RESUMEN
Aspergillus fumigatus is a filamentous fungus that can infect the lungs of patients with immunosuppression and/or underlying lung diseases. The mortality associated with chronic and invasive aspergillosis infections remain very high, despite availability of antifungal treatments. In the last decade, there has been a worrisome emergence and spread of resistance to the first-line antifungals, the azoles. The mortality caused by resistant isolates is even higher, and patient management is complicated as the therapeutic options are reduced. Nevertheless, treatment failure is also common in patients infected with azole-susceptible isolates, which can be due to several non-mutually exclusive reasons, such as poor drug absorption. In addition, the phenomena of tolerance or persistence, where susceptible pathogens can survive the action of an antimicrobial for extended periods, have been associated with treatment failure in bacterial infections, and their occurrence in fungal infections already proposed. Here, we demonstrate that some isolates of A. fumigatus display persistence to voriconazole. A subpopulation of the persister isolates can survive for extended periods and even grow at low rates in the presence of supra-MIC of voriconazole and seemingly other azoles. Persistence cannot be eradicated with adjuvant drugs or antifungal combinations and seemed to reduce the efficacy of treatment for certain individuals in a Galleria mellonella model of infection. Furthermore, persistence implies a distinct transcriptional profile, demonstrating that it is an active response. We propose that azole persistence might be a relevant and underestimated factor that could influence the outcome of infection in human aspergillosis. IMPORTANCE The phenomena of antibacterial tolerance and persistence, where pathogenic microbes can survive for extended periods in the presence of cidal drug concentrations, have received significant attention in the last decade. Several mechanisms of action have been elucidated, and their relevance for treatment failure in bacterial infections demonstrated. In contrast, our knowledge of antifungal tolerance and, in particular, persistence is still very limited. In this study, we have characterized the response of the prominent fungal pathogen Aspergillus fumigatus to the first-line therapy antifungal voriconazole. We comprehensively show that some isolates display persistence to this fungicidal antifungal and propose various potential mechanisms of action. In addition, using an alternative model of infection, we provide initial evidence to suggest that persistence may cause treatment failure in some individuals. Therefore, we propose that azole persistence is an important factor to consider and further investigate in A. fumigatus.
RESUMEN
Glucocorticoids (Gcs) potently inhibit inflammation, and regulate liver energy metabolism, often acting in a hypoxic environment. We now show hypoxic conditions open a specific GR cistrome, and prevent access of GR to part of the normoxic GR cistrome. Motif analysis identified enrichment of KLF4 binding sites beneath those peaks of GR binding exclusive to normoxia, implicating KLF4 as a pioneer, or co-factor under these conditions. Hypoxia reduced KLF4 expression, however, knockdown of KLF4 did not impair GR recruitment. KLF4 is a known target of microRNAs 103 and 107, both of which are induced by hypoxia. Expression of mimics to either microRNA103, or microRNA107 inhibited GR transactivation of normoxic target genes, thereby replicating the hypoxic effect. Therefore, studies in hypoxia reveal that microRNAs 103 and 107 are potent regulators of GR function. We have now identified a new pathway linking hypoxia through microRNAs 103 and 107 to regulation of GR function.
Asunto(s)
Hipoxia de la Célula/fisiología , MicroARNs/fisiología , Receptores de Glucocorticoides/fisiología , Dexametasona/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Glucocorticoides/farmacología , Células HEK293 , Células HeLa , Humanos , Hipoxia/genética , Hipoxia/metabolismo , Hipoxia/patología , Factor 4 Similar a Kruppel , MicroARNs/genética , Transducción de Señal/efectos de los fármacos , Transducción de Señal/genéticaRESUMEN
A subset of lung adenocarcinomas is driven by the EML4-ALK translocation. Even though ALK inhibitors in the clinic lead to excellent initial responses, acquired resistance to these inhibitors due to on-target mutations or parallel pathway alterations is a major clinical challenge. Exploring these mechanisms of resistance, we found that EML4-ALK cells parental or resistant to crizotinib, ceritinib or alectinib are remarkably sensitive to inhibition of CDK7/12 with THZ1 and CDK9 with alvocidib or dinaciclib. These compounds robustly induce apoptosis through transcriptional inhibition and downregulation of anti-apoptotic genes. Importantly, alvocidib reduced tumour progression in xenograft mouse models. In summary, our study takes advantage of the transcriptional addiction hypothesis to propose a new treatment strategy for a subset of patients with acquired resistance to first-, second- and third-generation ALK inhibitors.
