RESUMEN
The pluripotent state of embryonic stem cells (ESCs) is produced by active transcription of genes that control cell identity and repression of genes encoding lineage-specifying developmental regulators. Here, we use ESC cohesin ChIA-PET data to identify the local chromosomal structures at both active and repressed genes across the genome. The results produce a map of enhancer-promoter interactions and reveal that super-enhancer-driven genes generally occur within chromosome structures that are formed by the looping of two interacting CTCF sites co-occupied by cohesin. These looped structures form insulated neighborhoods whose integrity is important for proper expression of local genes. We also find that repressed genes encoding lineage-specifying developmental regulators occur within insulated neighborhoods. These results provide insights into the relationship between transcriptional control of cell identity genes and control of local chromosome structure.
Asunto(s)
Cromosomas de los Mamíferos/metabolismo , Células Madre Embrionarias/metabolismo , Animales , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Inmunoprecipitación de Cromatina , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/citología , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Ratones , Especificidad de Órganos , Células Madre Pluripotentes/metabolismo , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADN , CohesinasRESUMEN
BMP and Wnt signaling pathways control essential cellular responses through activation of the transcription factors SMAD (BMP) and TCF (Wnt). Here, we show that regeneration of hematopoietic lineages following acute injury depends on the activation of each of these signaling pathways to induce expression of key blood genes. Both SMAD1 and TCF7L2 co-occupy sites with master regulators adjacent to hematopoietic genes. In addition, both SMAD1 and TCF7L2 follow the binding of the predominant lineage regulator during differentiation from multipotent hematopoietic progenitor cells to erythroid cells. Furthermore, induction of the myeloid lineage regulator C/EBPα in erythroid cells shifts binding of SMAD1 to sites newly occupied by C/EBPα, whereas expression of the erythroid regulator GATA1 directs SMAD1 loss on nonerythroid targets. We conclude that the regenerative response mediated by BMP and Wnt signaling pathways is coupled with the lineage master regulators to control the gene programs defining cellular identity.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Hematopoyesis , Transducción de Señal , Vía de Señalización Wnt , Animales , Proteínas de Unión al ADN/metabolismo , Humanos , Regeneración , Proteína Smad1/metabolismo , Proteína 2 Similar al Factor de Transcripción 7/metabolismo , Pez CebraRESUMEN
The genetic circuits that allow cancer cells to evade destruction by the host immune system remain poorly understood1-3. Here, to identify a phenotypically robust core set of genes and pathways that enable cancer cells to evade killing mediated by cytotoxic T lymphocytes (CTLs), we performed genome-wide CRISPR screens across a panel of genetically diverse mouse cancer cell lines that were cultured in the presence of CTLs. We identify a core set of 182 genes across these mouse cancer models, the individual perturbation of which increases either the sensitivity or the resistance of cancer cells to CTL-mediated toxicity. Systematic exploration of our dataset using genetic co-similarity reveals the hierarchical and coordinated manner in which genes and pathways act in cancer cells to orchestrate their evasion of CTLs, and shows that discrete functional modules that control the interferon response and tumour necrosis factor (TNF)-induced cytotoxicity are dominant sub-phenotypes. Our data establish a central role for genes that were previously identified as negative regulators of the type-II interferon response (for example, Ptpn2, Socs1 and Adar1) in mediating CTL evasion, and show that the lipid-droplet-related gene Fitm2 is required for maintaining cell fitness after exposure to interferon-γ (IFNγ). In addition, we identify the autophagy pathway as a conserved mediator of the evasion of CTLs by cancer cells, and show that this pathway is required to resist cytotoxicity induced by the cytokines IFNγ and TNF. Through the mapping of cytokine- and CTL-based genetic interactions, together with in vivo CRISPR screens, we show how the pleiotropic effects of autophagy control cancer-cell-intrinsic evasion of killing by CTLs and we highlight the importance of these effects within the tumour microenvironment. Collectively, these data expand our knowledge of the genetic circuits that are involved in the evasion of the immune system by cancer cells, and highlight genetic interactions that contribute to phenotypes associated with escape from killing by CTLs.
