Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 36
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
1.
Methods Mol Biol ; 2021 Oct 06.
Artigo em Inglês | MEDLINE | ID: mdl-34611821

RESUMO

Embryonic stem cells (ESCs) are derived from the inner cell mass of the preimplantation blastocyst and can be maintained indefinitely in vitro without losing their properties. Given their self-renewal and pluripotency, ESCs not only represent a key tool to study early embryonic development in a dish, but also an unlimited source of material for tissue replacement in regenerative medicine. Loss-of-function assays using RNA interference are a powerful tool to understand the roles of specific genes and are facilitated by lentiviral-mediated delivery of vector-encoded shRNAs which allows long-term silencing of single or multiple genes. Here, we describe the steps for rapid and cost-effective production and testing of lentiviral particles with vector-encoded shRNAs for gene silencing in ESCs. This protocol can be easily adapted for loss-of-function assays in other pluripotent cells or culture conditions of interest.

2.
J Hepatol ; 2021 Sep 20.
Artigo em Inglês | MEDLINE | ID: mdl-34555423

RESUMO

BACKGROUND & AIMS: Autophagy-related gene 3 (ATG3) is an enzyme mainly known for its actions in the LC3 lipidation process, which is essential for autophagy. Whether ATG3 plays a role in lipid metabolism or contributes to nonalcoholic fatty liver disease (NAFLD) remains unknown. METHODS: By performing a liver proteomic analysis from mice with genetic manipulation of hepatic p63, a regulator of fatty acid metabolism, we identified ATG3 as a new target downstream of p63. ATG3 was evaluated in liver samples of patients with NAFLD. Further, genetic manipulation of ATG3 was performed in human hepatocyte cell lines, primary hepatocytes and in the liver of mice. RESULTS: ATG3 expression is induced in the liver of animal models and patients with NAFLD (both steatosis and NASH) compared with those without liver disease. Moreover, genetic knockdown of ATG3 in mice and human hepatocytes ameliorates p63- and diet-induced steatosis, while its overexpression increases the lipid load in hepatocytes. The inhibition of hepatic ATG3 improves fatty acid metabolism by reducing c-Jun N-terminal protein kinase 1 (JNK1), which increases sirtuin 1 (SIRT1), carnitine palmitoiltransferase I (CPT1a), and mitochondrial function. Hepatic knockdown of SIRT1 and CPT1a blunts the effects of ATG3 on mitochondrial activity. Unexpectedly, these effects are independent of an autophagic action. CONCLUSIONS: Collectively, these findings indicate that ATG3 is a novel protein implicated in the development of steatosis. LAY SUMMARY: We show that autophagy-related gene 3 (ATG3) contributes to the progression of NAFLD in humans and mice. Hepatic knockdown of ATG3 ameliorates the development of NAFLD, by stimulating SIRT1, CPT1a and mitochondrial function. Thus, ATG3 is an important factor implicated in steatosis.

3.
Nat Commun ; 12(1): 5068, 2021 08 20.
Artigo em Inglês | MEDLINE | ID: mdl-34417460

RESUMO

p53 regulates several signaling pathways to maintain the metabolic homeostasis of cells and modulates the cellular response to stress. Deficiency or excess of nutrients causes cellular metabolic stress, and we hypothesized that p53 could be linked to glucose maintenance. We show here that upon starvation hepatic p53 is stabilized by O-GlcNAcylation and plays an essential role in the physiological regulation of glucose homeostasis. More specifically, p53 binds to PCK1 promoter and regulates its transcriptional activation, thereby controlling hepatic glucose production. Mice lacking p53 in the liver show a reduced gluconeogenic response during calorie restriction. Glucagon, adrenaline and glucocorticoids augment protein levels of p53, and administration of these hormones to p53 deficient human hepatocytes and to liver-specific p53 deficient mice fails to increase glucose levels. Moreover, insulin decreases p53 levels, and over-expression of p53 impairs insulin sensitivity. Finally, protein levels of p53, as well as genes responsible of O-GlcNAcylation are elevated in the liver of type 2 diabetic patients and positively correlate with glucose and HOMA-IR. Overall these results indicate that the O-GlcNAcylation of p53 plays an unsuspected key role regulating in vivo glucose homeostasis.


