RESUMEN
Eukaryotic transcription factors recognize specific DNA sequence motifs, but are also endowed with generic, non-specific DNA-binding activity. How these binding modes are integrated to determine select transcriptional outputs remains unresolved. We addressed this question by site-directed mutagenesis of the Myc transcription factor. Impairment of non-specific DNA backbone contacts caused pervasive loss of genome interactions and gene regulation, associated with increased intra-nuclear mobility of the Myc protein in murine cells. In contrast, a mutant lacking base-specific contacts retained DNA-binding and mobility profiles comparable to those of the wild-type protein, but failed to recognize its consensus binding motif (E-box) and could not activate Myc-target genes. Incidentally, this mutant gained weak affinity for an alternative motif, driving aberrant activation of different genes. Altogether, our data show that non-specific DNA binding is required to engage onto genomic regulatory regions; sequence recognition in turn contributes to transcriptional activation, acting at distinct levels: stabilization and positioning of Myc onto DNA, and-unexpectedly-promotion of its transcriptional activity. Hence, seemingly pervasive genome interaction profiles, as detected by ChIP-seq, actually encompass diverse DNA-binding modalities, driving defined, sequence-dependent transcriptional responses.
Asunto(s)
ADN/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Factores de Transcripción/metabolismo , Secuencia de Bases/genética , Secuencia de Bases/fisiología , Sitios de Unión , ADN/genética , Regulación de la Expresión Génica/genética , Regulación de la Expresión Génica/fisiología , Estabilidad Proteica , Proteínas Proto-Oncogénicas c-myc/genética , Factores de Transcripción/genéticaRESUMEN
Overexpression of the MYC transcription factor causes its widespread interaction with regulatory elements in the genome but leads to the up- and down-regulation of discrete sets of genes. The molecular determinants of these selective transcriptional responses remain elusive. Here, we present an integrated time-course analysis of transcription and mRNA dynamics following MYC activation in proliferating mouse fibroblasts, based on chromatin immunoprecipitation, metabolic labeling of newly synthesized RNA, extensive sequencing, and mathematical modeling. Transcriptional activation correlated with the highest increases in MYC binding at promoters. Repression followed a reciprocal scenario, with the lowest gains in MYC binding. Altogether, the relative abundance (henceforth, "share") of MYC at promoters was the strongest predictor of transcriptional responses in diverse cell types, predominating over MYC's association with the corepressor ZBTB17 (also known as MIZ1). MYC activation elicited immediate loading of RNA polymerase II (RNAPII) at activated promoters, followed by increases in pause-release, while repressed promoters showed opposite effects. Gains and losses in RNAPII loading were proportional to the changes in the MYC share, suggesting that repression by MYC may be partly indirect, owing to competition for limiting amounts of RNAPII. Secondary to the changes in RNAPII loading, the dynamics of elongation and pre-mRNA processing were also rapidly altered at MYC regulated genes, leading to the transient accumulation of partially or aberrantly processed mRNAs. Altogether, our results shed light on how overexpressed MYC alters the various phases of the RNAPII cycle and the resulting transcriptional response.
Asunto(s)
Regiones Promotoras Genéticas/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/metabolismo , Precursores del ARN/biosíntesis , Transcripción Genética/fisiología , Animales , Línea Celular Transformada , Ratones , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Proteínas Inhibidoras de STAT Activados/genética , Proteínas Inhibidoras de STAT Activados/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , ARN Polimerasa II/genética , Precursores del ARN/genética , Procesamiento Postranscripcional del ARN/fisiología , Ubiquitina-Proteína LigasasRESUMEN
The c-myc proto-oncogene product, Myc, is a transcription factor that binds thousands of genomic loci. Recent work suggested that rather than up- and downregulating selected groups of genes, Myc targets all active promoters and enhancers in the genome (a phenomenon termed 'invasion') and acts as a general amplifier of transcription. However, the available data did not readily discriminate between direct and indirect effects of Myc on RNA biogenesis. We addressed this issue with genome-wide chromatin immunoprecipitation and RNA expression profiles during B-cell lymphomagenesis in mice, in cultured B cells and fibroblasts. Consistent with long-standing observations, we detected general increases in total RNA or messenger RNA copies per cell (hereby termed 'amplification') when comparing actively proliferating cells with control quiescent cells: this was true whether cells were stimulated by mitogens (requiring endogenous Myc for a proliferative response) or by deregulated, oncogenic Myc activity. RNA amplification and promoter/enhancer invasion by Myc were separable phenomena that could occur without one another. Moreover, whether or not associated with RNA amplification, Myc drove the differential expression of distinct subsets of target genes. Hence, although having the potential to interact with all active or poised regulatory elements in the genome, Myc does not directly act as a global transcriptional amplifier. Instead, our results indicate that Myc activates and represses transcription of discrete gene sets, leading to changes in cellular state that can in turn feed back on global RNA production and turnover.
