RESUMEN
In vitro modeling of human disease has recently become feasible with induced pluripotent stem cell (iPSC) technology. Here, we established patient-derived iPSCs from a Li-Fraumeni syndrome (LFS) family and investigated the role of mutant p53 in the development of osteosarcoma (OS). LFS iPSC-derived osteoblasts (OBs) recapitulated OS features including defective osteoblastic differentiation as well as tumorigenic ability. Systematic analyses revealed that the expression of genes enriched in LFS-derived OBs strongly correlated with decreased time to tumor recurrence and poor patient survival. Furthermore, LFS OBs exhibited impaired upregulation of the imprinted gene H19 during osteogenesis. Restoration of H19 expression in LFS OBs facilitated osteoblastic differentiation and repressed tumorigenic potential. By integrating human imprinted gene network (IGN) into functional genomic analyses, we found that H19 mediates suppression of LFS-associated OS through the IGN component DECORIN (DCN). In summary, these findings demonstrate the feasibility of studying inherited human cancer syndromes with iPSCs.
Asunto(s)
Redes Reguladoras de Genes , Células Madre Pluripotentes Inducidas/citología , Síndrome de Li-Fraumeni/complicaciones , Osteosarcoma/etiología , Adolescente , Adulto , Animales , Niño , Decorina/metabolismo , Femenino , Humanos , Síndrome de Li-Fraumeni/genética , Síndrome de Li-Fraumeni/patología , Masculino , Células Madre Mesenquimatosas/metabolismo , Ratones , Modelos Biológicos , Trasplante de Neoplasias , Osteoblastos/citología , Osteoblastos/metabolismo , Osteogénesis , Osteosarcoma/genética , Osteosarcoma/patología , ARN Largo no Codificante/metabolismo , Trasplante Heterólogo , Proteína p53 Supresora de Tumor/metabolismoRESUMEN
The embryonic stem (ES) cell transcriptional and chromatin-modifying networks are critical for self-renewal maintenance. However, it remains unclear whether these networks functionally interact and, if so, what factors mediate such interactions. Here, we show that WD repeat domain 5 (Wdr5), a core member of the mammalian Trithorax (trxG) complex, positively correlates with the undifferentiated state and is a regulator of ES cell self-renewal. We demonstrate that Wdr5, an "effector" of H3K4 methylation, interacts with the pluripotency transcription factor Oct4. Genome-wide protein localization and transcriptome analyses demonstrate overlapping gene regulatory functions between Oct4 and Wdr5. The Oct4-Sox2-Nanog circuitry and trxG cooperate in activating transcription of key self-renewal regulators, and furthermore, Wdr5 expression is required for the efficient formation of induced pluripotent stem (iPS) cells. We propose an integrated model of transcriptional and epigenetic control, mediated by select trxG members, for the maintenance of ES cell self-renewal and somatic cell reprogramming.
Asunto(s)
Células Madre Embrionarias/metabolismo , Redes Reguladoras de Genes , Proteínas/metabolismo , Animales , Inmunoprecipitación de Cromatina , Células Madre Embrionarias/citología , N-Metiltransferasa de Histona-Lisina , Histonas/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Metilación , Ratones , Proteína de la Leucemia Mieloide-Linfoide/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Análisis de Secuencia de ADN , Activación TranscripcionalRESUMEN
The commitment of stem cells to differentiate into osteoblasts is a highly regulated and complex process that involves the coordination of extrinsic signals and intrinsic transcriptional machinery. While rodent osteoblastic differentiation has been extensively studied, research on human osteogenesis has been limited by cell sources and existing models. Here, we systematically dissect human pluripotent stem cell-derived osteoblasts to identify functional membrane proteins and their downstream transcriptional networks involved in human osteogenesis. Our results reveal an enrichment of type II transmembrane serine protease CORIN in humans but not rodent osteoblasts. Functional analyses demonstrated that CORIN depletion significantly impairs osteogenesis. Genome-wide chromatin immunoprecipitation enrichment and mechanistic studies show that p38 MAPK-mediated CCAAT enhancer binding protein delta (CEBPD) upregulation is required for CORIN-modulated osteogenesis. Contrastingly, the type I transmembrane heparan sulfate proteoglycan SDC1 enriched in mesenchymal stem cells exerts a negative regulatory effect on osteogenesis through a similar mechanism. Chromatin immunoprecipitation-seq, bulk and single-cell transcriptomes, and functional validations indicated that CEBPD plays a critical role in controlling osteogenesis. In summary, our findings uncover previously unrecognized CORIN-mediated CEBPD transcriptomic networks in driving human osteoblast lineage commitment.
