RESUMEN
CREB-binding protein (CBP, CREBBP, KAT3A) and its closely related paralogue p300 (EP300, KAT3B), together termed p300/CBP, are histone/lysine acetyl-transferases that control gene expression by modifying chromatin-associated proteins. Here, we report roles for both of these chromatin-modifying enzymes in mouse sex determination, the process by which the embryonic gonad develops into a testis or an ovary. By targeting gene ablation to embryonic gonadal somatic cells using an inducible Cre line, we show that gonads lacking either gene exhibit major abnormalities of XY gonad development at 14.5 dpc, including partial sex reversal. Embryos lacking three out of four functional copies of p300/Cbp exhibit complete XY gonadal sex reversal and have greatly reduced expression of the key testis-determining genes Sry and Sox9. An analysis of histone acetylation at the Sry promoter in mutant gonads at 11.5 dpc shows a reduction in levels of the positive histone mark H3K27Ac. Our data suggest a role for CBP/p300 in testis determination mediated by control of histone acetylation at the Sry locus and reveal a novel element in the epigenetic control of Sry and mammalian sex determination. They also suggest possible novel causes of human disorders of sex development (DSD).
Asunto(s)
Proteína de Unión a CREB/deficiencia , Trastornos del Desarrollo Sexual/metabolismo , Proteína p300 Asociada a E1A/deficiencia , Histonas/metabolismo , Procesos de Determinación del Sexo/fisiología , Proteína de la Región Y Determinante del Sexo/genética , Testículo/embriología , Acetilación , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Trastornos del Desarrollo Sexual/genética , Proteína p300 Asociada a E1A/genética , Proteína p300 Asociada a E1A/metabolismo , Femenino , Masculino , Ratones , Ovario/embriología , Ovario/metabolismo , Regiones Promotoras Genéticas , Proteína de la Región Y Determinante del Sexo/metabolismo , Testículo/metabolismoRESUMEN
CREBBP is targeted by inactivating mutations in follicular lymphoma (FL) and diffuse large B-cell lymphoma (DLBCL). Here, we provide evidence from transgenic mouse models that Crebbp deletion results in deficits in B-cell development and can cooperate with Bcl2 overexpression to promote B-cell lymphoma. Through transcriptional and epigenetic profiling of these B cells, we found that Crebbp inactivation was associated with broad transcriptional alterations, but no changes in the patterns of histone acetylation at the proximal regulatory regions of these genes. However, B cells with Crebbp inactivation showed high expression of Myc and patterns of altered histone acetylation that were localized to intragenic regions, enriched for Myc DNA binding motifs, and showed Myc binding. Through the analysis of CREBBP mutations from a large cohort of primary human FL and DLBCL, we show a significant difference in the spectrum of CREBBP mutations in these 2 diseases, with higher frequencies of nonsense/frameshift mutations in DLBCL compared with FL. Together, our data therefore provide important links between Crebbp inactivation and Bcl2 dependence and show a role for Crebbp inactivation in the induction of Myc expression. We suggest this may parallel the role of CREBBP frameshift/nonsense mutations in DLBCL that result in loss of the protein, but may contrast the role of missense mutations in the lysine acetyltransferase domain that are more frequently observed in FL and yield an inactive protein.
Asunto(s)
Linfocitos B/patología , Proteína de Unión a CREB/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Proteínas Proto-Oncogénicas c-bcl-2/genética , Animales , Epigénesis Genética , Eliminación de Gen , Humanos , Linfoma Folicular/genética , Ratones , Ratones Transgénicos , MutaciónRESUMEN
The liver has multiple functions that preserve homeostasis. Liver diseases are debilitating, costly and often result in death. Elucidating the developmental mechanisms that establish the liver's architecture or generate the cellular diversity of this organ should help advance the prevention, diagnosis and treatment of hepatic diseases. We previously reported that migration of early hepatic precursors away from the gut epithelium requires the activity of the homeobox gene Prox1. Here, we show that Prox1 is a novel regulator of cell differentiation and morphogenesis during hepatogenesis. Prox1 ablation in bipotent hepatoblasts dramatically reduced the expression of multiple hepatocyte genes and led to very defective hepatocyte morphogenesis. As a result, abnormal epithelial structures expressing hepatocyte and cholangiocyte markers or resembling ectopic bile ducts developed in the Prox1-deficient liver parenchyma. By contrast, excessive commitment of hepatoblasts into cholangiocytes, premature intrahepatic bile duct morphogenesis, and biliary hyperplasia occurred in periportal areas of Prox1-deficient livers. Together, these abnormalities indicate that Prox1 activity is necessary to correctly allocate cell fates in liver precursors. These results increase our understanding of differentiation anomalies in pathological conditions and will contribute to improving stem cell protocols in which differentiation is directed towards hepatocytes and cholangiocytes.
