RESUMEN
BACKGROUND: Intercostal nerve block (ICNB) has long been used in thoracic surgery. Local anesthetic thoracoscopy (LAT) is performed under conscious sedation with local anesthesia at the port insertion site. This alone, however, does not anesthetize the parietal pleura from where biopsies are taken and patients can experience pain. OBJECTIVES: To compare LAT with multilevel ICNB versus standard care to determine whether it reduces pain during and post-LAT, its effect on analgesia use, the hospital length of stay (LOS), and related complications. METHODS: Prospective analysis of patients undergoing LAT between January and June 2021. In the ICNB group, levobupivacaine/xylocaine is administered at the angle of the rib immediately before LAT (up to 5 rib spaces). Visual Analog Score for pain (0 to 100 mm) was measured at 1 and 2 hours post-LAT and daily including analgesia use. RESULTS: Twenty patients (10 ICNB vs. 10 standard care group). The mean age is 68 years with 70% males. Visual Analog Score for pain in the ICNB group reduced by 55 mm at 1 and 2 hours post-LAT and 45 mm at day 1 ( P <0.05) (minimal clinically important difference >16 mm]. Median LOS was reduced by 50% in the ICNB group ( P <0.05). Paracetamol use reduced by 56% ( P <0.05). CONCLUSION: ICNB not only significantly reduces postprocedure pain but also reduces LOS.
Asunto(s)
Anestésicos Locales , Bloqueo Nervioso , Masculino , Humanos , Anciano , Femenino , Anestésicos Locales/uso terapéutico , Nervios Intercostales , Dolor Postoperatorio/tratamiento farmacológico , Dolor Postoperatorio/prevención & control , Dolor Postoperatorio/etiología , Bloqueo Nervioso/efectos adversos , Bloqueo Nervioso/métodos , Toracoscopía/efectos adversosRESUMEN
DNA looping is vital for establishing many enhancer-promoter interactions. While CTCF is known to anchor many cohesin-mediated loops, the looped chromatin fiber appears to predominantly exist in a poorly characterized actively extruding state. To better characterize extruding chromatin loop structures, we used CTCF MNase HiChIP data to determine both CTCF binding at high resolution and 3D contact information. Here we present FactorFinder, a tool that identifies CTCF binding sites at near base-pair resolution. We leverage this substantial advance in resolution to determine that the fully extruded (CTCF-CTCF) state is rare genome-wide with locus-specific variation from ~1-10%. We further investigate the impact of chromatin state on loop extrusion dynamics, and find that active enhancers and RNA Pol II impede cohesin extrusion, facilitating an enrichment of enhancer-promoter contacts in the partially extruded loop state. We propose a model of topological regulation whereby the transient, partially extruded states play active roles in transcription.
RESUMEN
Enhancers possess both structural elements mediating promoter looping and functional elements mediating gene expression. Traditional models of enhancer-mediated gene regulation imply genomic overlap or immediate adjacency of these elements. We test this model by combining densely-tiled CRISPRa screening with nucleosome-resolution Region Capture Micro-C topology analysis. Using this integrated approach, we comprehensively define the cis-regulatory landscape for the tumor suppressor PTEN, identifying and validating 10 distinct enhancers and defining their 3D spatial organization. Unexpectedly, we identify several long-range functional enhancers whose promoter proximity is facilitated by chromatin loop anchors several kilobases away, and demonstrate that accounting for this spatial separation improves the computational prediction of validated enhancers. Thus, we propose a new model of enhancer organization incorporating spatial separation of essential functional and structural components.
RESUMEN
Amplification of MDM2 on supernumerary chromosomes is a common mechanism of P53 inactivation across tumors. Here, we investigated the impact of MDM2 overexpression on chromatin, gene expression, and cellular phenotypes in liposarcoma. Three independent regulatory circuits predominate in aggressive, dedifferentiated tumors. RUNX and AP-1 family transcription factors bind mesenchymal gene enhancers. P53 and MDM2 co-occupy enhancers and promoters associated with P53 signaling. When highly expressed, MDM2 also binds thousands of P53-independent growth and stress response genes, whose promoters engage in multi-way topological interactions. Overexpressed MDM2 concentrates within nuclear foci that co-localize with PML and YY1 and could also contribute to P53-independent phenotypes associated with supraphysiologic MDM2. Importantly, we observe striking cell-to-cell variability in MDM2 copy number and expression in tumors and models. Whereas liposarcoma cells are generally sensitive to MDM2 inhibitors and their combination with pro-apoptotic drugs, MDM2-high cells tolerate them and may underlie the poor clinical efficacy of these agents.