RESUMEN
The RB1 gene is frequently mutated in human cancers but its role in tumorigenesis remains incompletely defined. Using an induced pluripotent stem cell (iPSC) model of hereditary retinoblastoma (RB), we report that the spliceosome is an up-regulated target responding to oncogenic stress in RB1-mutant cells. By investigating transcriptomes and genome occupancies in RB iPSCderived osteoblasts (OBs), we discover that both E2F3a, which mediates spliceosomal gene expression, and pRB, which antagonizes E2F3a, coregulate more than one-third of spliceosomal genes by cobinding to their promoters or enhancers. Pharmacological inhibition of the spliceosome in RB1-mutant cells leads to global intron retention, decreased cell proliferation, and impaired tumorigenesis. Tumor specimen studies and genome-wide TCGA (The Cancer Genome Atlas) expression profile analyses support the clinical relevance of pRB and E2F3a in modulating spliceosomal gene expression in multiple cancer types including osteosarcoma (OS). High levels of pRB/E2F3aregulated spliceosomal genes are associated with poor OS patient survival. Collectively, these findings reveal an undiscovered connection between pRB, E2F3a, the spliceosome, and tumorigenesis, pointing to the spliceosomal machinery as a potentially widespread therapeutic vulnerability of pRB-deficient cancers.
Asunto(s)
Neoplasias Óseas , Carcinogénesis , Factor de Transcripción E2F3 , Regulación Neoplásica de la Expresión Génica , Células Madre Pluripotentes Inducidas , Osteosarcoma , Proteínas de Unión a Retinoblastoma , Empalmosomas , Ubiquitina-Proteína Ligasas , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Carcinogénesis/genética , Factor de Transcripción E2F3/genética , Factor de Transcripción E2F3/metabolismo , Genes de Retinoblastoma , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Mutación , Osteosarcoma/genética , Osteosarcoma/patología , Neoplasias de la Retina/genética , Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/genética , Proteínas de Unión a Retinoblastoma/metabolismo , Empalmosomas/genética , Empalmosomas/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismoRESUMEN
The pluripotent state is not solely governed by the action of the core transcription factors OCT4, SOX2, and NANOG, but also by a series of co-transcriptional and post-transcriptional events, including alternative splicing (AS) and the interaction of RNA-binding proteins (RBPs) with defined subpopulations of RNAs. Zinc Finger Protein 207 (ZFP207) is an essential transcription factor for mammalian embryonic development. Here, we employ multiple functional analyses to characterize its role in mouse embryonic stem cells (ESCs). We find that ZFP207 plays a pivotal role in ESC maintenance, and silencing of Zfp207 leads to severe neuroectodermal differentiation defects. In striking contrast to human ESCs, mouse ZFP207 does not transcriptionally regulate neuronal and stem cell-related genes but exerts its effects by controlling AS networks and by acting as an RBP. Our study expands the role of ZFP207 in maintaining ESC identity, and underscores the functional versatility of ZFP207 in regulating neural fate commitment.