Asunto(s)
Resistencia a Antineoplásicos/efectos de los fármacos , Resistencia a Antineoplásicos/genética , Neoplasias Pulmonares/tratamiento farmacológico , Neoplasias Pulmonares/genética , Proteínas de Fusión Oncogénica/genética , Inhibidores de Proteínas Quinasas/farmacología , Transcripción Genética/efectos de los fármacos , Animales , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Carcinoma de Pulmón de Células no Pequeñas/genética , Femenino , Humanos , Ratones , Proteínas de Fusión Oncogénica/antagonistas & inhibidores , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
High-grade serous ovarian carcinoma is characterised by TP53 mutation and extensive chromosome instability (CIN). Because our understanding of CIN mechanisms is based largely on analysing established cell lines, we developed a workflow for generating ex vivo cultures from patient biopsies to provide models that support interrogation of CIN mechanisms in cells not extensively cultured in vitro. Here, we describe a "living biobank" of ovarian cancer models with extensive replicative capacity, derived from both ascites and solid biopsies. Fifteen models are characterised by p53 profiling, exome sequencing and transcriptomics, and karyotyped using single-cell whole-genome sequencing. Time-lapse microscopy reveals catastrophic and highly heterogeneous mitoses, suggesting that analysis of established cell lines probably underestimates mitotic dysfunction in advanced human cancers. Drug profiling reveals cisplatin sensitivities consistent with patient responses, demonstrating that this workflow has potential to generate personalized avatars with advantages over current pre-clinical models and the potential to guide clinical decision making.
Asunto(s)
Bancos de Muestras Biológicas , Mitosis/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Inestabilidad Cromosómica , Resistencia a Antineoplásicos , Femenino , Expresión Génica , Perfilación de la Expresión Génica , Técnicas Histológicas/métodos , Humanos , Imagenología Tridimensional , Hibridación Fluorescente in Situ , Técnicas In Vitro , Cariotipificación , Modelos Biológicos , Mutación , Neoplasias Ováricas/tratamiento farmacológico , Paclitaxel/farmacología , Análisis de la Célula Individual , Imagen de Lapso de Tiempo , Proteína p53 Supresora de Tumor/genética , Secuenciación del ExomaRESUMEN
Animal embryogenesis is initiated by maternal factors, but zygotic genome activation (ZGA) shifts regulatory control to the embryo during blastula stages. ZGA is thought to be mediated by maternally provided transcription factors (TFs), but few such TFs have been identified in vertebrates. Here we report that NF-Y and TALE TFs bind zebrafish genomic elements associated with developmental control genes already at ZGA. In particular, co-regulation by NF-Y and TALE is associated with broadly acting genes involved in transcriptional control, while regulation by either NF-Y or TALE defines genes in specific developmental processes, such that NF-Y controls a cilia gene expression program while TALE controls expression of hox genes. We also demonstrate that NF-Y and TALE-occupied genomic elements function as enhancers during embryogenesis. We conclude that combinatorial use of NF-Y and TALE at developmental enhancers permits the establishment of distinct gene expression programs at zebrafish ZGA.
Asunto(s)
Factor de Unión a CCAAT/metabolismo , Expresión Génica , Genoma , Proteínas de Homeodominio/metabolismo , Activación Transcripcional , Pez Cebra/embriología , Cigoto/metabolismo , Animales , Cilios/genética , Desarrollo Embrionario/genética , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/genética , Masculino , Proteínas de Pez CebraRESUMEN
The glucocorticoid receptor (GR) is a major drug target in inflammatory disease. However, chronic glucocorticoid (GC) treatment leads to disordered energy metabolism, including increased weight gain, adiposity, and hepatosteatosis - all programs modulated by the circadian clock. We demonstrated that while antiinflammatory GC actions were maintained irrespective of dosing time, the liver was significantly more GC sensitive during the day. Temporal segregation of GC action was underpinned by a physical interaction of GR with the circadian transcription factor REVERBa and co-binding with liver-specific hepatocyte nuclear transcription factors (HNFs) on chromatin. REVERBa promoted efficient GR recruitment to chromatin during the day, acting in part by maintaining histone acetylation, with REVERBa-dependent GC responses providing segregation of carbohydrate and lipid metabolism. Importantly, deletion of Reverba inverted circadian liver GC sensitivity and protected mice from hepatosteatosis induced by chronic GC administration. Our results reveal a mechanism by which the circadian clock acts through REVERBa in liver on elements bound by HNF4A/HNF6 to direct GR action on energy metabolism.