Asunto(s)
Genoma/genética , Genómica , Neoplasias/genética , Neoplasias/inmunología , Linfocitos T Citotóxicos/inmunología , Escape del Tumor/genética , Escape del Tumor/inmunología , Animales , Autofagia , Línea Celular Tumoral , Femenino , Genes Relacionados con las Neoplasias/genética , Humanos , Interferón gamma/inmunología , Masculino , Ratones , FN-kappa B/metabolismo , Reproducibilidad de los Resultados , Transducción de SeñalRESUMEN
Studying cancer metabolism gives insight into tumorigenic survival mechanisms and susceptibilities. In melanoma, we identify HEXIM1, a transcription elongation regulator, as a melanoma tumor suppressor that responds to nucleotide stress. HEXIM1 expression is low in melanoma. Its overexpression in a zebrafish melanoma model suppresses cancer formation, while its inactivation accelerates tumor onset in vivo. Knockdown of HEXIM1 rescues zebrafish neural crest defects and human melanoma proliferation defects that arise from nucleotide depletion. Under nucleotide stress, HEXIM1 is induced to form an inhibitory complex with P-TEFb, the kinase that initiates transcription elongation, to inhibit elongation at tumorigenic genes. The resulting alteration in gene expression also causes anti-tumorigenic RNAs to bind to and be stabilized by HEXIM1. HEXIM1 plays an important role in inhibiting cancer cell-specific gene transcription while also facilitating anti-cancer gene expression. Our study reveals an important role for HEXIM1 in coupling nucleotide metabolism with transcriptional regulation in melanoma.
Asunto(s)
Melanoma/metabolismo , Factor B de Elongación Transcripcional Positiva/genética , Pirimidinas/metabolismo , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Animales , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Humanos , Melanoma/genética , Melanoma/patología , Melanoma Experimental , Proteínas Oncogénicas/genética , Factores de Transcripción , Transcripción Genética , Proteínas Supresoras de Tumor/genética , Pez Cebra/genética , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
Adult stem cells must limit their rate of protein synthesis, but the underlying mechanisms remain largely unexplored. Differences in protein synthesis among hematopoietic stem cells (HSCs) and progenitor cells did not correlate with differences in proteasome activity, total RNA content, mRNA content, or cell division rate. However, adult HSCs had more hypophosphorylated eukaryotic initiation factor 4E-binding protein 1 (4E-BP1) and 4E-BP2 as compared with most other hematopoietic progenitors. Deficiency for 4E-BP1 and 4E-BP2 significantly increased global protein synthesis in HSCs, but not in other hematopoietic progenitors, and impaired their reconstituting activity, identifying a mechanism that promotes HSC maintenance by attenuating protein synthesis.
Asunto(s)
Proteínas Portadoras/metabolismo , Factores Eucarióticos de Iniciación/metabolismo , Células Madre Hematopoyéticas/metabolismo , Fosfoproteínas/metabolismo , Biosíntesis de Proteínas/genética , Proteínas Adaptadoras Transductoras de Señales , Animales , Proteínas Portadoras/genética , Proteínas de Ciclo Celular , Diferenciación Celular/genética , Factores Eucarióticos de Iniciación/genética , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfoproteínas/genética , Fosforilación , Complejo de la Endopetidasa Proteasomal/metabolismo , Eliminación de SecuenciaRESUMEN
Coordination of cellular metabolism is essential for optimal T cell responses. Here, we identify cytosolic acetyl-CoA production as an essential metabolic node for CD8 T cell function in vivo. We show that CD8 T cell responses to infection depend on acetyl-CoA derived from citrate via the enzyme ATP citrate lyase (ACLY). However, ablation of ACLY triggers an alternative, acetate-dependent pathway for acetyl-CoA production mediated by acyl-CoA synthetase short-chain family member 2 (ACSS2). Mechanistically, acetate fuels both the TCA cycle and cytosolic acetyl-CoA production, impacting T cell effector responses, acetate-dependent histone acetylation, and chromatin accessibility at effector gene loci. When ACLY is functional, ACSS2 is not required, suggesting acetate is not an obligate metabolic substrate for CD8 T cell function. However, loss of ACLY renders CD8 T cells dependent on acetate (via ACSS2) to maintain acetyl-CoA production and effector function. Together, ACLY and ACSS2 coordinate cytosolic acetyl-CoA production in CD8 T cells to maintain chromatin accessibility and T cell effector function.