Assuntos
Acetilglucosamina/metabolismo , Glucose/metabolismo , Fígado/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Restrição Calórica , Linhagem Celular , Colforsina/farmacologia , Diabetes Mellitus Tipo 2/complicações , Diabetes Mellitus Tipo 2/metabolismo , Epinefrina/metabolismo , Glucagon/metabolismo , Glucocorticoides/metabolismo , Gluconeogênese/efeitos dos fármacos , Glicosilação , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Hidrocortisona/metabolismo , Hiperglicemia/complicações , Hiperglicemia/metabolismo , Resistência à Insulina , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Fígado/efeitos dos fármacos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Obesidade/complicações , Obesidade/metabolismo , Fosfoenolpiruvato Carboxiquinase (GTP)/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica/efeitos dos fármacos , Estabilidade Proteica/efeitos dos fármacos , Ácido Pirúvico/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcrição Genética/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética
4.
Cell Stem Cell ; 28(10): 1868-1883.e11, 2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34038708

RESUMO

Topological-associated domains (TADs) are thought to be relatively stable across cell types, although some TAD reorganization has been observed during cellular differentiation. However, little is known about the mechanisms through which TAD reorganization affects cell fate or how master transcription factors affect TAD structures during cell fate transitions. Here, we show extensive TAD reorganization during somatic cell reprogramming, which is correlated with gene transcription and changes in cellular identity. Manipulating TAD reorganization promotes reprogramming, and the dynamics of concentrated chromatin loops in OCT4 phase separated condensates contribute to TAD reorganization. Disrupting OCT4 phase separation attenuates TAD reorganization and reprogramming, which can be rescued by fusing an intrinsically disordered region (IDR) to OCT4. We developed an approach termed TAD reorganization-based multiomics analysis (TADMAN), which identified reprogramming regulators. Together, these findings elucidate a role and mechanism of TAD reorganization, regulated by OCT4 phase separation, in cellular reprogramming.

5.
Methods Mol Biol ; 2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33689163

RESUMO

The reprogramming of somatic cells into induced pluripotent stem cells (iPSCs) has proven to be a powerful system creating new opportunities to interrogate molecular mechanisms controlling cell fate determination. Under standard conditions, the generation of iPSCs upon overexpression of OCT4, SOX2, KLF4, and c-MYC (OSKM) is generally slow and inefficient due to the presence of barriers that confer resistance to cell fate changes. Hyperactivated endoplasmic reticulum (ER) stress has emerged as a major reprogramming barrier that impedes the initial mesenchymal-to-epithelial transition (MET) step to form iPSCs from mesenchymal somatic cells. Here, we describe several systems to detect ER stress in the context of OSKM reprogramming and chemical interventions to modulate this process for improving iPSC formation.

6.
Hepatology ; 73(2): 606-624, 2021 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-32329085

RESUMO

BACKGROUND AND AIMS: G protein-coupled receptor (GPR) 55 is a putative cannabinoid receptor, and l-α-lysophosphatidylinositol (LPI) is its only known endogenous ligand. Although GPR55 has been linked to energy homeostasis in different organs, its specific role in lipid metabolism in the liver and its contribution to the pathophysiology of nonalcoholic fatty liver disease (NAFLD) remains unknown. APPROACH AND RESULTS: We measured (1) GPR55 expression in the liver of patients with NAFLD compared with individuals without obesity and without liver disease, as well as animal models with steatosis and nonalcoholic steatohepatitis (NASH), and (2) the effects of LPI and genetic disruption of GPR55 in mice, human hepatocytes, and human hepatic stellate cells. Notably, we found that circulating LPI and liver expression of GPR55 were up-regulated in patients with NASH. LPI induced adenosine monophosphate-activated protein kinase activation of acetyl-coenzyme A carboxylase (ACC) and increased lipid content in human hepatocytes and in the liver of treated mice by inducing de novo lipogenesis and decreasing ß-oxidation. The inhibition of GPR55 and ACCα blocked the effects of LPI, and the in vivo knockdown of GPR55 was sufficient to improve liver damage in mice fed a high-fat diet and in mice fed a methionine-choline-deficient diet. Finally, LPI promoted the initiation of hepatic stellate cell activation by stimulating GPR55 and activation of ACC. CONCLUSIONS: The LPI/GPR55 system plays a role in the development of NAFLD and NASH by activating ACC.

