RESUMEN
Master transcription factors Oct4, Sox2, and Nanog bind enhancer elements and recruit Mediator to activate much of the gene expression program of pluripotent embryonic stem cells (ESCs). We report here that the ESC master transcription factors form unusual enhancer domains at most genes that control the pluripotent state. These domains, which we call super-enhancers, consist of clusters of enhancers that are densely occupied by the master regulators and Mediator. Super-enhancers differ from typical enhancers in size, transcription factor density and content, ability to activate transcription, and sensitivity to perturbation. Reduced levels of Oct4 or Mediator cause preferential loss of expression of super-enhancer-associated genes relative to other genes, suggesting how changes in gene expression programs might be accomplished during development. In other more differentiated cells, super-enhancers containing cell-type-specific master transcription factors are also found at genes that define cell identity. Super-enhancers thus play key roles in the control of mammalian cell identity.
Asunto(s)
Linaje de la Célula , Células Madre Embrionarias/metabolismo , Elementos de Facilitación Genéticos , Complejo Mediador/metabolismo , Factores de Transcripción/metabolismo , Animales , Linfocitos B/metabolismo , Línea Celular , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Humanos , Ratones , Transcripción GenéticaRESUMEN
Gene expression analysis is a widely used and powerful method for investigating the transcriptional behavior of biological systems, for classifying cell states in disease, and for many other purposes. Recent studies indicate that common assumptions currently embedded in experimental and analytical practices can lead to misinterpretation of global gene expression data. We discuss these assumptions and describe solutions that should minimize erroneous interpretation of gene expression data from multiple analysis platforms.
Asunto(s)
Perfilación de la Expresión Génica/métodos , Estudio de Asociación del Genoma Completo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Proteínas Proto-Oncogénicas c-myc/genética , Análisis de Secuencia de ARN , Transcripción GenéticaRESUMEN
Elevated expression of the c-Myc transcription factor occurs frequently in human cancers and is associated with tumor aggression and poor clinical outcome. The effect of high levels of c-Myc on global gene regulation is poorly understood but is widely thought to involve newly activated or repressed "Myc target genes." We report here that in tumor cells expressing high levels of c-Myc the transcription factor accumulates in the promoter regions of active genes and causes transcriptional amplification, producing increased levels of transcripts within the cell's gene expression program. Thus, rather than binding and regulating a new set of genes, c-Myc amplifies the output of the existing gene expression program. These results provide an explanation for the diverse effects of oncogenic c-Myc on gene expression in different tumor cells and suggest that transcriptional amplification reduces rate-limiting constraints for tumor cell growth and proliferation.
Asunto(s)
Regulación Neoplásica de la Expresión Génica , Neoplasias/genética , Proteínas Proto-Oncogénicas c-myc/metabolismo , Línea Celular Tumoral , Proliferación Celular , Elementos de Facilitación Genéticos , Humanos , Neoplasias/patología , Regiones Promotoras Genéticas , Transcripción GenéticaRESUMEN
MYC contributes to the pathogenesis of a majority of human cancers, yet strategies to modulate the function of the c-Myc oncoprotein do not exist. Toward this objective, we have targeted MYC transcription by interfering with chromatin-dependent signal transduction to RNA polymerase, specifically by inhibiting the acetyl-lysine recognition domains (bromodomains) of putative coactivator proteins implicated in transcriptional initiation and elongation. Using a selective small-molecule bromodomain inhibitor, JQ1, we identify BET bromodomain proteins as regulatory factors for c-Myc. BET inhibition by JQ1 downregulates MYC transcription, followed by genome-wide downregulation of Myc-dependent target genes. In experimental models of multiple myeloma, a Myc-dependent hematologic malignancy, JQ1 produces a potent antiproliferative effect associated with cell-cycle arrest and cellular senescence. Efficacy of JQ1 in three murine models of multiple myeloma establishes the therapeutic rationale for BET bromodomain inhibition in this disease and other malignancies characterized by pathologic activation of c-Myc.