Asunto(s)
ATP Citrato (pro-S)-Liasa , Acetatos , Acetilcoenzima A , Linfocitos T CD8-positivos , Cromatina , Ratones Endogámicos C57BL , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Animales , Cromatina/metabolismo , Acetilcoenzima A/metabolismo , ATP Citrato (pro-S)-Liasa/metabolismo , ATP Citrato (pro-S)-Liasa/genética , Ratones , Acetatos/metabolismo , Acetato CoA Ligasa/metabolismo , Acetato CoA Ligasa/genética , Acetilación , Ratones Noqueados , Citosol/metabolismo , Histonas/metabolismoRESUMEN
Protein structural fluctuations occur over a wide spatial scale, ranging from minute, picometer-scale displacements, to large, interdomain motions and partial unfolding. While large-scale protein structural changes and their effects on protein function have been the focus of much recent attention, small-scale fluctuations have been less well studied, and are generally assumed to have proportionally smaller effects. Here we use the bacterial photoreceptor photoactive yellow protein (PYP) to test if subtle structural changes do, indeed, imply equally subtle functional effects. We flash froze crystals of PYP to trap the protein's conformational ensemble, and probed the molecules in this ensemble for their ability to facilitate PYP's biological function (i.e., light-driven isomerization of its chromophore). Our results indicate that the apparently homogeneous structural state observed in a 0.82 A crystal structure in fact comprises an ensemble of conformational states, in which subpopulations with nearly identical structures display dramatically different functional properties.
Asunto(s)
Proteínas Bacterianas/química , Fotorreceptores Microbianos/química , Proteínas Bacterianas/efectos de la radiación , Sitios de Unión , Cristalografía por Rayos X , Congelación , Isomerismo , Luz , Modelos Moleculares , Fotoquímica , Fotorreceptores Microbianos/efectos de la radiación , Conformación ProteicaRESUMEN
Agents targeting metabolic pathways form the backbone of standard oncology treatments, though a better understanding of differential metabolic dependencies could instruct more rationale-based therapeutic approaches. We performed a chemical biology screen that revealed a strong enrichment in sensitivity to a novel dihydroorotate dehydrogenase (DHODH) inhibitor, AG-636, in cancer cell lines of hematologic versus solid tumor origin. Differential AG-636 activity translated to the in vivo setting, with complete tumor regression observed in a lymphoma model. Dissection of the relationship between uridine availability and response to AG-636 revealed a divergent ability of lymphoma and solid tumor cell lines to survive and grow in the setting of depleted extracellular uridine and DHODH inhibition. Metabolic characterization paired with unbiased functional genomic and proteomic screens pointed to adaptive mechanisms to cope with nucleotide stress as contributing to response to AG-636. These findings support targeting of DHODH in lymphoma and other hematologic malignancies and suggest combination strategies aimed at interfering with DNA-damage response pathways.
Asunto(s)
Antineoplásicos/farmacología , Inhibidores Enzimáticos/farmacología , Neoplasias Hematológicas/metabolismo , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Pirimidinas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Daño del ADN/efectos de los fármacos , Dihidroorotato Deshidrogenasa , Genómica/métodos , Neoplasias Hematológicas/tratamiento farmacológico , Neoplasias Hematológicas/etiología , Neoplasias Hematológicas/patología , Humanos , Estadificación de Neoplasias , Proteómica/métodosRESUMEN
Aberrant metabolism of cancer cells is well appreciated, but the identification of cancer subsets with specific metabolic vulnerabilities remains challenging. We conducted a chemical biology screen and identified a subset of neuroendocrine tumors displaying a striking pattern of sensitivity to inhibition of the cholesterol biosynthetic pathway enzyme squalene epoxidase (SQLE). Using a variety of orthogonal approaches, we demonstrate that sensitivity to SQLE inhibition results not from cholesterol biosynthesis pathway inhibition, but rather surprisingly from the specific and toxic accumulation of the SQLE substrate, squalene. These findings highlight SQLE as a potential therapeutic target in a subset of neuroendocrine tumors, particularly small cell lung cancers.
Asunto(s)
Antineoplásicos/farmacología , Sistemas de Liberación de Medicamentos , Ensayos de Selección de Medicamentos Antitumorales , Escualeno-Monooxigenasa/antagonistas & inhibidores , Escualeno-Monooxigenasa/metabolismo , Antineoplásicos/química , Línea Celular Tumoral , Colesterol/biosíntesis , Eliminación de Gen , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , HumanosRESUMEN
In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease.