7.
Nat Commun ; 11(1): 4956, 2020 10 02.
Artigo em Inglês | MEDLINE | ID: mdl-33009383

RESUMO

Tet-enzyme-mediated 5-hydroxymethylation of cytosines in DNA plays a crucial role in mouse embryonic stem cells (ESCs). In RNA also, 5-hydroxymethylcytosine (5hmC) has recently been evidenced, but its physiological roles are still largely unknown. Here we show the contribution and function of this mark in mouse ESCs and differentiating embryoid bodies. Transcriptome-wide mapping in ESCs reveals hundreds of messenger RNAs marked by 5hmC at sites characterized by a defined unique consensus sequence and particular features. During differentiation a large number of transcripts, including many encoding key pluripotency-related factors (such as Eed and Jarid2), show decreased cytosine hydroxymethylation. Using Tet-knockout ESCs, we find Tet enzymes to be partly responsible for deposition of 5hmC in mRNA. A transcriptome-wide search further reveals mRNA targets to which Tet1 and Tet2 bind, at sites showing a topology similar to that of 5hmC sites. Tet-mediated RNA hydroxymethylation is found to reduce the stability of crucial pluripotency-promoting transcripts. We propose that RNA cytosine 5-hydroxymethylation by Tets is a mark of transcriptome flexibility, inextricably linked to the balance between pluripotency and lineage commitment.


Assuntos
5-Metilcitosina/análogos & derivados , Diferenciação Celular , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , RNA/metabolismo , 5-Metilcitosina/metabolismo , Animais , Especificidade de Anticorpos/imunologia , Sequência de Bases , Corpos Embrioides/metabolismo , Camundongos , Modelos Biológicos , Células-Tronco Pluripotentes/metabolismo , Ligação Proteica , Estabilidade de RNA/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transcriptoma/genética
8.
STAR Protoc ; 1(2)2020 Sep 18.
Artigo em Inglês | MEDLINE | ID: mdl-32995755

RESUMO

RNA-binding proteins are key regulators of cell identity and function, which underscores the need for unbiased and versatile protocols to identify and characterize novel protein-RNA interactions. Here, we describe a simple and cost-effective in vitro RNA immunoprecipitation (iv-RIP) method to assess the direct or indirect RNA-binding ability of any protein of interest. The versatility of this method relies on the adaptability of the immunoprecipitation conditions and the choice of the RNA, which exponentially broadens the range of potential applications. For complete details on the use and execution of this protocol, please refer to Guallar et al. (2020).

9.
Cell Stem Cell ; 27(2): 300-314.e11, 2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32396862

RESUMO

RNA editing of adenosine to inosine (A to I) is catalyzed by ADAR1 and dramatically alters the cellular transcriptome, although its functional roles in somatic cell reprogramming are largely unexplored. Here, we show that loss of ADAR1-mediated A-to-I editing disrupts mesenchymal-to-epithelial transition (MET) during induced pluripotent stem cell (iPSC) reprogramming and impedes acquisition of induced pluripotency. Using chemical and genetic approaches, we show that absence of ADAR1-dependent RNA editing induces aberrant innate immune responses through the double-stranded RNA (dsRNA) sensor MDA5, unleashing endoplasmic reticulum (ER) stress and hindering epithelial fate acquisition. We found that A-to-I editing impedes MDA5 sensing and sequestration of dsRNAs encoding membrane proteins, which promote ER homeostasis by activating the PERK-dependent unfolded protein response pathway to consequently facilitate MET. This study therefore establishes a critical role for ADAR1 and its A-to-I editing activity during cell fate transitions and delineates a key regulatory layer underlying MET to control efficient reprogramming.


Assuntos
Células-Tronco Pluripotentes Induzidas , Edição de RNA , Adenosina Desaminase/genética , Adenosina Desaminase/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Inosina/metabolismo , RNA de Cadeia Dupla
10.
Life Sci Alliance ; 3(5)2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284354

RESUMO

BMAL1 is essential for the regulation of circadian rhythms in differentiated cells and adult stem cells, but the molecular underpinnings of its function in pluripotent cells, which hold a great potential in regenerative medicine, remain to be addressed. Here, using transient and permanent loss-of-function approaches in mouse embryonic stem cells (ESCs), we reveal that although BMAL1 is dispensable for the maintenance of the pluripotent state, its depletion leads to deregulation of transcriptional programs linked to cell differentiation commitment. We further confirm that depletion of Bmal1 alters the differentiation potential of ESCs in vitro. Mechanistically, we demonstrate that BMAL1 participates in the regulation of energy metabolism maintaining a low mitochondrial function which is associated with pluripotency. Loss-of-function of Bmal1 leads to the deregulation of metabolic gene expression associated with a shift from glycolytic to oxidative metabolism. Our results highlight the important role that BMAL1 plays at the exit of pluripotency in vitro and provide evidence implicating a non-canonical circadian function of BMAL1 in the metabolic control for cell fate determination.