Asunto(s)
Antineoplásicos/farmacología , Descubrimiento de Drogas , Mieloma Múltiple/tratamiento farmacológico , Proteínas Proto-Oncogénicas c-myc/antagonistas & inhibidores , Animales , Antineoplásicos/química , Azepinas/química , Azepinas/farmacología , Benzodiazepinas/química , Benzodiazepinas/farmacología , Línea Celular Tumoral , Modelos Animales de Enfermedad , Humanos , Ratones , Proteínas Nucleares/química , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-myc/genética , Activación Transcripcional/efectos de los fármacos , Triazoles/química , Triazoles/farmacologíaRESUMEN
Recruitment of the RNA polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA-binding transcription factors is well recognized as a key regulatory step in gene expression. We report here that promoter-proximal pausing is a general feature of transcription by Pol II in mammalian cells and thus an additional step where regulation of gene expression occurs. This suggests that some transcription factors recruit the transcription apparatus to promoters, whereas others effect promoter-proximal pause release. Indeed, we find that the transcription factor c-Myc, a key regulator of cellular proliferation, plays a major role in Pol II pause release rather than Pol II recruitment at its target genes. We discuss the implications of these results for the role of c-Myc amplification in human cancer.
Asunto(s)
Proteínas Proto-Oncogénicas c-myc/metabolismo , ARN Polimerasa II/metabolismo , Transcripción Genética , Animales , Proliferación Celular , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/metabolismo , Humanos , Ratones , Proteínas Nucleares/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismo , Factores de Elongación TranscripcionalRESUMEN
We identified three retinoid-related orphan receptor gamma t (RORγt)-specific inhibitors that suppress T helper 17 (Th17) cell responses, including Th17-cell-mediated autoimmune disease. We systemically characterized RORγt binding in the presence and absence of drugs with corresponding whole-genome transcriptome sequencing. RORγt acts as a direct activator of Th17 cell signature genes and a direct repressor of signature genes from other T cell lineages; its strongest transcriptional effects are on cis-regulatory sites containing the RORα binding motif. RORγt is central in a densely interconnected regulatory network that shapes the balance of T cell differentiation. Here, the three inhibitors modulated the RORγt-dependent transcriptional network to varying extents and through distinct mechanisms. Whereas one inhibitor displaced RORγt from its target loci, the other two inhibitors affected transcription predominantly without removing DNA binding. Our work illustrates the power of a system-scale analysis of transcriptional regulation to characterize potential therapeutic compounds that inhibit pathogenic Th17 cells and suppress autoimmunity.
Asunto(s)
Bencenoacetamidas/farmacología , Compuestos de Bencidrilo/farmacología , Digoxina/farmacología , Encefalomielitis Autoinmune Experimental/tratamiento farmacológico , Redes Reguladoras de Genes/efectos de los fármacos , Compuestos Heterocíclicos de 4 o más Anillos/farmacología , Esclerosis Múltiple/tratamiento farmacológico , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/antagonistas & inhibidores , Subgrupos de Linfocitos T/efectos de los fármacos , Células Th17/efectos de los fármacos , Androstenoles/química , Animales , Bencenoacetamidas/química , Compuestos de Bencidrilo/química , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Linaje de la Célula/efectos de los fármacos , Citocinas/metabolismo , Digoxina/química , Encefalomielitis Autoinmune Experimental/inmunología , Compuestos Heterocíclicos de 4 o más Anillos/química , Humanos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Esclerosis Múltiple/inmunología , Glicoproteína Mielina-Oligodendrócito/inmunología , Miembro 3 del Grupo F de la Subfamilia 1 de Receptores Nucleares/genética , Fragmentos de Péptidos/inmunología , Unión Proteica/efectos de los fármacos , Relación Estructura-Actividad , Biología de Sistemas , Subgrupos de Linfocitos T/inmunología , Células Th17/inmunología , Transcripción Genética/efectos de los fármacos , Activación Transcripcional/efectos de los fármacosRESUMEN
Developmental transitions are guided by master regulatory transcription factors. During adipogenesis, a transcriptional cascade culminates in the expression of PPARγ and C/EBPα, which orchestrate activation of the adipocyte gene expression program. However, the coactivators controlling PPARγ and C/EBPα expression are less well characterized. Here, we show the bromodomain-containing protein, BRD4, regulates transcription of PPARγ and C/EBPα. Analysis of BRD4 chromatin occupancy reveals that induction of adipogenesis in 3T3L1 fibroblasts provokes dynamic redistribution of BRD4 to de novo super-enhancers proximal to genes controlling adipocyte differentiation. Inhibition of the bromodomain and extraterminal domain (BET) family of bromodomain-containing proteins impedes BRD4 occupancy at these de novo enhancers and disrupts transcription of Pparg and Cebpa, thereby blocking adipogenesis. Furthermore, silencing of these BRD4-occupied distal regulatory elements at the Pparg locus by CRISPRi demonstrates a critical role for these enhancers in the control of Pparg gene expression and adipogenesis in 3T3L1s. Together, these data establish BET bromodomain proteins as time- and context-dependent coactivators of the adipocyte cell state transition.