Asunto(s)
Proliferación Celular , Transformación Celular Neoplásica/genética , Regulación Neoplásica de la Expresión Génica , Linfoma de Células B/genética , Linfoma de Células B/patología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Transcripción Genética , Animales , Linfocitos B/metabolismo , Linfocitos B/patología , Transformación Celular Neoplásica/patología , Cromatina/genética , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , Progresión de la Enfermedad , Regulación hacia Abajo/genética , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica , Regulación Neoplásica de la Expresión Génica/genética , Genoma/genética , Linfoma de Células B/metabolismo , Masculino , Ratones , Mitógenos/farmacología , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Regulación hacia Arriba/genéticaRESUMEN
The regulation of miRNAs is critical to the definition of cell identity and behavior in normal physiology and disease. To date, the dynamics of miRNA degradation and the mechanisms involved in remain largely obscure, in particular, in higher organisms. Here, we developed a pulse-chase approach based on metabolic RNA labeling to calculate miRNA decay rates at genome-wide scale in mammalian cells. Our analysis revealed heterogeneous miRNA half-lives, with many species behaving as stable molecules (T1/2> 24 h), while others, including passenger miRNAs and a number (25/129) of guide miRNAs, are quickly turned over (T1/2= 4-14 h). Decay rates were coupled with other features, including genomic organization, transcription rates, structural heterogeneity (isomiRs), and target abundance, measured through quantitative experimental approaches. This comprehensive analysis highlighted functional mechanisms that mediate miRNA degradation, as well as the importance of decay dynamics in the regulation of the miRNA pool under both steady-state conditions and during cell transitions.
Asunto(s)
MicroARNs/genética , Animales , Proteínas Argonautas/metabolismo , Fibroblastos , Regulación de la Expresión Génica , Estudio de Asociación del Genoma Completo , Ratones , MicroARNs/metabolismo , Interferencia de ARN , Estabilidad del ARN , Ribonucleasa III/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
Expression of the Myc oncoprotein is downregulated in response to stress signals to allow cells to cease proliferation and escape apoptosis, but the mechanisms involved in this process are poorly understood. Cell cycle arrest in response to DNA damage requires downregulation of Myc via a p53-independent signaling pathway. Here we have used siRNA screening of the human kinome to identify MAPKAPK5 (MK5, PRAK) as a negative regulator of Myc expression. MK5 regulates translation of Myc, since it is required for expression of miR-34b and miR-34c that bind to the 3'UTR of MYC. MK5 activates miR-34b/c expression via phosphorylation of FoxO3a, thereby promoting nuclear localization of FoxO3a and enabling it to induce miR-34b/c expression and arrest proliferation. Expression of MK5 in turn is directly activated by Myc, forming a negative feedback loop. MK5 is downregulated in colon carcinomas, arguing that this feedback loop is disrupted during colorectal tumorigenesis.
