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Single-cell bisulfite sequencing (scBS) is a technique that enables the assessment of DNA methylation at single-base pair and single-cell resolution. The analysis of large datasets obtained from scBS requires preprocessing to reduce the data size, improve the signal-to-noise ratio and provide interpretability. Typically, this is achieved by dividing the genome into large tiles and averaging the methylation signals within each tile. Here we demonstrate that this coarse-graining approach can lead to signal dilution. We propose improved strategies to identify more informative regions for methylation quantification and a more accurate quantitation method than simple averaging. Our approach enables better discrimination of cell types and other features of interest and reduces the need for large numbers of cells. We also present an approach to detect differentially methylated regions between groups of cells and demonstrate its ability to identify biologically meaningful regions that are associated with genes involved in the core functions of specific cell types. Finally, we present the software tool MethSCAn for scBS data analysis ( https://anders-biostat.github.io/MethSCAn ).
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Current approaches to lineage tracing of stem cell clones require genetic engineering or rely on sparse somatic DNA variants, which are difficult to capture at single-cell resolution. Here, we show that targeted single-cell measurements of DNA methylation at single-CpG resolution deliver joint information about cellular differentiation state and clonal identities. We develop EPI-clone, a droplet-based method for transgene-free lineage tracing, and apply it to study hematopoiesis, capturing hundreds of clonal trajectories across almost 100,000 single-cells. Using ground-truth genetic barcodes, we demonstrate that EPI-clone accurately identifies clonal lineages throughout hematopoietic differentiation. Applied to unperturbed hematopoiesis, we describe an overall decline of clonal complexity during murine ageing and the expansion of rare low-output stem cell clones. In aged human donors, we identified expanded hematopoietic clones with and without genetic lesions, and various degrees of clonal complexity. Taken together, EPI-clone enables accurate and transgene-free single-cell lineage tracing at scale.
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OBJECTIVE: To analyze the ability to evaluate changes over time of individual lesions of incomplete or complete retinal pigment epithelium (RPE) and outer retinal atrophy (iRORA and cRORA, respectively) in patients with intermediate age-related macular degeneration (iAMD). DESIGN: OCT images from patients enrolled in Proxima B clinical trial (NCT02399072) were utilized. PARTICIPANTS: Patients enrolled in the Proxima B clinical trial, from the cohort with geographic atrophy (GA) in 1 eye and iAMD in the other eye at baseline, were included. METHODS: Junior and senior readers analyzed OCT images for the qualitative presence of 9 distinct early atrophic features (presence of zone of choroidal hypertransmission, attenuation and/or disruption of RPE, disruption of ellipsoid zone [EZ] and external limiting membrane [ELM], outer nuclear layer [ONL] thinning, outer plexiform layer [OPL]/inner nuclear layer [INL] subsidence, and hyporeflective wedge-shaped band). If deemed "present," 7 features were quantified with a predefined tolerance level of 50 µm (diameter for the zone of choroidal hypertransmission, zone of attenuation and/or disruption of the RPE, outer retinal thickness left/right vertical diameter, outer retinal thickness thinnest vertical diameter, annotation of EZ, and ELM disruption). MAIN OUTCOME MEASURES: Interreader agreements for qualitative assessments (κ-type statistics) and quantitative measurements (Bland-Altman statistics) were assessed. Progression of the lesion features over time was described. RESULTS: Moderate agreement was found for presence of choroidal hypertransmission (κ = 0.54), followed by ELM disruption (κ = 0.58), OPL/INL subsidence (κ = 0.46), and a hyporeflective wedge-shaped band (κ = 0.47). Quantification measurements showed that choroidal hypertransmission had the highest agreement, whereas RPE attenuation/disruption had the lowest agreement. Longitudinal adjudicated changes for quantitative measurements of lesion progression showed that choroidal hypertransmission and ELM disruption showed significant progression, whereas EZ disruption and RPE attenuation/disruption did not. CONCLUSIONS: The ability to evaluate changes over time for specific features of iRORA and cRORA was explored. The most robust biomarker was found to be choroidal hypertransmission, followed by ELM disruption and the qualitative markers of OPL/INL subsidence, as well as a wedge-shaped band. Disease progression over time could be assessed by some, but not all, spectral-domain OCT features that were explored. FINANCIAL DISCLOSURE(S): Proprietary or commercial disclosure may be found in the Footnotes and Disclosures at the end of this article.
