy in which mCARM1 was introduced in the morpholino-treated embryos exhibited recovery of the sensory defect phenotype. This study thus establishes the functional cooperation between arginine methylation and microRNA expression in the neuronal developmental process, with potential implications in sensory development pathways.

Asunto(s)

Astrocitos/metabolismo , Proteínas de Homeodominio/genética , MicroARNs/genética , Proteína-Arginina N-Metiltransferasas/genética , Activación Transcripcional , Animales , Arginina/metabolismo , Línea Celular , Línea Celular Tumoral , Linaje de la Célula/genética , Embrión no Mamífero/embriología , Embrión no Mamífero/metabolismo , Cuerpos Embrioides/metabolismo , Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Redes Reguladoras de Genes , Histonas/metabolismo , Proteínas de Homeodominio/metabolismo , Humanos , Immunoblotting , Metilación , MicroARNs/metabolismo , Microscopía Fluorescente , Proteína Homeótica Nanog , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , Unión Proteica , Proteína-Arginina N-Metiltransferasas/metabolismo , Pez Cebra/embriología , Pez Cebra/genética

RESUMEN

The acetylation of histone and non-histone proteins controls a great deal of cellular functions, thereby affecting the entire organism, including the brain. Acetylation modifications are mediated through histone acetyltransferases (HAT) and deacetylases (HDAC), and the balance of these enzymes regulates neuronal homeostasis, maintaining the pre-existing acetyl marks responsible for the global chromatin structure, as well as regulating specific dynamic acetyl marks that respond to changes and facilitate neurons to encode and strengthen long-term events in the brain circuitry (e.g., memory formation). Unfortunately, the dysfunction of these finely-tuned regulations might lead to pathological conditions, and the deregulation of the HAT/HDAC balance has been implicated in neurological disorders. During the last decade, research has focused on HDAC inhibitors that induce a histone hyperacetylated state to compensate acetylation deficits. The use of these inhibitors as a therapeutic option was efficient in several animal models of neurological disorders. The elaboration of new cell-permeant HAT activators opens a new era of research on acetylation regulation. Although pathological animal models have not been tested yet, HAT activator molecules have already proven to be beneficial in ameliorating brain functions associated with learning and memory, and adult neurogenesis in wild-type animals. Thus, HAT activator molecules contribute to an exciting area of research.

Asunto(s)

Acetiltransferasas/genética , Histonas/genética , Enfermedades del Sistema Nervioso/terapia , Neuronas/metabolismo , Acetilación , Acetiltransferasas/metabolismo , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Enfermedades del Sistema Nervioso/genética , Enfermedades del Sistema Nervioso/metabolismo

RESUMEN

Although the brain functions of specific acetyltransferases such as the CREB-binding protein (CBP) and p300 have been well documented using mutant transgenic mice models, studies based on their direct pharmacological activation are still missing due to the lack of cell-permeable activators. Here we present a small-molecule (TTK21) activator of the histone acetyltransferases CBP/p300, which, when conjugated to glucose-based carbon nanosphere (CSP), passed the blood-brain barrier, induced no toxicity, and reached different parts of the brain. After intraperitoneal administration in mice, CSP-TTK21 significantly acetylated histones in the hippocampus and frontal cortex. Remarkably, CSP-TTK21 treatment promoted the formation of long and highly branched doublecortin-positive neurons in the subgranular zone of the dentate gyrus and reduced BrdU incorporation, suggesting that CBP/p300 activation favors maturation and differentiation of adult neuronal progenitors. In addition, mRNA levels of the neuroD1 differentiation marker and BDNF, a neurotrophin required for the terminal differentiation of newly generated neurons, were both increased in the hippocampus concomitantly with an enrichment of acetylated-histone on their proximal promoter. Finally, we found that CBP/p300 activation during a spatial training, while not improving retention of a recent memory, resulted in a significant extension of memory duration. This report is the first evidence for CBP/p300-mediated histone acetylation in the brain by an activator molecule, which has beneficial implications for the brain functions of adult neurogenesis and long-term memory. We propose that direct stimulation of acetyltransferase function could be useful in terms of therapeutic options for brain diseases.