Asunto(s)
Conductos Biliares/patología , Linaje de la Célula , Eliminación de Gen , Hepatocitos/metabolismo , Hepatocitos/patología , Células Madre/metabolismo , Proteínas Supresoras de Tumor/deficiencia , Envejecimiento/metabolismo , Animales , Animales Recién Nacidos , Recuento de Células , Linaje de la Célula/genética , Coristoma/patología , Células Epiteliales/metabolismo , Células Epiteliales/patología , Feto/metabolismo , Regulación del Desarrollo de la Expresión Génica , Factor Nuclear 4 del Hepatocito/metabolismo , Proteínas de Homeodominio/metabolismo , Hígado/embriología , Hígado/metabolismo , Ratones , Factor de Transcripción SOX9/metabolismo , Transducción de Señal/genética , Células Madre/patología , Factor de Crecimiento Transformador beta/metabolismo , Proteínas Supresoras de Tumor/metabolismoRESUMEN
B-cell non-Hodgkin's lymphoma comprises biologically and clinically distinct diseases the pathogenesis of which is associated with genetic lesions affecting oncogenes and tumour-suppressor genes. We report here that the two most common types--follicular lymphoma and diffuse large B-cell lymphoma--harbour frequent structural alterations inactivating CREBBP and, more rarely, EP300, two highly related histone and non-histone acetyltransferases (HATs) that act as transcriptional co-activators in multiple signalling pathways. Overall, about 39% of diffuse large B-cell lymphoma and 41% of follicular lymphoma cases display genomic deletions and/or somatic mutations that remove or inactivate the HAT coding domain of these two genes. These lesions usually affect one allele, suggesting that reduction in HAT dosage is important for lymphomagenesis. We demonstrate specific defects in acetylation-mediated inactivation of the BCL6 oncoprotein and activation of the p53 tumour suppressor. These results identify CREBBP/EP300 mutations as a major pathogenetic mechanism shared by common forms of B-cell non-Hodgkin's lymphoma, with direct implications for the use of drugs targeting acetylation/deacetylation mechanisms.
Asunto(s)
Acetiltransferasas/genética , Acetiltransferasas/metabolismo , Proteína de Unión a CREB/genética , Proteína p300 Asociada a E1A/genética , Linfoma de Células B/enzimología , Linfoma de Células B/genética , Mutación/genética , Acetilcoenzima A/metabolismo , Acetilación , Acetiltransferasas/química , Acetiltransferasas/deficiencia , Animales , Secuencia de Bases , Proteína de Unión a CREB/química , Proteína de Unión a CREB/deficiencia , Proteína de Unión a CREB/metabolismo , Células Cultivadas , Proteínas de Unión al ADN/metabolismo , Proteína p300 Asociada a E1A/química , Proteína p300 Asociada a E1A/deficiencia , Proteína p300 Asociada a E1A/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HEK293 , Histona Acetiltransferasas/química , Histona Acetiltransferasas/deficiencia , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Humanos , Linfoma de Células B/patología , Linfoma Folicular/enzimología , Linfoma Folicular/genética , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/enzimología , Linfoma de Células B Grandes Difuso/genética , Linfoma de Células B Grandes Difuso/patología , Ratones , Mutación Missense/genética , Polimorfismo de Nucleótido Simple/genética , Unión Proteica , Estructura Terciaria de Proteína/genética , Proteínas Proto-Oncogénicas c-bcl-6 , Recurrencia , Eliminación de Secuencia/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
Relapsed acute lymphoblastic leukaemia (ALL) is a leading cause of death due to disease in young people, but the biological determinants of treatment failure remain poorly understood. Recent genome-wide profiling of structural DNA alterations in ALL have identified multiple submicroscopic somatic mutations targeting key cellular pathways, and have demonstrated substantial evolution in genetic alterations from diagnosis to relapse. However, DNA sequence mutations in ALL have not been analysed in detail. To identify novel mutations in relapsed ALL, we resequenced 300 genes in matched diagnosis and relapse samples from 23 patients with ALL. This identified 52 somatic non-synonymous mutations in 32 genes, many of which were novel, including the transcriptional coactivators CREBBP and NCOR1, the transcription factors ERG, SPI1, TCF4 and TCF7L2, components of the Ras signalling pathway, histone genes, genes involved in histone modification (CREBBP and CTCF), and genes previously shown to be targets of recurring DNA copy number alteration in ALL. Analysis of an extended cohort of 71 diagnosis-relapse cases and 270 acute leukaemia cases that did not relapse found that 18.3% of relapse cases had sequence or deletion mutations of CREBBP, which encodes the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CREBBP, also known as CBP). The mutations were either present at diagnosis or acquired at relapse, and resulted in truncated alleles or deleterious substitutions in conserved residues of the histone acetyltransferase domain. Functionally, the mutations impaired histone acetylation and transcriptional regulation of CREBBP targets, including glucocorticoid responsive genes. Several mutations acquired at relapse were detected in subclones at diagnosis, suggesting that the mutations may confer resistance to therapy. These results extend the landscape of genetic alterations in leukaemia, and identify mutations targeting transcriptional and epigenetic regulation as a mechanism of resistance in ALL.