Asunto(s)
Neoplasias Colorrectales/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Línea Celular Tumoral , Neoplasias Colorrectales/enzimología , Regulación hacia Abajo , Retroalimentación Fisiológica , Proteína Forkhead Box O3 , Factores de Transcripción Forkhead/genética , Factores de Transcripción Forkhead/metabolismo , Células HCT116 , Células HeLa , Humanos , Péptidos y Proteínas de Señalización Intracelular/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
BACKGROUND & AIMS: Liver regeneration after partial hepatectomy (PH) increases the protein folding burden at the endoplasmic reticulum of remnant hepatocytes, resulting in induction of the unfolded protein response. We investigated the role of the core unfolded protein response transcription factor X-box binding protein 1 (XBP1) in liver regeneration using genome-wide chromatin immunoprecipitation analysis. METHODS: We performed studies with C57Bl6-J (control) and interleukin 6-knockout mice. Mice underwent PH or sham surgeries. In some mice, hepatic expression of XBP1 was knocked down by injection of adenoviral vectors encoding small hairpin RNAs against Xbp1 messenger RNA. Liver tissues were collected before surgery and at 6 and 48 hours after surgery and analyzed by chromatin immunoprecipitation followed by sequencing. We also performed functional analyses of HepG2 cells. RESULTS: Expression of XBP1 by hepatocytes increased immediately after PH (priming phase of liver regeneration) in control mice, but this effect was delayed in interleukin 6-deficient mice. In mice with knockdown of XBP1, we observed of liver tissue persistent endoplasmic reticulum stress, defects in acute-phase response, and increased hepatocellular damage, compared with control mice. Chromatin immunoprecipitation analyses of liver tissue showed that at 6 hours after PH, liver XBP1 became bound to a large set of genes implicated in proteostasis, the acute-phase response, metabolism, and the DNA damage response (DDR). At this time point, XBP1 bound the promoter of the signal transducer and activator of transcription 3 gene (Stat3). Livers of XBP1-knockdown mice showed reduced expression of STAT3 and had lower levels of STAT3 phosphorylation at Ser727, a modification that promotes cell proliferation and the DDR. Regenerating livers from XBP1-knockdown mice expressed high levels of a marker of DNA double-strand breaks, phosphorylated histone 2A, member X (H2AX), compared with control mice. The inhibition of XBP1 expression caused a reduced up-regulation of DDR messenger RNAs in regenerating hepatocytes. CONCLUSION: In livers of mice, we found that PH induces expression of XBP1, and that this activity requires interleukin 6. XBP1 expression regulates the unfolded protein response, acute-phase response, and DDR in hepatocytes. In regenerating livers, XBP1 deficiency leads to endoplasmic reticulum stress and DNA damage.
Asunto(s)
Reacción de Fase Aguda/genética , Daño del ADN/genética , Estrés del Retículo Endoplásmico/genética , Regeneración Hepática/genética , Hígado/metabolismo , Respuesta de Proteína Desplegada/genética , Proteína 1 de Unión a la X-Box/genética , Animales , Células Hep G2 , Hepatectomía , Humanos , Interleucina-6/genética , Ratones , Ratones Noqueados , Fosforilación , Factor de Transcripción STAT3/metabolismoRESUMEN
The ST18 gene has been proposed to act either as a tumor suppressor or as an oncogene in different human cancers, but direct evidence for its role in tumorigenesis has been lacking thus far. Here, we demonstrate that ST18 is critical for tumor progression and maintenance in a mouse model of liver cancer, based on oncogenic transformation and adoptive transfer of primary precursor cells (hepatoblasts). ST18 messenger RNA (mRNA) and protein were detectable neither in normal liver nor in cultured hepatoblasts, but were readily expressed after subcutaneous engraftment and tumor growth. ST18 expression in liver cells was induced by inflammatory cues, including acute or chronic inflammation in vivo, as well as coculture with macrophages in vitro. Knocking down the ST18 mRNA in transplanted hepatoblasts delayed tumor progression. Induction of ST18 knockdown in pre-established tumors caused rapid tumor involution associated with pervasive morphological changes, proliferative arrest, and apoptosis in tumor cells, as well as depletion of tumor-associated macrophages, vascular ectasia, and hemorrhage. Reciprocally, systemic depletion of macrophages in recipient animals had very similar phenotypic consequences, impairing either tumor development or maintenance, and suppressing ST18 expression in hepatoblasts. Finally, RNA sequencing of ST18-depleted tumors before involution revealed down-regulation of inflammatory response genes, pointing to the suppression of nuclear factor kappa B-dependent transcription. CONCLUSION: ST18 expression in epithelial cells is induced by tumor-associated macrophages, contributing to the reciprocal feed-forward loop between both cell types in liver tumorigenesis. Our findings warrant the exploration of means to interfere with ST18-dependent epithelium-macrophage interactions in a therapeutic setting. (Hepatology 2017;65:1708-1719).