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Degeneración Macular , Epitelio Pigmentado de la Retina , Humanos , Epitelio Pigmentado de la Retina/patología , Angiografía con Fluoresceína , Degeneración Macular/patología , Retina/patología , Tomografía de Coherencia Óptica/métodos , AtrofiaRESUMEN
In the present work, we aim at developing an in vitro release assay to predict circulation times of hydrophobic drugs loaded into polymeric micelles (PM), upon intravenous (i.v.) administration. PM based on poly (ethylene glycol)-b-poly (N-2-benzoyloxypropyl methacrylamide) (mPEG-b-p(HPMA-Bz)) block copolymer were loaded with a panel of hydrophobic anti-cancer drugs and characterized for size, loading efficiency and release profile in different release media. Circulation times in mice of two selected drugs loaded in PM were evaluated and compared to the in vitro release profile. Release of drugs from PM was evaluated over 7 days in PBS containing Triton X-100 and in PBS containing albumin at physiological concentration (40 g/L). The results were utilized to identify crucial molecular features of the studied hydrophobic drugs leading to better micellar retention. For the best and the worst retained drugs in the in vitro assays (ABT-737 and BCI, respectively), the circulation of free and entrapped drugs into PM was examined after i.v. administration in mice. We found in vivo drug retention at 24 h post-injection similar to the retention found in the in vitro assays. This demonstrates that in vitro release assay in buffers supplemented with albumin, and to a lesser degree Triton X-100, can be employed to predict the in vivo circulation kinetics of drugs loaded in PM. Utilizing media containing acceptor molecules for hydrophobic compounds, provide a first screen to understand the stability of drug-loaded PM in the circulation and, therefore, can contribute to the reduction of animals used for circulation kinetics studies.
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Portadores de Fármacos , Micelas , Albúminas , Animales , Portadores de Fármacos/química , Liberación de Fármacos , Ratones , Octoxinol , Polietilenglicoles/química , Polímeros/químicaRESUMEN
INTRODUCTION: This retrospective analysis assessed geographic atrophy (GA) progression in fellow eyes from the Proxima B trial intermediate age-related macular degeneration (iAMD) subcohort using high-resolution multimodal imaging anchored on optical coherence tomography (OCT). METHODS: Thirty-two patients from the Proxima B iAMD subcohort were assessed; all had GA with no macular neovascularization (MNV) in the contralateral eye. Imaging data, including color fundus photography, fluorescein angiography, near-infrared reflectance, fundus autofluorescence (FAF), and spectral-domain OCT, were obtained. Features preceding progression/conversion to advanced AMD (drusen, reticular pseudodrusen [RPD], MNV, incomplete/complete retinal pigment epithelium and outer retinal atrophy [iRORA/cRORA]) were assessed. RESULTS: Of 30 fellow eyes with available follow-up images, 12 converted to GA (FAF), 2 converted to MNV, and 16 were nonconverters during the review period (median: 17.8 months). iRORA/cRORA features (present in all converters at baseline) were identified on OCT in eyes that progressed to GA. Median time interval from iRORA to cRORA and from cRORA to GA was 7 months each. GA development/progression was either drusen- or RPD-associated (n = 6 each). Eyes with baseline RPD showed faster GA progression versus eyes with drusen (1.49 vs. 0.38 mm2/year). CONCLUSIONS: RPD presence was associated with rapid GA lesion enlargement and may provide an early indication of faster GA progression.
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Atrofia Geográfica , Atrofia Geográfica/diagnóstico , Humanos , Estudios RetrospectivosRESUMEN
Clinical evidence suggests alterations in receptor activator of NF-κB (RANK) signaling are key contributors to B cell autoimmunity and malignancy, but the pathophysiological consequences of aberrant B cell-intrinsic RANK signaling remain unknown. We generated mice that express a human lymphoma-derived, hyperactive RANKK240E variant in B lymphocytes in vivo. Forced RANK signaling disrupted B cell tolerance and induced a fully penetrant systemic lupus erythematosus-like disease in addition to the development of chronic lymphocytic leukemia (CLL). Importantly, RANKK240E transgenic CLL cells as well as CLL cells of independent murine and of human origin depend on microenvironmental RANK ligand (RANKL) for tumor cell survival. Consequently, inhibition of the RANKL-RANK axis with anti-RANKL antibodies killed murine and human CLL cells in vitro and in vivo. These results establish pathological B cell-intrinsic RANK signaling as a potential driver of autoimmunity and B cell malignancy, and they suggest the exploitation of clinically available anti-RANKL compounds for CLL treatment.