Asunto(s)
Cromosomas Humanos/genética , Células Madre Pluripotentes/metabolismo , Factor de Unión a CCCTC , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , ADN/química , ADN/metabolismo , Enfermedad/genética , Elementos de Facilitación Genéticos , Regulación de la Expresión Génica , Células Madre Embrionarias Humanas/metabolismo , Humanos , Elementos Aisladores/genética , MicroARNs/metabolismo , Conformación de Ácido Nucleico , Proteínas Represoras , Factores de Transcripción/metabolismo , CohesinasRESUMEN
Although aberrant metabolism in tumors has been well described, the identification of cancer subsets with particular metabolic vulnerabilities has remained challenging. Here, we conducted an siRNA screen focusing on enzymes involved in the tricarboxylic acid (TCA) cycle and uncovered a striking range of cancer cell dependencies on OGDH, the E1 subunit of the alpha-ketoglutarate dehydrogenase complex. Using an integrative metabolomics approach, we identified differential aspartate utilization, via the malate-aspartate shuttle, as a predictor of whether OGDH is required for proliferation in 3D culture assays and for the growth of xenograft tumors. These findings highlight an anaplerotic role of aspartate and, more broadly, suggest that differential nutrient utilization patterns can identify subsets of cancers with distinct metabolic dependencies for potential pharmacological intervention.
Asunto(s)
Ácido Aspártico/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Neoplasias/metabolismo , Animales , Línea Celular Tumoral , Respiración de la Célula/efectos de los fármacos , Ciclo del Ácido Cítrico/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Técnicas de Silenciamiento del Gen , Humanos , ARN Interferente Pequeño/metabolismoRESUMEN
Oncogenes are activated through well-known chromosomal alterations such as gene fusion, translocation, and focal amplification. In light of recent evidence that the control of key genes depends on chromosome structures called insulated neighborhoods, we investigated whether proto-oncogenes occur within these structures and whether oncogene activation can occur via disruption of insulated neighborhood boundaries in cancer cells. We mapped insulated neighborhoods in T cell acute lymphoblastic leukemia (T-ALL) and found that tumor cell genomes contain recurrent microdeletions that eliminate the boundary sites of insulated neighborhoods containing prominent T-ALL proto-oncogenes. Perturbation of such boundaries in nonmalignant cells was sufficient to activate proto-oncogenes. Mutations affecting chromosome neighborhood boundaries were found in many types of cancer. Thus, oncogene activation can occur via genetic alterations that disrupt insulated neighborhoods in malignant cells.
Asunto(s)
Aberraciones Cromosómicas , Regulación Leucémica de la Expresión Génica , Leucemia-Linfoma Linfoblástico de Células T Precursoras/genética , Proto-Oncogenes/genética , Eliminación de Secuencia , Translocación Genética , Mapeo Cromosómico , Células HEK293 , Humanos , Mutación , Activación TranscripcionalRESUMEN
The "cancerized field" concept posits that cancer-prone cells in a given tissue share an oncogenic mutation, but only discreet clones within the field initiate tumors. Most benign nevi carry oncogenic BRAF(V600E) mutations but rarely become melanoma. The zebrafish crestin gene is expressed embryonically in neural crest progenitors (NCPs) and specifically reexpressed in melanoma. Live imaging of transgenic zebrafish crestin reporters shows that within a cancerized field (BRAF(V600E)-mutant; p53-deficient), a single melanocyte reactivates the NCP state, revealing a fate change at melanoma initiation in this model. NCP transcription factors, including sox10, regulate crestin expression. Forced sox10 overexpression in melanocytes accelerated melanoma formation, which is consistent with activation of NCP genes and super-enhancers leading to melanoma. Our work highlights NCP state reemergence as a key event in melanoma initiation.