RESUMEN
Epigenetic lesions that disrupt regulatory elements represent potential cancer drivers. However, we lack experimental models for validating their tumorigenic impact. Here, we model aberrations arising in isocitrate dehydrogenase-mutant gliomas, which exhibit DNA hypermethylation. We focus on a CTCF insulator near the PDGFRA oncogene that is recurrently disrupted by methylation in these tumors. We demonstrate that disruption of the syntenic insulator in mouse oligodendrocyte progenitor cells (OPCs) allows an OPC-specific enhancer to contact and induce Pdgfra, thereby increasing proliferation. We show that a second lesion, methylation-dependent silencing of the Cdkn2a tumor suppressor, cooperates with insulator loss in OPCs. Coordinate inactivation of the Pdgfra insulator and Cdkn2a drives gliomagenesis in vivo. Despite locus synteny, the insulator is CpG-rich only in humans, a feature that may confer human glioma risk but complicates mouse modeling. Our study demonstrates the capacity of recurrent epigenetic lesions to drive OPC proliferation in vitro and gliomagenesis in vivo.
Asunto(s)
Neoplasias Encefálicas , Epigénesis Genética , Glioma , Animales , Humanos , Ratones , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Metilación de ADN , Glioma/genética , Glioma/patología , Isocitrato Deshidrogenasa/genética , Mutación , Oncogenes , Receptor alfa de Factor de Crecimiento Derivado de Plaquetas/genéticaRESUMEN
BACKGROUND: Local anesthetic thoracoscopy (LAT) is important in the diagnosis of unilateral pleural effusions. Indwelling pleural catheters (IPC) can be inserted during LAT if a nonexpandable lung is suspected. Subcutaneous emphysema (SCE) is a known complication and is associated with increased morbidity and length of stay. It is unclear however if the incidence of SCE is affected if IPC is inserted through a separate incision to the LAT port. We aim to establish the incidence and grading of SCE when IPC is inserted during LAT and to determine if the site of IPC placement influences this. METHODS: Retrospective analysis of LAT electronic records and radiology images over 8 years in a University Hospital. The incidence of SCE was assessed during admission and follow-up with the severity of SCE graded 0 to 4 (0 none; 1 at IPC site; 2 ipsilateral chest wall; 3 ipsilateral neck; 4 contralateral chest wall). RESULTS: 55 combined LAT and IPC procedures were performed. In 28 patients the IPC was inserted through the LAT port and in 27 the IPC was inserted in a separate intercostal space (ICS) to the LAT port. On day zero, the incidence of any SCE was lower if the IPC was inserted using a separate ICS to the LAT port compared with the same site as the LAT port( P =0.01). This was similarly reduced on discharge chest radiographs and subsequent follow-up. CONCLUSION: IPC insertion at LAT using a separate ICS to the LAT port is associated with a reduction in the incidence of SCE during admission and follow-up.
Asunto(s)
Derrame Pleural Maligno , Enfisema Subcutáneo , Humanos , Anestésicos Locales , Estudios Retrospectivos , Derrame Pleural Maligno/etiología , Catéteres de Permanencia/efectos adversos , Toracoscopía/efectos adversos , Enfisema Subcutáneo/diagnóstico por imagen , Enfisema Subcutáneo/epidemiología , Enfisema Subcutáneo/etiología , Pleurodesia/métodosRESUMEN
Global methylation changes in aging cells affect cancer risk and tissue homeostasis.
Asunto(s)
Envejecimiento , Metilación de ADN , Epigénesis Genética , Mitosis , Neoplasias , Humanos , Envejecimiento/genética , Senescencia Celular , Epigenómica , Neoplasias/genéticaRESUMEN
The genome can be divided into two spatially segregated compartments, A and B, which partition active and inactive chromatin states. While constitutive heterochromatin is predominantly located within the B compartment near the nuclear lamina, facultative heterochromatin marked by H3K27me3 spans both compartments. How epigenetic modifications, compartmentalization, and lamina association collectively maintain heterochromatin architecture remains unclear. Here we develop Lamina-Inducible Methylation and Hi-C (LIMe-Hi-C) to jointly measure chromosome conformation, DNA methylation, and lamina positioning. Through LIMe-Hi-C, we identify topologically distinct sub-compartments with high levels of H3K27me3 and differing degrees of lamina association. Inhibition of Polycomb repressive complex 2 (PRC2) reveals that H3K27me3 is essential for sub-compartment segregation. Unexpectedly, PRC2 inhibition promotes lamina association and constitutive heterochromatin spreading into H3K27me3-marked B sub-compartment regions. Consistent with this repositioning, genes originally marked with H3K27me3 in the B compartment, but not the A compartment, remain largely repressed, suggesting that constitutive heterochromatin spreading can compensate for H3K27me3 loss at a transcriptional level. These findings demonstrate that Polycomb sub-compartments and their antagonism with lamina association are fundamental features of genome structure. More broadly, by jointly measuring nuclear position and Hi-C contacts, our study demonstrates how compartmentalization and lamina association represent distinct but interdependent modes of heterochromatin regulation.