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Autoinmunidad/inmunología , Linfocitos B/metabolismo , Leucemia Linfocítica Crónica de Células B/metabolismo , Receptor Activador del Factor Nuclear kappa-B/metabolismo , Animales , Linfocitos B/inmunología , Línea Celular Tumoral , Supervivencia Celular/inmunología , Femenino , Células HEK293 , Humanos , Leucemia Linfocítica Crónica de Células B/inmunología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , FN-kappa B/metabolismo , Transducción de Señal/inmunologíaRESUMEN
Acute myeloid leukemia (AML) represents the most common acute leukemia among adults. Despite recent progress in diagnosis and treatment, long-term outcome remains unsatisfactory. The success of allogeneic stem cell transplantation underscores the immunoresponsive nature of AML, creating the basis for further exploiting immunotherapies. However, emerging evidence suggests that AML, similar to other malignant entities, employs a variety of mechanisms to evade immunosurveillance. In light of this, T-cell inhibitory myeloid-derived suppressor cells (MDSC) are gaining interest as key facilitators of immunoescape. Accumulation of CD14+HLA-DRlow monocytic MDSCs has been described in newly diagnosed AML patients, and deciphering the underlying mechanisms could help to improve anti-AML immunity. Here, we report that conventional monocytes readily take-up AML-derived extracellular vesicles (EV) and subsequently undergo MDSC differentiation. They acquired an CD14+HLA-DRlow phenotype, expressed the immunomodulatory indoleamine-2,3-dioxygenase, and upregulated expression of genes characteristic for MDSCs, such as S100A8/9 and cEBPß. The Akt/mTOR pathway played a critical role in the AML-EV-induced phenotypical and functional transition of monocytes. Generated MDSCs displayed a glycolytic switch, which rendered them more susceptible toward glycolytic inhibitors. Furthermore, palmitoylated proteins on the AML-EV surface activated Toll-like receptor 2 as the initiating event of Akt/mTOR-dependent induction of MDSC. Therefore, targeting protein palmitoylation in AML blasts could block MDSC accumulation to improve immune responses. SIGNIFICANCE: These findings indicate that targeting protein palmitoylation in AML could interfere with the leukemogenic potential and block MDSC accumulation to improve immunity.
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Vesículas Extracelulares/metabolismo , Leucemia Mieloide Aguda/patología , Células Supresoras de Origen Mieloide/patología , Transducción de Señal/fisiología , Escape del Tumor/fisiología , Adulto , Anciano , Diferenciación Celular/fisiología , Células Cultivadas , Vesículas Extracelulares/inmunología , Femenino , Humanos , Leucemia Mieloide Aguda/inmunología , Leucemia Mieloide Aguda/metabolismo , Lipoilación , Masculino , Persona de Mediana Edad , Células Supresoras de Origen Mieloide/inmunología , Células Supresoras de Origen Mieloide/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Receptor Toll-Like 2/metabolismoRESUMEN
Acute myeloid leukemia (AML) is the most common acute leukemia amongst adults with a 5-year overall survival lower than 30%. Emerging evidence suggest that immune alterations favor leukemogenesis and/or AML relapse thereby negatively impacting disease outcome. Over the last years myeloid derived suppressor cells (MDSCs) have been gaining momentum in the field of cancer research. MDSCs are a heterogeneous cell population morphologically resembling either monocytes or granulocytes and sharing some key features including myeloid origin, aberrant (immature) phenotype, and immunosuppressive activity. Increasing evidence suggests that accumulating MDSCs are involved in hampering anti-tumor immune responses and immune-based therapies. Here, we demonstrate increased frequencies of CD14+ monocytic MDSCs in newly diagnosed AML that co-express CD33 but lack HLA-DR (HLA-DRlo). AML-blasts induce HLA-DRlo cells from healthy donor-derived monocytes in vitro that suppress T-cells and express indoleamine-2,3-dioxygenase (IDO). We investigated whether a CD33/CD3-bispecific BiTE® antibody construct (AMG 330) with pre-clinical activity against AML-blasts by redirection of T-cells can eradicate CD33+ MDSCs. In fact, T-cells eliminate IDO+CD33+ MDSCs in the presence of AMG 330. Depletion of total CD14+ cells (including MDSCs) in peripheral blood mononuclear cells from AML patients did not enhance AMG 330-triggered T-cell activation and expansion, but boosted AML-blast lysis. This finding was corroborated in experiments showing that adding MDSCs into co-cultures of T- and AML-cells reduced AML-blast killing, while IDO inhibition promotes AMG 330-mediated clearance of AML-blasts. Taken together, our results suggest that AMG 330 may achieve anti-leukemic efficacy not only through T-cell-mediated cytotoxicity against AML-blasts but also against CD33+ MDSCs, suggesting that it is worth exploring the predictive role of MDSCs for responsiveness towards an AMG 330-based therapy.