Assuntos
Fatores de Transcrição ARNTL/metabolismo , Células-Tronco Pluripotentes/metabolismo , Fatores de Transcrição ARNTL/fisiologia , Animais , Diferenciação Celular/fisiologia , Ritmo Circadiano/genética , Metabolismo Energético/fisiologia , Expressão Gênica/genética , Glicólise , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Camundongos , Mitocôndrias/genética , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologia
11.
Cell Stem Cell ; 26(2): 234-250.e7, 2020 02 06.
Artigo em Inglês | MEDLINE | ID: mdl-32032525

RESUMO

Mouse embryonic stem cells (ESCs) sporadically express preimplantation two-cell-stage (2C) transcripts, including MERVL endogenous retrovirus and Zscan4 cluster genes. Such 2C-like cells (2CLCs) can contribute to both embryonic and extraembryonic tissues when reintroduced into early embryos, although the molecular mechanism underlying such an expanded 2CLC potency remains elusive. We examine global nucleosome occupancy and gene expression in 2CLCs and identified miR-344 as the noncoding molecule that positively controls 2CLC potency. We find that activation of endogenous MERVL or miR-344-2 alone is sufficient to induce 2CLCs with activation of 2C genes and an expanded potency. Mechanistically, miR-344 is activated by DUX and post-transcriptionally represses ZMYM2 and its partner LSD1, and ZMYM2 recruits LSD1/HDAC corepressor complex to MERVL LTR for transcriptional repression. Consistently, zygotic depletion of Zmym2 compromises the totipotency-to-pluripotency transition during early development. Our studies establish the previously unappreciated DUX-miR-344-Zmym2/Lsd1 axis that controls MERVL for expanded stem cell potency.


Assuntos
Retrovirus Endógenos , MicroRNAs , Animais , Retrovirus Endógenos/genética , Camundongos , MicroRNAs/genética , Células-Tronco Embrionárias Murinas , Zigoto
12.
Nat Metab ; 1(8): 811-829, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31579887

RESUMO

Dopamine signaling is a crucial part of the brain reward system and can affect feeding behavior. Dopamine receptors are also expressed in the hypothalamus, which is known to control energy metabolism in peripheral tissues. Here we show that pharmacological or chemogenetic stimulation of dopamine receptor 2 (D2R) expressing cells in the lateral hypothalamic area (LHA) and the zona incerta (ZI) decreases body weight and stimulates brown fat activity in rodents in a feeding-independent manner. LHA/ZI D2R stimulation requires an intact sympathetic nervous system and orexin system to exert its action and involves inhibition of PI3K in the LHA/ZI. We further demonstrate that, as early as 3 months after onset of treatment, patients treated with the D2R agonist cabergoline experience an increase in energy expenditure that persists for one year, leading to total body weight and fat loss through a prolactin-independent mechanism. Our results may provide a mechanistic explanation for how clinically used D2R agonists act in the CNS to regulate energy balance.


Assuntos
Tecido Adiposo Marrom/metabolismo , Dopamina/metabolismo , Hipotálamo/metabolismo , Transdução de Sinais , Termogênese/fisiologia , Animais , Bromocriptina/administração & dosagem , Bromocriptina/farmacologia , Feminino , Humanos , Hipotálamo/efeitos dos fármacos , Injeções Intraventriculares , Masculino , Ratos
13.
Diabetes ; 68(12): 2210-2222, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31530579