Asunto(s)
Carcinogénesis/genética , Regulación del Desarrollo de la Expresión Génica , Regulación Neoplásica de la Expresión Génica , Melanoma Experimental/genética , Melanoma/genética , Cresta Neural/metabolismo , Neoplasias Cutáneas/genética , Pez Cebra , Animales , Animales Modificados Genéticamente , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Genes Reporteros , Proteínas Fluorescentes Verdes/genética , Melanocitos/metabolismo , Mutación , Proteínas del Tejido Nervioso/genética , Proteínas Proto-Oncogénicas B-raf/genética , Factores de Transcripción SOXE/genética , Proteína p53 Supresora de Tumor/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Hundreds of transcription factors (TFs) are expressed in each cell type, but cell identity can be induced through the activity of just a small number of core TFs. Systematic identification of these core TFs for a wide variety of cell types is currently lacking and would establish a foundation for understanding the transcriptional control of cell identity in development, disease, and cell-based therapy. Here, we describe a computational approach that generates an atlas of candidate core TFs for a broad spectrum of human cells. The potential impact of the atlas was demonstrated via cellular reprogramming efforts where candidate core TFs proved capable of converting human fibroblasts to retinal pigment epithelial-like cells. These results suggest that candidate core TFs from the atlas will prove a useful starting point for studying transcriptional control of cell identity and reprogramming in many human cell types.
Asunto(s)
Reprogramación Celular , Células Epiteliales/citología , Fibroblastos/citología , Epitelio Pigmentado de la Retina/citología , Factores de Transcripción/genética , Línea Celular , Simulación por Computador , Células Epiteliales/metabolismo , Fibroblastos/metabolismo , Humanos , Epitelio Pigmentado de la Retina/metabolismoRESUMEN
Overexpression of transcription factors has been used to directly reprogram somatic cells into a range of other differentiated cell types, including multipotent neural stem cells (NSCs), that can be used to generate neurons and glia. However, the ability to maintain the NSC state independent of the inducing factors and the identity of the somatic donor cells remain two important unresolved issues in transdifferentiation. Here we used transduction of doxycycline-inducible transcription factors to generate stable tripotent NSCs. The induced NSCs (iNSCs) maintained their characteristics in the absence of exogenous factor expression and were transcriptionally, epigenetically, and functionally similar to primary brain-derived NSCs. Importantly, we also generated tripotent iNSCs from multiple adult cell types, including mature liver and B cells. Our results show that self-maintaining proliferative neural cells can be induced from nonectodermal cells by expressing specific combinations of transcription factors.
Asunto(s)
Linfocitos B/citología , Linaje de la Célula , Transdiferenciación Celular , Hepatocitos/citología , Células-Madre Neurales/citología , Animales , Linfocitos B/metabolismo , Linaje de la Célula/genética , Transdiferenciación Celular/genética , Reprogramación Celular , Análisis por Conglomerados , Epigénesis Genética , Expresión Génica , Perfilación de la Expresión Génica , Hepatocitos/metabolismo , Ratones , Células-Madre Neurales/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
A vast number of small-molecule ligands, including therapeutic drugs under development and in clinical use, elicit their effects by binding specific proteins associated with the genome. An ability to map the direct interactions of a chemical entity with chromatin genome-wide could provide important insights into chemical perturbation of cellular function. Here we describe a method that couples ligand-affinity capture and massively parallel DNA sequencing (Chem-seq) to identify the sites bound by small chemical molecules throughout the human genome. We show how Chem-seq can be combined with ChIP-seq to gain unique insights into the interaction of drugs with their target proteins throughout the genome of tumor cells. These methods will be broadly useful to enhance understanding of therapeutic action and to characterize the specificity of chemical entities that interact with DNA or genome-associated proteins.
Asunto(s)
Cromatina/genética , ADN/genética , Proteínas/genética , Factores de Transcripción/genética , Sitios de Unión/genética , Genoma Humano , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Ligandos , Unión Proteica/genéticaRESUMEN
Embryonic stem cells (ESCs) of mice and humans have distinct molecular and biological characteristics, raising the question of whether an earlier, "naive" state of pluripotency may exist in humans. Here we took a systematic approach to identify small molecules that support self-renewal of naive human ESCs based on maintenance of endogenous OCT4 distal enhancer activity, a molecular signature of ground state pluripotency. Iterative chemical screening identified a combination of five kinase inhibitors that induces and maintains OCT4 distal enhancer activity when applied directly to conventional human ESCs. These inhibitors generate human pluripotent cells in which transcription factors associated with the ground state of pluripotency are highly upregulated and bivalent chromatin domains are depleted. Comparison with previously reported naive human ESCs indicates that our conditions capture a distinct pluripotent state in humans that closely resembles that of mouse ESCs. This study presents a framework for defining the culture requirements of naive human pluripotent cells.
