RESUMEN
Skeletal myogenesis involves highly coordinated steps that integrate developmental cues at the chromatin of muscle progenitors. Here, we identify Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of muscle-specific gene expression and myoblast differentiation. The mode of action of Mybbp1a was linked to promoter regulation as illustrated by its interaction with MyoD at the genomic regions of silent muscle-specific genes as well as its negative effect on MyoD-mediated transcriptional activity. We propose that Mybbp1a exerts its repressive role by inducing a less permissible chromatin structure following recruitment of negative epigenetic modifiers such as HDAC1/2 and Suv39h1. At the onset of differentiation, Mybbp1a undergoes a promoter disengagement that may be due to the differentiation-responsive, miR-546-mediated downregulation of Mybbp1a expression. Moreover, such alteration gave rise to promoter enrichment of activators and histone acetylation, an epigenetic status amenable to gene activation. Together, these findings unveil a hitherto unrecognized transcriptional co-repressor role of Mybbp1a in proliferating muscle progenitor cells, and highlight an epigenetic mechanism by which Mybbp1a and miR-546 interplay to control myoblast differentiation transition.
Asunto(s)
Proteínas Portadoras/metabolismo , Silenciador del Gen , Desarrollo de Músculos/genética , Proteínas Nucleares/metabolismo , Regiones Promotoras Genéticas , Animales , Proteínas Portadoras/genética , Células Cultivadas , Proteínas de Unión al ADN , Regulación hacia Abajo , Expresión Génica , Humanos , Ratones , MicroARNs/metabolismo , Músculo Esquelético/metabolismo , Proteína MioD/genética , Proteína MioD/metabolismo , Proteínas Nucleares/genética , Proteínas de Unión al ARN , Factores de TranscripciónRESUMEN
EDD (E3 isolated by differential display) was initially isolated as a progestin-regulated gene in breast cancer cells, and represents the human ortholog of the Drosophila melanogaster hyperplastic discs gene (hyd). It encodes a highly conserved and predominantly nuclear ubiquitin E3 ligase of the HECT family, with potential multifunctional roles in development and tumorigenesis. In this study, we further examined the largely uncharacterized role of EDD in transcriptional regulation by uncovering the spectrum of its direct target genes at a genome-wide level. Use of a systematic approach that integrates gene expression and chromatin binding profiling identified several candidate EDD-target genes, one of which is ACVRL1, a TGF-ß receptor with functional implications in blood vessel development. Further characterization revealed a negative regulation of ACVRL1 gene expression by EDD that is exerted at the promoter. Consistent with the aberrant upregulation of ACVRL1 and downstream Smad signaling, abrogation of EDD led to deregulated vessel development and endothelial cell motility. Collectively, these results extended the known cellular roles of EDD to critical functions in transcriptional regulation as well as angiogenesis, and may provide mechanistic explanations for EDD's tumorigenic and developmental roles.
Asunto(s)
Receptores de Activinas Tipo II/genética , Movimiento Celular , Genómica , Regiones Promotoras Genéticas/genética , Activación Transcripcional , Ubiquitina-Proteína Ligasas/metabolismo , Receptores de Activinas Tipo II/metabolismo , Western Blotting , Quimiotaxis , Inmunoprecipitación de Cromatina , Técnica del Anticuerpo Fluorescente Indirecta , Células HeLa , Humanos , Técnicas para Inmunoenzimas , Luciferasas/metabolismo , Unión Proteica , ARN Mensajero/genética , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transfección , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
BACKGROUND: Transcription of the ribosomal RNA gene repeats by Pol I occurs in the nucleolus and is a fundamental step in ribosome biogenesis and protein translation. Due to tight coordination between ribosome biogenesis and cell proliferation, transcription of rRNA and stable maintenance of rDNA clusters are thought to be under intricate control by intercalated mechanisms, particularly at the epigenetic level. METHODS AND RESULTS: Here we identify the nucleolar protein Myb-binding protein 1a (Mybbp1a) as a novel negative regulator of rRNA expression. Suppression of rDNA transcription by Mybbp1a was linked to promoter regulation as illustrated by its binding to the chromatin around the hypermethylated, inactive rDNA gene promoters. Our data further showed that downregulation of Mybbp1a abrogated the local DNA methylation levels and histone marks associated with gene silencing, and altered the promoter occupancy of various factors such UBF and HDACs, consequently leading to elevated rRNA expression. Mechanistically, we propose that Mybbp1a maintains rDNA repeats in a silenced state while in association with the negative epigenetic modifiers HDAC1/2. CONCLUSIONS: Results from our present work reveal a previously unrecognized co-repressor role of Mybbp1a in rRNA expression. They are further consistent with the scenario that Mybbp1a is an integral constituent of the rDNA epigenetic regulation that underlies the balanced state of rDNA clusters.