Asunto(s)
Adipocitos/fisiología , Tejido Adiposo/citología , Regulación de la Expresión Génica/fisiología , Proteínas Nucleares/metabolismo , Factores de Transcripción/metabolismo , Adipogénesis , Tejido Adiposo/fisiología , Animales , Diferenciación Celular , Masculino , RatonesRESUMEN
MicroRNAs (miRNAs) are critical to proliferation, differentiation, and development. Here, we characterize gene expression in murine Dicer-null adult mesenchymal stem cell lines, a fibroblast cell type. Loss of Dicer leads to derepression of let-7 targets at levels that exceed 10-fold to 100-fold with increases in transcription. Direct and indirect targets of this miRNA belong to a mid-gestation embryonic program that encompasses known oncofetal genes as well as oncogenes not previously associated with an embryonic state. Surprisingly, this mid-gestation program represents a distinct period that occurs between the pluripotent state of the inner cell mass at embryonic day 3.5 (E3.5) and the induction of let-7 upon differentiation at E10.5. Within this mid-gestation program, we characterize the let-7 target Nr6a1, an embryonic transcriptional repressor that regulates gene expression in adult fibroblasts following miRNA loss. In total, let-7 is required for the continual suppression of embryonic gene expression in adult cells, a mechanism that may underlie its tumor-suppressive function.
Asunto(s)
Fibroblastos/citología , Regulación de la Expresión Génica , MicroARNs/genética , MicroARNs/metabolismo , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/genética , Miembro 1 del Grupo A de la Subfamilia 6 de Receptores Nucleares/metabolismo , Animales , Antígenos de Neoplasias/metabolismo , Línea Celular , ARN Helicasas DEAD-box/genética , ARN Helicasas DEAD-box/metabolismo , Fibroblastos/metabolismo , Eliminación de Gen , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Unión Proteica , Ribonucleasa III/genética , Ribonucleasa III/metabolismoRESUMEN
Tumour oncogenes include transcription factors that co-opt the general transcriptional machinery to sustain the oncogenic state, but direct pharmacological inhibition of transcription factors has so far proven difficult. However, the transcriptional machinery contains various enzymatic cofactors that can be targeted for the development of new therapeutic candidates, including cyclin-dependent kinases (CDKs). Here we present the discovery and characterization of a covalent CDK7 inhibitor, THZ1, which has the unprecedented ability to target a remote cysteine residue located outside of the canonical kinase domain, providing an unanticipated means of achieving selectivity for CDK7. Cancer cell-line profiling indicates that a subset of cancer cell lines, including human T-cell acute lymphoblastic leukaemia (T-ALL), have exceptional sensitivity to THZ1. Genome-wide analysis in Jurkat T-ALL cells shows that THZ1 disproportionally affects transcription of RUNX1 and suggests that sensitivity to THZ1 may be due to vulnerability conferred by the RUNX1 super-enhancer and the key role of RUNX1 in the core transcriptional regulatory circuitry of these tumour cells. Pharmacological modulation of CDK7 kinase activity may thus provide an approach to identify and treat tumour types that are dependent on transcription for maintenance of the oncogenic state.