Asunto(s)
Cromatina/metabolismo , Relojes Circadianos/efectos de los fármacos , Hígado Graso/metabolismo , Glucocorticoides/efectos adversos , Hígado/metabolismo , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/metabolismo , Animales , Cromatina/genética , Cromatina/patología , Relojes Circadianos/genética , Metabolismo Energético/efectos de los fármacos , Metabolismo Energético/genética , Hígado Graso/inducido químicamente , Hígado Graso/genética , Hígado Graso/patología , Glucocorticoides/farmacología , Células HEK293 , Humanos , Hígado/patología , Ratones , Ratones Noqueados , Miembro 1 del Grupo D de la Subfamilia 1 de Receptores Nucleares/genética , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismoRESUMEN
TALE factors are broadly expressed embryonically and known to function in complexes with transcription factors (TFs) like Hox proteins at gastrula/segmentation stages, but it is unclear if such generally expressed factors act by the same mechanism throughout embryogenesis. We identify a TALE-dependent gene regulatory network (GRN) required for anterior development and detect TALE occupancy associated with this GRN throughout embryogenesis. At blastula stages, we uncover a novel functional mode for TALE factors, where they occupy genomic DECA motifs with nearby NF-Y sites. We demonstrate that TALE and NF-Y form complexes and regulate chromatin state at genes of this GRN. At segmentation stages, GRN-associated TALE occupancy expands to include HEXA motifs near PBX:HOX sites. Hence, TALE factors control a key GRN, but utilize distinct DNA motifs and protein partners at different stages - a strategy that may also explain their oncogenic potential and may be employed by other broadly expressed TFs.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica , Genes Esenciales/genética , Proteínas de Homeodominio/genética , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Animales , Blástula/embriología , Blástula/metabolismo , Factor de Unión a CCAAT/genética , Factor de Unión a CCAAT/metabolismo , Técnicas de Silenciamiento del Gen , Redes Reguladoras de Genes , Proteínas de Homeodominio/metabolismo , Unión Proteica , Pez Cebra/embriología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/metabolismoRESUMEN
Nephrotic syndrome (NS) occurs when the glomerular filtration barrier becomes excessively permeable leading to massive proteinuria. In childhood NS, immune system dysregulation has been implicated and increasing evidence points to the central role of podocytes in the pathogenesis. Children with NS are typically treated with an empiric course of glucocorticoid (Gc) therapy; a class of steroids that are activating ligands for the glucocorticoid receptor (GR) transcription factor. Although Gc-therapy has been the cornerstone of NS management for decades, the mechanism of action, and target cell, remain poorly understood. We tested the hypothesis that Gc acts directly on the podocyte to produce clinically useful effects without involvement of the immune system. In human podocytes, we demonstrated that the basic GR-signalling mechanism is intact and that Gc induced an increase in podocyte barrier function. Defining the GR-cistrome identified Gc regulation of motility genes. These findings were functionally validated with live-cell imaging. We demonstrated that treatment with Gc reduced the activity of the pro-migratory small GTPase regulator Rac1. Furthermore, Rac1 inhibition had a direct, protective effect on podocyte barrier function. Our studies reveal a new mechanism for Gc action directly on the podocyte, with translational relevance to designing new selective synthetic Gc molecules.