RESUMO

Melanin-concentrating hormone (MCH) is an important regulator of food intake, glucose metabolism, and adiposity. However, the mechanisms mediating these actions remain largely unknown. We used pharmacological and genetic approaches to show that the sirtuin 1 (SIRT1)/FoxO1 signaling pathway in the hypothalamic arcuate nucleus (ARC) mediates MCH-induced feeding, adiposity, and glucose intolerance. MCH reduces proopiomelanocortin (POMC) neuronal activity, and the SIRT1/FoxO1 pathway regulates the inhibitory effect of MCH on POMC expression. Remarkably, the metabolic actions of MCH are compromised in mice lacking SIRT1 specifically in POMC neurons. Of note, the actions of MCH are independent of agouti-related peptide (AgRP) neurons because inhibition of γ-aminobutyric acid receptor in the ARC did not prevent the orexigenic action of MCH, and the hypophagic effect of MCH silencing was maintained after chemogenetic stimulation of AgRP neurons. Central SIRT1 is required for MCH-induced weight gain through its actions on the sympathetic nervous system. The central MCH knockdown causes hypophagia and weight loss in diet-induced obese wild-type mice; however, these effects were abolished in mice overexpressing SIRT1 fed a high-fat diet. These data reveal the neuronal basis for the effects of MCH on food intake, body weight, and glucose metabolism and highlight the relevance of SIRT1/FoxO1 pathway in obesity.


Assuntos
Adiposidade/efeitos dos fármacos , Proteína Forkhead Box O1/metabolismo , Intolerância à Glucose/metabolismo , Hiperfagia/metabolismo , Hormônios Hipotalâmicos/farmacologia , Melaninas/farmacologia , Neurônios/efeitos dos fármacos , Hormônios Hipofisários/farmacologia , Pró-Opiomelanocortina/metabolismo , Sirtuína 1/metabolismo , Adiposidade/fisiologia , Animais , Proteína Forkhead Box O1/genética , Intolerância à Glucose/genética , Hiperfagia/genética , Hipotálamo/efeitos dos fármacos , Hipotálamo/metabolismo , Masculino , Camundongos , Camundongos Knockout , Neurônios/metabolismo , Técnicas de Patch-Clamp , Ratos Sprague-Dawley , Sirtuína 1/genética
14.
Blood Adv ; 3(16): 2499-2511, 2019 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-31455666

RESUMO

Erythroid maturation requires the concerted action of a core set of transcription factors. We previously identified the Krüppel-type zinc finger transcription factor Zfp148 (also called ZBP-89) as an interacting partner of the master erythroid transcription factor GATA1. Here we report the conditional knockout of Zfp148 in mice. Global loss of Zfp148 results in perinatal lethality from nonhematologic causes. Selective Zfp148 loss within the hematopoietic system results in a mild microcytic and hypochromic anemia, mildly impaired erythroid maturation, and delayed recovery from phenylhydrazine-induced hemolysis. Based on the mild erythroid phenotype of these mice compared with GATA1-deficient mice, we hypothesized that additional factor(s) may complement Zfp148 function during erythropoiesis. We show that Zfp281 (also called ZBP-99), another member of the Zfp148 transcription factor family, is highly expressed in murine and human erythroid cells. Zfp281 knockdown by itself results in partial erythroid defects. However, combined deficiency of Zfp148 and Zfp281 causes a marked erythroid maturation block. Zfp281 physically associates with GATA1, occupies many common chromatin sites with GATA1 and Zfp148, and regulates a common set of genes required for erythroid cell differentiation. These findings uncover a previously unknown role for Zfp281 in erythroid development and suggest that it functionally overlaps with that of Zfp148 during erythropoiesis.


Assuntos
Diferenciação Celular/genética , Proteínas de Ligação a DNA/genética , Células Eritroides/citologia , Células Eritroides/metabolismo , Eritropoese/genética , Fatores de Transcrição/genética , Animais , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/metabolismo , Fator de Transcrição GATA1/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Genótipo , Humanos , Camundongos , Camundongos Knockout , Ligação Proteica , Fatores de Transcrição/metabolismo , gama-Globinas/genética , gama-Globinas/metabolismo
15.
Diabetes ; 67(11): 2213-2226, 2018 11.
Artigo em Inglês | MEDLINE | ID: mdl-30104247

RESUMO

AMPK is a cellular gauge that is activated under conditions of low energy, increasing energy production and reducing energy waste. Current evidence links hypothalamic AMPK with the central regulation of energy balance. However, it is unclear whether targeting hypothalamic AMPK has beneficial effects in obesity. Here, we show that genetic inhibition of AMPK in the ventromedial nucleus of the hypothalamus (VMH) protects against high-fat diet (HFD)-induced obesity by increasing brown adipose tissue (BAT) thermogenesis and subsequently energy expenditure. Notably, this effect depends upon the AMPKα1 isoform in steroidogenic factor 1 (SF1) neurons of the VMH, since mice bearing selective ablation of AMPKα1 in SF1 neurons display resistance to diet-induced obesity, increased BAT thermogenesis, browning of white adipose tissue, and improved glucose and lipid homeostasis. Overall, our findings point to hypothalamic AMPK in specific neuronal populations as a potential druggable target for the treatment of obesity and associated metabolic disorders.


Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Dieta Hiperlipídica/efeitos adversos , Neurônios/metabolismo , Obesidade/metabolismo , Fatores de Processamento de RNA/metabolismo , Núcleo Hipotalâmico Ventromedial/metabolismo , Proteínas Quinases Ativadas por AMP/genética , Tecido Adiposo Marrom/metabolismo , Animais , Composição Corporal/fisiologia , Metabolismo Energético/fisiologia , Masculino , Obesidade/etiologia , Obesidade/genética , Consumo de Oxigênio/fisiologia , Ratos , Ratos Sprague-Dawley
16.
Stem Cell Reports ; 10(4): 1324-1339, 2018 04 10.
Artigo em Inglês | MEDLINE | ID: mdl-29503092

RESUMO

Yin Yang 1 (YY1) regulates early embryogenesis and adult tissue formation. However, the role of YY1 in stem cell regulation remains unclear. YY1 has a Polycomb group (PcG) protein-dependent role in mammalian cells. The PcG-independent functions of YY1 are also reported, although their underlying mechanism is still undefined. This paper reports the role of YY1 and BAF complex in the OCT4-mediated pluripotency network in mouse embryonic stem cells (mESCs). The interaction between YY1 and BAF complex promotes mESC proliferation and pluripotency. Knockdown of Yy1 or Smarca4, the core component of the BAF complex, downregulates pluripotency markers and upregulates several differentiation markers. Moreover, YY1 enriches at both promoter and super-enhancer regions to stimulate transcription. Thus, this study elucidates the role of YY1 in regulating pluripotency through its interaction with OCT4 and the BAF complex and the role of BAF complex in integrating YY1 into the core pluripotency network.


Assuntos
Elementos Facilitadores Genéticos/genética , Células-Tronco Embrionárias Murinas/metabolismo , Complexos Multiproteicos/metabolismo , Regiões Promotoras Genéticas , Transcrição Genética , Fator de Transcrição YY1/metabolismo , Animais , Linhagem Celular , Linhagem da Célula , Proliferação de Células , Redes e Vias Metabólicas , Camundongos , Modelos Biológicos , Fator 3 de Transcrição de Octâmero/metabolismo , Ligação Proteica , Mapas de Interação de Proteínas
17.
Nat Genet ; 50(3): 443-451, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29483655

RESUMO

Ten-eleven translocation (TET) proteins play key roles in the regulation of DNA-methylation status by oxidizing 5-methylcytosine (5mC) to generate 5-hydroxymethylcytosine (5hmC), which can both serve as a stable epigenetic mark and participate in active demethylation. Unlike the other members of the TET family, TET2 does not contain a DNA-binding domain, and it remains unclear how it is recruited to chromatin. Here we show that TET2 is recruited by the RNA-binding protein Paraspeckle component 1 (PSPC1) through transcriptionally active loci, including endogenous retroviruses (ERVs) whose long terminal repeats (LTRs) have been co-opted by mammalian genomes as stage- and tissue-specific transcriptional regulatory modules. We found that PSPC1 and TET2 contribute to ERVL and ERVL-associated gene regulation by both transcriptional repression via histone deacetylases and post-transcriptional destabilization of RNAs through 5hmC modification. Our findings provide evidence for a functional role of transcriptionally active ERVs as specific docking sites for RNA epigenetic modulation and gene regulation.


Assuntos
Cromatina/metabolismo , Proteínas de Ligação a DNA/metabolismo , Retrovirus Endógenos/fisiologia , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas de Ligação a RNA/metabolismo , RNA/fisiologia , Animais , Células Cultivadas , Cromatina/genética , Metilação de DNA , Epigênese Genética/fisiologia , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Ligação Proteica
18.
Nat Genet ; 50(1): 96-105, 2018 01.
Artigo em Inglês | MEDLINE | ID: mdl-29203909