Asunto(s)
Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Fenilendiaminas/farmacología , Leucemia-Linfoma Linfoblástico de Células T Precursoras/enzimología , Pirimidinas/farmacología , Antineoplásicos/farmacología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Quinasas Ciclina-Dependientes/antagonistas & inhibidores , Cisteína/metabolismo , Humanos , Células Jurkat , Fosforilación/efectos de los fármacosRESUMEN
Most human genes are loaded with promoter-proximally paused RNA polymerase II (Pol II) molecules that are poised for release into productive elongation by P-TEFb. We present evidence that Gdown1, the product of the POLR2M gene that renders Pol II responsive to Mediator, is involved in Pol II elongation control. During in vitro transcription, Gdown1 specifically blocked elongation stimulation by TFIIF, inhibited the termination activity of TTF2, and influenced pausing factors NELF and DSIF, but did not affect the function of TFIIS or the mRNA capping enzyme. Without P-TEFb, Gdown1 led to the production of stably paused polymerases in the presence of nuclear extract. Supporting these mechanistic insights, ChIP-Seq demonstrated that Gdown1 mapped over essentially all poised polymerases across the human genome. Our results establish that Gdown1 stabilizes poised polymerases while maintaining their responsiveness to P-TEFb and suggest that Mediator overcomes a Gdown1-mediated block of initiation by allowing TFIIF function.
Asunto(s)
ARN Polimerasa II/fisiología , Células HeLa , Humanos , ARN Polimerasa II/metabolismo , Factores de Transcripción TFII/metabolismo , Transcripción GenéticaRESUMEN
Melanoma is a tumour of transformed melanocytes, which are originally derived from the embryonic neural crest. It is unknown to what extent the programs that regulate neural crest development interact with mutations in the BRAF oncogene, which is the most commonly mutated gene in human melanoma. We have used zebrafish embryos to identify the initiating transcriptional events that occur on activation of human BRAF(V600E) (which encodes an amino acid substitution mutant of BRAF) in the neural crest lineage. Zebrafish embryos that are transgenic for mitfa:BRAF(V600E) and lack p53 (also known as tp53) have a gene signature that is enriched for markers of multipotent neural crest cells, and neural crest progenitors from these embryos fail to terminally differentiate. To determine whether these early transcriptional events are important for melanoma pathogenesis, we performed a chemical genetic screen to identify small-molecule suppressors of the neural crest lineage, which were then tested for their effects on melanoma. One class of compound, inhibitors of dihydroorotate dehydrogenase (DHODH), for example leflunomide, led to an almost complete abrogation of neural crest development in zebrafish and to a reduction in the self-renewal of mammalian neural crest stem cells. Leflunomide exerts these effects by inhibiting the transcriptional elongation of genes that are required for neural crest development and melanoma growth. When used alone or in combination with a specific inhibitor of the BRAF(V600E) oncogene, DHODH inhibition led to a marked decrease in melanoma growth both in vitro and in mouse xenograft studies. Taken together, these studies highlight developmental pathways in neural crest cells that have a direct bearing on melanoma formation.