Asunto(s)
Glucocorticoides/farmacología , Podocitos/efectos de los fármacos , Prednisolona/farmacología , Sustancias Protectoras/farmacología , Puromicina Aminonucleósido/antagonistas & inhibidores , Receptores de Glucocorticoides/genética , Proteína de Unión al GTP rac1/genética , Antimetabolitos Antineoplásicos/toxicidad , Transporte Biológico/efectos de los fármacos , Línea Celular Transformada , Membrana Celular/efectos de los fármacos , Movimiento Celular/efectos de los fármacos , Impedancia Eléctrica , Perfilación de la Expresión Génica , Regulación de la Expresión Génica , Humanos , Análisis por Micromatrices , Podocitos/citología , Podocitos/metabolismo , Puromicina Aminonucleósido/toxicidad , Receptores de Glucocorticoides/antagonistas & inhibidores , Receptores de Glucocorticoides/metabolismo , Transducción de Señal , Transcriptoma , Proteína de Unión al GTP rac1/antagonistas & inhibidores , Proteína de Unión al GTP rac1/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
Cleft palate is a common congenital disorder that affects up to 1 in 2500 live births and results in considerable morbidity to affected individuals and their families. The aetiology of cleft palate is complex with both genetic and environmental factors implicated. Mutations in the transcription factor p63 are one of the major individual causes of cleft palate; however, the gene regulatory networks in which p63 functions remain only partially characterized. Our findings demonstrate that p63 functions as an essential regulatory molecule in the spatio-temporal control of palatal epithelial cell fate to ensure appropriate fusion of the palatal shelves. Initially, p63 induces periderm formation and controls its subsequent maintenance to prevent premature adhesion between adhesion-competent, intra-oral epithelia. Subsequently, TGFß3-induced down-regulation of p63 in the medial edge epithelia of the palatal shelves is a pre-requisite for palatal fusion by facilitating periderm migration from, and reducing the proliferative potential of, the midline epithelial seam thereby preventing cleft palate.
Asunto(s)
Fisura del Paladar/genética , Redes Reguladoras de Genes/genética , Fosfoproteínas/genética , Transactivadores/genética , Factor de Crecimiento Transformador beta3/genética , Animales , Movimiento Celular/genética , Proliferación Celular/genética , Fisura del Paladar/fisiopatología , Modelos Animales de Enfermedad , Células Epiteliales/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Mutación , Fosfoproteínas/biosíntesis , Transducción de Señal/genética , Transactivadores/biosíntesisRESUMEN
The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/biosíntesis , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero , Desarrollo Embrionario/genética , Factor de Crecimiento Epidérmico/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Nucleares , Transducción de Señal/genéticaRESUMEN
In protein synthesis translation factor eIF2 binds initiator tRNA to ribosomes and facilitates start codon selection. eIF2 GDP/GTP status is regulated by eIF5 (GAP and GDI functions) and eIF2B (GEF and GDF activities), while eIF2α phosphorylation in response to diverse signals is a major point of translational control. Here we characterize a growth suppressor mutation in eIF2ß that prevents eIF5 GDI and alters cellular responses to reduced eIF2B activity, including control of GCN4 translation. By monitoring the binding of fluorescent nucleotides and initiator tRNA to purified eIF2 we show that the eIF2ß mutation does not affect intrinsic eIF2 affinities for these ligands, neither does it interfere with eIF2 binding to 43S pre-initiation complex components. Instead we show that the eIF2ß mutation prevents eIF5 GDI stabilizing nucleotide binding to eIF2, thereby altering the off-rate of GDP from eIF2â¢GDP/eIF5 complexes. This enables cells to grow with reduced eIF2B GEF activity but impairs activation of GCN4 targets in response to amino acid starvation. These findings provide support for the importance of eIF5 GDI activity in vivo and demonstrate that eIF2ß acts in concert with eIF5 to prevent premature release of GDP from eIF2γ and thereby ensure tight control of protein synthesis initiation.
