RESUMO
Some transcription factors that specifically bind double-stranded DNA appear to also function as RNA-binding proteins. Here, we demonstrate that the transcription factor Sox2 is able to directly bind RNA in vitro as well as in mouse and human cells. Sox2 targets RNA via a 60-amino-acid RNA binding motif (RBM) positioned C-terminally of the DNA binding high mobility group (HMG) box. Sox2 can associate with RNA and DNA simultaneously to form ternary RNA/Sox2/DNA complexes. Deletion of the RBM does not affect selection of target genes but mitigates binding to pluripotency related transcripts, switches exon usage and impairs the reprogramming of somatic cells to a pluripotent state. Our findings designate Sox2 as a multi-functional factor that associates with RNA whilst binding to cognate DNA sequences, suggesting that it may co-transcriptionally regulate RNA metabolism during somatic cell reprogramming.
Assuntos
Reprogramação Celular/genética , DNA/metabolismo , RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Motivos de Aminoácidos , Animais , Células Cultivadas , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Ligação Proteica , Domínios Proteicos , Splicing de RNA , Fatores de Transcrição SOXB1/químicaRESUMO
We combine the labeling of newly transcribed RNAs with 5-ethynyluridine with the characterization of bound proteins. This approach, named capture of the newly transcribed RNA interactome using click chemistry (RICK), systematically captures proteins bound to a wide range of RNAs, including nascent RNAs and traditionally neglected nonpolyadenylated RNAs. RICK has identified mitotic regulators amongst other novel RNA-binding proteins with preferential affinity for nonpolyadenylated RNAs, revealed a link between metabolic enzymes/factors and nascent RNAs, and expanded the known RNA-bound proteome of mouse embryonic stem cells. RICK will facilitate an in-depth interrogation of the total RNA-bound proteome in different cells and systems.
Assuntos
Química Click/métodos , Proteoma/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/metabolismo , Animais , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala/métodos , Humanos , Espectrometria de Massas/métodos , Camundongos , Mapas de Interação de Proteínas , RNA/genética , Proteínas de Ligação a RNA/genética , Uridina/análogos & derivados , Uridina/químicaRESUMO
Megadose vitamin C (Vc) is one of the most enduring alternative treatments for diverse human diseases and is deeply engrafted in popular culture. Preliminary studies in the 1970s described potent effects of Vc on prolonging the survival of patients with terminal cancer, but these claims were later criticized. An improved knowledge of the pharmacokinetics of Vc and recent reports using cancer cell lines have renewed the interest in this subject. Despite these findings, using Vc as an adjuvant for anticancer therapy remains questionable, among other things because there is no proper mechanistic understanding. Here, we show that a Warburg effect triggered by activation of the hypoxia-inducible factor (HIF) pathway greatly enhances Vc-induced toxicity in multiple cancer cell lines, including von Hippel-Lindau (VHL)-defective renal cancer cells. HIF increases the intracellular uptake of oxidized Vc through its transcriptional target glucose transporter 1 (GLUT1), synergizing with the uptake of its reduced form through sodium-dependent Vc transporters. The resulting high levels of intracellular Vc induce oxidative stress and massive DNA damage, which then causes metabolic exhaustion by depleting cellular ATP reserves. HIF-positive cells are particularly sensitive to Vc-induced ATP reduction because they mostly rely on the rather inefficient glycolytic pathway for energy production. Thus, our experiments link Vc-induced toxicity and cancer metabolism, providing a new explanation for the preferential effect of Vc on cancer cells.