Asunto(s)
Empalme Alternativo , Proteínas de Ciclo Celular/metabolismo , Proteínas de Unión al ADN/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , ARN , Animales , Diferenciación Celular/genética , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , ARN/metabolismoRESUMEN
Rothmund-Thomson syndrome (RTS) is an autosomal recessive genetic disorder characterized by poikiloderma, small stature, skeletal anomalies, sparse brows/lashes, cataracts, and predisposition to cancer. Type 2 RTS patients with biallelic RECQL4 pathogenic variants have multiple skeletal anomalies and a significantly increased incidence of osteosarcoma. Here, we generated RTS patient-derived induced pluripotent stem cells (iPSCs) to dissect the pathological signaling leading to RTS patient-associated osteosarcoma. RTS iPSC-derived osteoblasts showed defective osteogenic differentiation and gain of in vitro tumorigenic ability. Transcriptome analysis of RTS osteoblasts validated decreased bone morphogenesis while revealing aberrantly upregulated mitochondrial respiratory complex I gene expression. RTS osteoblast metabolic assays demonstrated elevated mitochondrial respiratory complex I function, increased oxidative phosphorylation (OXPHOS), and increased ATP production. Inhibition of mitochondrial respiratory complex I activity by IACS-010759 selectively suppressed cellular respiration and cell proliferation of RTS osteoblasts. Furthermore, systems analysis of IACS-010759-induced changes in RTS osteoblasts revealed that chemical inhibition of mitochondrial respiratory complex I impaired cell proliferation, induced senescence, and decreased MAPK signaling and cell cycle associated genes, but increased H19 and ribosomal protein genes. In summary, our study suggests that mitochondrial respiratory complex I is a potential therapeutic target for RTS-associated osteosarcoma and provides future insights for clinical treatment strategies.
Asunto(s)
Complejo I de Transporte de Electrón/genética , Osteosarcoma/genética , ARN Largo no Codificante/genética , RecQ Helicasas/genética , Síndrome Rothmund-Thomson/genética , Adenosina Trifosfato/biosíntesis , Proliferación Celular/efectos de los fármacos , Respiración de la Célula/efectos de los fármacos , Senescencia Celular/genética , Complejo I de Transporte de Electrón/antagonistas & inhibidores , Regulación del Desarrollo de la Expresión Génica/genética , Humanos , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Células Madre Pluripotentes Inducidas/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/genética , Mutación/genética , Osteoblastos/efectos de los fármacos , Osteogénesis/genética , Osteosarcoma/complicaciones , Osteosarcoma/patología , Oxadiazoles/farmacología , Fosforilación Oxidativa/efectos de los fármacos , Piperidinas/farmacología , Síndrome Rothmund-Thomson/complicaciones , Síndrome Rothmund-Thomson/patologíaRESUMEN
Nanog facilitates embryonic stem cell self-renewal and induced pluripotent stem cell generation during the final stage of reprogramming. From a genome-wide small interfering RNA screen using a Nanog-GFP reporter line, we discovered opposing effects of Snai1 and Snai2 depletion on Nanog promoter activity. We further discovered mutually repressive expression profiles and opposing functions of Snai1 and Snai2 during Nanog-driven reprogramming. We found that Snai1, but not Snai2, is both a transcriptional target and protein partner of Nanog in reprogramming. Ectopic expression of Snai1 or depletion of Snai2 greatly facilitates Nanog-driven reprogramming. Snai1 (but not Snai2) and Nanog cobind to and transcriptionally activate pluripotency-associated genes including Lin28 and miR-290-295. Ectopic expression of miR-290-295 cluster genes partially rescues reprogramming inefficiency caused by Snai1 depletion. Our study thus uncovers the interplay between Nanog and mesenchymal factors Snai1 and Snai2 in the transcriptional regulation of pluripotency-associated genes and miRNAs during the Nanog-driven reprogramming process.
Asunto(s)
Proteínas de Homeodominio/fisiología , Factores de Transcripción/fisiología , Animales , Sitios de Unión , Diferenciación Celular/genética , Línea Celular , Regulación de la Expresión Génica , Células HEK293 , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Células Madre Pluripotentes Inducidas , Ratones , Proteína Homeótica Nanog , Regiones Promotoras Genéticas , Interferencia de ARN , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Osteosarcoma (OS), the most common primary bone tumor, is highly metastatic with high chemotherapeutic resistance and poor survival rates. Using induced pluripotent stem cells (iPSCs) generated from Li-Fraumeni syndrome (LFS) patients, we investigate an oncogenic role of secreted frizzled-related protein 2 (SFRP2) in p53 mutation-associated OS development. Interestingly, we find that high SFRP2 expression in OS patient samples correlates with poor survival. Systems-level analyses identified that expression of SFRP2 increases during LFS OS development and can induce angiogenesis. Ectopic SFRP2 overexpression in normal osteoblast precursors is sufficient to suppress normal osteoblast differentiation and to promote OS phenotypes through induction of oncogenic molecules such as FOXM1 and CYR61 in a ß-catenin-independent manner. Conversely, inhibition of SFRP2, FOXM1, or CYR61 represses the tumorigenic potential. In summary, these findings demonstrate the oncogenic role of SFRP2 in the development of p53 mutation-associated OS and that inhibition of SFRP2 is a potential therapeutic strategy.