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Leucemia Mieloide Aguda/inmunología , Monocitos/metabolismo , Células Supresoras de Origen Mieloide/inmunología , Lectina 3 Similar a Ig de Unión al Ácido Siálico/metabolismo , Femenino , Humanos , Leucemia Mieloide Aguda/patología , MasculinoRESUMEN
It is well established that the stromal niche exerts a protective effect on chronic lymphocytic leukemia (CLL) cells, thereby also affecting their drug sensitivity. One hallmark of malignant cells is metabolic reprogramming, which is mostly represented by a glycolytic shift known as the Warburg effect. Because treatment resistance can be linked to metabolic alterations, we investigated whether bone marrow stromal cells impact the bioenergetics of primary CLL cells. In fact, stromal contact led to an increase of aerobic glycolysis and the cells' overall glycolytic capacity accompanied by an increased glucose uptake, expression of glucose transporter, and glycolytic enzymes. Activation of Notch signaling and of its direct transcriptional target c-Myc contributed to this metabolic switch. Based on these observations, CLL cells' acquired increased glucose dependency as well as Notch-c-Myc signaling could be therapeutically exploited in an effort to overcome stroma-mediated drug resistance.
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Glucólisis , Leucemia Linfocítica Crónica de Células B/metabolismo , Proteínas Proto-Oncogénicas c-myc/fisiología , Receptores Notch/fisiología , Células del Estroma/metabolismo , Aerobiosis/genética , Células de la Médula Ósea/metabolismo , Respiración de la Célula/genética , Células Cultivadas , Glucólisis/genética , Humanos , Leucemia Linfocítica Crónica de Células B/patología , Transducción de Señal/fisiologíaRESUMEN
Molecular models of cell fate specification typically focus on the activation of specific lineage programs. However, the concurrent repression of unwanted transcriptional networks is also essential to stabilize certain cellular identities, as shown in a number of diverse systems and phyla. Here, we demonstrate that this dual requirement also holds true in the context of Drosophila myogenesis. By integrating genetics and genomics, we identified a new role for the pleiotropic transcriptional repressor Tramtrack69 in myoblast specification. Drosophila muscles are formed through the fusion of two discrete cell types: founder cells (FCs) and fusion-competent myoblasts (FCMs). When tramtrack69 is removed, FCMs appear to adopt an alternative muscle FC-like fate. Conversely, ectopic expression of this repressor phenocopies muscle defects seen in loss-of-function lame duck mutants, a transcription factor specific to FCMs. This occurs through Tramtrack69-mediated repression in FCMs, whereas Lame duck activates a largely distinct transcriptional program in the same cells. Lineage-specific factors are therefore not sufficient to maintain FCM identity. Instead, their identity appears more plastic, requiring the combination of instructive repressive and activating programs to stabilize cell fate.
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Diferenciación Celular/fisiología , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Regulación del Desarrollo de la Expresión Génica/fisiología , Desarrollo de Músculos/fisiología , Mioblastos/fisiología , Factores Reguladores Miogénicos/metabolismo , Proteínas Represoras/metabolismo , Animales , Inmunoprecipitación de Cromatina , Drosophila/genética , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación Fluorescente in Situ , Mesodermo/fisiología , Mioblastos/metabolismoRESUMEN
Myeloid-derived suppressor cells (MDSCs) represent a heterogeneous population that shares certain characteristics including an aberrant myeloid phenotype and the ability to suppress T cells. MDSCs have been predominantly studied in malignant diseases and findings suggest involvement in tumor-associated immune suppression. Chronic lymphocytic leukemia (CLL) is the leukemia with the highest incidence among adults. Immune defects occur already at early disease stages and impact the clinical course. We assessed presence, frequency, association to other immune parameters, and functional properties of circulating CD14(+) cells lacking HLA-DR expression (HLA-DR(lo)) in patients with untreated CLL. These monocytic cells represent one of the best-defined human MDSC subsets. Frequency of CD14(+)HLA-DR(lo) cells was significantly increased in CLL patients. Furthermore, MDSCs suppressed in vitro T-cell activation and induced suppressive regulatory T cells (TRegs). The MDSC-mediated modulation of T cells could be attributed to their increased indoleamine 2,3-dioxygenase (IDO) activity. CLL cells induced IDO(hi) MDSCs from healthy donor monocytes suggesting bidirectional crosstalk between CLL-cells, MDSCs, and TRegs. Overall, we identified a MDSC population that expands in CLL. The exact mechanisms responsible for such accumulation remain to be elucidated and it will be of interest to test whether antagonizing suppressive functions of CLL MDSCs could represent a mean for enhancing immune responses.