Asunto(s)
Heterocromatina , Histonas , Núcleo Celular/metabolismo , Metilación de ADN , Heterocromatina/genética , Heterocromatina/metabolismo , Histonas/metabolismo , Complejo Represivo Polycomb 2/genética , Complejo Represivo Polycomb 2/metabolismo , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismoRESUMEN
BACKGROUND: Chest drain displacement is a common clinical problem that occurs in 9-42% of cases and results in treatment failure or additional pleural procedures conferring unnecessary risk. A novel chest drain with an integrated intrapleural balloon may reduce the risk of displacement. METHODS: A prospective randomised controlled trial comparing the balloon drain to standard care (12â F chest drain with no balloon) with the primary outcome of objectively defined unintentional or accidental chest drain displacement. RESULTS: 267 patients were randomised (primary outcome data available in 257, 96.2%). Displacement occurred less frequently using the balloon drain (displacement 5 of 128, 3.9%; standard care displacement 13 of 129, 10.1%) but this was not statistically significant (OR for drain displacement 0.36, 95% CI 0.13-1.0, Chi-squared 1â degree of freedom (df)=2.87, p=0.09). Adjusted analysis to account for minimisation factors and use of drain sutures demonstrated balloon drains were independently associated with reduced drain fall-out rate (adjusted OR 0.27, 95% CI 0.08-0.87, p=0.028). Adverse events were higher in the balloon arm than the standard care arm (balloon drain 59 of 131, 45.0%; standard care 18 of 132, 13.6%; Chi-squared 1â df=31.3, p<0.0001). CONCLUSION: Balloon drains reduce displacement compared with standard drains independent of the use of sutures but are associated with increased adverse events specifically during drain removal. The potential benefits of the novel drain should be weighed against the risks, but may be considered in practices where sutures are not routinely used.
Asunto(s)
Drenaje , Procedimientos Quirúrgicos Torácicos , Tubos Torácicos , Remoción de Dispositivos/efectos adversos , Drenaje/efectos adversos , Humanos , Estudios ProspectivosRESUMEN
The COVID-19 pandemic has created new challenges for management of pleural diseases. As resources and staff have been redirected to manage acutely unwell COVID-19 patients, routine medical practice and service provision for pleural diseases have been severely disrupted. We recognised the impact this had for patients with pleural diseases, who can be highly vulnerable to infection and often have conditions for which treatment cannot be safely delayed. The pleural service was reviewed in a tertiary centre, focusing on the changes that allowed maintenance of a service whilst maximising patient and staff safety, with the aim that these service transformations can be adopted elsewhere to improve care for pleural patients during and beyond COVID-19.
Asunto(s)
COVID-19 , Humanos , Pandemias , SARS-CoV-2 , TriajeRESUMEN
Chromosomes are positioned nonrandomly inside the nucleus to coordinate with their transcriptional activity. The molecular mechanisms that dictate the global genome organization and the nuclear localization of individual chromosomes are not fully understood. We introduce a polymer model to study the organization of the diploid human genome. It is data-driven because all parameters can be derived from Hi-C data; it is also a mechanistic model because the energy function is explicitly written out based on a few biologically motivated hypotheses. These two features distinguish the model from existing approaches and make it useful both for reconstructing genome structures and for exploring the principles of genome organization. We carried out extensive validations to show that simulated genome structures reproduce a wide variety of experimental measurements, including chromosome radial positions and spatial distances between homologous pairs. Detailed mechanistic investigations support the importance of both specific interchromosomal interactions and centromere clustering for chromosome positioning. We anticipate the polymer model, when combined with Hi-C experiments, to be a powerful tool for investigating large-scale rearrangements in genome structure upon cell differentiation and tumor progression.