Asunto(s)
Neoplasias Óseas/genética , Carcinogénesis/genética , Síndrome de Li-Fraumeni/patología , Proteínas de la Membrana/genética , Osteosarcoma/genética , Proteína p53 Supresora de Tumor/genética , Animales , Neoplasias Óseas/patología , Línea Celular Tumoral , Proteína 61 Rica en Cisteína/antagonistas & inhibidores , Proteína 61 Rica en Cisteína/genética , Proteína Forkhead Box M1/antagonistas & inhibidores , Proteína Forkhead Box M1/genética , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome de Li-Fraumeni/genética , Masculino , Proteínas de la Membrana/antagonistas & inhibidores , Ratones , Ratones Desnudos , Neovascularización Patológica/genética , Osteoblastos/citología , Osteosarcoma/patologíaRESUMEN
The Kruppel-like transcription factor zinc finger protein (ZNF)217 (mouse homolog ZFP217) contributes to tumorigenesis by dysregulating gene expression programs. The newly discovered molecular function of ZFP217 in controlling N6-methyladenosine (m6A) deposition in embryonic stem cells (ESCs) sheds new light on the role of this transcription factor in tumor development.
Asunto(s)
Adenosina/análogos & derivados , Carcinogénesis/genética , Regulación Neoplásica de la Expresión Génica , Procesamiento Postranscripcional del ARN , ARN Mensajero/metabolismo , Transactivadores/metabolismo , Adenosina/metabolismo , Animales , Cadherinas/genética , Cadherinas/metabolismo , Carcinogénesis/metabolismo , Carcinogénesis/patología , Cromatina/química , Cromatina/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteína Potenciadora del Homólogo Zeste 2/genética , Proteína Potenciadora del Homólogo Zeste 2/metabolismo , Histona Desacetilasas/genética , Histona Desacetilasas/metabolismo , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Metilación , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Células Madre Embrionarias de Ratones/patología , ARN Mensajero/genética , Transactivadores/genética , Transcripción GenéticaRESUMEN
Proinflammatory cytokine TNFα plays critical roles in promoting malignant cell proliferation, angiogenesis, and tumor metastasis in many cancers. However, the mechanism of TNFα-mediated tumor development remains unclear. Here, we show that IKKα, an important downstream kinase of TNFα, interacts with and phosphorylates FOXA2 at S107/S111, thereby suppressing FOXA2 transactivation activity and leading to decreased NUMB expression, and further activates the downstream NOTCH pathway and promotes cell proliferation and tumorigenesis. Moreover, we found that levels of IKKα, pFOXA2 (S107/111), and activated NOTCH1 were significantly higher in hepatocellular carcinoma tumors than in normal liver tissues and that pFOXA2 (S107/111) expression was positively correlated with IKKα and activated NOTCH1 expression in tumor tissues. Therefore, dysregulation of NUMB-mediated suppression of NOTCH1 by TNFα/IKKα-associated FOXA2 inhibition likely contributes to inflammation-mediated cancer pathogenesis. Here, we report a TNFα/IKKα/FOXA2/NUMB/NOTCH1 pathway that is critical for inflammation-mediated tumorigenesis and may provide a target for clinical intervention in human cancer.