Asunto(s)
Proteína de Unión a CREB/genética , Mutación/genética , Leucemia-Linfoma Linfoblástico de Células Precursoras/genética , Acetilación , Proteína de Unión a CREB/química , Proteína de Unión a CREB/metabolismo , Resistencia a Antineoplásicos/genética , Epigénesis Genética/genética , Regulación Neoplásica de la Expresión Génica , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Humanos , Estructura Terciaria de Proteína/genética , RecurrenciaRESUMEN
In the fasted state, increases in catecholamine signaling promote adipocyte function via the protein kinase A-mediated phosphorylation of cyclic AMP response element binding protein (CREB). CREB activity is further up-regulated in obesity, despite reductions in catecholamine signaling, where it contributes to the development of insulin resistance. Here we show that obesity promotes the CREB binding protein (CBP)-mediated acetylation of CREB at Lys136 in adipose. Under lean conditions, CREB acetylation was low due to an association with the energy-sensing NAD(+)-dependent deacetylase SirT1; amounts of acetylated CREB were increased in obesity, when SirT1 undergoes proteolytic degradation. Whereas CREB phosphorylation stimulated an association with the KIX domain of CBP, Lys136 acetylation triggered an interaction with the CBP bromodomain (BRD) that augmented recruitment of this coactivator to the promoter. Indeed, coincident Ser133 phosphorylation and Lys136 acetylation of CREB stimulated the formation of a ternary complex with the KIX and BRD domains of CBP by NMR analysis. As disruption of the CREB:BRD complex with a CBP-specific BRD inhibitor blocked effects of CREB acetylation on target gene expression, our results demonstrate how changes in nutrient status modulate cellular gene expression in response to hormonal signals.
Asunto(s)
Adipocitos/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Obesidad/metabolismo , Transducción de Señal , Células 3T3-L1 , Acetilación , Animales , Proteína de Unión a CREB/genética , Proteína de Unión a CREB/metabolismo , Células Cultivadas , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Immunoblotting , Lisina/genética , Lisina/metabolismo , Ratones , Ratones Noqueados , Ratones Obesos , Mutación , Obesidad/genética , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosforilación , Regiones Promotoras Genéticas/genética , Unión Proteica , Sirtuina 1/genética , Sirtuina 1/metabolismoRESUMEN
Genome-wide distribution of histone H3K18 and H3K27 acetyltransferases, CBP (CREBBP) and p300 (EP300), is used to map enhancers and promoters, but whether these elements functionally require CBP/p300 remains largely uncertain. Here we compared global CBP recruitment with gene expression in wild-type and CBP/p300 double-knockout (dKO) fibroblasts. ChIP-seq using CBP-null cells as a control revealed nearby CBP recruitment for 20% of constitutively-expressed genes, but surprisingly, three-quarters of these genes were unaffected or slightly activated in dKO cells. Computationally defined enhancer-promoter-units (EPUs) having a CBP peak near the enhancer-like element were more predictive, with CBP/p300 deletion attenuating expression of 40% of such constitutively-expressed genes. Examining signal-responsive (Hypoxia Inducible Factor) genes showed that 97% were within 50 kilobases of an inducible CBP peak, and 70% of these required CBP/p300 for full induction. Unexpectedly, most inducible CBP peaks occurred near signal-nonresponsive genes. Finally, single-cell expression analysis revealed additional context dependence where some signal-responsive genes were not uniformly dependent on CBP/p300 in individual cells. While CBP/p300 was needed for full induction of some genes in single-cells, for other genes CBP/p300 increased the probability of maximal expression. Thus, target gene context influences the transcriptional requirement for CBP/p300, possibly by multiple mechanisms.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Activación Transcripcional , 2,2'-Dipiridil/farmacología , Animales , Proteína de Unión a CREB/genética , Células Cultivadas , Inmunoprecipitación de Cromatina , Elementos de Facilitación Genéticos , Genoma , Ratones , Regiones Promotoras Genéticas , Elementos de Respuesta , Análisis de la Célula Individual , Sitio de Iniciación de la Transcripción , Transcripción Genética , Factores de Transcripción p300-CBP/genética , Factores de Transcripción p300-CBP/metabolismoRESUMEN
Histone acetyltransferases (HATs) GCN5 and PCAF (GCN5/PCAF) and CBP and p300 (CBP/p300) are transcription co-activators. However, how these two distinct families of HATs regulate gene activation remains unclear. Here, we show deletion of GCN5/PCAF in cells specifically and dramatically reduces acetylation on histone H3K9 (H3K9ac) while deletion of CBP/p300 specifically and dramatically reduces acetylations on H3K18 and H3K27 (H3K18/27ac). A ligand for nuclear receptor (NR) PPARδ induces sequential enrichment of H3K18/27ac, RNA polymerase II (Pol II) and H3K9ac on PPARδ target gene Angptl4 promoter, which correlates with a robust Angptl4 expression. Inhibiting transcription elongation blocks ligand-induced H3K9ac, but not H3K18/27ac, on the Angptl4 promoter. Finally, we show GCN5/PCAF and GCN5/PCAF-mediated H3K9ac correlate with, but are surprisingly dispensable for, NR target gene activation. In contrast, CBP/p300 and their HAT activities are essential for ligand-induced Pol II recruitment on, and activation of, NR target genes. These results highlight the substrate and site specificities of HATs in cells, demonstrate the distinct roles of GCN5/PCAF- and CBP/p300-mediated histone acetylations in gene activation, and suggest an important role of CBP/p300-mediated H3K18/27ac in NR-dependent transcription.
