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1.
iScience ; 27(5): 109782, 2024 May 17.
Artigo em Inglês | MEDLINE | ID: mdl-38711449

RESUMO

Ten-eleven translocation (TET) proteins are DNA dioxygenases that mediate active DNA demethylation. TET3 is the most highly expressed TET protein in thymic developing T cells. TET3, either independently or in cooperation with TET1 or TET2, has been implicated in T cell lineage specification by regulating DNA demethylation. However, TET-deficient mice exhibit complex phenotypes, suggesting that TET3 exerts multifaceted roles, potentially by interacting with other proteins. We performed liquid chromatography with tandem mass spectrometry in primary developing T cells to identify TET3 interacting partners in endogenous, in vivo conditions. We discover TET3 interacting partners. Our data establish that TET3 participates in a plethora of fundamental biological processes, such as transcriptional regulation, RNA polymerase elongation, splicing, DNA repair, and DNA replication. This resource brings in the spotlight emerging functions of TET3 and sets the stage for systematic studies to dissect the precise mechanistic contributions of TET3 in shaping T cell biology.

2.
STAR Protoc ; 3(4): 101839, 2022 12 16.
Artigo em Inglês | MEDLINE | ID: mdl-36386867

RESUMO

Here, we describe steps to isolate mature thymic T cell subsets, namely CD4 single positive (SP), CD8 SP, and invariant natural killer T (iNKT) cells starting from murine total thymocytes using fluorescence-activated cell sorting. We detail protocols to study gene expression by RNA-seq and assess binding of transcription factors across the genome using CUT&RUN. This approach deciphers the molecular principles that govern T cell lineage specification and function. This protocol works well with limited starting material. For complete details on the use and execution of this protocol, please refer to Äijö et al. (2022).1.


Assuntos
Subpopulações de Linfócitos T , Fatores de Transcrição , Camundongos , Animais , Citometria de Fluxo , RNA-Seq , Fatores de Transcrição/genética , Expressão Gênica
3.
Front Immunol ; 13: 940995, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35990681

RESUMO

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.


Assuntos
Dioxigenases , Células T Matadoras Naturais , Animais , Linhagem da Célula , Metilação de DNA , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Dioxigenases/genética , Camundongos , Células T Matadoras Naturais/metabolismo , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Transcrição/metabolismo
4.
Metabolism ; 87: 36-47, 2018 10.
Artigo em Inglês | MEDLINE | ID: mdl-29928895

RESUMO

BACKGROUND: High Density Lipoprotein (HDL) and its main protein component, apolipoprotein A-I (apoA-I), have numerous atheroprotective functions on various tissues including the endothelium. Therapies based on reconstituted HDL containing apoA-I (rHDL-apoA-I) have been used successfully in patients with acute coronary syndrome, peripheral vascular disease or diabetes but very little is known about the genomic effects of rHDL-apoA-I and how they could contribute to atheroprotection. OBJECTIVE: The present study aimed to understand the endothelial signaling pathways and the genes that may contribute to rHDL-apoA-I-mediated atheroprotection. METHODS: Human aortic endothelial cells (HAECs) were treated with rHDL-apoA-I and their total RNA was analyzed with whole genome microarrays. Validation of microarray data was performed using multiplex RT-qPCR. The expression of ANGPTL4 in EA.hy926 endothelial cells was determined by RT-qPCR and Western blotting. The contribution of signaling kinases and transcription factors in ANGPTL4 gene regulation by HDL-apoA-I was assessed by RT-qPCR, Western blotting and immunofluorescence using chemical inhibitors or siRNA-mediated gene silencing. RESULTS: It was found that 410 transcripts were significantly changed in the presence of rHDL-apoA-I and that angiopoietin like 4 (ANGPTL4) was one of the most upregulated and biologically relevant molecules. In validation experiments rHDL-apoA-I, as well as natural HDL from human healthy donors or from transgenic mice overexpressing human apoA-I (TgHDL-apoA-I), increased ANGPTL4 mRNA and protein levels. ANGPTL4 gene induction by HDL was direct and was blocked in the presence of inhibitors for the AKT or the p38 MAP kinases. TgHDL-apoA-I caused phosphorylation of the transcription factor forkhead box O1 (FOXO1) and its translocation from the nucleus to the cytoplasm. Importantly, a FOXO1 inhibitor or a FOXO1-specific siRNA enhanced ANGPTL4 expression, whereas administration of TgHDL-apoA-I in the presence of the FOXO1 inhibitor or the FOXO1-specific siRNA did not induce further ANGPTL4 expression. These data suggest that FOXO1 functions as an inhibitor of ANGPTL4, while HDL-apoA-I blocks FOXO1 activity and induces ANGPTL4 through the activation of AKT. CONCLUSION: Our data provide novel insights into the global molecular effects of HDL-apoA-I on endothelial cells and identify ANGPTL4 as a putative mediator of the atheroprotective functions of HDL-apoA-I on the artery wall, with notable therapeutic potential.