Asunto(s)
Carcinoma Hepatocelular/metabolismo , Transformación Celular Neoplásica/metabolismo , Factor Nuclear 3-beta del Hepatocito/genética , Quinasa I-kappa B/metabolismo , Neoplasias Hepáticas/metabolismo , Receptor Notch1/metabolismo , Animales , Carcinoma Hepatocelular/patología , Proliferación Celular , Regulación Neoplásica de la Expresión Génica , Factor Nuclear 3-beta del Hepatocito/metabolismo , Humanos , Neoplasias Hepáticas Experimentales/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Modelos Biológicos , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Fosforilación , Receptor Notch1/genética , Transducción de Señal , Factor de Necrosis Tumoral alfa/metabolismoRESUMEN
In 2006, Noble Prize laureate Shinya Yamanaka discovered that a set of transcription factors can reprogram terminally differentiated somatic cells to a pluripotent stem cell state. Since then, induced pluripotent stem cells (iPSCs) have come into the public spotlight. Amidst a growing field of promising clinical uses of iPSCs in recent years, cancer disease modeling has emerged as a particularly promising and rapidly translatable application of iPSCs. Technological advances in genome editing over the past few years have facilitated increasingly rapid progress in generation of iPSCs with clearly defined genetic backgrounds to complement existing patient-derived models. Improved protocols for differentiation of iPSCs, engineered iPSCs and embryonic stem cells (ESCs) now permit the study of disease biology in the majority of somatic cell types. Here, we highlight current efforts to create patient-derived iPSC disease models to study various cancer types. We review the advantages and current challenges of using iPSCs in cancer disease modeling.
Asunto(s)
Células Madre Embrionarias , Células Madre Pluripotentes Inducidas , Células Madre Pluripotentes , Diferenciación Celular , Humanos , Neoplasias , Factores de TranscripciónRESUMEN
IkappaB kinase beta (IKKbeta) is involved in tumor development and progression through activation of the nuclear factor (NF)-kappaB pathway. However, the molecular mechanism that regulates IKKbeta degradation remains largely unknown. Here, we show that a Cullin 3 (CUL3)-based ubiquitin ligase, Kelch-like ECH-associated protein 1 (KEAP1), is responsible for IKKbeta ubiquitination. Depletion of KEAP1 led to the accumulation and stabilization of IKKbeta and to upregulation of NF-kappaB-derived tumor angiogenic factors. A systematic analysis of the CUL3, KEAP1, and RBX1 genomic loci revealed a high percentage of genome loss and missense mutations in human cancers that failed to facilitate IKKbeta degradation. Our results suggest that the dysregulation of KEAP1-mediated IKKbeta ubiquitination may contribute to tumorigenesis.
Asunto(s)
Quinasa I-kappa B/metabolismo , Péptidos y Proteínas de Señalización Intracelular/fisiología , FN-kappa B/metabolismo , Transducción de Señal/fisiología , Animales , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Línea Celular , Línea Celular Tumoral , Proteínas Cullin/genética , Proteínas Cullin/metabolismo , Variaciones en el Número de Copia de ADN/genética , Femenino , Expresión Génica/efectos de los fármacos , Expresión Génica/genética , Humanos , Quinasa I-kappa B/genética , Interleucina-8/genética , Estimación de Kaplan-Meier , Proteína 1 Asociada A ECH Tipo Kelch , Ratones , Mutación/fisiología , Neoplasias/genética , Neoplasias/metabolismo , Neovascularización Fisiológica/genética , Unión Proteica/fisiología , Dominios y Motivos de Interacción de Proteínas/fisiología , ARN Interferente Pequeño/genética , Transducción de Señal/efectos de los fármacos , Factor de Transcripción ReIA/metabolismo , Transfección , Factor de Necrosis Tumoral alfa/farmacología , Ubiquitinación/fisiologíaRESUMEN
The generation of reprogrammed induced pluripotent stem cells (iPSCs) from patients with defined genetic disorders holds the promise of increased understanding of the aetiologies of complex diseases and may also facilitate the development of novel therapeutic interventions. We have generated iPSCs from patients with LEOPARD syndrome (an acronym formed from its main features; that is, lentigines, electrocardiographic abnormalities, ocular hypertelorism, pulmonary valve stenosis, abnormal genitalia, retardation of growth and deafness), an autosomal-dominant developmental disorder belonging to a relatively prevalent class of inherited RAS-mitogen-activated protein kinase signalling diseases, which also includes Noonan syndrome, with pleomorphic effects on several tissues and organ systems. The patient-derived cells have a mutation in the PTPN11 gene, which encodes the SHP2 phosphatase. The iPSCs have been extensively characterized and produce multiple differentiated cell lineages. A major disease phenotype in patients with LEOPARD syndrome is hypertrophic cardiomyopathy. We show that in vitro-derived cardiomyocytes from LEOPARD syndrome iPSCs are larger, have a higher degree of sarcomeric organization and preferential localization of NFATC4 in the nucleus when compared with cardiomyocytes derived from human embryonic stem cells or wild-type iPSCs derived from a healthy brother of one of the LEOPARD syndrome patients. These features correlate with a potential hypertrophic state. We also provide molecular insights into signalling pathways that may promote the disease phenotype.
