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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.
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
12.
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
13.
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
14.
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
15.
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
16.
Stem Cell Reports ; 8(5): 1115-1123, 2017 05 09.
Artigo em Inglês | MEDLINE | ID: mdl-28457890

RESUMO

The core pluripotency transcription factor NANOG is critical for embryonic stem cell (ESC) self-renewal and somatic cell reprogramming. Although NANOG is phosphorylated at multiple residues, the role of NANOG phosphorylation in ESC self-renewal is incompletely understood, and no information exists regarding its functions during reprogramming. Here we report our findings that NANOG phosphorylation is beneficial, although nonessential, for ESC self-renewal, and that loss of phosphorylation enhances NANOG activity in reprogramming. Mutation of serine 65 in NANOG to alanine (S65A) alone has the most significant impact on increasing NANOG reprogramming capacity. Mechanistically, we find that pluripotency regulators (ESRRB, OCT4, SALL4, DAX1, and TET1) are transcriptionally primed and preferentially associated with NANOG S65A at the protein level due to presumed structural alterations in the N-terminal domain of NANOG. These results demonstrate that a single phosphorylation site serves as a critical interface for controlling context-dependent NANOG functions in pluripotency and reprogramming.


Assuntos
Reprogramação Celular , Células-Tronco Embrionárias/citologia , Células-Tronco Pluripotentes Induzidas/citologia , Mutação de Sentido Incorreto , Proteína Homeobox Nanog/metabolismo , Processamento de Proteína Pós-Traducional , Animais , Linhagem Celular , Células Cultivadas , Receptor Nuclear Órfão DAX-1/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células-Tronco Embrionárias/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Camundongos , Proteína Homeobox Nanog/química , Proteína Homeobox Nanog/genética , Fator 3 de Transcrição de Octâmero/metabolismo , Fosforilação , Domínios Proteicos , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Estrogênio/metabolismo , Fatores de Transcrição/metabolismo
17.
Cell Rep ; 18(7): 1713-1726, 2017 02 14.
Artigo em Inglês | MEDLINE | ID: mdl-28199843

RESUMO

Although SIN3A is required for the survival of early embryos and embryonic stem cells (ESCs), the role of SIN3A in the maintenance and establishment of pluripotency remains unclear. Here, we find that the SIN3A/HDAC corepressor complex maintains ESC pluripotency and promotes the generation of induced pluripotent stem cells (iPSCs). Members of the SIN3A/HDAC corepressor complex are enriched in an extended NANOG interactome and function in transcriptional coactivation in ESCs. We also identified a critical role for SIN3A and HDAC2 in efficient reprogramming of somatic cells. Mechanistically, NANOG and SIN3A co-occupy transcriptionally active pluripotency genes in ESCs and also co-localize extensively at their genome-wide targets in pre-iPSCs. Additionally, both factors are required to directly induce a synergistic transcriptional program wherein pluripotency genes are activated and reprogramming barrier genes are repressed. Our findings indicate a transcriptional regulatory role for a major HDAC-containing complex in promoting pluripotency.


Assuntos
Proteínas Correpressoras/metabolismo , Histona Desacetilase 2/metabolismo , Células-Tronco Pluripotentes Induzidas/metabolismo , Proteína Homeobox Nanog/metabolismo , Proteínas Repressoras/metabolismo , Animais , Reprogramação Celular/genética , Reprogramação Celular/fisiologia , Células-Tronco Embrionárias/metabolismo , Células-Tronco Embrionárias/fisiologia , Feminino , Regulação da Expressão Gênica no Desenvolvimento/genética , Genes Homeobox/genética , Genoma/genética , Células-Tronco Pluripotentes Induzidas/fisiologia , Camundongos , Complexo Correpressor Histona Desacetilase e Sin3 , Transcrição Genética/genética
18.
Cell Stem Cell ; 19(3): 355-69, 2016 09 01.
Artigo em Inglês | MEDLINE | ID: mdl-27345836

RESUMO

Pluripotency is increasingly recognized as a spectrum of cell states defined by their growth conditions. Although naive and primed pluripotency states have been characterized molecularly, our understanding of events regulating state acquisition is wanting. Here, we performed comparative RNA sequencing of mouse embryonic stem cells (ESCs) and defined a pluripotent cell fate (PCF) gene signature associated with acquisition of naive and primed pluripotency. We identify Zfp281 as a key transcriptional regulator for primed pluripotency that also functions as a barrier toward achieving naive pluripotency in both mouse and human ESCs. Mechanistically, Zfp281 interacts with Tet1, but not Tet2, and its direct transcriptional target, miR-302/367, to negatively regulate Tet2 expression to establish and maintain primed pluripotency. Conversely, ectopic Tet2 alone, but not Tet1, efficiently reprograms primed cells toward naive pluripotency. Our study reveals a molecular circuitry in which opposing functions of Tet1 and Tet2 control acquisition of alternative pluripotent states.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Células-Tronco Pluripotentes/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Linhagem da Célula/genética , Epigênese Genética , Perfilação da Expressão Gênica , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologia , Interferência de RNA , Transcrição Genética
19.
Genome Biol ; 17: 104, 2016 05 16.
Artigo em Inglês | MEDLINE | ID: mdl-27184958

RESUMO

A new study shows how RNA-induced silencing complex (RISC)-mediated posttranscriptional regulation of chromatin remodelers allows for tight control of the naïve-to-primed pluripotency transition.


Assuntos
Proteínas Argonauta , Complexo de Inativação Induzido por RNA/genética , Interferência de RNA
20.
Cell Stem Cell ; 16(6): 653-68, 2015 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-25936917

RESUMO

Super-enhancers (SEs) are large clusters of transcriptional enhancers that are co-occupied by multiple lineage-specific transcription factors driving expression of genes that define cell identity. In embryonic stem cells (ESCs), SEs are highly enriched for the core pluripotency factors Oct4, Sox2, and Nanog. In this study, we sought to dissect the molecular control mechanism of SE activity in pluripotency and reprogramming. Starting from a protein interaction network surrounding Sox2, we identified Tex10 as a key pluripotency factor that plays a functionally significant role in ESC self-renewal, early embryo development, and reprogramming. Tex10 is enriched at SEs in a Sox2-dependent manner and coordinates histone acetylation and DNA demethylation at SEs. Tex10 activity is also important for pluripotency and reprogramming in human cells. Our study therefore highlights Tex10 as a core component of the pluripotency network and sheds light on its role in epigenetic control of SE activity for cell fate determination.


Assuntos
Reprogramação Celular/genética , Elementos Facilitadores Genéticos/genética , Epigênese Genética , Proteínas Nucleares/metabolismo , Células-Tronco Pluripotentes/metabolismo , Animais , Autorrenovação Celular , Desenvolvimento Embrionário/genética , Regulação da Expressão Gênica no Desenvolvimento , Células-Tronco Embrionárias Humanas/citologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Camundongos , Células-Tronco Embrionárias Murinas/citologia , Células-Tronco Embrionárias Murinas/metabolismo , Células-Tronco Pluripotentes/citologia , Ligação Proteica , RNA/genética , RNA/metabolismo , Fatores de Transcrição SOXB1/metabolismo , Transcrição Genética
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