Asunto(s)
Factor 2B Eucariótico de Iniciación/metabolismo , Inhibidores de Disociación de Guanina Nucleótido/metabolismo , Biosíntesis de Proteínas , Proteínas Represoras/metabolismo , Secuencia de Aminoácidos , Secuencia Conservada , Factor 2B Eucariótico de Iniciación/química , Factor 2B Eucariótico de Iniciación/genética , Evolución Molecular , Inhibidores de Disociación de Guanina Nucleótido/química , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Modelos Moleculares , Mutación , Unión Proteica , Conformación Proteica , ARN de Transferencia/genética , ARN de Transferencia/metabolismo , Proteínas Represoras/química , Levaduras/efectos de los fármacos , Levaduras/genética , Levaduras/metabolismoRESUMEN
Microsatellites are useful tools for ecologists and conservationist biologists, but are taxa-specific and traditionally expensive and time-consuming to develop. New methods using next-generation sequencing (NGS) have reduced these problems, but the plethora of software available for processing NGS data may cause confusion and difficulty for researchers new to the field of bioinformatics. We developed a bioinformatics pipeline for microsatellite development from Illumina paired-end sequences, which is packaged in the open-source bioinformatics tool Galaxy. This optimises and streamlines the design of a microsatellite panel and provides a user-friendly graphical user interface. The pipeline utilises existing programs along with our own novel program and wrappers to: quality-filter and trim reads (Trimmomatic); generate sequence quality reports (FastQC); identify potentially-amplifiable microsatellite loci (Pal_finder); design primers (Primer3); assemble pairs of reads to enhance marker amplification success rates (PANDAseq); and filter optimal loci (Pal_filter). The complete pipeline is freely available for use via a pre-configured Galaxy instance, accessible at https://palfinder.ls.manchester.ac.uk.
RESUMEN
Hox transcription factors (TFs) are essential for vertebrate development, but how these evolutionary conserved proteins function in vivo remains unclear. Because Hox proteins have notoriously low binding specificity, they are believed to bind with cofactors, mainly homeodomain TFs Pbx and Meis, to select their specific targets. We mapped binding of Meis, Pbx, and Hoxa2 in the branchial arches, a series of segments in the developing vertebrate head. Meis occupancy is largely similar in Hox-positive and -negative arches. Hoxa2, which specifies second arch (IIBA) identity, recognizes a subset of Meis prebound sites that contain Hox motifs. Importantly, at these sites Meis binding is strongly increased. This enhanced Meis binding coincides with active enhancers, which are linked to genes highly expressed in the IIBA and regulated by Hoxa2. These findings show that Hoxa2 operates as a tissue-specific cofactor, enhancing Meis binding to specific sites that provide the IIBA with its anatomical identity.
Asunto(s)
Región Branquial/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/metabolismo , Animales , Línea Celular , Ratones , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Gross chromosomal rearrangements have the potential to be evolutionarily advantageous to an adapting organism. The generation of a hybrid species increases opportunity for recombination by bringing together two homologous genomes. We sought to define the location of genomic rearrangements in three strains of Saccharomyces pastorianus, a natural lager-brewing yeast hybrid of Saccharomyces cerevisiae and Saccharomyces eubayanus, using whole genome shotgun sequencing. Each strain of S. pastorianus has lost species-specific portions of its genome and has undergone extensive recombination, producing chimeric chromosomes. We predicted 30 breakpoints that we confirmed at the single nucleotide level by designing species-specific primers that flank each breakpoint, and then sequencing the PCR product. These rearrangements are the result of recombination between areas of homology between the two subgenomes, rather than repetitive elements such as transposons or tRNAs. Interestingly, 28/30 S. cerevisiae-S. eubayanus recombination breakpoints are located within genic regions, generating chimeric genes. Furthermore we show evidence for the reuse of two breakpoints, located in HSP82 and KEM1, in strains of proposed independent origin.
Asunto(s)
Quimerismo , Cromosomas Fúngicos , Regulación Fúngica de la Expresión Génica , Genoma Fúngico , Saccharomyces cerevisiae/genética , Saccharomyces/genética , Secuencia de Bases , Puntos de Rotura del Cromosoma , Mapeo Cromosómico , ADN de Hongos/genética , Exorribonucleasas/genética , Proteínas HSP90 de Choque Térmico/genética , Recombinación Homóloga , Hibridación Genética , Datos de Secuencia Molecular , Proteínas de Saccharomyces cerevisiae/genética , Análisis de Secuencia de ADNRESUMEN
The regulation of gene expression is central to developmental programs and largely depends on the binding of sequence-specific transcription factors with cis-regulatory elements in the genome. Hox transcription factors specify the spatial coordinates of the body axis in all animals with bilateral symmetry, but a detailed knowledge of their molecular function in instructing cell fates is lacking. Here, we used chromatin immunoprecipitation with massively parallel sequencing (ChIP-seq) to identify Hoxa2 genomic locations in a time and space when it is actively instructing embryonic development in mouse. Our data reveals that Hoxa2 has large genome coverage and potentially regulates thousands of genes. Sequence analysis of Hoxa2-bound regions identifies high occurrence of two main classes of motifs, corresponding to Hox and Pbx-Hox recognition sequences. Examination of the binding targets of Hoxa2 faithfully captures the processes regulated by Hoxa2 during embryonic development; in addition, it uncovers a large cluster of potential targets involved in the Wnt-signaling pathway. In vivo examination of canonical Wnt-ß-catenin signaling reveals activity specifically in Hoxa2 domain of expression, and this is undetectable in Hoxa2 mutant embryos. The comprehensive mapping of Hoxa2-binding sites provides a framework to study Hox regulatory networks in vertebrate developmental processes.