Asunto(s)
Melanoma/genética , Melanoma/patología , Cresta Neural/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Transcripción Genética , Sustitución de Aminoácidos , Animales , Animales Modificados Genéticamente , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Linaje de la Célula/efectos de los fármacos , Dihidroorotato Deshidrogenasa , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Genes p53/genética , Humanos , Isoxazoles/farmacología , Isoxazoles/uso terapéutico , Leflunamida , Melanoma/tratamiento farmacológico , Melanoma/enzimología , Ratones , Cresta Neural/efectos de los fármacos , Cresta Neural/metabolismo , Cresta Neural/patología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/antagonistas & inhibidores , Proteínas Proto-Oncogénicas B-raf/química , Proteínas Proto-Oncogénicas B-raf/genética , Proteínas Proto-Oncogénicas B-raf/metabolismo , Ratas , Células Madre/citología , Células Madre/efectos de los fármacos , Células Madre/patología , Transcripción Genética/efectos de los fármacos , Transcripción Genética/fisiología , Ensayos Antitumor por Modelo de Xenoinjerto , Pez Cebra/embriología , Pez Cebra/genéticaRESUMEN
Transcription factors that play key roles in regulating embryonic stem (ES) cell state have been identified, but the chromatin regulators that help maintain ES cells are less well understood. A high-throughput shRNA screen was used to identify novel chromatin regulators that influence ES cell state. Loss of histone H3 Lys 9 (H3K9) methyltransferases, particularly SetDB1, had the most profound effects on ES cells. Chromatin immunoprecipitation (ChIP) coupled with massively parallel DNA sequencing (ChIP-Seq) and functional analysis revealed that SetDB1 and histone H3K9-methylated nucleosomes occupy and repress genes encoding developmental regulators. These SetDB1-occupied genes are a subset of the "bivalent" genes, which contain nucleosomes with H3K4me3 (H3K4 trimethylation) and H3K27me3 modifications catalyzed by Trithorax and Polycomb group proteins, respectively. These genes are subjected to repression by both Polycomb group proteins and SetDB1, and loss of either regulator can destabilize ES cell state.
Asunto(s)
Células Madre Embrionarias/citología , Células Madre Embrionarias/enzimología , Regulación del Desarrollo de la Expresión Génica , Metiltransferasas/metabolismo , Proteína Metiltransferasas/metabolismo , Animales , Cromatina/metabolismo , Inmunoprecipitación de Cromatina , N-Metiltransferasa de Histona-Lisina , Ratones , Nucleosomas/enzimologíaRESUMEN
Transcription factors control cell-specific gene expression programs through interactions with diverse coactivators and the transcription apparatus. Gene activation may involve DNA loop formation between enhancer-bound transcription factors and the transcription apparatus at the core promoter, but this process is not well understood. Here we report that mediator and cohesin physically and functionally connect the enhancers and core promoters of active genes in murine embryonic stem cells. Mediator, a transcriptional coactivator, forms a complex with cohesin, which can form rings that connect two DNA segments. The cohesin-loading factor Nipbl is associated with mediator-cohesin complexes, providing a means to load cohesin at promoters. DNA looping is observed between the enhancers and promoters occupied by mediator and cohesin. Mediator and cohesin co-occupy different promoters in different cells, thus generating cell-type-specific DNA loops linked to the gene expression program of each cell.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ensamble y Desensamble de Cromatina/genética , Cromatina/genética , Cromatina/metabolismo , Proteínas Cromosómicas no Histona/metabolismo , Células Madre Embrionarias/metabolismo , Regulación de la Expresión Génica/genética , Complejo Mediador/metabolismo , Animales , Proteínas de Ciclo Celular/genética , Células Cultivadas , Cromatina/química , Proteínas Cromosómicas no Histona/genética , ADN/química , ADN/genética , ADN/metabolismo , Células Madre Embrionarias/citología , Elementos de Facilitación Genéticos/genética , Fibroblastos , Complejo Mediador/genética , Ratones , Conformación de Ácido Nucleico , Especificidad de Órganos , Regiones Promotoras Genéticas/genética , Unión Proteica , CohesinasRESUMEN
Aire is a transcriptional regulator that induces expression of peripheral tissue antigens (PTA) in thymic medullary epithelial cells (MECs), driving immunological self-tolerance in differentiating T cells. To elucidate its mechanistic pathways, we examined its transcriptional impact in MECs in vivo by microarray analysis with mRNA-spanning probes. This analysis revealed initiation of Aire-activated genes to be comparable in Aire-deficient and wild-type MECs, but with a block to elongation after 50-100 bp in the absence of Aire, suggesting activation by release of stalled polymerases by Aire. In contrast, patterns of activation by transcription factors such as Klf4 were consistent with regulation of initiation. Mapping of Aire and RNA polymerase-II (Pol-II) by ChIP and high-throughput sequencing (ChIP-seq) revealed that Aire bound all Pol-II-rich transcriptional start sites (TSS), irrespective of its eventual effect. However, the genes it preferentially activated were characterized by a relative surfeit of stalled polymerases at the TSS, which resolved once Aire was introduced into cells. Thus, transcript mapping and ChIP-seq data indicate that Aire activates ectopic transcription not through specific recognition of PTA gene promoters but by releasing stalled polymerases.