Asunto(s)

Proteína de Unión a CREB/metabolismo , Activadores de Enzimas/farmacología , Memoria/efectos de los fármacos , Neurogénesis/efectos de los fármacos , Factores de Transcripción p300-CBP/metabolismo , Acetiltransferasas/metabolismo , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Encéfalo/crecimiento & desarrollo , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Recuento de Células , Núcleo Celular/metabolismo , Inmunoprecipitación de Cromatina , Dendritas/metabolismo , Dendritas/ultraestructura , Técnica del Anticuerpo Fluorescente , Hipocampo/citología , Hipocampo/metabolismo , Histona Acetiltransferasas/metabolismo , Histonas/aislamiento & purificación , Inmunohistoquímica , Masculino , Ratones , Ratones Endogámicos C57BL , Nanosferas , Neuronas/metabolismo , Neuronas/ultraestructura , Reacción en Cadena en Tiempo Real de la Polimerasa

RESUMEN

The recent developments in the field of epigenetics have changed the way the covalent modifications were perceived from mere chemical tags to important biological recruiting platforms as well as decisive factors in the process of transcriptional regulation and gene expression. Over the years, the parallel investigations in the area of epigenetics and disease have also shown the significance of the epigenetic modifications as important regulatory nodes that exhibit dysfunction in disease states. In the present scenario where epigenetic therapy is also being considered at par with the conventional therapeutic strategies, this article reviews the role of histone acetylation as an epigenetic mark involved in different biological processes associated with normal as well as abnormal gene expression states, modulation of this acetylation by small molecules and warrants the possibility of acetylation as a therapeutic target.

Asunto(s)

Epigénesis Genética/efectos de los fármacos , Inhibidores de Histona Desacetilasas/uso terapéutico , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Acetilación , Animales , Regulación de la Expresión Génica/efectos de los fármacos , Predisposición Genética a la Enfermedad , Humanos , Fenotipo , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genética , Transcripción Genética/efectos de los fármacos

Asunto(s)

Biología/educación , Química/educación , Investigación/educación , Bioquímica/economía , Bioquímica/educación , Biología/economía , Química/economía , ADN/química , Sistemas de Liberación de Medicamentos , Descubrimiento de Drogas , India , Péptidos/química , Péptidos/metabolismo , Investigación/economía , Universidades

RESUMEN

Chromatin modifications have gained immense significance in the past few decades as key regulators of gene expression. The enzymes responsible for these modifications along with the other non-histone proteins, remodeling factors and small RNAs modulate the chromatin dynamicity, which in turn directs the chromatin function. A concerted action of different modifying enzymes catalyzes these modifications, which are read by effector modules and converted to functional outcomes by various protein complexes. Several small molecules in the physiological system such as acetyl CoA, NAD(+), and ATP are actively involved in regulating these functional outcomes. Recent understanding in the field of epigenetics indicate the possibility of the existence of a network, 'the epigenetic language' involving cross talk among different modifications that could regulate cellular processes like transcription, replication and repair. Hence, these modifications are essential for the cellular homeostasis, and any alteration in this balance leads to a pathophysiological condition or disease manifestation. Therefore, it is becoming more evident that modulators of these modifying enzymes could be an attractive therapeutic strategy, popularly referred to as 'Epigenetic therapy.' Although this field is currently monopolized by DNA methylation and histone deacetylase inhibitors, this review highlights the modulators of the other modifications namely histone acetylation, lysine methylation and arginine methylation and argues in favor of their therapeutic potential.

Asunto(s)

Ensamble y Desensamble de Cromatina/efectos de los fármacos , Cromatina/metabolismo , Activadores de Enzimas/farmacología , Inhibidores Enzimáticos/farmacología , Histonas/metabolismo , Procesamiento Proteico-Postraduccional/efectos de los fármacos , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Acetilación/efectos de los fármacos , Adenosina Trifosfato/química , Adenosina Trifosfato/metabolismo , Animales , Cromatina/química , Reparación del ADN/efectos de los fármacos , Replicación del ADN/efectos de los fármacos , Activadores de Enzimas/química , Activadores de Enzimas/uso terapéutico , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/uso terapéutico , Histonas/química , Humanos , Metilación/efectos de los fármacos , NAD/química , NAD/metabolismo , Transcripción Genética/efectos de los fármacos