Asunto(s)
ADN Ribosómico/genética , Epigénesis Genética , Proteínas Nucleares/genética , Proteínas de Transporte Nucleocitoplasmático/genética , ARN Ribosómico/genética , Animales , Proliferación Celular , Cromatina/genética , Metilación de ADN/genética , Proteínas de Unión al ADN , Regulación de la Expresión Génica/genética , Silenciador del Gen , Células HeLa , Histona Desacetilasa 1/genética , Humanos , Ratones , Proteínas de Unión al ARN , Factores de Transcripción , Transcripción GenéticaRESUMEN
Differentiated neurons and glia are acquired from immature precursors via transcriptional controls exerted by factors such as proteins in the family of Glial Cells Missing (Gcm). Mammalian Gcm proteins mediate neural stem cell induction, placenta and parathyroid development, whereas Drosophila Gcm proteins act as a key switch to determine neuronal and glial cell fates and regulate hemocyte development. The present study reports a hypoparathyroidism-associated mutation R59L that alters Drosophila Gcm (Gcm) protein stability, rendering it unstable, and hyperubiquitinated via the ubiquitin-proteasome system (UPS). GcmR59L interacts with the Slimb-based SCF complex and Protein Kinase C (PKC), which possibly plays a role in its phosphorylation, hence altering ubiquitination. Additionally, R59L causes reduced Gcm protein levels in a manner independent of the PEST domain signaling protein turnover. GcmR59L proteins bind DNA, functionally activate transcription, and induce glial cells, yet at a less efficient level. Finally, overexpression of either wild-type human Gcmb (hGcmb) or hGcmb carrying the conserved hypoparathyroidism mutation only slightly affects gliogenesis, indicating differential regulatory mechanisms in human and flies. Taken together, these findings demonstrate the significance of this disease-associated mutation in controlling Gcm protein stability via UPS, hence advance our understanding on how glial formation is regulated.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/metabolismo , Hipoparatiroidismo/patología , Neuroglía/metabolismo , Factores de Transcripción/metabolismo , Animales , ADN/metabolismo , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/antagonistas & inhibidores , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Humanos , Hipoparatiroidismo/metabolismo , Leupeptinas/farmacología , Neuroglía/citología , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Fosforilación/efectos de los fármacos , Polimorfismo de Nucleótido Simple , Complejo de la Endopetidasa Proteasomal/metabolismo , Unión Proteica , Proteína Quinasa C/metabolismo , Estabilidad Proteica , Factores de Transcripción/química , Factores de Transcripción/genética , Ubiquitina/metabolismo , Ubiquitinación/efectos de los fármacosRESUMEN
Cellular senescence is a permanent proliferative arrest triggered by genome instability or aberrant growth stresses, acting as a protective or even tumor-suppressive mechanism. While several key aspects of gene regulation have been known to program this cessation of cell growth, the involvement of the epigenetic regulation has just emerged but remains largely unresolved. Using a systems approach that is based on targeted gene profiling, we uncovered known and novel chromatin modifiers with putative link to the senescent state of the cells. Among these, we identified SETD8 as a new target as well as a key regulator of the cellular senescence signaling. Knockdown of SETD8 triggered senescence induction in proliferative culture, irrespectively of the p53 status of the cells; ectopic expression of this epigenetic writer alleviated the extent doxorubicin-induced cellular senescence. This repressive effect of SETD8 in senescence was mediated by directly maintaining the silencing mark H4K20me1 at the locus of the senescence switch gene p21. Further in support of this regulatory link, depletion of p21 reversed this SETD8-mediated cellular senescence. Additionally, we found that PPARγ acts upstream and regulates SETD8 expression in proliferating cells. Downregulation of PPARγ coincided with the senescence induction, while its activation inhibited the progression of this process. Viewed together, our findings delineated a new epigenetic pathway through which the PPARγ-SETD8 axis directly silences p21 expression and consequently impinges on its senescence-inducing function. This implies that SETD8 may be part of a cell proliferation checkpoint mechanism and has important implications in antitumor therapeutics.