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Leucemia Linfocítica Crónica de Células B/inmunología , Activación de Linfocitos , Células Mieloides/inmunología , Linfocitos T Reguladores/inmunología , Adulto , Línea Celular Tumoral , Femenino , Regulación de la Expresión Génica/inmunología , Antígenos HLA-DR/inmunología , Humanos , Indolamina-Pirrol 2,3,-Dioxigenasa/inmunología , Leucemia Linfocítica Crónica de Células B/patología , Receptores de Lipopolisacáridos/inmunología , Masculino , Células Mieloides/patología , Linfocitos T Reguladores/patologíaRESUMEN
Genomic structural variation (SV) is a major determinant for phenotypic variation. Although it has been extensively studied in humans, the nucleotide resolution structure of SVs within the widely used model organism Drosophila remains unknown. We report a highly accurate, densely validated map of unbalanced SVs comprising 8962 deletions and 916 tandem duplications in 39 lines derived from short-read DNA sequencing in a natural population (the "Drosophila melanogaster Genetic Reference Panel," DGRP). Most SVs (>90%) were inferred at nucleotide resolution, and a large fraction was genotyped across all samples. Comprehensive analyses of SV formation mechanisms using the short-read data revealed an abundance of SVs formed by mobile element and nonhomologous end-joining-mediated rearrangements, and clustering of variants into SV hotspots. We further observed a strong depletion of SVs overlapping genes, which, along with population genetics analyses, suggests that these SVs are often deleterious. We inferred several gene fusion events also highlighting the potential role of SVs in the generation of novel protein products. Expression quantitative trait locus (eQTL) mapping revealed the functional impact of our high-resolution SV map, with quantifiable effects at >100 genic loci. Our map represents a resource for population-level studies of SVs in an important model organism.
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Drosophila melanogaster/genética , Genoma de los Insectos , Variación Estructural del Genoma , Análisis de Secuencia de ADN/métodos , Animales , Mapeo Cromosómico , Femenino , Regulación de la Expresión Génica , Genotipo , Desequilibrio de Ligamiento , Masculino , Polimorfismo de Nucleótido Simple , Sitios de Carácter CuantitativoRESUMEN
Cell fate decisions are driven through the integration of inductive signals and tissue-specific transcription factors (TFs), although the details on how this information converges in cis remain unclear. Here, we demonstrate that the five genetic components essential for cardiac specification in Drosophila, including the effectors of Wg and Dpp signaling, act as a collective unit to cooperatively regulate heart enhancer activity, both in vivo and in vitro. Their combinatorial binding does not require any specific motif orientation or spacing, suggesting an alternative mode of enhancer function whereby cooperative activity occurs with extensive motif flexibility. A fraction of enhancers co-occupied by cardiogenic TFs had unexpected activity in the neighboring visceral mesoderm but could be rendered active in heart through single-site mutations. Given that cardiac and visceral cells are both derived from the dorsal mesoderm, this "dormant" TF binding signature may represent a molecular footprint of these cells' developmental lineage.
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Drosophila melanogaster/citología , Redes Reguladoras de Genes , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Elementos de Facilitación Genéticos , Regulación del Desarrollo de la Expresión Génica , Mesodermo/citología , Mesodermo/metabolismo , Miocardio/citología , Miocardio/metabolismo , Factores de Transcripción/metabolismoRESUMEN
Development requires the establishment of precise patterns of gene expression, which are primarily controlled by transcription factors binding to cis-regulatory modules. Although transcription factor occupancy can now be identified at genome-wide scales, decoding this regulatory landscape remains a daunting challenge. Here we used a novel approach to predict spatio-temporal cis-regulatory activity based only on in vivo transcription factor binding and enhancer activity data. We generated a high-resolution atlas of cis-regulatory modules describing their temporal and combinatorial occupancy during Drosophila mesoderm development. The binding profiles of cis-regulatory modules with characterized expression were used to train support vector machines to predict five spatio-temporal expression patterns. In vivo transgenic reporter assays demonstrate the high accuracy of these predictions and reveal an unanticipated plasticity in transcription factor binding leading to similar expression. This data-driven approach does not require previous knowledge of transcription factor sequence affinity, function or expression, making it widely applicable.