Asunto(s)
Desarrollo Embrionario/genética , Proteínas de Homeodominio/metabolismo , Vía de Señalización Wnt/genética , Animales , Sitios de Unión , Región Branquial/metabolismo , Inmunoprecipitación de Cromatina , Genoma , Secuenciación de Nucleótidos de Alto Rendimiento , Proteínas de Homeodominio/genética , Ratones , Análisis de Secuencia de ADN , beta Catenina/metabolismoRESUMEN
The transcriptional control circuitry in eukaryotic cells is complex and is orchestrated by combinatorially acting transcription factors. Forkhead transcription factors often function in concert with heterotypic transcription factors to specify distinct transcriptional programs. Here, we demonstrate that FOXK2 participates in combinatorial transcriptional control with the AP-1 transcription factor. FOXK2 binding regions are widespread throughout the genome and are often coassociated with AP-1 binding motifs. FOXK2 acts to promote AP-1-dependent gene expression changes in response to activation of the AP-1 pathway. In this context, FOXK2 is required for the efficient recruitment of AP-1 to chromatin. Thus, we have uncovered an important new molecular mechanism that controls AP-1-dependent gene expression.
Asunto(s)
Factores de Transcripción Forkhead/metabolismo , Factor de Transcripción AP-1/genética , Activación Transcripcional , Secuencia de Bases , Sitios de Unión , Línea Celular , Cromatina/genética , Cromatina/metabolismo , Factores de Transcripción Forkhead/genética , Genoma Humano , Humanos , Unión Proteica , ARN Interferente Pequeño/genética , Factor de Transcripción AP-1/metabolismoRESUMEN
ETS-domain transcription factors play important roles in controlling gene expression in a variety of different contexts; however, these proteins bind to very similar sites and it is unclear how in vivo specificity is achieved. In silico analysis is unlikely to reveal specific targets for individual family members and direct experimental approaches are therefore required. Here, we take advantage of an inducible dominant-negative expression system to identify a group of novel target genes for the ETS-domain transcription factor Elk-1. Elk-1 is thought to mainly function through cooperation with a second transcription factor SRF, but the targets we identify are largely SRF-independent. Furthermore, we demonstrate that there is a high degree of overlapping, cell type-specific, target gene binding by Elk-1 and other ETS-domain transcription factors. Our results are therefore consistent with the notion that there is a high degree of functional redundancy in target gene regulation by ETS-domain transcription factors in addition to the specific target gene regulation that can be dictated through heterotypic interactions exemplified by the Elk-1-SRF complex.
Asunto(s)
Regiones Promotoras Genéticas , Proteína Elk-1 con Dominio ets/metabolismo , Sitios de Unión , Línea Celular , Regulación de la Expresión Génica , Humanos , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas c-ets/química , Proteínas Proto-Oncogénicas c-ets/metabolismo , Factor de Respuesta Sérica/metabolismo , Proteína Elk-1 con Dominio ets/químicaRESUMEN
Transcription factors play an important role in orchestrating the activation of specific networks of genes through targeting their proximal promoter and distal enhancer regions. However, it is unclear how the specificity of downstream responses is maintained by individual members of transcription-factor families and, in most cases, what their target repertoire is. We have used ChIP-chip analysis to identify the target genes of the ETS-domain transcription factor ELK1. Two distinct modes of ELK1 target gene selection are identified; the first involves redundant promoter binding with other ETS-domain family members; the second occurs through combinatorial binding with a second transcription factor SRF, which specifies a unique group of target genes. One of the most prominent groups of genes forming the ELK1 target network includes classes involved in core gene expression control, namely, components of the basal transcriptional machinery, the spliceosome and the ribosome. Amongst the set of genes encoding the basal transcription machinery components, are a functionally linked subset of GTFs and TAFs. Our study, therefore, reveals an unsuspected level of coordinate regulation of components of the core gene expression control machinery and also identifies two different modes of promoter targeting through binding with a second transcription factor or redundant binding with other ETS-domain family members.
