RESUMEN
Alternative splicing (AS) plays important roles in embryonic stem cell (ESC) differentiation. In this study, we first identified transcripts that display specific AS patterns in pluripotent human ESCs (hESCs) relative to differentiated cells. One of these encodes T-cell factor 3 (TCF3), a transcription factor that plays important roles in ESC differentiation. AS creates two TCF3 isoforms, E12 and E47, and we identified two related splicing factors, heterogeneous nuclear ribonucleoproteins (hnRNPs) H1 and F (hnRNP H/F), that regulate TCF3 splicing. We found that hnRNP H/F levels are high in hESCs, leading to high E12 expression, but decrease during differentiation, switching splicing to produce elevated E47 levels. Importantly, hnRNP H/F knockdown not only recapitulated the switch in TCF3 AS but also destabilized hESC colonies and induced differentiation. Providing an explanation for this, we show that expression of known TCF3 target E-cadherin, critical for maintaining ESC pluripotency, is repressed by E47 but not by E12.
Asunto(s)
Empalme Alternativo , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Cadherinas/metabolismo , Células Madre Embrionarias/metabolismo , Ribonucleoproteína Heterogénea-Nuclear Grupo F-H/metabolismo , Antígenos CD , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Cadherinas/genética , Diferenciación Celular/genética , Línea Celular , Células Madre Embrionarias/citología , Exones , Regulación de la Expresión Génica , Humanos , Precursores del ARN/química , ARN Mensajero/química , Secuencias Reguladoras de Ácido RibonucleicoRESUMEN
Mutant p53 impacts the expression of numerous genes at the level of transcription to mediate oncogenesis. We identified vascular endothelial growth factor receptor 2 (VEGFR2), the primary functional VEGF receptor that mediates endothelial cell vascularization, as a mutant p53 transcriptional target in multiple breast cancer cell lines. Up-regulation of VEGFR2 mediates the role of mutant p53 in increasing cellular growth in two-dimensional (2D) and three-dimensional (3D) culture conditions. Mutant p53 binds near the VEGFR2 promoter transcriptional start site and plays a role in maintaining an open conformation at that location. Relatedly, mutant p53 interacts with the SWI/SNF complex, which is required for remodeling the VEGFR2 promoter. By both querying individual genes regulated by mutant p53 and performing RNA sequencing, the results indicate that >40% of all mutant p53-regulated gene expression is mediated by SWI/SNF. We surmise that mutant p53 impacts transcription of VEGFR2 as well as myriad other genes by promoter remodeling through interaction with and likely regulation of the SWI/SNF chromatin remodeling complex. Therefore, not only might mutant p53-expressing tumors be susceptible to anti VEGF therapies, impacting SWI/SNF tumor suppressor function in mutant p53 tumors may also have therapeutic potential.
Asunto(s)
Neoplasias de la Mama/fisiopatología , Ensamble y Desensamble de Cromatina/genética , Regulación Neoplásica de la Expresión Génica , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Receptor 2 de Factores de Crecimiento Endotelial Vascular/genética , Receptor 2 de Factores de Crecimiento Endotelial Vascular/metabolismo , Línea Celular Tumoral , Proteínas Cromosómicas no Histona/metabolismo , Células HT29 , Humanos , Células MCF-7 , Mutación/genética , Nucleosomas/metabolismo , Regiones Promotoras Genéticas/genética , Unión Proteica , Conformación Proteica , Factores de Transcripción/metabolismoRESUMEN
The p53 tumor suppressor protein, a sequence-specific DNA binding transcription factor, regulates the expression of a large number of genes, in response to various forms of cellular stress. Although the protein coding target genes of p53 have been well studied, less is known about its role in regulating long noncoding genes and their functional relevance to cancer. Here we report the genome-wide identification of a large set (>1,000) of long noncoding RNAs (lncRNA), which are putative p53 targets in a colon cancer cell line and in human patient datasets from five different common types of cancer. These lncRNAs have not been annotated by other studies of normal unstressed systems. In the colon cancer cell line, a high proportion of these lncRNAs are uniquely induced by different chemotherapeutic agents that activate p53, whereas others are induced by more than one agent tested. Further, subsets of these lncRNAs independently predict overall and disease-free survival of patients across the five different common cancer types. Interestingly, both genetic alterations and patient survival associated with different lncRNAs are unique to each cancer tested, indicating extraordinary tissue-specific variability in the p53 noncoding response. The newly identified noncoding p53 target genes have allowed us to construct a classifier for tumor diagnosis and prognosis. IMPLICATIONS: Our results not only identify myriad p53-regulated long noncoding (lncRNA), they also reveal marked drug-induced, as well as tissue- and tumor-specific heterogeneity in these putative p53 targets and our findings have enabled the construction of robust classifiers for diagnosis and prognosis.