Asunto(s)
Senescencia Celular/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Epigénesis Genética , Fibroblastos/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , PPAR gamma/genética , Puntos de Control del Ciclo Celular/efectos de los fármacos , Puntos de Control del Ciclo Celular/genética , Puntos de Control del Ciclo Celular/efectos de la radiación , Línea Celular Tumoral , Senescencia Celular/efectos de los fármacos , Senescencia Celular/efectos de la radiación , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Daño del ADN , Doxorrubicina/farmacología , Fibroblastos/citología , Fibroblastos/efectos de los fármacos , Fibroblastos/efectos de la radiación , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Humanos , Peróxido de Hidrógeno/farmacología , Pulmón/citología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/efectos de la radiación , PPAR gamma/metabolismo , Cultivo Primario de Células , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Rayos UltravioletaRESUMEN
Adenosine-to-inosine RNA editing constitutes a crucial component of the cellular transcriptome and critically underpins organism survival and development. While recent high-throughput approaches have provided comprehensive documentation of the RNA editome, its functional output remains mostly unresolved, particularly for events in the non-coding regions. Gene ontology analysis of the known RNA editing targets unveiled a preponderance of genes related to apoptosis regulation, among which proto-oncogenes XIAP and MDM2 encode two the most abundantly edited transcripts. To further decode this potential functional connection, here we showed that the main RNA editor ADAR1 directly targets this 3' UTR editing of XIAP and MDM2, and further exerts a negative regulation on the expression of their protein products. This post-transcriptional silencing role was mediated via the inverted Alu elements in the 3' UTR but independent of alteration in transcript stability or miRNA targeting. Rather, we discovered that ADAR1 competes transcript occupancy with the RNA shuttling factor STAU1 to facilitate nuclear retention of the XIAP and MDM2 mRNAs. As a consequence, ADAR1 may acquire functionality in part by conferring spatial distribution and translation efficiency of the target transcripts. Finally, abrogation of ADAR1 expression or catalytic activity elicited a XIAP-dependent suppression of apoptotic response, whereas ectopic expression reversed this protective effect on cell death. Together, our results extended the known functions of ADAR1 and RNA editing to the critical fine-tuning of the intracellular apoptotic signaling and also provided mechanistic explanation for ADAR1's roles in development and tumorigenesis.
Asunto(s)
Regiones no Traducidas 3'/genética , Adenosina Desaminasa/metabolismo , Apoptosis/genética , Edición de ARN/genética , Proteínas de Unión al ARN/metabolismo , Adenosina Desaminasa/genética , Elementos Alu/genética , Secuencia de Bases , Citoprotección/genética , Proteínas del Citoesqueleto/metabolismo , Células HEK293 , Células Hep G2 , Humanos , Proteínas Proto-Oncogénicas c-mdm2/genética , Transporte de ARN , ARN Mensajero/genética , ARN Mensajero/metabolismo , Proteínas de Unión al ARN/genética , Transcripción Genética , Proteína Inhibidora de la Apoptosis Ligada a X/genéticaRESUMEN
Triiodothyronine (T3) and its receptor (TR) modulate several physiological processes, including cell development, proliferation, differentiation and metabolism. The regulatory mechanism of T3/TR involves binding to the thyroid hormone response element (TRE) within the target gene promoter. However, the number of target genes directly regulated by TRα1 and the specific pathways of TR-regulated target genes remain largely unknown. Here, we expressed TRα1 in a HepG2 cell line and used chromatin immunoprecipitation coupled with microarray to determine the genes that are directly regulated by TRα1 and also involved in cell metabolism and proliferation. Our analysis identified E74-like factor 2 (ELF2), a transcription factor associated with tumor growth, as a direct target downregulated by T3/TR. Overexpression of ELF2 enhanced tumor cell proliferation, and conversely, its knockdown suppressed tumor growth. Additionally, ELF2 restored the proliferative ability of hepatoma cells inhibited by T3/TR. Our findings collectively support a potential role of T3/TR in tumor growth inhibition through regulation of ELF2.
Asunto(s)
Carcinoma Hepatocelular/patología , Regulación Neoplásica de la Expresión Génica/genética , Neoplasias Hepáticas/patología , Receptores de Hormona Tiroidea/metabolismo , Factores de Transcripción/metabolismo , Triyodotironina/metabolismo , Inmunoprecipitación de Cromatina/métodos , Células Hep G2 , HumanosRESUMEN
BACKGROUND: Human umbilical mesenchymal stem cells (HUMSCs) isolated from Wharton's jelly of the umbilical cord can be easily obtained and processed compared with embryonic or bone marrow stem cells. These cells may be a valuable source in the repair of spinal cord injury. METHODOLOGY/PRINCIPAL FINDINGS: We examine the effects of HUMSC transplantation after complete spinal cord transection in rats. Approximately 5x10(5) HUMSCs were transplanted into the lesion site. Three groups of rats were implanted with either untreated HUMSCs (referred to as the stem cell group), or HUMSCs treated with neuronal conditioned medium (NCM) for either three days or six days (referred to as NCM-3 and NCM-6 days, respectively). The control group received no HUMSCs in the transected spinal cord. Three weeks after transplantation, significant improvements in locomotion were observed in all the three groups receiving HUMSCs (stem cell, NCM-3 and NCM-6 days groups). This recovery was accompanied by increased numbers of regenerated axons in the corticospinal tract and neurofilament-positive fibers around the lesion site. There were fewer microglia and reactive astrocytes in both the rostral and caudal stumps of the spinal cord in the stem cell group than in the control group. Transplanted HUMSCs survived for 16 weeks and produced large amounts of human neutrophil-activating protein-2, neurotrophin-3, basic fibroblast growth factor, glucocorticoid induced tumor necrosis factor receptor, and vascular endothelial growth factor receptor 3 in the host spinal cord, which may help spinal cord repair. CONCLUSIONS/SIGNIFICANCE: Transplantation of HUMSCs is beneficial to wound healing after spinal cord injury in rats.