Assuntos
Ácido Ascórbico/farmacologia , Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Citotoxinas/farmacologia , Dano ao DNA , Neoplasias/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 1/metabolismo , Células HeLa , Humanos , Neoplasias/genética , Neoplasias/metabolismo , Neoplasias/patologia , Estresse Oxidativo/genética , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/genética , Proteína Supressora de Tumor Von Hippel-Lindau/genética , Proteína Supressora de Tumor Von Hippel-Lindau/metabolismoRESUMO
Many human diseases share a developmental origin that manifests during childhood or maturity. Aneuploid syndromes are caused by supernumerary or reduced number of chromosomes and represent an extreme example of developmental disease, as they have devastating consequences before and after birth. Investigating how alterations in gene dosage drive these conditions is relevant because it might help treat some clinical aspects. It may also provide explanations as to how quantitative differences in gene expression determine phenotypic diversity and disease susceptibility among natural populations. Here, we aimed to produce induced pluripotent stem cell (iPSC) lines that can be used to improve our understanding of aneuploid syndromes. We have generated iPSCs from monosomy X [Turner syndrome (TS)], trisomy 8 (Warkany syndrome 2), trisomy 13 (Patau syndrome) and partial trisomy 11;22 (Emanuel syndrome), using either skin fibroblasts from affected individuals or amniocytes from antenatal diagnostic tests. These cell lines stably maintain the karyotype of the donors and behave like embryonic stem cells in all tested assays. TS iPSCs were used for further studies including global gene expression analysis and tissue-specific directed differentiation. Multiple clones displayed lower levels of the pseudoautosomal genes ASMTL and PPP2R3B than the controls. Moreover, they could be transformed into neural-like, hepatocyte-like and heart-like cells, but displayed insufficient up-regulation of the pseudoautosomal placental gene CSF2RA during embryoid body formation. These data support that abnormal organogenesis and early lethality in TS are not caused by a tissue-specific differentiation blockade, but rather involves other abnormalities including impaired placentation.
Assuntos
Aneuploidia , Transtornos Cromossômicos/genética , Células-Tronco Pluripotentes Induzidas/citologia , Diferenciação Celular , Células Cultivadas , Transtornos Cromossômicos/metabolismo , Transtornos Cromossômicos/fisiopatologia , Feminino , Expressão Gênica , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Lactente , Masculino , Modelos GenéticosRESUMO
Directed hepatocyte differentiation from human induced pluripotent stem cells (iPSCs) potentially provides a unique platform for modeling liver genetic diseases and performing drug-toxicity screening in vitro. Wilson's disease is a genetic disease caused by mutations in the ATP7B gene, whose product is a liver transporter protein responsible for coordinated copper export into bile and blood. Interestingly, the spectrum of ATP7B mutations is vast and can influence clinical presentation (a variable spectrum of hepatic and neural manifestations), though the reason is not well understood. We describe the generation of iPSCs from a Chinese patient with Wilson's disease that bears the R778L Chinese hotspot mutation in the ATP7B gene. These iPSCs were pluripotent and could be readily differentiated into hepatocyte-like cells that displayed abnormal cytoplasmic localization of mutated ATP7B and defective copper transport. Moreover, gene correction using a self-inactivating lentiviral vector that expresses codon optimized-ATP7B or treatment with the chaperone drug curcumin could reverse the functional defect in vitro. Hence, our work describes an attractive model for studying the pathogenesis of Wilson's disease that is valuable for screening compounds or gene therapy approaches aimed to correct the abnormality. In the future, once relevant safety concerns (including the stability of the mature liver-like phenotype) and technical issues for the transplantation procedure are solved, hepatocyte-like cells from similarly genetically corrected iPSCs could be an option for autologous transplantation in Wilson's disease.
Assuntos
Adenosina Trifosfatases/genética , Proteínas de Transporte de Cátions/genética , Curcumina/uso terapêutico , Terapia Genética , Hepatócitos/metabolismo , Degeneração Hepatolenticular/tratamento farmacológico , Degeneração Hepatolenticular/patologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Adenosina Trifosfatases/metabolismo , Adenosina Trifosfatases/uso terapêutico , Sequência de Bases , Proteínas de Transporte de Cátions/metabolismo , Proteínas de Transporte de Cátions/uso terapêutico , Cobre/metabolismo , ATPases Transportadoras de Cobre , Humanos , Masculino , Pessoa de Meia-Idade , Chaperonas Moleculares/uso terapêutico , Dados de Sequência Molecular , Mutação/genética , Transporte Proteico , Frações Subcelulares/metabolismoRESUMO
MicroRNAs (miRNAs) are emerging critical regulators of cell function that frequently reside in clusters throughout the genome. They influence a myriad of cell functions, including the generation of induced pluripotent stem cells, also termed reprogramming. Here, we have successfully delivered entire miRNA clusters into reprogramming fibroblasts using retroviral vectors. This strategy avoids caveats associated with transient transfection of chemically synthesized miRNA mimics. Overexpression of 2 miRNA clusters, 106a-363 and in particular 302-367, allowed potent increases in induced pluripotent stem cell generation efficiency in mouse fibroblasts using 3 exogenous factors (Sox2, Klf4, and Oct4). Pathway analysis highlighted potential relevant effectors, including mesenchymal-to-epithelial transition, cell cycle, and epigenetic regulators. Further study showed that miRNA cluster 302-367 targeted TGFß receptor 2, promoted increased E-cadherin expression, and accelerated mesenchymal-to-epithelial changes necessary for colony formation. Our work thus provides an interesting alternative for improving reprogramming using miRNAs and adds new evidence for the emerging relationship between pluripotency and the epithelial phenotype.
