RESUMO
The differentiation of human blood monocytes (MO), the post-mitotic precursors of macrophages (MAC) and dendritic cells (moDC), is accompanied by the active turnover of DNA methylation, but the extent, consequences and mechanisms of DNA methylation changes remain unclear. Here, we profile and compare epigenetic landscapes during IL-4/GM-CSF-driven MO differentiation across the genome and detect several thousand regions that are actively demethylated during culture, both with or without accompanying changes in chromatin accessibility or transcription factor (TF) binding. We further identify TF that are globally associated with DNA demethylation processes. While interferon regulatory factor 4 (IRF4) is found to control hallmark dendritic cell functions with less impact on DNA methylation, early growth response 2 (EGR2) proves essential for MO differentiation as well as DNA methylation turnover at its binding sites. We also show that ERG2 interacts with the 5mC hydroxylase TET2, and its consensus binding sequences show a characteristic DNA methylation footprint at demethylated sites with or without detectable protein binding. Our findings reveal an essential role for EGR2 as epigenetic pioneer in human MO and suggest that active DNA demethylation can be initiated by the TET2-recruiting TF both at stable and transient binding sites.
Assuntos
Proteína 2 de Resposta de Crescimento Precoce/metabolismo , Monócitos/metabolismo , Sítios de Ligação , Células Cultivadas , Sequenciamento de Cromatina por Imunoprecipitação , Desmetilação do DNA , Metilação de DNA/genética , Metilação de DNA/fisiologia , Proteína 2 de Resposta de Crescimento Precoce/química , Proteína 2 de Resposta de Crescimento Precoce/genética , Humanos , Immunoblotting , Imunoprecipitação , Espectrometria de Massas , Ligação Proteica , RNA-SeqRESUMO
Diabetes mellitus type II (DM) and immune cell infiltration determine patient outcome in many tumor entities. Here we studied a possible link between the metabolic and immune cell status of OSCC patients. Glucose transporter (GLUT) 1 mRNA expression was elevated in all tumor samples, whereas other glycolytic markers such as lactate dehydrogenase (LDH) A or monocarboxylate transporter (MCT) 1 were increased in tumor samples from patients with diabetes and these patients had a significantly worse prognosis compared to non-diabetic patients. Analyses of immune cell infiltration in tumors from diabetic and non-diabetic patients revealed an increased leukocyte (CD45+) infiltration compared to normal mucosa only in non-diabetic patients. In line, the amount of CD3+ T cells per mm2 tumor tissue, was elevated in patients without diabetes and crucial for patient outcome in OSCC patients without diabetes, as compared to healthy mucosa using fluorescence immunohistochemistry in tissue microarrays of 229 patients. Our results demonstrate that diabetes is a prognostic factor for OSCC patients and associates with decreased leukocyte and CD3+ infiltration indicating that metabolic differences between diabetic and non-diabetic patients may alter tumor-infiltrating T cells and thereby determine patient outcome.
Assuntos
Diabetes Mellitus Tipo 2/epidemiologia , Linfócitos do Interstício Tumoral/imunologia , Neoplasias Bucais/mortalidade , Carcinoma de Células Escamosas de Cabeça e Pescoço/mortalidade , Linfócitos T/imunologia , Adulto , Complexo CD3/metabolismo , Diabetes Mellitus Tipo 2/imunologia , Diabetes Mellitus Tipo 2/metabolismo , Transportador de Glucose Tipo 1/metabolismo , Humanos , Imuno-Histoquímica , Estimativa de Kaplan-Meier , Linfócitos do Interstício Tumoral/metabolismo , Masculino , Pessoa de Meia-Idade , Transportadores de Ácidos Monocarboxílicos/metabolismo , Mucosa Bucal/imunologia , Mucosa Bucal/patologia , Mucosa Bucal/cirurgia , Neoplasias Bucais/imunologia , Neoplasias Bucais/metabolismo , Neoplasias Bucais/cirurgia , Valor Preditivo dos Testes , Prognóstico , Medição de Risco/métodos , Medição de Risco/estatística & dados numéricos , Fatores de Risco , Carcinoma de Células Escamosas de Cabeça e Pescoço/imunologia , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/cirurgia , Simportadores/metabolismo , Linfócitos T/metabolismo , Efeito Warburg em OncologiaRESUMO
An amendment to this paper has been published and can be accessed via a link at the top of the paper.
RESUMO
Establishing gene regulatory networks during differentiation or reprogramming requires master or pioneer transcription factors (TFs) such as PU.1, a prototype master TF of hematopoietic lineage differentiation. To systematically determine molecular features that control its activity, here we analyze DNA-binding in vitro and genome-wide in vivo across different cell types with native or ectopic PU.1 expression. Although PU.1, in contrast to classical pioneer factors, is unable to access nucleosomal target sites in vitro, ectopic induction of PU.1 leads to the extensive remodeling of chromatin and redistribution of partner TFs. De novo chromatin access, stable binding, and redistribution of partner TFs both require PU.1's N-terminal acidic activation domain and its ability to recruit SWI/SNF remodeling complexes, suggesting that the latter may collect and distribute co-associated TFs in conjunction with the non-classical pioneer TF PU.1.