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Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Modelos Genéticos , Factores de Transcripción/metabolismo , Animales , Animales Modificados Genéticamente , Inteligencia Artificial , Inmunoprecipitación de Cromatina , Secuencia Conservada/genética , Bases de Datos Genéticas , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica/genética , Genes Reporteros/genética , Mesodermo/embriología , Mesodermo/metabolismo , Unión Proteica , Factores de TiempoRESUMEN
OBJECTIVES: The extent of adverse myocardial remodeling contributes essentially to the prognosis after myocardial infarction (MI). In this study we investigated whether inhibition of "mammalian target of rapamycin" (mTOR) attenuates left ventricular (LV) remodeling after MI. BACKGROUND: Therapeutic strategies to inhibit remodeling are currently limited to inhibition of neurohumoral activation. The mTOR-dependent signaling mechanisms are centrally involved in remodeling processes and provide new therapeutic opportunities. METHODS: Everolimus (RAD) treatment was initiated on the day after or 3 days after induction of myocardial infarction (MI) in rats. RESULTS: After 28 days, RAD-treated animals had reduced post-MI remodeling, with improved LV function and smaller LV end-diastolic diameters (8.9 + or - 0.3 mm vs. 11.4 + or - 0.2 mm, p < 0.05), end-diastolic volumes (304 + or - 30 microl vs. 414 + or - 16 microl, p < 0.05), and cardiac myocyte size (-40% vs. vehicle, p < 0.05). Infarct size was significantly reduced compared with vehicle-treated animals. The mTOR inhibition increased autophagy and concomitantly decreased proteasome activity in the border zone of the infarcted myocardium. Measurement of autophagic flux demonstrated that RAD did not decrease autophagosome clearance. When RAD treatment was initiated 3 days after MI, adverse remodeling was still attenuated and increased autophagy was still present. Sustained improvement of LV function was observed 3 months after MI, even when RAD treatment was discontinued after 1 month. CONCLUSIONS: Inhibition of mTOR is a potential therapeutic strategy to limit infarct size and to attenuate adverse LV remodeling after MI.
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Inmunosupresores/farmacología , Péptidos y Proteínas de Señalización Intracelular/antagonistas & inhibidores , Infarto del Miocardio/fisiopatología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Sirolimus/análogos & derivados , Remodelación Ventricular/efectos de los fármacos , Animales , Factor Natriurético Atrial/metabolismo , Autofagia/efectos de los fármacos , Diástole/fisiología , Ecocardiografía , Everolimus , Ventrículos Cardíacos/diagnóstico por imagen , Péptidos y Proteínas de Señalización Intracelular/fisiología , Masculino , Proteínas Asociadas a Microtúbulos/fisiología , Miocitos Cardíacos/patología , FN-kappa B/efectos de los fármacos , Fosforilación , Proteínas Serina-Treonina Quinasas/fisiología , Ratas , Ratas Wistar , Proteínas Quinasas S6 Ribosómicas 70-kDa/efectos de los fármacos , Sirolimus/farmacología , Serina-Treonina Quinasas TOR , Regulación hacia Arriba , Disfunción Ventricular Izquierda/fisiopatología , Remodelación Ventricular/fisiologíaRESUMEN
Smooth muscle plays a prominent role in many fundamental processes and diseases, yet our understanding of the transcriptional network regulating its development is very limited. The FoxF transcription factors are essential for visceral smooth muscle development in diverse species, although their direct regulatory role remains elusive. We present a transcriptional map of Biniou (a FoxF transcription factor) and Bagpipe (an Nkx factor) activity, as a first step to deciphering the developmental program regulating Drosophila visceral muscle development. A time course of chromatin immunoprecipitatation followed by microarray analysis (ChIP-on-chip) experiments and expression profiling of mutant embryos reveal a dynamic map of in vivo bound enhancers and direct target genes. While Biniou is broadly expressed, it regulates enhancers driving temporally and spatially restricted expression. In vivo reporter assays indicate that the timing of Biniou binding is a key trigger for the time span of enhancer activity. Although bagpipe and biniou mutants phenocopy each other, their regulatory potential is quite different. This network architecture was not apparent from genetic studies, and highlights Biniou as a universal regulator in all visceral muscle, regardless of its developmental origin or subsequent function. The regulatory connection of a number of Biniou target genes is conserved in mice, suggesting an ancient wiring of this developmental program.