Asunto(s)
ARN Polimerasas Dirigidas por ADN/metabolismo , Células Epiteliales/metabolismo , Regulación de la Expresión Génica/inmunología , Autotolerancia/inmunología , Linfocitos T/inmunología , Timo/citología , Factores de Transcripción/metabolismo , Animales , Línea Celular , Inmunoprecipitación de Cromatina , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Factor 4 Similar a Kruppel , Ratones , Ratones Endogámicos C57BL , Análisis por Micromatrices , Linfocitos T/citología , Proteína AIRERESUMEN
A striking characteristic of a Rab protein is its steady-state localization to the cytosolic surface of a particular subcellular membrane. In this study, we have undertaken a combined bioinformatic and experimental approach to examine the evolutionary conservation of Rab protein localization. A comprehensive primary sequence classification shows that 10 out of the 11 Rab proteins identified in the yeast (Saccharomyces cerevisiae) genome can be grouped within a major subclass, each comprising multiple Rab orthologs from diverse species. We compared the locations of individual yeast Rab proteins with their localizations following ectopic expression in mammalian cells. Our results suggest that green fluorescent protein-tagged Rab proteins maintain localizations across large evolutionary distances and that the major known player in the Rab localization pathway, mammalian Rab-GDI, is able to function in yeast. These findings enable us to provide insight into novel gene functions and classify the uncharacterized Rab proteins Ypt10p (YBR264C) as being involved in endocytic function and Ypt11p (YNL304W) as being localized to the endoplasmic reticulum, where we demonstrate it is required for organelle inheritance.
Asunto(s)
Biología Computacional , Proteínas de Unión al GTP/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Unión al GTP rab/metabolismo , Animales , Células Cultivadas , Retículo Endoplásmico/metabolismo , Fluorescencia , Proteínas Fluorescentes Verdes/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Células HeLa , Humanos , Análisis de Componente Principal , Transporte de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Saccharomyces cerevisiae/citologíaRESUMEN
BACKGROUND: The appendage domain of the gammaCOP subunit of the COPI vesicle coat bears a striking structural resemblance to adaptin-family appendages despite limited primary sequence homology. Both the gammaCOP appendage domain and an equivalent region on betaCOP contain the FxxxW motif; the conservation of this motif suggested the existence of a functional appendage domain in betaCOP. RESULTS: Sequence comparisons in combination with structural prediction tools show that the fold of the COOH-terminus of Sec26p is strongly predicted to closely mimic that of adaptin-family appendages. Deletion of the appendage domain of Sec26p results in inviability in yeast, over-expression of the deletion construct is dominant negative and mutagenesis of this region identifies residues critical for function. The ArfGAP Glo3p was identified via suppression screening as a potential downstream modulator of Sec26p in a manner that is independent of the GAP activity of Glo3p but requires the presence of the COOH-terminal ISS motifs. CONCLUSION: Together, these results indicate an essential function for the predicted betaCOP appendage and suggest that both COPI appendages perform a biologically active regulatory role with a structure related to adaptin-family appendage domains.