Asunto(s)
Diploidia , Polímeros , Núcleo Celular/genética , Cromosomas/genética , Genoma Humano , HumanosRESUMEN
Widespread changes to DNA methylation and chromatin are well documented in cancer, but the fate of higher-order chromosomal structure remains obscure. Here we integrated topological maps for colon tumors and normal colons with epigenetic, transcriptional, and imaging data to characterize alterations to chromatin loops, topologically associated domains, and large-scale compartments. We found that spatial partitioning of the open and closed genome compartments is profoundly compromised in tumors. This reorganization is accompanied by compartment-specific hypomethylation and chromatin changes. Additionally, we identify a compartment at the interface between the canonical A and B compartments that is reorganized in tumors. Remarkably, similar shifts were evident in non-malignant cells that have accumulated excess divisions. Our analyses suggest that these topological changes repress stemness and invasion programs while inducing anti-tumor immunity genes and may therefore restrain malignant progression. Our findings call into question the conventional view that tumor-associated epigenomic alterations are primarily oncogenic.
Asunto(s)
Cromatina/metabolismo , Cromosomas/metabolismo , Neoplasias Colorrectales/genética , Neoplasias Colorrectales/metabolismo , Metilación de ADN , Epigénesis Genética , Regulación Neoplásica de la Expresión Génica/genética , División Celular , Senescencia Celular/genética , Secuenciación de Inmunoprecipitación de Cromatina , Cromosomas/genética , Estudios de Cohortes , Neoplasias Colorrectales/mortalidad , Neoplasias Colorrectales/patología , Biología Computacional , Metilación de ADN/genética , Epigenómica , Células HCT116 , Humanos , Hibridación Fluorescente in Situ , Microscopía Electrónica de Transmisión , Simulación de Dinámica Molecular , RNA-Seq , Análisis Espacial , Proteínas Supresoras de Tumor/genética , Proteínas Supresoras de Tumor/metabolismoRESUMEN
Myogenic differentiation (MD) has been claimed to be a poor prognostic factor in dedifferentiated liposarcoma (DDLPS). To validate this, the prognostic significance of MD in a uniformly treated cohort of DDLPS was assessed. A cohort of patients that have been uniformly treated at one institution for DDLPS of the retroperitoneum and pelvis were stained with smooth muscle actin (SMA) and desmin and semiquantitatively scored for staining focality and strength. Clinical and survival data was collected, and the prognostic significance of MD was evaluated. A total of 50 patients with uniformly treated DDLPS were evaluated. SMA (P=0.052) and a combined score of MD (SMA+desmin) showed a statistically significant decrease in 5-year disease-free survival (P=0.002) in univariate analysis and in multivariate testing combined MD trended toward significance (P=0.052). Combined MD was associated with a decreased OS in multivariate analysis (P=0.004). In a uniformly treated cohort of DDLPS stained for myogenic markers, a combined myogenic score was associated with poor overall survival in multivariate analysis. However, the difference in groups was slight and the clinical application is limited.
Asunto(s)
Liposarcoma/patología , Actinas/análisis , Adulto , Anciano , Anciano de 80 o más Años , Biomarcadores de Tumor/análisis , Diferenciación Celular , Desmina/análisis , Supervivencia sin Enfermedad , Femenino , Humanos , Liposarcoma/mortalidad , Masculino , Persona de Mediana Edad , Células Musculares/patología , PronósticoRESUMEN
Objective Posterior fossa meningiomas are surgically challenging tumors that are associated with high morbidity and mortality. We sought to investigate the anatomical distribution of clinically actionable mutations in posterior fossa meningioma to facilitate identifying patients amenable for systemic targeted therapy trials. Methods Targeted sequencing of clinically targetable AKT1 , SMO , and PIK3CA mutations was performed in 61 posterior fossa meningioma using Illumina NextSeq 500 to a target depth of >500 × . Samples were further interrogated for 53 cancer-relevant RNA fusions by the Archer FusionPlex panel to detect gene rearrangements. Results AKT 1 ( E17K ) mutations were detected in five cases (8.2%), four in the foramen magnum and one in the cerebellopontine angle. In contrast, none of the posterior fossa tumors harbored an SMO ( L412F ) or a PIK3CA ( E545K ) mutation. Notably, the majority of foramen magnum meningiomas (4/7, 57%) harbored an AKT1 mutation. In addition, common clinically targetable gene fusions were not detected in any of the cases. Conclusion A large subset of foramen magnum meningiomas harbor AKT1 E17K mutations and are therefore potentially amenable to targeted medical therapy. Genotyping of foramen magnum meningiomas may enable more therapeutic alternatives and guide their treatment decision process.