RESUMEN

Neurodegenerative diseases, such as polyglutamine-related diseases, amyotrophic lateral sclerosis, and Alzheimer's disease are accompanied by transcriptional dysfunctions, leading to neuronal death. It is becoming more evident that the chromatin acetylation status is impaired during the lifetime of neurons, by a common mechanism related to the loss of function of histone acetyltransferase (HAT) activity. Notably, the HAT termed cAMP response element binding protein (CREB)-binding protein (CBP) was shown to display neuroprotective functions. Several other HATs have now been shown to participate in basic but vital neuronal functions. In addition, there is increasing evidence of several HATs (including CBP), as essential regulators of neuronal plasticity and memory formation processes. In order to counteract neuronal loss and/or memory deficits in neurodegenerative diseases, the current therapeutic strategies involve the use of small molecules antagonizing histone deacetylase (HDAC) activity (i.e. HDAC inhibitors). Although this strategy lacks specificity, some of these molecules display promising therapeutic properties. With the rapidly evolving literature on HATs and their respective functions in neuronal survival and memory formation, it seems essential to envisage direct stimulation of the acetyltransferase function as a new therapeutic tool in neurodegenerative diseases. In this review, we will highlight the present understanding and the future prospects of such therapeutic approach.

Asunto(s)

Cromatina/metabolismo , Activadores de Enzimas/uso terapéutico , Histona Acetiltransferasas/metabolismo , Memoria/efectos de los fármacos , Enfermedades Neurodegenerativas/tratamiento farmacológico , Plasticidad Neuronal/efectos de los fármacos , Animales , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Activadores de Enzimas/química , Histona Acetiltransferasas/química , Inhibidores de Histona Desacetilasas/química , Inhibidores de Histona Desacetilasas/uso terapéutico , Humanos , Enfermedades Neurodegenerativas/enzimología , Enfermedades Neurodegenerativas/patología , Neuronas/enzimología , Neuronas/patología

Asunto(s)

Epigénesis Genética , Lisina/metabolismo , Metabolismo , Acetilación , Humanos , Terapéutica/métodos

RESUMEN

Methylation of the arginine residues of histones by methyltransferases has important consequences for chromatin structure and gene regulation; however, the molecular mechanism(s) of methyltransferase regulation is still unclear, as is the biological significance of methylation at particular arginine residues. Here, we report a novel specific inhibitor of coactivator-associated arginine methyltransferase 1 (CARM1; also known as PRMT4) that selectively inhibits methylation at arginine 17 of histone H3 (H3R17). Remarkably, this plant-derived inhibitor, called TBBD (ellagic acid), binds to the substrate (histone) preferentially at the signature motif, "KAPRK," where the proline residue (Pro-16) plays a critical role for interaction and subsequent enzyme inhibition. In a promoter-specific context, inhibition of H3R17 methylation represses expression of p21, a p53-responsive gene, thus implicating a possible role for H3 Arg-17 methylation in tumor suppressor function. These data establish TBBD as a novel specific inhibitor of arginine methylation and demonstrate substrate sequence-directed inhibition of enzyme activity by a small molecule and its physiological consequence.

Asunto(s)

Arginina/metabolismo , Ácido Elágico/metabolismo , Histonas/metabolismo , Proteína-Arginina N-Metiltransferasas/antagonistas & inhibidores , Proteína-Arginina N-Metiltransferasas/metabolismo , Secuencia de Aminoácidos , Animales , Línea Celular , Ácido Elágico/química , Regulación de la Expresión Génica , Histonas/química , Histonas/genética , Humanos , Lythraceae/química , Metilación , Modelos Moleculares , Datos de Secuencia Molecular , Estructura Molecular , Mutagénesis Sitio-Dirigida , Prolina/metabolismo , Estructura Terciaria de Proteína , Proteína-Arginina N-Metiltransferasas/química , Proteína-Arginina N-Metiltransferasas/genética , Termodinámica , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Xenopus laevis

RESUMEN

Lysine acetyltransferases (KATs), p300 (KAT3B), and its close homologue CREB-binding protein (KAT3A) are probably the most widely studied KATs with well documented roles in various cellular processes. Hence, the dysfunction of p300 may result in the dysregulation of gene expression leading to the manifestation of many disorders. The acetyltransferase activity of p300/CREB-binding protein is therefore considered as a target for new generation therapeutics. We describe here a natural compound, plumbagin (RTK1), isolated from Plumbago rosea root extract, that inhibits histone acetyltransferase activity potently in vivo. Interestingly, RTK1 specifically inhibits the p300-mediated acetylation of p53 but not the acetylation by another acetyltransferase, p300/CREB-binding protein -associated factor, PCAF, in vivo. RTK1 inhibits p300 histone acetyltransferase activity in a noncompetitive manner. Docking studies and site-directed mutagenesis of the p300 histone acetyltransferase domain suggest that a single hydroxyl group of RTK1 makes a hydrogen bond with the lysine 1358 residue of this domain. In agreement with this, we found that indeed the hydroxyl group-substituted plumbagin derivatives lost the acetyltransferase inhibitory activity. This study describes for the first time the chemical entity (hydroxyl group) required for the inhibition of acetyltransferase activity.