Asunto(s)
Carcinoma Hepatocelular/etiología , Neoplasias Hepáticas Experimentales/etiología , Factores de Transcripción/metabolismo , Animales , Carcinoma Hepatocelular/metabolismo , Neoplasias Hepáticas Experimentales/metabolismo , Ratones Endogámicos C57BLRESUMEN
Deregulated expression of the MYC oncoprotein contributes to the genesis of many human tumours, yet strategies to exploit this for a rational tumour therapy are scarce. MYC promotes cell growth and proliferation, and alters cellular metabolism to enhance the provision of precursors for phospholipids and cellular macromolecules. Here we show in human and murine cell lines that oncogenic levels of MYC establish a dependence on AMPK-related kinase 5 (ARK5; also known as NUAK1) for maintaining metabolic homeostasis and for cell survival. ARK5 is an upstream regulator of AMPK and limits protein synthesis via inhibition of the mammalian target of rapamycin 1 (mTORC1) signalling pathway. ARK5 also maintains expression of mitochondrial respiratory chain complexes and respiratory capacity, which is required for efficient glutamine metabolism. Inhibition of ARK5 leads to a collapse of cellular ATP levels in cells expressing deregulated MYC, inducing multiple pro-apoptotic responses as a secondary consequence. Depletion of ARK5 prolongs survival in MYC-driven mouse models of hepatocellular carcinoma, demonstrating that targeting cellular energy homeostasis is a valid therapeutic strategy to eliminate tumour cells that express deregulated MYC.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Genes myc/genética , Proteínas Quinasas/metabolismo , Proteínas Represoras/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Adenosina Trifosfato/metabolismo , Animales , Apoptosis , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patología , Línea Celular Tumoral , Respiración de la Célula , Supervivencia Celular , Transformación Celular Neoplásica/genética , Modelos Animales de Enfermedad , Doxiciclina/farmacología , Transporte de Electrón , Glutamina/metabolismo , Homeostasis , Humanos , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Ratones , Mitocondrias/metabolismo , Complejos Multiproteicos , Proteína Oncogénica p55(v-myc)/genética , Proteína Oncogénica p55(v-myc)/metabolismo , Biosíntesis de Proteínas , Proteínas Quinasas/deficiencia , Proteínas Quinasas/genética , Proteínas/antagonistas & inhibidores , Proteínas/metabolismo , Interferencia de ARN , Proteínas Represoras/antagonistas & inhibidores , Proteínas Represoras/deficiencia , Proteínas Represoras/genética , Transducción de Señal , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
MOTIVATION: Cellular mRNA levels originate from the combined action of multiple regulatory processes, which can be recapitulated by the rates of pre-mRNA synthesis, pre-mRNA processing and mRNA degradation. Recent experimental and computational advances set the basis to study these intertwined levels of regulation. Nevertheless, software for the comprehensive quantification of RNA dynamics is still lacking. RESULTS: INSPEcT is an R package for the integrative analysis of RNA- and 4sU-seq data to study the dynamics of transcriptional regulation. INSPEcT provides gene-level quantification of these rates, and a modeling framework to identify which of these regulatory processes are most likely to explain the observed mRNA and pre-mRNA concentrations. Software performance is tested on a synthetic dataset, instrumental to guide the choice of the modeling parameters and the experimental design. AVAILABILITY AND IMPLEMENTATION: INSPEcT is submitted to Bioconductor and is currently available as Supplementary Additional File S1. CONTACT: mattia.pelizzola@iit.it SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Precursores del ARN/genética , Estabilidad del ARN/genética , ARN Mensajero/metabolismo , ARN/genética , Análisis de Secuencia de ARN/métodos , Programas Informáticos , Algoritmos , Animales , Células Cultivadas , Regulación de la Expresión Génica , Ratones , ARN Mensajero/química , ARN Mensajero/genética , Transcripción GenéticaRESUMEN