Asunto(s)
Vesículas Cubiertas por Proteínas de Revestimiento/genética , Vesículas Cubiertas por Proteínas de Revestimiento/metabolismo , Proteína Coatómero/química , Proteína Coatómero/genética , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Subunidades alfa de Complejo de Proteína Adaptadora/genética , Subunidades alfa de Complejo de Proteína Adaptadora/metabolismo , Secuencias de Aminoácidos/genética , Secuencia de Bases/genética , Vesículas Cubiertas por Proteínas de Revestimiento/ultraestructura , Retículo Endoplásmico Rugoso/metabolismo , Proteínas Activadoras de GTPasa/genética , Proteínas Activadoras de GTPasa/metabolismo , Regulación Fúngica de la Expresión Génica/genética , Aparato de Golgi/metabolismo , Mutagénesis Sitio-Dirigida , Mutación/genética , Estructura Terciaria de Proteína/genética , Transporte de Proteínas/fisiología , Proteínas de Saccharomyces cerevisiae/metabolismoRESUMEN
The regulation of membrane trafficking events in the secretory and endocytic pathways by Rab GTPases requires the cycling and activation of a Rab protein. The cycle of nucleotide binding and hydrolysis of Rab proteins is accompanied by a physical cycle of membrane translocation. An open question in membrane traffic remains how the cycle of Rab GTPase function is coupled to regulatory inputs from other cellular processes. This chapter describes the principles and methodologies used to identify the physiological regulators that influence Rab-mediated membrane traffic.
Asunto(s)
Exocitosis/fisiología , Histona Acetiltransferasas/fisiología , Factores de Elongación de Péptidos/fisiología , Proteínas de Saccharomyces cerevisiae/fisiología , Animales , Formación de Anticuerpos , Pollos/inmunología , Yema de Huevo/inmunología , Histona Acetiltransferasas/inmunología , Factores de Elongación de Péptidos/inmunología , Proteínas de Saccharomyces cerevisiae/inmunología , Proteínas de Unión al GTP rab/antagonistas & inhibidores , Proteínas de Unión al GTP rab/fisiologíaRESUMEN
Most transcription factors specify the subset of genes that will be actively transcribed in the cell by stimulating transcription initiation at these genes, but MYC has a fundamentally different role. MYC binds E-box sites in the promoters of active genes and stimulates recruitment of the elongation factor P-TEFb and thus transcription elongation. Consequently, rather than specifying the set of genes that will be transcribed in any particular cell, MYC's predominant role is to increase the production of transcripts from active genes. This increase in the transcriptional output of the cell's existing gene expression program, called transcriptional amplification, has a profound effect on proliferation and other behaviors of a broad range of cells. Transcriptional amplification may reduce rate-limiting constraints for tumor cell proliferation and explain MYC's broad oncogenic activity among diverse tissues.
Asunto(s)
Genes myc/fisiología , Proteínas Proto-Oncogénicas c-myc/metabolismo , Elongación de la Transcripción Genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/genética , Factores de Transcripción Básicos con Cremalleras de Leucinas y Motivos Hélice-Asa-Hélice/metabolismo , Amplificación de Genes , Regulación Neoplásica de la Expresión Génica , Humanos , Regiones Promotoras Genéticas , Proteínas Proto-Oncogénicas c-myc/genética , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismoRESUMEN
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
Diffuse large B cell lymphoma (DLBCL) is a biologically heterogeneous and clinically aggressive disease. Here, we explore the role of bromodomain and extra-terminal domain (BET) proteins in DLBCL, using integrative chemical genetics and functional epigenomics. We observe highly asymmetric loading of bromodomain 4 (BRD4) at enhancers, with approximately 33% of all BRD4 localizing to enhancers at 1.6% of occupied genes. These super-enhancers prove particularly sensitive to bromodomain inhibition, explaining the selective effect of BET inhibitors on oncogenic and lineage-specific transcriptional circuits. Functional study of genes marked by super-enhancers identifies DLBCLs dependent on OCA-B and suggests a strategy for discovering unrecognized cancer dependencies. Translational studies performed on a comprehensive panel of DLBCLs establish a therapeutic rationale for evaluating BET inhibitors in this disease.