Asunto(s)
Proteína p300 Asociada a E1A/metabolismo , Histonas/metabolismo , PPAR delta/metabolismo , Activación Transcripcional/fisiología , Factores de Transcripción p300-CBP/metabolismo , Acetilación , Proteína 4 Similar a la Angiopoyetina , Angiopoyetinas/genética , Animales , Western Blotting , Inmunoprecipitación de Cromatina , Proteína p300 Asociada a E1A/genética , Técnicas de Inactivación de Genes , Humanos , Espectrometría de Masas , Ratones , PPAR delta/agonistas , Regiones Promotoras Genéticas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Especificidad por Sustrato , Tiazoles/metabolismo , Tiazoles/farmacología , Activación Transcripcional/efectos de los fármacos , Factores de Transcripción p300-CBP/genéticaRESUMEN
Lens induction is a classical embryologic model to study cell fate determination. It has been proposed earlier that specific changes in core histone modifications accompany the process of cell fate specification and determination. The lysine acetyltransferases CBP and p300 function as principal enzymes that modify core histones to facilitate specific gene expression. Herein, we performed conditional inactivation of both CBP and p300 in the ectodermal cells that give rise to the lens placode. Inactivation of both CBP and p300 resulted in the dramatic discontinuation of all aspects of lens specification and organogenesis, resulting in aphakia. The CBP/p300(-/-) ectodermal cells are viable and not prone to apoptosis. These cells showed reduced expression of Six3 and Sox2, while expression of Pax6 was not upregulated, indicating discontinuation of lens induction. Consequently, expression of αB- and αA-crystallins was not initiated. Mutant ectoderm exhibited markedly reduced levels of histone H3 K18 and K27 acetylation, subtly increased H3 K27me3 and unaltered overall levels of H3 K9ac and H3 K4me3. Our data demonstrate that CBP and p300 are required to establish lens cell-type identity during lens induction, and suggest that posttranslational histone modifications are integral to normal cell fate determination in the mammalian lens.
Asunto(s)
Proteína de Unión a CREB/fisiología , Proteína p300 Asociada a E1A/fisiología , Histonas/metabolismo , Cristalino/embriología , Acetilación , Animales , Apoptosis , Proteína de Unión a CREB/genética , Proteína p300 Asociada a E1A/genética , Inducción Embrionaria , Expresión Génica , Cristalino/anatomía & histología , Cristalino/enzimología , Ratones , Mutación , Procesamiento Proteico-Postraduccional , Fase SRESUMEN
It remains uncertain how the DNA sequence of mammalian genes influences the transcriptional response to extracellular signals. Here, we show that the number of CREB-binding sites (CREs) affects whether the related histone acetyltransferases (HATs) CREB-binding protein (CBP) and p300 are required for endogenous gene transcription. Fibroblasts with both CBP and p300 knocked-out had strongly attenuated histone H4 acetylation at CREB-target genes in response to cyclic-AMP, yet transcription was not uniformly inhibited. Interestingly, dependence on CBP/p300 was often different between reporter plasmids and endogenous genes. Transcription in the absence of CBP/p300 correlated with endogenous genes having more CREs, more bound CREB, and more CRTC2 (a non-HAT coactivator of CREB). Indeed, CRTC2 rescued cAMP-inducible expression for certain genes in CBP/p300 null cells and contributed to the CBP/p300-independent expression of other targets. Thus, endogenous genes with a greater local concentration and diversity of coactivators tend to have more resilient-inducible expression. This model suggests how gene expression patterns could be tuned by altering coactivator availability rather than by changing signal input or transcription factor levels.