RESUMO

In mammals, all somatic development originates from lineage segregation in early embryos. However, the dynamics of transcriptomes and epigenomes acting in concert with initial cell fate commitment remains poorly characterized. Here we report a comprehensive investigation of transcriptomes and base-resolution methylomes for early lineages in peri- and postimplantation mouse embryos. We found allele-specific and lineage-specific de novo methylation at CG and CH sites that led to differential methylation between embryonic and extraembryonic lineages at promoters of lineage regulators, gene bodies, and DNA-methylation valleys. By using Hi-C experiments to define chromatin architecture across the same developmental period, we demonstrated that both global demethylation and remethylation in early development correlate with chromatin compartments. Dynamic local methylation was evident during gastrulation, which enabled the identification of putative regulatory elements. Finally, we found that de novo methylation patterning does not strictly require implantation. These data reveal dynamic transcriptomes, DNA methylomes, and 3D chromatin landscapes during the earliest stages of mammalian lineage specification.


Assuntos
Metilação de DNA , Embrião de Mamíferos/metabolismo , Epigênese Genética , Alelos , Animais , Blastocisto/metabolismo , Linhagem da Célula/genética , Cromatina/química , Implantação do Embrião , Endoderma/metabolismo , Gastrulação/genética , Camadas Germinativas/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas , Sequências Reguladoras de Ácido Nucleico , Transcriptoma
19.
Elife ; 62017 11 23.
Artigo em Inglês | MEDLINE | ID: mdl-29168693

RESUMO

Pluripotency is defined by a cell's potential to differentiate into any somatic cell type. How pluripotency is transited during embryo implantation, followed by cell lineage specification and establishment of the basic body plan, is poorly understood. Here we report the transcription factor Zfp281 functions in the exit from naive pluripotency occurring coincident with pre-to-post-implantation mouse embryonic development. By characterizing Zfp281 mutant phenotypes and identifying Zfp281 gene targets and protein partners in developing embryos and cultured pluripotent stem cells, we establish critical roles for Zfp281 in activating components of the Nodal signaling pathway and lineage-specific genes. Mechanistically, Zfp281 cooperates with histone acetylation and methylation complexes at target gene enhancers and promoters to exert transcriptional activation and repression, as well as epigenetic control of epiblast maturation leading up to anterior-posterior axis specification. Our study provides a comprehensive molecular model for understanding pluripotent state progressions in vivo during mammalian embryonic development.


Assuntos
Epigênese Genética , Regulação da Expressão Gênica no Desenvolvimento , Camadas Germinativas/embriologia , Proteína Nodal/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Animais , Células Cultivadas , Deleção de Genes , Camundongos , Células-Tronco Pluripotentes/fisiologia , Fatores de Transcrição/genética
20.
Stem Cell Reports ; 9(3): 913-926, 2017 09 12.
Artigo em Inglês | MEDLINE | ID: mdl-28781078

RESUMO

Reprogramming somatic cells to induced pluripotent stem cells (iPSCs) is a long and inefficient process. A thorough understanding of the molecular mechanisms underlying reprogramming is paramount for efficient generation and safe application of iPSCs in medicine. While intensive efforts have been devoted to identifying reprogramming facilitators and barriers, a full repertoire of such factors, as well as their mechanistic actions, is poorly defined. Here, we report that NAC1, a pluripotency-associated factor and NANOG partner, is required for establishment of pluripotency during reprogramming. Mechanistically, NAC1 is essential for proper expression of E-cadherin by a dual regulatory mechanism: it facilitates NANOG binding to the E-cadherin promoter and fine-tunes its expression; most importantly, it downregulates the E-cadherin repressor ZEB1 directly via transcriptional repression and indirectly via post-transcriptional activation of the miR-200 miRNAs. Our study thus uncovers a previously unappreciated role for the pluripotency regulator NAC1 in promoting efficient somatic cell reprogramming.


Assuntos
Proteínas Cdh1/metabolismo , Reprogramação Celular , Proteínas do Tecido Nervoso/metabolismo , Proteínas Repressoras/metabolismo , Homeobox 1 de Ligação a E-box em Dedo de Zinco/metabolismo , Animais , Citoproteção , Regulação para Baixo/genética , Expressão Gênica , Células HEK293 , Humanos , Células-Tronco Pluripotentes Induzidas/citologia , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Modelos Biológicos , Proteína Homeobox Nanog/metabolismo , Fenótipo , Regiões Promotoras Genéticas/genética , Transgenes , Homeobox 1 de Ligação a E-box em Dedo de Zinco/genética
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...