Assuntos
MicroRNAs/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Animais , Adesão Celular , Células Epiteliais/citologia , Feminino , Fibroblastos/citologia , Fibroblastos/metabolismo , Células-Tronco Pluripotentes Induzidas/citologia , Fator 4 Semelhante a Kruppel , Masculino , Mesoderma/citologia , Camundongos , MicroRNAs/genética , Fenótipo , Células-Tronco/citologiaRESUMO
Forced expression of selected transcription factors can transform somatic cells into embryonic stem cell (ESC)-like cells, termed induced pluripotent stem cells (iPSCs). There is no consensus regarding the preferred tissue from which to harvest donor cells for reprogramming into iPSCs, and some donor cell types may be more prone than others to accumulation of epigenetic imprints and somatic cell mutations. Here, we present a simple, reproducible, noninvasive method for generating human iPSCs from renal tubular cells present in urine. This procedure eliminates many problems associated with other protocols, and the resulting iPSCs display an excellent ability to differentiate. These data suggest that urine may be a preferred source for generating iPSCs.
Assuntos
Células-Tronco Pluripotentes Induzidas , Túbulos Renais/citologia , Urina/citologia , Idoso , Feminino , Técnicas de Transferência de Genes , Humanos , Masculino , Adulto JovemRESUMO
The recent discovery of induced pluripotent stem cell (iPSC) technology provides an invaluable tool for creating in vitro representations of human genetic conditions. This is particularly relevant for those diseases that lack adequate animal models or where the species comparison is difficult, e.g. imprinting diseases such as the neurogenetic disorder Prader-Willi syndrome (PWS). However, recent reports have unveiled transcriptional and functional differences between iPSCs and embryonic stem cells that in cases are attributable to imprinting errors. This has suggested that human iPSCs may not be useful to model genetic imprinting diseases. Here, we describe the generation of iPSCs from a patient with PWS bearing a partial translocation of the paternally expressed chromosome 15q11-q13 region to chromosome 4. The resulting iPSCs match all standard criteria of bona fide reprogramming and could be readily differentiated into tissues derived from the three germ layers, including neurons. Moreover, these iPSCs retain a high level of DNA methylation in the imprinting center of the maternal allele and show concomitant reduced expression of the disease-associated small nucleolar RNA HBII-85/SNORD116. These results indicate that iPSCs may be a useful tool to study PWS and perhaps other genetic imprinting diseases as well.
Assuntos
Metilação de DNA , Impressão Genômica , Células-Tronco Pluripotentes Induzidas/metabolismo , Modelos Biológicos , Síndrome de Prader-Willi/metabolismo , Desdiferenciação Celular/genética , Células Cultivadas , Cromossomos Humanos Par 15/genética , Cromossomos Humanos Par 15/metabolismo , Cromossomos Humanos Par 4/genética , Cromossomos Humanos Par 4/metabolismo , Humanos , Células-Tronco Pluripotentes Induzidas/patologia , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/patologia , RNA Nuclear Pequeno/biossíntese , RNA Nuclear Pequeno/genética , Translocação Genética/genéticaRESUMO
The umbilical cord and placenta are extra-embryonic tissues of particular interest for regenerative medicine. They share an early developmental origin and are a source of vast amounts of cells with multilineage differentiation potential that are poorly immunogenic and without controversy. Moreover, these cells are likely exempt from incorporated mutations when compared with juvenile or adult donor cells such as skin fibroblasts or keratinocytes. Here we report the efficient generation of induced pluripotent stem cells (iPSCs) from mesenchymal cells of the umbilical cord matrix (up to 0.4% of the cells became reprogrammed) and the placental amniotic membrane (up to 0.1%) using exogenous factors and a chemical mixture. iPSCs from these 2 tissues homogeneously showed human embryonic stem cell (hESC)-like characteristics including morphology, positive staining for alkaline phosphatase, normal karyotype, and expression of hESC-like markers including Nanog, Rex1, Oct4, TRA-1-60, TRA-1-80, SSEA-3, and SSEA-4. Selected clones also formed embryonic bodies and teratomas containing derivatives of the 3 germ layers, and could as well be readily differentiated into functional motor neurons. Among other things, our cell lines may prove useful for comparisons between iPSCs derived from multiple tissues regarding the extent of the epigenetic reprogramming, differentiation ability, stability of the resulting lineages, and the risk of associated abnormalities.