Asunto(s)
Proteína de Unión a CREB/fisiología , Proteína p300 Asociada a E1A/fisiología , Histona Acetiltransferasas/metabolismo , Transactivadores/metabolismo , Transcripción Genética , Acetilación , Animales , Sitios de Unión , Biomarcadores/metabolismo , Western Blotting , Células Cultivadas , Inmunoprecipitación de Cromatina , AMP Cíclico/farmacología , Embrión de Mamíferos/citología , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Histonas/metabolismo , Integrasas/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas/genética , ARN Mensajero/genética , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Transactivadores/antagonistas & inhibidores , Transactivadores/genética , Factores de TranscripciónRESUMEN
CBP can function as a tumor suppressor, but the mechanisms that govern oncogenesis in its absence are unknown. Here we show that CBP inactivation in mouse thymocytes leads to lymphoma. Although CBP has been implicated in the transactivation functions of p53, development of these tumors does not seem to involve loss of p53 activity. CBP-null tumors show reduced levels of p27Kip1 and increased levels of cyclin E and Skp2, two oncoproteins that can promote p27Kip1 proteolysis. Reduction of p27Kip1 by introduction of a p27Kip1-null allele into CBP knockout mice accelerates lymphomagenesis and seems to obviate the requirement for Skp2 and cyclin E upregulation. These data suggest that CBP loss mediates lymphomagenesis in cooperation with a mechanism that reduces p27Kip1 abundance.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Linfoma de Células T/metabolismo , Proteínas Nucleares/metabolismo , Proteínas Represoras , Linfocitos T/metabolismo , Transactivadores/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Proteína de Unión a CREB , Clonación Molecular , Ciclina E/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Daño del ADN/fisiología , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Genes myc/fisiología , Predisposición Genética a la Enfermedad , Linfoma de Células T/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Noqueados , Microscopía Fluorescente , Proteínas Nucleares/genética , Complejo Represivo Polycomb 1 , Proteínas Proto-Oncogénicas/metabolismo , Receptores Notch , Proteínas Quinasas Asociadas a Fase-S/metabolismo , Linfocitos T/patología , Transactivadores/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Regulación hacia Arriba/fisiologíaRESUMEN
Histone acetylation plays a critical role during long-term memory formation. Several studies have demonstrated that the histone acetyltransferase (HAT) CBP is required during long-term memory formation, but the involvement of other HAT proteins has not been extensively investigated. The HATs CBP and p300 have at least 400 described interacting proteins including transcription factors known to play a role in long-term memory formation. Thus, CBP and p300 constitute likely candidates for transcriptional coactivators in memory formation. In this study, we took a loss-of-function approach to evaluate the role of p300 in long-term memory formation. We used conditional knock-out mice in which the deletion of p300 is restricted to the postnatal phase and to subregions of the forebrain. We found that p300 is required for the formation of long-term recognition memory and long-term contextual fear memory in the CA1 area of the hippocampus and cortical areas.
Asunto(s)
Encéfalo/fisiología , Memoria a Largo Plazo/fisiología , Factores de Transcripción p300-CBP/metabolismo , Animales , Western Blotting , Condicionamiento Clásico/fisiología , Miedo/fisiología , Inmunohistoquímica , Aprendizaje por Laberinto/fisiología , Ratones , Ratones Noqueados , Factores de Transcripción p300-CBP/deficiencia , Factores de Transcripción p300-CBP/genéticaRESUMEN
Glucose homeostasis is regulated systemically by hormones such as insulin and glucagon, and at the cellular level by energy status. Glucagon enhances glucose output from the liver during fasting by stimulating the transcription of gluconeogenic genes via the cyclic AMP-inducible factor CREB (CRE binding protein). When cellular ATP levels are low, however, the energy-sensing kinase AMPK inhibits hepatic gluconeogenesis through an unknown mechanism. Here we show that hormonal and energy-sensing pathways converge on the coactivator TORC2 (transducer of regulated CREB activity 2) to modulate glucose output. Sequestered in the cytoplasm under feeding conditions, TORC2 is dephosphorylated and transported to the nucleus where it enhances CREB-dependent transcription in response to fasting stimuli. Conversely, signals that activate AMPK attenuate the gluconeogenic programme by promoting TORC2 phosphorylation and blocking its nuclear accumulation. Individuals with type 2 diabetes often exhibit fasting hyperglycaemia due to elevated gluconeogenesis; compounds that enhance TORC2 phosphorylation may offer therapeutic benefits in this setting.