Asunto(s)

Inhibidores Enzimáticos/química , Naftoquinonas/química , Raíces de Plantas/química , Plumbaginaceae/química , Factores de Transcripción p300-CBP/antagonistas & inhibidores , Acetilación , Animales , Antineoplásicos Fitogénicos/química , Antineoplásicos Fitogénicos/farmacología , Línea Celular Tumoral , Inhibidores Enzimáticos/farmacología , Humanos , Masculino , Ratones , Mutagénesis Sitio-Dirigida , Naftoquinonas/farmacología , Estructura Terciaria de Proteína/genética , Proteína p53 Supresora de Tumor/química , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Factores de Transcripción p300-CBP/química , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismo

RESUMEN

DNA-binding anticancer agents cause alteration in chromatin structure and dynamics. We report the dynamic interaction of the DNA intercalator and potential anticancer plant alkaloid, sanguinarine (SGR), with chromatin. Association of SGR with different levels of chromatin structure was enthalpy driven with micromolar dissociation constant. Apart from DNA, it binds with comparable affinity with core histones and induces chromatin aggregation. The dual binding property of SGR leads to inhibition of core histone modifications. Although it potently inhibits H3K9 methylation by G9a in vitro, H3K4 and H3R17 methylation are more profoundly inhibited in cells. SGR inhibits histone acetylation both in vitro and in vivo. It does not affect the in vitro transcription from DNA template but significantly represses acetylation-dependent chromatin transcription. SGR-mediated repression of epigenetic marks and the alteration of chromatin geography (nucleography) also result in the modulation of global gene expression. These data, conclusively, show an anticancer DNA binding intercalator as a modulator of chromatin modifications and transcription in the chromatin context.

Asunto(s)

Benzofenantridinas/metabolismo , Cromatina/genética , Cromatina/metabolismo , Isoquinolinas/metabolismo , Acetilación/efectos de los fármacos , Animales , Cromatina/química , ADN/química , ADN/genética , ADN/metabolismo , Epigénesis Genética , Células HeLa , Histonas/química , Histonas/genética , Histonas/metabolismo , Humanos , Metilación/efectos de los fármacos , Conformación Molecular , Ratas , Transcripción Genética

RESUMEN

In this report, we demonstrate glucose-derived carbon nanospheres to be an emerging class of intracellular carriers. The surfaces of these spheres are highly functionalized and do not need any further modification. Besides, the intrinsic fluorescence property of carbon nanospheres helps in tracking their cellular localization without any additional fluorescent tags. The spheres are found to target the nucleus of the mammalian cells, causing no toxicity. Interestingly, the in vivo experiments show that these nanospheres have an important ability to cross the blood-brain barrier and localize in the brain besides getting localized in the liver and the spleen. There is also evidence to show that they are continuously being removed from these tissues over time. Furthermore, these nanospheres were used as a carrier for the membrane-impermeable molecule CTPB (N-(4-chloro-3-trifluoromethylphenyl)-2-ethoxybenzamide), the only known small-molecule activator of histone acetyltransferase (HAT) p300. Biochemical analyses such as Western blotting, immunohistochemistry, and gene expression analysis show the induction of the hyperacetylation of histone acetyltransferase (HAT) p300 (autoacetylation) as well as histones both in vitro and in vivo and the activation of HAT-dependent transcription upon CTPB delivery. These results establish an alternative path for the activation of gene expression mediated by the induction of HAT activity instead of histone deacetylase (HDAC) inhibition.

Asunto(s)

Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Colorantes Fluorescentes/farmacología , Regulación de la Expresión Génica , Técnicas Genéticas , Terapia Genética/métodos , Vectores Genéticos/genética , Nanotubos de Carbono/química , Células HeLa , Histonas/metabolismo , Humanos , Hígado/metabolismo , Microscopía Fluorescente/métodos , Nanosferas/química , Bazo/metabolismo