Assuntos
Proteína 4 Semelhante a Angiopoietina/biossíntese , Apolipoproteína A-I/farmacologia , Células Endoteliais/metabolismo , Proteína Forkhead Box O1/metabolismo , Lipoproteínas HDL/farmacologia , Proteína Oncogênica v-akt/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proteína 4 Semelhante a Angiopoietina/efeitos dos fármacos , Proteína 4 Semelhante a Angiopoietina/genética , Animais , Proteína Forkhead Box O1/efeitos dos fármacos , Proteína Forkhead Box O1/genética , Expressão Gênica/efeitos dos fármacos , Inativação Gênica , Voluntários Saudáveis , Humanos , Camundongos , Camundongos Transgênicos , Análise em Microsséries , Proteína Oncogênica v-akt/efeitos dos fármacos , Fosfatidilinositol 3-Quinases/efeitos dos fármacos , Transporte Proteico/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia
5.
Biochem Biophys Res Commun ; 469(3): 573-9, 2016 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-26692490

RESUMO

Liver X Receptors (LXRs) are sterol-activated transcription factors that play major roles in cellular cholesterol homeostasis, HDL biogenesis and reverse cholesterol transport. The aim of the present study was to investigate the mechanisms that control the expression of the human LXRα gene in hepatic cells. A series of reporter plasmids containing consecutive 5' deletions of the hLXRα promoter upstream of the luciferase gene were constructed and the activity of each construct was measured in HepG2 cells. This analysis showed that the activity of the human LXRα promoter was significantly reduced by deleting the -111 to -42 region suggesting the presence of positive regulatory elements in this short proximal fragment. Bioinformatics data including motif search and ChIP-Seq revealed the presence of a potential binding motif for Hepatocyte Nuclear Factor 4 α (HNF-4α) in this area. Overexpression of HNF-4α in HEK 293T cells increased the expression of all LXRα promoter constructs except -42/+384. In line, silencing the expression of endogenous HNF-4α in HepG2 cells was associated with reduced LXRα protein levels and reduced activity of the -111/+384 LXRα promoter but not of the -42/+384 promoter. Using ChiP assays in HepG2 cells combined with DNAP assays we mapped the novel HNF-4α specific binding motif (H4-SBM) in the -50 to -40 region of the human LXRα promoter. A triple mutation in this H4-SBM abolished HNF-4α binding and reduced the activity of the promoter to 65% relative to the wild type. Furthermore, the mutant promoter could not be transactivated by HNF-4α. In conclusion, our data indicate that HNF-4α may have a wider role in cell and plasma cholesterol homeostasis by controlling the expression of LXRα in hepatic cells.


Assuntos
Regulação da Expressão Gênica/genética , Fator 4 Nuclear de Hepatócito/genética , Fator 4 Nuclear de Hepatócito/metabolismo , Receptores Nucleares Órfãos/genética , Receptores Nucleares Órfãos/metabolismo , Ativação Transcricional/genética , Sítios de Ligação , Células Hep G2 , Fator 4 Nuclear de Hepatócito/química , Humanos , Receptores X do Fígado , Receptores Nucleares Órfãos/química , Ligação Proteica
6.
J Rheumatol ; 42(9): 1652-60, 2015 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-26233507