Asunto(s)
Células Madre Pluripotentes Inducidas/patología , Síndrome LEOPARD/patología , Modelos Biológicos , Medicina de Precisión , Adulto , Diferenciación Celular , Línea Celular , Linaje de la Célula , Células Cultivadas , Células Madre Embrionarias/metabolismo , Activación Enzimática , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Perfilación de la Expresión Génica , Proteínas de Homeodominio/genética , Humanos , Células Madre Pluripotentes Inducidas/enzimología , Células Madre Pluripotentes Inducidas/metabolismo , Síndrome LEOPARD/tratamiento farmacológico , Síndrome LEOPARD/metabolismo , Masculino , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Factores de Transcripción NFATC/genética , Factores de Transcripción NFATC/metabolismo , Proteína Homeótica Nanog , Factor 3 de Transcripción de Unión a Octámeros/genética , Fosfoproteínas/análisis , Reacción en Cadena de la Polimerasa , Proteína Tirosina Fosfatasa no Receptora Tipo 11/genética , Proteína Tirosina Fosfatasa no Receptora Tipo 11/metabolismo , Factores de Transcripción SOXB1/genéticaRESUMEN
BACKGROUND: Chemical or small interfering (si) RNA screens measure the effects of many independent experimental conditions, each applied to a population of cells (e.g., all of the cells in a well). High-content screens permit a readout (e.g., fluorescence, luminescence, cell morphology) from each cell in the population. Most analysis approaches compare the average effect on each population, precluding identification of outliers that affect the distribution of the reporter in the population but not its average. Other approaches only measure changes to the distribution with a single parameter, precluding accurate distinction and clustering of interesting outlier distributions. RESULTS: We describe a methodology to identify outlier conditions by considering the cell-level measurements from each condition as a sample of an underlying distribution. With appropriate selection of a distance metric, all effects can be embedded in a fixed-dimensionality Euclidean basis, facilitating identification and clustering of biologically interesting outliers. We demonstrate that measurement of distances with the Hellinger distance metric offers substantial computational efficiencies over alternative metrics. We validate this methodology using an RNA interference (RNAi) screen in mouse embryonic stem cells (ESC) with a Nanog reporter. The methodology clusters effects of multiple control siRNAs into their true identities better than conventional approaches describing the median cell fluorescence or the commonly used Kolmogorov-Smirnov distance between the observed fluorescence distribution and the null distribution. It identifies outlier genes with effects on the reporter distribution that would have been missed by other methods. Among them, siRNA targeting Chek1 leads to a wider Nanog reporter fluorescence distribution. Similarly, siRNA targeting Med14 or Med27 leads to a narrower Nanog reporter fluorescence distribution. We confirm the roles of these three genes in regulating pluripotency by mRNA expression and alkaline phosphatase staining using independent short hairpin (sh) RNAs. CONCLUSIONS: Using our methodology, we describe each experimental condition by a probability distribution. Measuring distances between probability distributions permits a multivariate rather than univariate readout. Clustering points derived from these distances allows us to obtain greater biological insight than methods based solely on single parameters. We find several outliers from a mouse ESC RNAi screen that we confirm to be pluripotency regulators. Many of these outliers would have been missed by other analysis methods.