Asunto(s)
Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Ayuno/metabolismo , Glucosa/metabolismo , Transactivadores/metabolismo , Proteínas Quinasas Activadas por AMP , Animales , Células Cultivadas , Retroalimentación Fisiológica , Gluconeogénesis , Hepatocitos/metabolismo , Homeostasis , Humanos , Hígado/citología , Hígado/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Complejos Multienzimáticos/metabolismo , Fosfoenolpiruvato Carboxiquinasa (GTP)/genética , Fosforilación , Proteínas Serina-Treonina Quinasas/metabolismo , Ratas , Ratas Sprague-Dawley , Transactivadores/deficiencia , Transactivadores/genética , Factores de TranscripciónRESUMEN
The global transcriptional coactivators CREB-binding protein (CBP) and the closely related p300 interact with over 312 proteins, making them among the most heavily connected hubs in the known mammalian protein-protein interactome. It is largely uncertain, however, if these interactions are important in specific cell lineages of adult animals, as homozygous null mutations in either CBP or p300 result in early embryonic lethality in mice. Here we describe a Cre/LoxP conditional p300 null allele (p300flox) that allows for the temporal and tissue-specific inactivation of p300. We used mice carrying p300flox and a CBP conditional knockout allele (CBPflox) in conjunction with an Lck-Cre transgene to delete CBP and p300 starting at the CD4- CD8- double-negative thymocyte stage of T-cell development. Loss of either p300 or CBP led to a decrease in CD4+ CD8+ double-positive thymocytes, but an increase in the percentage of CD8+ single-positive thymocytes seen in CBP mutant mice was not observed in p300 mutants. T cells completely lacking both CBP and p300 did not develop normally and were nonexistent or very rare in the periphery, however. T cells lacking CBP or p300 had reduced tumor necrosis factor alpha gene expression in response to phorbol ester and ionophore, while signal-responsive gene expression in CBP- or p300-deficient macrophages was largely intact. Thus, CBP and p300 each supply a surprising degree of redundant coactivation capacity in T cells and macrophages, although each gene has also unique properties in thymocyte development.
Asunto(s)
Linfocitos T CD4-Positivos/citología , Linfocitos T CD8-positivos/citología , Proteína de Unión a CREB/fisiología , Proteína p300 Asociada a E1A/fisiología , Timo/crecimiento & desarrollo , Alelos , Animales , Linfocitos T CD4-Positivos/efectos de los fármacos , Linfocitos T CD4-Positivos/metabolismo , Linfocitos T CD8-positivos/efectos de los fármacos , Linfocitos T CD8-positivos/metabolismo , Proteína de Unión a CREB/genética , Diferenciación Celular , Proteína p300 Asociada a E1A/genética , Eliminación de Gen , Expresión Génica , Ionóforos/farmacología , Recuento de Linfocitos , Linfoma de Células T/genética , Macrófagos/metabolismo , Ratones , Ratones Noqueados , Ésteres del Forbol/farmacología , Receptores de Antígenos de Linfocitos T , Bazo/citología , Bazo/crecimiento & desarrollo , Timo/citología , Transcripción Genética , Factor de Necrosis Tumoral alfa/genéticaRESUMEN
Histone deacetylase (HDAC) inhibitors increase histone acetylation and enhance both memory and synaptic plasticity. The current model for the action of HDAC inhibitors assumes that they alter gene expression globally and thus affect memory processes in a nonspecific manner. Here, we show that the enhancement of hippocampus-dependent memory and hippocampal synaptic plasticity by HDAC inhibitors is mediated by the transcription factor cAMP response element-binding protein (CREB) and the recruitment of the transcriptional coactivator and histone acetyltransferase CREB-binding protein (CBP) via the CREB-binding domain of CBP. Furthermore, we show that the HDAC inhibitor trichostatin A does not globally alter gene expression but instead increases the expression of specific genes during memory consolidation. Our results suggest that HDAC inhibitors enhance memory processes by the activation of key genes regulated by the CREB:CBP transcriptional complex.
Asunto(s)
Proteína de Unión a CREB/biosíntesis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/biosíntesis , Inhibidores de Histona Desacetilasas , Memoria/fisiología , Plasticidad Neuronal/fisiología , Activación Transcripcional/fisiología , Animales , Proteína de Unión a CREB/genética , Inhibidores Enzimáticos/farmacología , Femenino , Histona Desacetilasas/metabolismo , Ácidos Hidroxámicos/farmacología , Masculino , Memoria/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Plasticidad Neuronal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/enzimología , Activación Transcripcional/efectos de los fármacosRESUMEN
p300 (EP300) and CBP (CREBBP) are transcriptional coactivators with histone acetyltransferase activity. Various ß-cell transcription factors can recruit p300/CBP, and thus the coactivators could be important for ß-cell function and health in vivo. We hypothesized that p300/CBP contribute to the development and proper function of pancreatic islets. To test this, we bred and studied mice lacking p300/CBP in their islets. Mice lacking either p300 or CBP in islets developed glucose intolerance attributable to impaired insulin secretion, together with reduced α- and ß-cell area and islet insulin content. These phenotypes were exacerbated in mice with only a single copy of p300 or CBP expressed in islets. Removing p300 in pancreatic endocrine progenitors impaired proliferation of neonatal α- and ß-cells. Mice lacking all four copies of p300/CBP in pancreatic endocrine progenitors failed to establish α- and ß-cell mass postnatally. Transcriptomic analyses revealed significant overlaps between p300/CBP-downregulated genes and genes downregulated in Hnf1α-null islets and Nkx2.2-null islets, among others. Furthermore, p300/CBP are important for the acetylation of H3K27 at loci downregulated in Hnf1α-null islets. We conclude that p300 and CBP are limiting cofactors for islet development, and hence for postnatal glucose homeostasis, with some functional redundancy.