RESUMEN
Epigenetic aberrations are widespread in cancer, yet the underlying mechanisms and causality remain poorly understood1-3. A subset of gastrointestinal stromal tumours (GISTs) lack canonical kinase mutations but instead have succinate dehydrogenase (SDH) deficiency and global DNA hyper-methylation4,5. Here, we associate this hyper-methylation with changes in genome topology that activate oncogenic programs. To investigate epigenetic alterations systematically, we mapped DNA methylation, CTCF insulators, enhancers, and chromosome topology in KIT-mutant, PDGFRA-mutant and SDH-deficient GISTs. Although these respective subtypes shared similar enhancer landscapes, we identified hundreds of putative insulators where DNA methylation replaced CTCF binding in SDH-deficient GISTs. We focused on a disrupted insulator that normally partitions a core GIST super-enhancer from the FGF4 oncogene. Recurrent loss of this insulator alters locus topology in SDH-deficient GISTs, allowing aberrant physical interaction between enhancer and oncogene. CRISPR-mediated excision of the corresponding CTCF motifs in an SDH-intact GIST model disrupted the boundary between enhancer and oncogene, and strongly upregulated FGF4 expression. We also identified a second recurrent insulator loss event near the KIT oncogene, which is also highly expressed across SDH-deficient GISTs. Finally, we established a patient-derived xenograft (PDX) from an SDH-deficient GIST that faithfully maintains the epigenetics of the parental tumour, including hypermethylation and insulator defects. This PDX model is highly sensitive to FGF receptor (FGFR) inhibition, and more so to combined FGFR and KIT inhibition, validating the functional significance of the underlying epigenetic lesions. Our study reveals how epigenetic alterations can drive oncogenic programs in the absence of canonical kinase mutations, with implications for mechanistic targeting of aberrant pathways in cancers.
Asunto(s)
Carcinogénesis/genética , Aberraciones Cromosómicas , Tumores del Estroma Gastrointestinal/genética , Tumores del Estroma Gastrointestinal/patología , Oncogenes/genética , Succinato Deshidrogenasa/deficiencia , Animales , Sistemas CRISPR-Cas/genética , Metilación de ADN , Elementos de Facilitación Genéticos/genética , Epigénesis Genética , Factor 4 de Crecimiento de Fibroblastos/genética , Tumores del Estroma Gastrointestinal/enzimología , Humanos , Ratones , Mutación , Proteínas Proto-Oncogénicas c-kit/antagonistas & inhibidores , Receptores de Factores de Crecimiento de Fibroblastos/antagonistas & inhibidores , Succinato Deshidrogenasa/genéticaRESUMEN
The human genome folds to create thousands of intervals, called "contact domains," that exhibit enhanced contact frequency within themselves. "Loop domains" form because of tethering between two loci-almost always bound by CTCF and cohesin-lying on the same chromosome. "Compartment domains" form when genomic intervals with similar histone marks co-segregate. Here, we explore the effects of degrading cohesin. All loop domains are eliminated, but neither compartment domains nor histone marks are affected. Loss of loop domains does not lead to widespread ectopic gene activation but does affect a significant minority of active genes. In particular, cohesin loss causes superenhancers to co-localize, forming hundreds of links within and across chromosomes and affecting the regulation of nearby genes. We then restore cohesin and monitor the re-formation of each loop. Although re-formation rates vary greatly, many megabase-sized loops recovered in under an hour, consistent with a model where loop extrusion is rapid.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Núcleo Celular/genética , Proteínas Cromosómicas no Histona/metabolismo , Cromosomas/metabolismo , Genoma Humano , Proteínas Represoras/metabolismo , Factor de Unión a CCCTC , Línea Celular Tumoral , Proteínas de Unión al ADN , Elementos de Facilitación Genéticos , Código de Histonas , Humanos , Proteínas Nucleares/metabolismo , Nucleosomas/metabolismo , Fosfoproteínas/metabolismo , CohesinasRESUMEN
Many DNA-hypermethylated cancer genes are occupied by the Polycomb (PcG) repressor complex in embryonic stem cells (ESCs). Their prevalence in the full spectrum of cancers, the exact context of chromatin involved, and their status in adult cell renewal systems are unknown. Using a genome-wide analysis, we demonstrate that ~75% of hypermethylated genes are marked by PcG in the context of bivalent chromatin in both ESCs and adult stem/progenitor cells. A large number of these genes are key developmental regulators, and a subset, which we call the "DNA hypermethylation module," comprises a portion of the PcG target genes that are down-regulated in cancer. Genes with bivalent chromatin have a low, poised gene transcription state that has been shown to maintain stemness and self-renewal in normal stem cells. However, when DNA-hypermethylated in tumors, we find that these genes are further repressed. We also show that the methylation status of these genes can cluster important subtypes of colon and breast cancers. By evaluating the subsets of genes that are methylated in different cancers with consideration of their chromatin status in ESCs, we provide evidence that DNA hypermethylation preferentially targets the subset of PcG genes that are developmental regulators, and this may contribute to the stem-like state of cancer. Additionally, the capacity for global methylation profiling to cluster tumors by phenotype may have important implications for further refining tumor behavior patterns that may ultimately aid therapeutic interventions.