Asunto(s)
Biología Computacional/métodos , Proteínas de Homeodominio/genética , Interferencia de ARN , Animales , Diferenciación Celular/efectos de los fármacos , Línea Celular , Análisis por Conglomerados , Genes Reporteros , Genoma , Complejo Mediador/antagonistas & inhibidores , Complejo Mediador/genética , Complejo Mediador/metabolismo , Ratones , Células Madre Embrionarias de Ratones/citología , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Proteína Homeótica Nanog , Regiones Promotoras Genéticas , ARN Interferente Pequeño/metabolismo , Tretinoina/farmacologíaRESUMEN
The retinoblastoma (RB) transcriptional corepressor 1 (RB1) is a critical tumor suppressor gene, governing diverse cellular processes implicated in cancer biology. Dysregulation or deletion in RB1 contributes to the development and progression of various cancers, making it a prime target for therapeutic intervention. RB1's canonical function in cell cycle control and DNA repair mechanisms underscores its significance in restraining aberrant cell growth and maintaining genomic stability. Understanding the complex interplay between RB1 and cellular pathways is beneficial to fully elucidate its tumor-suppressive role across different cancer types and for therapeutic development. As a result, investigating vulnerabilities arising from RB1 deletion-associated mechanisms offers promising avenues for targeted therapy. Recently, several findings highlighted multiple methods as a promising strategy for combating tumor growth driven by RB1 loss, offering potential clinical benefits in various cancer types. This review summarizes the multifaceted role of RB1 in cancer biology and its implications for targeted therapy.
RESUMEN
The eukaryotic Mediator, comprising a large Core (cMED) and a dissociable CDK8 kinase module (CKM), regulates RNA Polymerase II (Pol II)-dependent transcription. cMED recruits Pol II and promotes pre-initiation complex (PIC) formation in a manner inhibited by the CKM, which is also implicated in post-initiation control of gene expression. Herein we report cryo-electron microscopy structures of the human complete Mediator and its CKM, which explains the basis for CKM inhibition of cMED-activated transcription. The CKM binds to cMED through an intrinsically disordered region (IDR) in MED13 and HEAT repeats in MED12. The CKM inhibits transcription by allocating its MED13 IDR to occlude binding of Pol II and MED26 to cMED and further obstructing cMED-PIC assembly through steric hindrance with TFIIH and the +1 nucleosome. Notably, MED12 binds to the cMED Hook, positioning CDK8 downstream of the transcription start site, which sheds new light on its stimulatory function in post-initiation events.
RESUMEN
Fetal liver and adult bone marrow hematopoietic stem cells (HSCs) renew or differentiate into committed progenitors to generate all blood cells. PRDM16 is involved in human leukemic translocations and is expressed highly in some karyotypically normal acute myeloblastic leukemias. As many genes involved in leukemogenic fusions play a role in normal hematopoiesis, we analyzed the role of Prdm16 in the biology of HSCs using Prdm16-deficient mice. We show here that, within the hematopoietic system, Prdm16 is expressed very selectively in the earliest stem and progenitor compartments, and, consistent with this expression pattern, is critical for the establishment and maintenance of the HSC pool during development and after transplantation. Prdm16 deletion enhances apoptosis and cycling of HSCs. Expression analysis revealed that Prdm16 regulates a remarkable number of genes that, based on knockout models, both enhance and suppress HSC function, and affect quiescence, cell cycling, renewal, differentiation, and apoptosis to various extents. These data suggest that Prdm16 may be a critical node in a network that contains negative and positive feedback loops and integrates HSC renewal, quiescence, apoptosis, and differentiation.