Asunto(s)
Proteína de Unión a CREB/metabolismo , Proliferación Celular , Proteína p300 Asociada a E1A/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Insulina/metabolismo , Células Madre/metabolismo , Acetilación , Animales , Animales Recién Nacidos , Glucemia/análisis , Proteína de Unión a CREB/genética , Tamaño de la Célula , Cruzamientos Genéticos , Proteína p300 Asociada a E1A/genética , Células Secretoras de Glucagón/citología , Células Secretoras de Glucagón/patología , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/metabolismo , Intolerancia a la Glucosa/patología , Histonas/metabolismo , Proteína Homeobox Nkx-2.2 , Insulina/metabolismo , Secreción de Insulina , Células Secretoras de Insulina/citología , Células Secretoras de Insulina/patología , Lisina , Ratones Noqueados , Ratones Transgénicos , Procesamiento Proteico-Postraduccional , Células Madre/citología , Células Madre/patologíaRESUMEN
Inactivating mutations of the CREBBP acetyltransferase are highly frequent in diffuse large B-cell lymphoma (DLBCL) and follicular lymphoma (FL), the two most common germinal center (GC)-derived cancers. However, the role of CREBBP inactivation in lymphomagenesis remains unclear. Here, we show that CREBBP regulates enhancer/super-enhancer networks with central roles in GC/post-GC cell fate decisions, including genes involved in signal transduction by the B-cell receptor and CD40 receptor, transcriptional control of GC and plasma cell development, and antigen presentation. Consistently, Crebbp-deficient B cells exhibit enhanced response to mitogenic stimuli and perturbed plasma cell differentiation. Although GC-specific loss of Crebbp was insufficient to initiate malignant transformation, compound Crebbp-haploinsufficient/BCL2-transgenic mice, mimicking the genetics of FL and DLBCL, develop clonal lymphomas recapitulating the features of the human diseases. These findings establish CREBBP as a haploinsufficient tumor-suppressor gene in GC B cells and provide insights into the mechanisms by which its loss contributes to lymphomagenesis.Significance: Loss-of-function mutations of CREBBP are common and early lesions in FL and DLBCL, suggesting a prominent role in lymphoma initiation. Our studies identify the cellular program by which reduced CREBBP dosage facilitates malignant transformation, and have direct implications for targeted lymphoma therapy based on drugs affecting CREBBP-mediated chromatin acetylation. Cancer Discov; 7(3); 322-37. ©2017 AACR.This article is highlighted in the In This Issue feature, p. 235.
Asunto(s)
Linfocitos B/patología , Proteína de Unión a CREB/genética , Genes Supresores de Tumor , Linfoma de Células B Grandes Difuso/genética , Animales , Linfocitos B/metabolismo , Proteína de Unión a CREB/metabolismo , Diferenciación Celular/genética , Cromatina/metabolismo , Elementos de Facilitación Genéticos , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica , Centro Germinal/patología , Haploinsuficiencia , Humanos , Linfoma Folicular/genética , Linfoma Folicular/patología , Linfoma de Células B Grandes Difuso/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Células Plasmáticas/efectos de los fármacos , Células Plasmáticas/patología , Proteínas Proto-Oncogénicas c-bcl-6/genética , Proteínas Proto-Oncogénicas c-bcl-6/metabolismoRESUMEN
Autism spectrum disorders (ASDs) are a group of neurodevelopmental afflictions characterized by repetitive behaviors, deficits in social interaction, and impaired communication skills. For most ASD patients, the underlying causes are unknown. Genetic mutations have been identified in about 25 percent of ASD cases, including mutations in epigenetic regulators, suggesting that dysregulated chromatin or DNA function is a critical component of ASD. Mutations in the histone acetyltransferase CREB binding protein (CBP, CREBBP) cause Rubinstein-Taybi Syndrome (RTS), a developmental disorder that includes ASD-like symptoms. Recently, genomic studies involving large numbers of ASD patient families have theoretically modeled CBP and its paralog p300 (EP300) as critical hubs in ASD-associated protein and gene interaction networks, and have identified de novo missense mutations in highly conserved residues of the CBP acetyltransferase and CH1 domains. Here we provide animal model evidence that supports this notion that CBP and its CH1 domain are relevant to autism. We show that mice with a deletion mutation in the CBP CH1 (TAZ1) domain (CBPΔCH1/ΔCH1) have an RTS-like phenotype that includes ASD-relevant repetitive behaviors, hyperactivity, social interaction deficits, motor dysfunction, impaired recognition memory, and abnormal synaptic plasticity. Our results therefore indicate that loss of CBP CH1 domain function contributes to RTS, and possibly ASD, and that this domain plays an essential role in normal motor function, cognition and social behavior. Although the key physiological functions affected by ASD-associated mutation of epigenetic regulators have been enigmatic, our findings are consistent with theoretical models involving CBP and p300 in ASD, and with a causative role for recently described ASD-associated CBP mutations.