Assuntos
Âmnio/metabolismo , Técnicas de Cultura de Células/métodos , Regulação da Expressão Gênica , Células-Tronco Mesenquimais/citologia , Células-Tronco Pluripotentes/citologia , Cordão Umbilical/metabolismo , Animais , Linhagem Celular , Células Cultivadas/citologia , Humanos , Cariotipagem , Células-Tronco Mesenquimais/metabolismo , Camundongos , Modelos Biológicos , Neurônios Motores/metabolismo , Técnicas de Patch-Clamp , Células-Tronco Pluripotentes/metabolismo , Cordão Umbilical/citologiaRESUMO
Application of synthetic nucleoside analogues to capture newly transcribed RNAs has unveiled key features of RNA metabolism. Whether this approach could be adapted to isolate the RNA-bound proteome (RNA interactome) was, however, unexplored. We have developed a new method (capture of the newly transcribed RNA interactome using click chemistry, or RICK) for the systematic identification of RNA-binding proteins based on the incorporation of 5-ethynyluridine into newly transcribed RNAs followed by UV cross-linking and click chemistry-mediated biotinylation. The RNA-protein adducts are then isolated by affinity capture using streptavidin-coated beads. Through high-throughput RNA sequencing and mass spectrometry, the RNAs and proteins can be elucidated globally. A typical RICK experimental procedure takes only 1 d, excluding the steps of cell preparation, 5-ethynyluridine labeling, validation (silver staining, western blotting, quantitative reverse-transcription PCR (qRT-PCR) or RNA sequencing (RNA-seq)) and proteomics. Major advantages of RICK are the capture of RNA-binding proteins interacting with any type of RNA and, particularly, the ability to discern between newly transcribed and steady-state RNAs through controlled labeling. Thanks to its versatility, RICK will facilitate the characterization of the total and newly transcribed RNA interactome in different cell types and conditions.
Assuntos
Química Click , RNA , Células HeLa , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Proteômica , Análise de Sequência de RNARESUMO
The generation of induced pluripotent stem cells through somatic cell reprogramming requires a global reorganization of cellular functions. This reorganization occurs in a multi-phased manner and involves a gradual revision of both the epigenome and transcriptome. Recent studies have shown that the large-scale transcriptional changes observed during reprogramming also apply to long non-coding RNAs (lncRNAs), a type of traditionally neglected RNA species that are increasingly viewed as critical regulators of cellular function. Deeper understanding of lncRNAs in reprogramming may not only help to improve this process but also have implications for studying cell plasticity in other contexts, such as development, aging, and cancer. In this review, we summarize the current progress made in profiling and analyzing the role of lncRNAs in various phases of somatic cell reprogramming, with emphasis on the re-establishment of the pluripotency gene network and X chromosome reactivation.
Assuntos
Reprogramação Celular/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , RNA Longo não Codificante/fisiologia , Animais , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , RNA Longo não Codificante/genética , Transcriptoma , Cromossomo XRESUMO
Mouse embryonic stem cells cultured with MEK (mitogen-activated protein kinase kinase) and GSK3 (glycogen synthase kinase 3) inhibitors (2i) more closely resemble the inner cell mass of preimplantation blastocysts than those cultured with SL [serum/leukemia inhibitory factor (LIF)]. The transcriptional mechanisms governing this pluripotent ground state are unresolved. Release of promoter-proximal paused RNA polymerase II (Pol2) is a multistep process necessary for pluripotency and cell cycle gene transcription in SL. We show that ß-catenin, stabilized by GSK3 inhibition in medium with 2i, supplies transcriptional coregulators at pluripotency loci. This selectively strengthens pluripotency loci and renders them addicted to transcription initiation for productive gene body elongation in detriment to Pol2 pause release. By contrast, cell cycle genes are not bound by ß-catenin, and proliferation/self-renewal remains tightly controlled by Pol2 pause release under 2i conditions. Our findings explain how pluripotency is reinforced in the ground state and also provide a general model for transcriptional resilience/adaptation upon network perturbation in other contexts.