Asunto(s)
Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Hematopoyesis/fisiología , Células Madre Hematopoyéticas/citología , Factores de Transcripción/metabolismo , Animales , Apoptosis/fisiología , Separación Celular , Proteínas de Unión al ADN/genética , Citometría de Flujo , Expresión Génica , Perfilación de la Expresión Génica , Genotipo , Células Madre Hematopoyéticas/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genéticaRESUMEN
Missense mutations in the DNA binding domain of p53 are characterized as structural or contact mutations based on their effect on the conformation of the protein. These mutations show gain-of-function (GOF) activities, such as promoting increased metastatic incidence compared with p53 loss, often mediated by the interaction of mutant p53 with a set of transcription factors. These interactions are largely context specific. To understand the mechanisms by which p53 DNA binding domain mutations drive osteosarcoma progression, we created mouse models, in which either the p53 structural mutant p53R172H or the contact mutant p53R245W are expressed specifically in osteoblasts, yielding osteosarcoma tumor development. Survival significantly decreased and metastatic incidence increased in mice expressing p53 mutants compared with p53-null mice, suggesting GOF. RNA sequencing of primary osteosarcomas revealed vastly different gene expression profiles between tumors expressing the missense mutants and p53-null tumors. Further, p53R172H and p53R245W each regulated unique transcriptomes and pathways through interactions with a distinct repertoire of transcription factors. Validation assays showed that p53R245W, but not p53R172H, interacts with KLF15 to drive migration and invasion in osteosarcoma cell lines and promotes metastasis in allogeneic transplantation models. In addition, analyses of p53R248W chromatin immunoprecipitation peaks showed enrichment of KLF15 motifs in human osteoblasts. Taken together, these data identify unique mechanisms of action of the structural and contact mutants of p53. SIGNIFICANCE: The p53 DNA binding domain contact mutant p53R245W, but not the structural mutant p53R172H, interacts with KLF15 to drive metastasis in somatic osteosarcoma, providing a potential vulnerability in tumors expressing p53R245W mutation.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Ratones , Humanos , Animales , Proteína p53 Supresora de Tumor/genética , Osteosarcoma/patología , Mutación , Ratones Noqueados , Neoplasias Óseas/genética , Neoplasias Óseas/patología , Factores de Transcripción/metabolismo , ADN , Línea Celular TumoralRESUMEN
The coronavirus disease (COVID-19) pandemic has increased pressure to develop low-cost, compact, user-friendly, and ubiquitous virus sensors for monitoring infection outbreaks in communities and preventing economic damage resulting from city lockdowns. As proof of concept, we developed a wearable paper-based virus sensor based on a molecular imprinting technique, using a conductive polyaniline (PANI) polymer to detect the lentivirus as a test sample. This sensor detected the lentivirus with a 4181 TU/mL detection limit in liquid and 0.33% to 2.90% detection efficiency in aerosols at distances ranging from 30 cm to 60 cm. For fabrication, a mixture of a PANI monomer solution and virus were polymerized together to form a conductive PANI sensing element on a polyethylene terephthalate (PET) paper substrate. The sensing element exhibited formation of virus recognition sites after the removal of the virus via ultrasound sonication. A dry measurement technique was established that showed aerosol virus detection by the molecularly imprinted sensors within 1.5 h of virus spraying. This was based on the mechanism via which dispensing virus droplets on the PANI sensing element induced hybridization of the virus and molecularly imprinted virus recognition templates in PANI, influencing the conductivity of the PANI film upon drying. Interestingly, the paper-based virus sensor was easily integrated with a wearable face mask for the detection of viruses in aerosols. Since the paper sensor with molecular imprinting of virus recognition sites showed excellent stability in dry conditions for long periods of time, unlike biological reagents, this wearable biosensor will offer an alternative approach to monitoring virus infections in communities.
RESUMEN
Cancer-relevant mutations in the oligomerization domain (OD) of the p53 tumor suppressor protein, unlike those in the DNA binding domain, have not been well elucidated. Here, we characterized the germline OD mutant p53(A347D), which occurs in cancer-prone Li-Fraumeni syndrome (LFS) patients. Unlike wild-type p53, mutant p53(A347D) cannot form tetramers and exists as a hyperstable dimeric protein. Further, p53(A347D) cannot bind or transactivate the majority of canonical p53 target genes. Isogenic cell lines harboring either p53(A347D) or no p53 yield comparable tumorigenic properties, yet p53(A347D) displays remarkable neomorphic activities. Cells bearing p53(A347D) possess a distinct transcriptional profile and undergo metabolic reprogramming. Further, p53(A347D) induces striking mitochondrial network aberration and associates with mitochondria to drive apoptotic cell death upon topoisomerase II inhibition in the absence of transcription. Thus, dimer-forming p53 demonstrates both loss-of-function (LOF) and gain-of-function (GOF) properties compared with the wild-type form of the protein. SIGNIFICANCE: A mutant p53 (A347D), which can only form dimers, is associated with increased cancer susceptibility in LFS individuals. We found that this mutant wields a double-edged sword, driving tumorigenesis through LOF while gaining enhanced apoptogenic activity as a new GOF, thereby yielding a potential vulnerability to select therapeutic approaches. See related commentary by Stieg et al., p. 1046. See related article by Gencel-Augusto et al., p. 1230. This article is highlighted in the In This Issue feature, p. 1027.