Asunto(s)
Metilación de ADN , Células Madre Embrionarias/metabolismo , Regulación Neoplásica de la Expresión Génica , Células Madre Mesenquimatosas/metabolismo , Neoplasias/genética , Línea Celular Tumoral , Cromatina/metabolismo , Análisis por Conglomerados , Islas de CpG , Epigénesis Genética , Perfilación de la Expresión Génica , Genes Relacionados con las Neoplasias , Genes Reguladores , Histonas/metabolismo , Humanos , Análisis de Secuencia por Matrices de Oligonucleótidos , Osteoblastos/metabolismo , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Proteínas Represoras/metabolismo , Análisis de Secuencia de ADNRESUMEN
Accumulating evidence points to a major role for chronic stress of cell renewal systems in the pathogenesis of important human diseases, including cancer, atherosclerosis and diabetes. Here we discuss emerging evidence that epigenetic abnormalities may make substantial contributions to these stress-induced pathologies. Although the mechanisms remain to be fully elucidated, we suggest that chronic stress can elicit heritable changes in the chromatin landscape that 'lock' cells in abnormal states, which then lead to disease. We emphasize the need to investigate epigenetic states in disease and links to stress and to consider how the knowledge gained through these studies may foster new means of disease prevention and management.
Asunto(s)
Enfermedad/etiología , Epigénesis Genética/fisiología , Estrés Fisiológico/fisiología , Proliferación Celular , Complicaciones de la Diabetes/genética , Enfermedad/genética , Humanos , Modelos Biológicos , Lesiones Precancerosas/genética , Lesiones Precancerosas/patología , Estrés Fisiológico/genéticaRESUMEN
MicroRNAs (miRNAs) are crucial for normal embryonic stem (ES) cell self-renewal and cellular differentiation, but how miRNA gene expression is controlled by the key transcriptional regulators of ES cells has not been established. We describe here the transcriptional regulatory circuitry of ES cells that incorporates protein-coding and miRNA genes based on high-resolution ChIP-seq data, systematic identification of miRNA promoters, and quantitative sequencing of short transcripts in multiple cell types. We find that the key ES cell transcription factors are associated with promoters for miRNAs that are preferentially expressed in ES cells and with promoters for a set of silent miRNA genes. This silent set of miRNA genes is co-occupied by Polycomb group proteins in ES cells and shows tissue-specific expression in differentiated cells. These data reveal how key ES cell transcription factors promote the ES cell miRNA expression program and integrate miRNAs into the regulatory circuitry controlling ES cell identity.
Asunto(s)
Células Madre Embrionarias/metabolismo , MicroARNs/genética , Transcripción Genética , Animales , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Regiones Promotoras Genéticas , Factores de Transcripción/metabolismoRESUMEN
Embryonic stem (ES) cells have a unique regulatory circuitry, largely controlled by the transcription factors Oct4, Sox2, and Nanog, which generates a gene expression program necessary for pluripotency and self-renewal. How external signals connect to this regulatory circuitry to influence ES cell fate is not known. We report here that a terminal component of the canonical Wnt pathway in ES cells, the transcription factor T-cell factor-3 (Tcf3), co-occupies promoters throughout the genome in association with the pluripotency regulators Oct4 and Nanog. Thus, Tcf3 is an integral component of the core regulatory circuitry of ES cells, which includes an autoregulatory loop involving the pluripotency regulators. Both Tcf3 depletion and Wnt pathway activation cause increased expression of Oct4, Nanog, and other pluripotency factors and produce ES cells that are refractory to differentiation. Our results suggest that the Wnt pathway, through Tcf3, brings developmental signals directly to the core regulatory circuitry of ES cells to influence the balance between pluripotency and differentiation.