The DNA damage response activates several pathways that stall the cell cycle and allow DNA repair. These consist of the well-characterized ATR (Ataxia telangiectasia and Rad-3 related)/CHK1 and ATM (Ataxia telangiectasia mutated)/CHK2 pathways in addition to a newly identified ATM/ATR/p38MAPK/MK2 checkpoint. Crucial to maintaining the integrity of the genome is the S-phase checkpoint that functions to prevent DNA replication until damaged DNA is repaired. Inappropriate expression of the proto-oncogene c-Myc is known to cause DNA damage. One mechanism by which c-Myc induces DNA damage is through binding directly to components of the prereplicative complex thereby promoting DNA synthesis, resulting in replication-associated DNA damage and checkpoint activation due to inappropriate origin firing. Here we show that following etoposide-induced DNA damage translation of c-Myc is repressed by miR-34c via a highly conserved target-site within the 3(') UTR. While miR-34c is induced by p53 following DNA damage, we show that in cells lacking p53 this is achieved by an alternative pathway which involves p38 MAPK signalling to MK2. The data presented here suggest that a major physiological target of miR-34c is c-Myc. Inhibition of miR-34c activity prevents S-phase arrest in response to DNA damage leading to increased DNA synthesis, DNA damage, and checkpoint activation in addition to that induced by etoposide alone, which are all reversed by subsequent c-Myc depletion. These data demonstrate that miR-34c is a critical regulator of the c-Myc expression following DNA damage acting downstream of p38 MAPK/MK2 and suggest that miR-34c serves to remove c-Myc to prevent inappropriate replication which may otherwise lead to genomic instability.
Asunto(s)
Daño del ADN , Replicación del ADN/fisiología , Péptidos y Proteínas de Señalización Intracelular/metabolismo , MicroARNs/biosíntesis , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Regiones no Traducidas 3' , Animales , Línea Celular , Replicación del ADN/genética , Células HeLa , Humanos , Sistema de Señalización de MAP Quinasas , Ratones , MicroARNs/genética , Proto-Oncogenes Mas , Fase S/genética , Fase S/fisiología , Proteína p53 Supresora de Tumor/deficiencia , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The molecular heterogeneity of human cancer cells at the level of signaling protein activities remains poorly understood. Using a panel of 64 colorectal (CRC) cancer cell lines the activity status of the MAP kinases Erk1 and Erk2 was investigated. Erk1/2 activity varied greatly within the CRC cell line panel and was not detectably associated with the speed of cell growth in 10 CRC lines analyzed. As expected, mutations in K-Ras or B-Raf were often, albeit not always, linked to high Erk1/2 activity. The phosphorylation of several known Erk1/2 targets investigated did not generally reflect Erk1/2 activity in the 10 CRC lines analyzed. However, the reduction of Erk1/2 activity with MEK inhibitors generally abolished cell growth but only led to an increase of cellular p27Kip1 levels in CRC cells with high Erk1/2 activity levels. The results indicate that high Erk1/2 activation is utilized by some CRC lines to override the cell cycle brake p27Kip1, while others presumably rely on different mechanisms in order to inactivate this important cell cycle brake. Such detailed knowledge of the molecular diversity of cancer cell signaling mechanisms may eventually help to develop molecularly targeted, patient-specific therapeutic strategies and treatments.