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Proteínas de Drosophila/metabolismo , Drosophila/embriología , Desarrollo Embrionario/genética , Redes Reguladoras de Genes , Músculo Liso/embriología , Factores de Transcripción/metabolismo , Animales , Inmunoprecipitación de Cromatina , Secuencia Conservada , Drosophila/genética , Proteínas de Drosophila/genética , Elementos de Facilitación Genéticos , Factores de Transcripción Forkhead , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Ratones , Mutación , Factores de Transcripción/genética , Vísceras/embriologíaRESUMEN
Large-scale microarray expression profiling studies have helped us to understand basic biological processes and to classify and predict the prognosis of cancers; they have also accelerated the identification of new drug targets. Affymetrix GeneChip probe arrays are high-density oligonucleotide microarrays that are available for many prokaryotic and eukaryotic species. Affymetrix human and mouse whole-genome microarrays analyze the expression level of up to 47,000 transcripts and variants. Each transcript is measured by 11 probe pairs, which consist of a perfect match 25mer oligonucleotide (PM) and a 25mer mismatch oligonucleotide (MM) that contains a single base pair mismatch in the central position. The PM/MM design is used for identification and subtraction of nonspecific hybridization and background signals. Advantages of Affymetrix GeneChip arrays include highly standardized array fabrication, as well as standardized target preparation, hybridization, and processing protocols, resulting in low technical variability and good reproducibility between experiments. This chapter describes the standard assay procedures for isolating total RNA from heart tissue, generating a biotin-labeled target for expression analysis, processing of Affymetrix GeneChip probe arrays using the Affymetrix instrument system, and quality control measures.
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Perfilación de la Expresión Génica/métodos , Corazón/fisiología , Modelos Genéticos , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Algoritmos , Animales , Regulación de la Expresión Génica/genética , Humanos , Ratones , Hibridación de Ácido Nucleico , ARN Mensajero/genética , RatasRESUMEN
Physical activity protects against cardiovascular disease, and physiological cardiac hypertrophy associated with regular exercise is usually beneficial, in marked contrast to pathological hypertrophy associated with disease. The p110alpha isoform of phosphoinositide 3-kinase (PI3K) plays a critical role in the induction of exercise-induced hypertrophy. Whether it or other genes activated in the athlete's heart might have an impact on cardiac function and survival in a setting of heart failure is unknown. To examine whether progressive exercise training and PI3K(p110alpha) activity affect survival and/or cardiac function in two models of heart disease, we subjected a transgenic mouse model of dilated cardiomyopathy (DCM) to swim training, genetically crossed cardiac-specific transgenic mice with increased or decreased PI3K(p110alpha) activity to the DCM model, and subjected PI3K(p110alpha) transgenics to acute pressure overload (ascending aortic constriction). Life-span, cardiac function, and molecular markers of pathological hypertrophy were examined. Exercise training and increased cardiac PI3K(p110alpha) activity prolonged survival in the DCM model by 15-20%. In contrast, reduced PI3K(p110alpha) activity drastically shortened lifespan by approximately 50%. Increased PI3K(p110alpha) activity had a favorable effect on cardiac function and fibrosis in the pressure-overload model and attenuated pathological growth. PI3K(p110alpha) signaling negatively regulated G protein-coupled receptor stimulated extracellular responsive kinase and Akt (via PI3K, p110gamma) activation in isolated cardiomyocytes. These findings suggest that exercise and enhanced PI3K(p110alpha) activity delay or prevent progression of heart disease, and that supraphysiologic activity can be beneficial. Identification of genes important for hypertrophy in the athlete's heart could offer new strategies for treating heart failure.