RESUMO
In the version of this Article originally published, in Fig. 2c, the '+' sign and 'OSKM' were superimposed in the label '+OSKM'. In Fig. 4e, in the labels, all instances of 'Ant' should have been 'Anti-'. And, in Fig. 7a, the label '0.0' was misplaced; it should have been on the colour scale bar. These figures have now been corrected in the online versions.
RESUMO
Somatic cell reprogramming by exogenous factors requires cooperation with transcriptional co-activators and co-repressors to effectively remodel the epigenetic environment. How this interplay is regulated remains poorly understood. Here, we demonstrate that NCoR/SMRT co-repressors bind to pluripotency loci to create a barrier to reprogramming with the four Yamanaka factors (OCT4, SOX2, KLF4 and c-MYC), and consequently, suppressing NCoR/SMRT significantly enhances reprogramming efficiency and kinetics. The core epigenetic subunit of the NCoR/SMRT complex, histone deacetylase 3 (HDAC3), contributes to the effects of NCoR/SMRT by inducing histone deacetylation at pluripotency loci. Among the Yamanaka factors, recruitment of NCoR/SMRT-HDAC3 to genomic loci is mostly facilitated by c-MYC. Hence, we describe how c-MYC is beneficial for the early phase of reprogramming but deleterious later. Overall, we uncover a role for NCoR/SMRT co-repressors in reprogramming and propose a dual function for c-MYC in this process.
Assuntos
Reprogramação Celular , Epigênese Genética , Células-Tronco Embrionárias Murinas/metabolismo , Correpressor 1 de Receptor Nuclear/metabolismo , Correpressor 2 de Receptor Nuclear/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Acetilação , Animais , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Histona Desacetilases/genética , Histona Desacetilases/metabolismo , Histonas/metabolismo , Humanos , Fator 4 Semelhante a Kruppel , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Camundongos , Camundongos Endogâmicos ICR , Correpressor 1 de Receptor Nuclear/genética , Correpressor 2 de Receptor Nuclear/genética , Fator 3 de Transcrição de Octâmero/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-myc/genética , Fatores de Transcrição SOXB1/genética , Fatores de Transcrição SOXB1/metabolismo , Transdução de Sinais , Fatores de TempoRESUMO
Dgcr8 knockout cells provide a great means to understand the function of microRNAs (miRNAs) in vitro and in vivo. Current strategies to study miRNA function in Dgcr8 knockout cells depend on transient transfection of chemically synthesized miRNA mimics, which is costly and not suitable for long-term study and genetic selection of miRNA function. Here, we developed a cost-effective DGCR8-independent stable miRNA expression (DISME) strategy based on a short hairpin RNA vector that can be precisely processed by DICER. Using DISME, we found that miR-294 promoted the formation of meso-endoderm lineages during embryonic stem cell differentiation. Furthermore, DISME allowed for a pooled screen of miRNA function and identified an miR-183-182 cluster of miRNAs promoting self-renewal and pluripotency in mouse embryonic stem cells. Altogether, our study demonstrates that DISME is a robust and cost-effective strategy that allows for long-term study and genetic selection of miRNA function in a Dgcr8 knockout background.
Assuntos
Diferenciação Celular , Regulação da Expressão Gênica no Desenvolvimento , MicroRNAs/genética , Células-Tronco Embrionárias Murinas/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Endoderma/citologia , Perfilação da Expressão Gênica/métodos , Mesoderma/citologia , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Proteínas de Ligação a RNA/genéticaRESUMO
Recent studies have boosted our understanding of long noncoding RNAs (lncRNAs) in numerous biological processes, but few have examined their roles in somatic cell reprogramming. Through expression profiling and functional screening, we have identified that the large intergenic noncoding RNA p21 (lincRNA-p21) impairs reprogramming. Notably, lincRNA-p21 is induced by p53 but does not promote apoptosis or cell senescence in reprogramming. Instead, lincRNA-p21 associates with the H3K9 methyltransferase SETDB1 and the maintenance DNA methyltransferase DNMT1, which is facilitated by the RNA-binding protein HNRNPK. Consequently, lincRNA-p21 prevents reprogramming by sustaining H3K9me3 and/or CpG methylation at pluripotency gene promoters. Our results provide insight into the role of lncRNAs in reprogramming and establish a novel link between p53 and heterochromatin regulation.