RESUMEN
It is unclear why some tissues are refractory to the mitogenic effects of the oncogene Myc. Here we show that Myc activation induces rapid transcriptional responses followed by proliferation in some, but not all, organs. Despite such disparities in proliferative response, Myc is bound to DNA at open elements in responsive (liver) and non-responsive (heart) tissues, but fails to induce a robust transcriptional and proliferative response in the heart. Using heart as an exemplar of a non-responsive tissue, we show that Myc-driven transcription is re-engaged in mature cardiomyocytes by elevating levels of the positive transcription elongation factor (P-TEFb), instating a large proliferative response. Hence, P-TEFb activity is a key limiting determinant of whether the heart is permissive for Myc transcriptional activation. These data provide a greater understanding of how Myc transcriptional activity is determined and indicate modification of P-TEFb levels could be utilised to drive regeneration of adult cardiomyocytes for the treatment of heart myopathies.
Asunto(s)
Miocardio/metabolismo , Proteínas Proto-Oncogénicas c-myc/genética , Transcripción Genética , Animales , Proliferación Celular/genética , Cromatina/metabolismo , Ciclina T/metabolismo , Ratones , Miocitos Cardíacos/metabolismo , Especificidad de Órganos , Fosforilación , Factor B de Elongación Transcripcional Positiva/metabolismo , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/metabolismo , Activación Transcripcional/genéticaRESUMEN
The Smyd2 protein (Set- and Mynd domain containing protein 2) is a methyl-transferase that can modify both histones and cytoplasmic proteins. Smyd2 is over-expressed in several cancer types and was shown to be limiting for tumor development in the pancreas. However, genetic evidence for a role of Smyd2 in other cancers or in mouse development was missing to date. Using germ line-deleted mouse strains, we now show that Smyd2 and the related protein Smyd3 are dispensable for normal development. Ablation of Smyd2 did not affect hematopoiesis, but retarded the development of leukemia promoted by MLL-AF9, a fusion oncogene associated with acute myeloid leukemia (AML) in humans. Smyd2-deleted leukemic cells showed a competitive disadvantage relative to wild-type cells, either in vitro or in vivo. The Smyd2 gene was directly activated by the oncogenic transcription factor Myc in either MLL9-AF9-induced leukemias, Myc-induced lymphomas, or fibroblasts. However, unlike leukemias, the development of lymphomas was not dependent upon Smyd2. Our data indicate that Smyd2 has a critical role downstream of Myc in AML.
Asunto(s)
Transformación Celular Neoplásica/genética , N-Metiltransferasa de Histona-Lisina/genética , Leucemia Mieloide Aguda/genética , Proteínas de Fusión Oncogénica/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Regulación Leucémica de la Expresión Génica , Hematopoyesis/genética , Leucemia Mieloide Aguda/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones NoqueadosRESUMEN
The c-myc proto-oncogene is activated by translocation in Burkitt's lymphoma and substitutions in codon 58 stabilize the Myc protein or augment its oncogenic potential. In wild-type Myc, phosphorylation of Ser 62 and Thr 58 provides a landing pad for the peptidyl prolyl-isomerase Pin1, which in turn promotes Ser 62 dephosphorylation and Myc degradation. However, the role of Pin1 in Myc-induced lymphomagenesis remains unknown. We show here that genetic ablation of Pin1 reduces lymphomagenesis in Eµ-myc transgenic mice. In both Pin1-deficient B-cells and MEFs, the proliferative response to oncogenic Myc was selectively impaired, with no alterations in Myc-induced apoptosis or mitogen-induced cell cycle entry. This proliferative defect wasn't attributable to alterations in either Ser 62 phosphorylation or Myc-regulated transcription, but instead relied on the activity of the ARF-p53 pathway. Pin1 silencing in lymphomas retarded disease progression in mice, making Pin1 an attractive therapeutic target in Myc-driven tumors.