RESUMO

OBJECTIVE: Ankylosing spondylitis (AS) is a chronic inflammatory disease associated with increased risk of cardiovascular disease (CVD). High-density lipoprotein (HDL) exerts a series of antiatherogenic properties and protects from CVD. We evaluated whether HDL antiatherogenic properties are impaired in patients with AS. METHODS: HDL (apoB-depleted serum) was isolated from 35 patients with AS and 35 age- and sex-matched controls. We measured the antioxidant capacity of HDL, the ability of HDL to induce cholesterol efflux, the activity of HDL-associated enzymes paraoxonase-1 (PON1) and myeloperoxidase (MPO), as well as the ability of HDL to induce Akt kinase activation. RESULTS: HDL from patients with AS had decreased antioxidant capacity and decreased ability to promote cholesterol efflux from macrophages compared to controls. HDL-associated PON1 activity was lower and HDL-associated MPO activity higher in patients with AS compared to controls. Higher MPO activity correlated positively with lower antioxidant capacity of HDL in patients with AS. In addition, HDL from patients with AS had impaired endothelial Akt kinase activating properties that were inversely correlated with the MPO/PON1 ratio and positively correlated with the cholesterol efflux capacity of HDL. CONCLUSION: HDL from patients with AS displays impaired antiatherogenic properties. Attenuation of HDL properties may constitute a link between AS and CVD.


Assuntos
Arildialquilfosfatase/metabolismo , Colesterol/metabolismo , Lipoproteínas HDL/metabolismo , Peroxidase/metabolismo , Espondilite Anquilosante/metabolismo , Adulto , Antirreumáticos/uso terapêutico , Feminino , Humanos , Infliximab/uso terapêutico , Masculino , Metotrexato/uso terapêutico , Metilprednisolona/uso terapêutico , Pessoa de Meia-Idade , Espondilite Anquilosante/tratamento farmacológico
7.
Endocrinology ; 152(4): 1274-83, 2011 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-21325398

RESUMO

Impaired insulin sensitivity (insulin resistance) is a common denominator in many metabolic disorders, exerting pleiotropic effects on skeletal muscle, liver, and adipose tissue function. Heme oxygenase-1 (HOX-1), the rate-limiting enzyme in heme catabolism, has recently been shown to confer an antidiabetic effect while regulating cellular redox-buffering capacity. Therefore, in the present study, we probed into the mechanisms underlying the effect of insulin on HOX-1 in C2 skeletal myoblasts. Hence, insulin was found to suppress C2 myoblasts viability via stimulation of oxidative stress, with HOX-1 counteracting this action. Insulin induced HOX-1 expression in a time- and dose-dependent manner, an effect attenuated by selective inhibitors of ERK1/2 (PD98059), Src (4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d] pyrimidine), and c-Jun terminal kinases 1 and 2 (SP600125) pathways. Furthermore, nuclear factor-κB role in insulin-induced HOX-1 up-regulation was verified, with ERK1/2, Src, and c-Jun terminal kinases 1 and 2 mediating p65-nuclear factor-κB subunit phosphorylation. Overall, our novel findings highlight for the first time the transduction mechanisms mediating HOX-1 induction in insulin-treated C2 myoblasts. This effect was established to be cell type specific because insulin failed to promote HOX-1 expression in HepG2 hepatoma cells. Deciphering the signaling networks involved in insulin-stimulated HOX-1 up-regulation is of prominent significance because it may potentially contribute to elucidation of the mechanisms involved in associated metabolic pathologies.


Assuntos
Heme Oxigenase-1/metabolismo , Insulina/farmacologia , Mioblastos Esqueléticos/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Animais , Antracenos/farmacologia , Antioxidantes/farmacologia , Ácido Ascórbico/farmacologia , Western Blotting , Caspase 3/metabolismo , Catalase/farmacologia , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Inibidores Enzimáticos/farmacologia , Flavonoides/farmacologia , Heme Oxigenase-1/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/antagonistas & inibidores , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Mioblastos Esqueléticos/efeitos dos fármacos , Mioblastos Esqueléticos/enzimologia , NF-kappa B/metabolismo , Fosforilação/efeitos dos fármacos , Protoporfirinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Transdução de Sinais/efeitos dos fármacos
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