Asunto(s)
Neoplasias , ARN Largo no Codificante , Proteína p53 Supresora de Tumor , Humanos , ARN Largo no Codificante/genética , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Neoplasias/genética , Neoplasias/patología , Línea Celular Tumoral , Regulación Neoplásica de la Expresión GénicaRESUMEN
Arterial Vascular smooth muscle cells (VSMCs) play a central role in the onset and progression of atherosclerosis. Upon exposure to pathological stimuli, they can take on alternative phenotypes that, among others, have been described as macrophage like, or foam cells. VSMC foam cells make up >50% of all arterial foam cells and have been suggested to retain an even higher proportion of the cell stored lipid droplets, further leading to apoptosis, secondary necrosis, and an inflammatory response. However, the mechanism of VSMC foam cell formation is still unclear. Here, it is identified that mechanical stimulation through hypertensive pressure alone is sufficient for the phenotypic switch. Hyperspectral stimulated Raman scattering imaging demonstrates rapid lipid droplet formation and changes to lipid metabolism and changes are confirmed in ABCA1, KLF4, LDLR, and CD68 expression, cell proliferation, and migration. Further, a mechanosignaling route is identified involving Piezo1, phospholipid, and arachidonic acid signaling, as well as epigenetic regulation, whereby CUT&Tag epigenomic analysis confirms changes in the cells (lipid) metabolism and atherosclerotic pathways. Overall, the results show for the first time that VSMC foam cell formation can be triggered by mechanical stimulation alone, suggesting modulation of mechanosignaling can be harnessed as potential therapeutic strategy.
Asunto(s)
Aterosclerosis , Células Espumosas , Humanos , Células Espumosas/metabolismo , Células Espumosas/patología , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/patología , Gotas Lipídicas/metabolismo , Gotas Lipídicas/patología , Transdiferenciación Celular , Epigénesis Genética , Aterosclerosis/genéticaRESUMEN
Next generation sequencing enables large-scale analysis of mRNA expression (RNA-seq), genome variance (whole genome or exome), and transcription factor binding (ChIP-seq). Here we describe a method that allows the identification of transcription factor-binding sites in the vicinity of nonprotein-coding genes.
Asunto(s)
Secuenciación de Inmunoprecipitación de Cromatina , Biología Computacional/métodos , Regulación de la Expresión Génica , ARN no Traducido/genética , Análisis de Secuencia de ADN , Programas Informáticos , Factores de Transcripción/genética , Bases de Datos Genéticas , Secuenciación de Nucleótidos de Alto Rendimiento , Navegador WebRESUMEN
A GGGGCC repeat expansion in the C9ORF72 (C9) gene is the most common known cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. Several mechanisms have been proposed to account for its toxicity, including the possibility that reduced C9 protein levels contribute to disease. To investigate this possibility, we examined the effects of reduced C9 levels in several cell systems. We first showed that C9 knockdown (KD) in U87 glioblastoma cells results in striking morphological changes, including vacuolization and alterations in cell size. Unexpectedly, RNA analysis revealed changes in expression of many genes, including genes involved in endothelin (EDN) signaling and immune system pathways and multiple glutamate cycling genes (e.g., EAAT2), which were verified in several cell models, including astrocytes and brain samples from C9-positive patients. Consistent with deregulation of the glutamate cycling genes, elevated intracellular glutamate was detected in both KD cells and patient astrocytes. Importantly, levels of mRNAs encoding EDN1 and its receptors, known to be elevated in ALS, were sharply increased by C9 KD, likely resulting from an observed activation of NF-κB signaling and/or a possible role of a C9 isoform in gene control.