Asunto(s)
Cardiomiopatía Dilatada/prevención & control , Cardiomiopatía Dilatada/fisiopatología , Cardiomiopatía Hipertrófica/prevención & control , Cardiomiopatía Hipertrófica/fisiopatología , Fosfatidilinositol 3-Quinasas/metabolismo , Esfuerzo Físico/fisiología , Animales , Cardiomiopatía Dilatada/patología , Cardiomiopatía Hipertrófica/patología , Fosfatidilinositol 3-Quinasa Clase I , Modelos Animales de Enfermedad , Femenino , Expresión Génica , Humanos , Masculino , Ratones , Ratones Noqueados , Ratones Transgénicos , Fosfatidilinositol 3-Quinasas/deficiencia , Fosfatidilinositol 3-Quinasas/genética , Condicionamiento Físico Animal , Transducción de SeñalRESUMEN
Thioredoxin1 (Trx1) inhibits hypertrophy and exhibits protective functions in the heart. To elucidate further the cardiac functions of Trx1, we used a DNA microarray analysis, with hearts from transgenic mice with cardiac- specific overexpression of Trx1 (Tg-Trx1, n = 4) and nontransgenic controls (n = 4). Expression of a large number of genes is regulated in Tg-Trx1, with a greater number of genes downregulated, versus upregulated, at high-fold changes. The peroxisome proliferator-activated receptor gamma coactivator-1alpha (PGC-1gamma) gene was among the top 50 significantly upregulated genes. By pathway analyses, we found that genes involved in both mitochondrial oxidative phosphorylation and the TCA cycle were upregulated in Tg-Trx1. We confirmed upregulation of cytochrome c oxidase (COX) components and mitochondrial transcription factor A in Tg-Trx1. The activity of citrate synthase and COX and the cardiac ATP content were significantly higher in Tg-Trx1. A transcription factor binding-site analysis showed that upregulated genes frequently contained binding sites for nuclear respiratory factor 1 (NRF1). Expression of NRF1 and PGC-1gamma was upregulated in Tg-Trx1, and Trx1 stimulated the transcriptional activity of NRF1 and NRF2 in cardiac myocytes. These results suggest that, in cardiac myocytes, Trx1 upregulates mitochondrial proteins and enhances mitochondrial functions, possibly through PGC-1alpha and NRFs.
Asunto(s)
Ciclo del Ácido Cítrico/genética , Proteínas Mitocondriales/genética , Fosforilación Oxidativa , Tiorredoxinas/genética , Regulación hacia Arriba/genética , Adenosina Trifosfato/metabolismo , Animales , Citrato (si)-Sintasa/genética , Citrato (si)-Sintasa/metabolismo , Citocromos c/genética , Citocromos c/metabolismo , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Complejo IV de Transporte de Electrones/genética , Complejo IV de Transporte de Electrones/metabolismo , Perfilación de la Expresión Génica , Proteínas del Grupo de Alta Movilidad/genética , Masculino , Ratones , Ratones Transgénicos , Mitocondrias/genética , Mitocondrias/metabolismo , Proteínas Mitocondriales/metabolismo , Miocardio/metabolismo , Factores Nucleares de Respiración/genética , Factores Nucleares de Respiración/metabolismo , Análisis de Secuencia por Matrices de Oligonucleótidos , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma , Transactivadores/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
BACKGROUND: The shape of a nucleus depends on the nuclear lamina, which is tightly associated with the inner nuclear membrane and on the interaction with the cytoskeleton. However, the mechanism connecting the differentiation state of a cell to the shape changes of its nucleus are not well understood. We investigated this question in early Drosophila embryos, where the nuclear shape changes from spherical to ellipsoidal together with a 2.5-fold increase in nuclear length during cellularization. RESULTS: We identified two genes, kugelkern and kurzkern, required for nuclear elongation. In kugelkern- and kurzkern-depleted embryos, the nuclei reach only half the length of the wild-type nuclei at the end of cellularization. The reduced nuclear size affects chromocenter formation as marked by Heterochromatin protein 1 and expression of a specific set of genes, including early zygotic genes. kugelkern contains a putative coiled-coil domain in the N-terminal half of the protein, a nuclear localization signal (NLS), and a C-terminal CxxM-motif. The carboxyterminal CxxM motif is required for the targeting of Kugelkern to the inner nuclear membrane, where it colocalizes with lamins. Depending on the farnesylation motif, expression of kugelkern in Drosophila embryos or Xenopus cells induces overproliferation of nuclear membrane. CONCLUSIONS: Kugelkern is so far the first nuclear protein, except for lamins, that contains a farnesylation site. Our findings suggest that Kugelkern is a rate-determining factor for nuclear size increase. We propose that association of farnesylated Kugelkern with the inner nuclear membrane induces expansion of nuclear surface area, allowing nuclear growth.