Asunto(s)
Regulación de la Expresión Génica , Redes Reguladoras de Genes , Proteína Elk-1 con Dominio ets/genética , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Línea Celular , Inmunoprecipitación de Cromatina , Factor de Transcripción de la Proteína de Unión a GA/genética , Factor de Transcripción de la Proteína de Unión a GA/metabolismo , Células HeLa , Humanos , Células Jurkat , Luciferasas/genética , Luciferasas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Interferencia de ARN , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factor de Respuesta Sérica/genética , Factor de Respuesta Sérica/metabolismo , Transfección , Proteína Elk-1 con Dominio ets/metabolismoRESUMEN
The discovery of JAK2V617F as an acquired mutation in the majority of patients with myeloproliferative disorders (MPDs) and the key role of the JAK2-STAT5 signaling cascade in normal hematopoiesis has focused attention on the downstream transcriptional targets of STAT5. Despite evidence of its vital role in normal erythropoiesis and its ability to recapitulate many of the features of myeloid malignancies, including the MPDs, few functionally validated targets of STAT5 have been described. Here we used a combination of comparative genomics and chromatin immunoprecipitation assays to identify ID1 as a novel target of the JAK2-STAT5 signaling axis in erythroid cells. STAT5 binds and transactivates a downstream enhancer of ID1, and ID1 expression levels correlate with the JAK2V617F mutation in both retrovirally transfected fetal liver cells and polycythemia vera patients. Knockdown and overexpression studies in a well-characterized erythroid differentiation assay from primary murine fetal liver cells demonstrated a survival-promoting action of ID1. This hitherto unrecognized function implicates ID1 in the expansion of erythroblasts during terminal differentiation and suggests that ID1 plays an important role in the pathogenesis of polycythemia vera. Furthermore, our findings contribute to an increasing body of evidence implicating ID proteins in a wider range of cellular functions than initially appreciated.
Asunto(s)
Células Eritroides/citología , Proteína 1 Inhibidora de la Diferenciación/metabolismo , Janus Quinasa 2/metabolismo , Factor de Transcripción STAT5/metabolismo , Animales , Separación Celular , Citometría de Flujo , Humanos , Janus Quinasa 2/genética , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos CBA , Retroviridae/genética , Transducción de SeñalRESUMEN
The basic helix-loop-helix transcription factor Scl/Tal1 controls the development and subsequent differentiation of hematopoietic stem cells (HSCs). However, because few Scl target genes have been validated to date, the underlying mechanisms have remained largely unknown. In this study, we have used ChIP-Seq technology (coupling chromatin immunoprecipitation with deep sequencing) to generate a genome-wide catalog of Scl-binding events in a stem/progenitor cell line, followed by validation using primary fetal liver cells and comprehensive transgenic mouse assays. Transgenic analysis provided in vivo validation of multiple new direct Scl target genes and allowed us to reconstruct an in vivo validated network consisting of 17 factors and their respective regulatory elements. By coupling ChIP-Seq in model cell lines with in vivo transgenic validation and sophisticated bioinformatic analysis, we have identified a widely applicable strategy for the reconstruction of stem cell regulatory networks in which biologic material is otherwise limiting. Moreover, in addition to revealing multiple previously unrecognized links to known HSC regulators, as well as novel links to genes not previously implicated in HSC function, comprehensive transgenic analysis of regulatory elements provided substantial new insights into the transcriptional control of several important hematopoietic regulators, including Cbfa2t3h/Eto2, Cebpe, Nfe2, Zfpm1/Fog1, Erg, Mafk, Gfi1b, and Myb.