Asunto(s)
Linfocitos B/metabolismo , Proliferación Celular/genética , Linfoma/genética , Peptidilprolil Isomerasa de Interacción con NIMA/genética , Proteínas Proto-Oncogénicas c-myc/genética , Animales , Células Cultivadas , Embrión de Mamíferos/citología , Fibroblastos/metabolismo , Perfilación de la Expresión Génica/métodos , Linfoma/metabolismo , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Interferencia de ARNRESUMEN
Tumors driven by activation of the transcription factor MYC generally show oncogene addiction. However, the gene expression programs that depend upon sustained MYC activity remain unknown. In this study, we employed a mouse model of liver carcinoma driven by a reversible tet-MYC transgene, combined with chromatin immunoprecipitation and gene expression profiling to identify MYC-dependent regulatory events. As previously reported, MYC-expressing mice exhibited hepatoblastoma- and hepatocellular carcinoma-like tumors, which regressed when MYC expression was suppressed. We further show that cellular transformation, and thus initiation of liver tumorigenesis, were impaired in mice harboring a MYC mutant unable to associate with the corepressor protein MIZ1 (ZBTB17). Notably, switching off the oncogene in advanced carcinomas revealed that MYC was required for the continuous activation and repression of distinct sets of genes, constituting no more than half of all genes deregulated during tumor progression and an even smaller subset of all MYC-bound genes. Altogether, our data provide the first detailed analysis of a MYC-dependent transcriptional program in a fully developed carcinoma and offer a guide to identifying the critical effectors contributing to MYC-driven tumor maintenance. Cancer Res; 76(12); 3463-72. ©2016 AACR.
Asunto(s)
Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Oncogenes , Proteínas Proto-Oncogénicas c-myc/fisiología , Transcripción Genética , Animales , Células Cultivadas , Regulación Neoplásica de la Expresión Génica , Humanos , Ratones , Regiones Promotoras GenéticasRESUMEN
Two opposing models have been proposed to describe the function of the MYC oncoprotein in shaping cellular transcriptomes: one posits that MYC amplifies transcription at all active loci; the other that MYC differentially controls discrete sets of genes, the products of which affect global transcript levels. Here, we argue that differential gene regulation by MYC is the sole unifying model that is consistent with all available data. Among other effects, MYC endows cells with physiological and metabolic changes that have the potential to feed back on global RNA production, processing and turnover. The field is progressing steadily towards a full characterization of the MYC-regulated genes and pathways that mediate these biological effects and - by the same token - endow MYC with its pervasive oncogenic potential.
Asunto(s)
Regulación Neoplásica de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas c-myc/genética , ARN Mensajero/metabolismo , HumanosRESUMEN
The gene expression programs regulated by the Myc transcription factor were evaluated by integrated genome-wide profiling of Myc binding sites, chromatin marks and RNA expression in several biological models. Our results indicate that Myc directly drives selective transcriptional regulation, which in certain physiological conditions may indirectly lead to RNA amplification. Here, we illustrate in detail the experimental design concerning the high-throughput sequencing data associated with our study (Sabò et al., Nature. (2014) 511:488-492) and the R scripts used for their computational analysis.
RESUMEN
The steroid hormone aldosterone is a major regulator of extracellular volume and blood pressure. Aldosterone effectors are for example the epithelial Na(+) channel (ENaC), the Na(+)-K(+)-ATPase and the proximal tubule Na(+)/H(+) exchanger isoform 3 (NHE3). The aim of this study was to investigate whether aldosterone acts directly on proximal tubule cells to stimulate NHE3 and if so whether the EGF-receptor (EGFR) is involved. For this purpose, primary human renal proximal tubule cells were exposed to aldosterone. NHE3 activity was determined from Na(+)- dependent pH-recovery, NHE3 surface expression was determined by biotinylation and immunoblotting. EGFR-expression was assessed by ELISA. pH(i)- measurements revealed an aldosterone-induced increase in NHE3 activity, which was inhibited by the mineralocorticoid receptor blocker spironolactone and by the EGFR-kinase inhibitor AG1478. Immunoprecipitation and immunoblot analysis showed an aldosterone-induced increase in NHE3 surface expression, which was also inhibited by spironolactone and AG1478. Furthermore, aldosterone enhanced EGFR-expression. In conclusion, aldosterone stimulates NHE3 in human proximal tubule cells. The underlying mechanisms include AG1478 inhibitable kinase and are paralleled by enhanced EGFR expression, which could be compatible with EGF-receptor-pathway-dependent surface expression and activity of NHE3 in human primary renal proximal tubule epithelial cells.