Asunto(s)
Trastorno Autístico/genética , Proteína de Unión a CREB/genética , Histona Acetiltransferasas/genética , Mutación , Síndrome de Rubinstein-Taybi/genética , Análisis de Varianza , Animales , Trastorno Autístico/enzimología , Trastorno Autístico/fisiopatología , Sitios de Unión/genética , Proteína de Unión a CREB/metabolismo , Anomalías Craneofaciales/genética , Anomalías Craneofaciales/fisiopatología , Hipocampo/metabolismo , Hipocampo/fisiopatología , Histona Acetiltransferasas/metabolismo , Humanos , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Aprendizaje por Laberinto/fisiología , Trastornos de la Memoria/genética , Trastornos de la Memoria/fisiopatología , Ratones de la Cepa 129 , Ratones Endogámicos C57BL , Ratones Noqueados , Actividad Motora/genética , Actividad Motora/fisiología , Fenotipo , Síndrome de Rubinstein-Taybi/enzimología , Síndrome de Rubinstein-Taybi/fisiopatología , Conducta SocialRESUMEN
MED23, a subunit of the Mediator coactivator complex, is important for the expression of a subset of MAPK/ERK pathway-responsive genes, the constituents of which vary between cell types for reasons that are not completely clear. MAPK/ERK pathway-dependent processes are essential for T-cell development and function, but whether MED23 has a role in this context is unknown. We generated Med23 conditional knockout mice and induced Med23 deletion in early T-cell development using the lineage specific Lck-Cre transgene. While the total cell number and distribution of cell populations in the thymuses of Med23flox/flox;Lck-Cre mice were essentially normal, MED23 null T-cells failed to efficiently populate the peripheral lymphoid organs. MED23 null thymocytes displayed decreased expression of the MAPK/ERK-responsive genes Egr1, Egr2, as well as of the membrane glycoprotein Cd52 (CAMPATH-1). MED23 null CD4 single-positive thymocytes also showed decreased expression of KLF2 (LKLF), a T-cell master regulatory transcription factor. Indeed, similarities between the phenotypes of mice lacking MED23 or KLF2 in T-cells suggest that KLF2 deficiency in MED23 null T-cells is one of their key defects. Mechanistic experiments using MED23 null MEFs further suggest that MED23 is required for full activity of the MAPK-responsive transcription factor MEF2, which has previously been shown to mediate Klf2 expression. In summary, our data indicate that MED23 has critical roles in enabling T-cells to populate the peripheral lymphoid organs, possibly by potentiating MEF2-dependent expression of the T-cell transcription factor KLF2.
Asunto(s)
Factores de Transcripción de Tipo Kruppel/metabolismo , Tejido Linfoide/metabolismo , Complejo Mediador/metabolismo , Linfocitos T/metabolismo , Animales , Apoptosis/genética , Western Blotting , Proliferación Celular , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Factores de Transcripción de Tipo Kruppel/genética , Tejido Linfoide/citología , Factores de Transcripción MEF2/genética , Factores de Transcripción MEF2/metabolismo , Complejo Mediador/genética , Ratones Noqueados , Ratones Transgénicos , Análisis de Secuencia por Matrices de Oligonucleótidos , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Timocitos/metabolismo , Timo/citología , Timo/metabolismoRESUMEN
T-regulatory (Treg) cells are important to immune homeostasis, and Treg cell deficiency or dysfunction leads to autoimmune disease. A histone/protein acetyltransferase (HAT), p300, was recently found to be important for Treg function and stability, but further insights into the mechanisms by which p300 or other HATs affect Treg biology are needed. Here we show that CBP, a p300 paralog, is also important in controlling Treg function and stability. Thus, while mice with Treg-specific deletion of CBP or p300 developed minimal autoimmune disease, the combined deletion of CBP and p300 led to fatal autoimmunity by 3 to 4 weeks of age. The effects of CBP and p300 deletion on Treg development are dose dependent and involve multiple mechanisms. CBP and p300 cooperate with several key Treg transcription factors that act on the Foxp3 promoter to promote Foxp3 production. CBP and p300 also act on the Foxp3 conserved noncoding sequence 2 (CNS2) region to maintain Treg stability in inflammatory environments by regulating pCREB function and GATA3 expression, respectively. Lastly, CBP and p300 regulate the epigenetic status and function of Foxp3. Our findings provide insights into how HATs orchestrate multiple aspects of Treg development and function and identify overlapping but also discrete activities for p300 and CBP in control of Treg cells.