Asunto(s)
Técnicas de Cultivo de Célula/métodos , Células Madre Pluripotentes/citología , Supervivencia Celular , Cromatina/metabolismo , Elementos de Facilitación Genéticos/genética , Perfilación de la Expresión Génica , Genes Reporteros , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Datos de Secuencia Molecular , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/metabolismo , TransgenesRESUMEN
We present a protocol for visualizing and quantifying single mRNA molecules in mammalian (mouse and human) tissues. In the approach described here, sets of about 50 short oligonucleotides, each labeled with a single fluorophore, are hybridized to target mRNAs in tissue sections. Each set binds to a single mRNA molecule and can be detected by fluorescence microscopy as a diffraction-limited spot. Tissue architecture is then assessed by counterstaining the sections with DNA dye (DAPI), and cell borders can be visualized with a dye-coupled antibody. Spots are detected automatically with custom-made software, which we make freely available. The mRNA molecules thus detected are assigned to single cells within a tissue semiautomatically by using a graphical user interface developed in our laboratory. In this protocol, we describe an example of quantitative analysis of mRNA levels and localization in mouse small intestine. The procedure (from tissue dissection to obtaining data sets) takes 3 d. Data analysis will require an additional 3-7 d, depending on the type of analysis.
Asunto(s)
Hibridación Fluorescente in Situ/métodos , ARN Mensajero/análisis , Programas Informáticos , Animales , Proteínas Fluorescentes Verdes/análisis , Humanos , Intestino Delgado/metabolismo , Ratones , Sondas de Oligonucleótidos , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Interfaz Usuario-ComputadorRESUMEN
While cytotoxic chemotherapy remains the hallmark of cancer treatment, intensive regimens fall short in many malignancies, including high-risk neuroblastoma. One alternative strategy is to therapeutically promote tumor differentiation. We created a gene expression signature to measure neuroblast maturation, adapted it to a high-throughput platform, and screened a diversity oriented synthesis-generated small-molecule library for differentiation inducers. We identified BRD8430, containing a nine-membered lactam, an ortho-amino anilide functionality, and three chiral centers, as a selective class I histone deacetylase (HDAC) inhibitor (HDAC1 > 2 > 3). Further investigation demonstrated that selective HDAC1/HDAC2 inhibition using compounds or RNA interference induced differentiation and decreased viability in neuroblastoma cell lines. Combined treatment with 13-cis retinoic acid augmented these effects and enhanced activation of retinoic acid signaling. Therefore, by applying a chemical genomic screening approach, we identified selective HDAC1/HDAC2 inhibition as a strategy to induce neuroblastoma differentiation.
Asunto(s)
Histona Desacetilasa 1/antagonistas & inhibidores , Histona Desacetilasa 2/antagonistas & inhibidores , Inhibidores de Histona Desacetilasas/farmacología , Lactamas/farmacología , Neuroblastoma/tratamiento farmacológico , Neuroblastoma/enzimología , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Histona Desacetilasa 1/genética , Histona Desacetilasa 1/metabolismo , Histona Desacetilasa 2/genética , Histona Desacetilasa 2/metabolismo , Inhibidores de Histona Desacetilasas/química , Humanos , Lactamas/química , Neuroblastoma/genética , Neuroblastoma/patología , Tretinoina/metabolismoRESUMEN
In mammalian embryonic stem cells, the acquisition of pluripotency is dependent on Nanog, but the in vivo analysis of Nanog has been hampered by its requirement for early mouse development. In an effort to examine the role of Nanog in vivo, we identified a zebrafish Nanog ortholog and found that its knockdown impaired endoderm formation. Genome-wide transcription analysis revealed that nanog-like morphants fail to develop the extraembryonic yolk syncytial layer (YSL), which produces Nodal, required for endoderm induction. We examined the genes that were regulated by Nanog-like and identified the homeobox gene mxtx2, which is both necessary and sufficient for YSL induction. Chromatin immunoprecipitation assays and genetic studies indicated that Nanog-like directly activates mxtx2, which, in turn, specifies the YSL lineage by directly activating YSL genes. Our study identifies a Nanog-like-Mxtx2-Nodal pathway and establishes a role for Nanog-like in regulating the formation of the extraembryonic tissue required for endoderm induction.