Assuntos
Montagem e Desmontagem da Cromatina/fisiologia , Redes Reguladoras de Genes , Hematopoese/genética , Nucleossomos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Sítios de Ligação/genética , Diferenciação Celular/genética , Linhagem Celular Tumoral , Proteínas Cromossômicas não Histona/metabolismo , DNA/metabolismo , Voluntários Saudáveis , Células-Tronco Hematopoéticas/fisiologia , Humanos , Leucaférese , Domínios Proteicos , RNA-SeqRESUMO
Alpha-syntrophin (SNTA) is a molecular adapter protein which is expressed in adipocytes. Knock-down of SNTA in 3T3-L1 preadipocytes increases cell proliferation, and differentiated adipocytes display small lipid droplets. These effects are both characteristics of healthy adipose tissue growth which is associated with metabolic improvements in obesity. To evaluate a role of SNTA in adipose tissue morphology and obesity associated metabolic dysfunction, SNTA deficient mice were fed a standard chow or a high fat diet. Mice deficient of SNTA had less fat mass and smaller adipocytes in obesity when compared to control animals. Accordingly, these animals did not develop liver steatosis and did not store excess triglycerides in skeletal muscle upon high fat diet feeding. SNTA-/- animals were protected from hyperinsulinemia and hepatic insulin resistance. Of note, body-weight, food uptake, and serum lipids were normal in the SNTA null mice. SNTA was induced in adipose tissues but not in the liver of diet induced obese and ob/ob mice. In human subcutaneous and visceral fat of seven patients SNTA was similarly expressed and was not associated with body mass index. Current data demonstrate beneficial effects of SNTA deficiency in obesity which is partly attributed to smaller adipocytes and reduced white adipose tissue mass. Higher SNTA protein in fat depots of obese mice may contribute to adipose tissue hypertrophy and ectopic lipid deposition which has to be confirmed in humans.
Assuntos
Adipócitos/patologia , Proteínas de Ligação ao Cálcio/fisiologia , Hipertrofia/prevenção & controle , Gordura Intra-Abdominal/patologia , Proteínas de Membrana/fisiologia , Proteínas Musculares/fisiologia , Obesidade/complicações , Triglicerídeos/metabolismo , Adipócitos/metabolismo , Animais , Células Cultivadas , Dieta Hiperlipídica/efeitos adversos , Hipertrofia/etiologia , Hipertrofia/patologia , Resistência à Insulina , Gordura Intra-Abdominal/metabolismo , Masculino , Camundongos , Camundongos Knockout , Camundongos Obesos , Obesidade/fisiopatologiaRESUMO
Adiponectin receptor 1 (AdipoR1) is one of the two signaling receptors of adiponectin with multiple beneficial effects in metabolic diseases. AdipoR1 C-terminal peptide is concordant with the consensus sequence of class I PSD-95, disc large, ZO-1 (PDZ) proteins, and screening of a liver yeast two hybrid library identified binding to ß2-syntrophin (SNTB2). Hybridization of a PDZ-domain array with AdipoR1 C-terminal peptide shows association with PDZ-domains of further proteins including ß1- and α-syntrophin (SNTA). Interaction of PDZ proteins and C-terminal peptides requires a free carboxy terminus next to the PDZ-binding region and is blocked by carboxy terminal added tags. N-terminal tagged AdipoR1 is more highly expressed than C-terminal tagged receptor suggesting that the free carboxy terminus may form a complex with PDZ proteins to regulate cellular AdipoR1 levels. The C- and N-terminal tagged AdipoR1 proteins are mainly localized in the cytoplasma. N-terminal but not C-terminal tagged AdipoR1 colocalizes with syntrophins in adiponectin incubated Huh7 cells. Adiponectin induced hepatic phosphorylation of AMPK and p38 MAPK which are targets of AdipoR1 is, however, not blocked in SNTA and SNTB2 deficient mice. Further, AdipoR1 protein is similarly abundant in the liver of knock-out and wild type mice when kept on a standard chow or a high fat diet. In summary these data suggest that AdipoR1 protein levels are regulated by so far uncharacterized class I PDZ proteins which are distinct from SNTA and SNTB2.
Assuntos
Proteínas Associadas à Distrofina/metabolismo , Hepatócitos/metabolismo , Domínios PDZ , Receptores de Adiponectina/metabolismo , Quinases Proteína-Quinases Ativadas por AMP , Animais , Linhagem Celular , Proteínas Associadas à Distrofina/química , Ativação Enzimática/fisiologia , Imunofluorescência , Humanos , Immunoblotting , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Proteínas Quinases/metabolismo , Receptores de Adiponectina/química , Transfecção , Técnicas do Sistema de Duplo-Híbrido , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the 'rules' for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25-45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.
Assuntos
Proteínas Proto-Oncogênicas/metabolismo , Transativadores/metabolismo , Sequência de Bases , Sítios de Ligação , Cromatina/química , Cromatina/metabolismo , Sequência Consenso , DNA/metabolismo , Desoxirribonuclease I , Humanos , Macrófagos/metabolismo , Monócitos/metabolismo , Motivos de Nucleotídeos , Ligação Proteica , Células-Tronco/metabolismoRESUMO
BACKGROUND: Cytosine methylation is a frequent epigenetic modification restricting the activity of gene regulatory elements. Whereas DNA methylation patterns are generally inherited during replication, both embryonic and somatic differentiation processes require the removal of cytosine methylation at specific gene loci to activate lineage-restricted elements. However, the exact mechanisms facilitating the erasure of DNA methylation remain unclear in many cases. RESULTS: We previously established human post-proliferative monocytes as a model to study active DNA demethylation. We now show, for several previously identified genomic sites, that the loss of DNA methylation during the differentiation of primary, post-proliferative human monocytes into dendritic cells is preceded by the local appearance of 5-hydroxymethylcytosine. Monocytes were found to express the methylcytosine dioxygenase Ten-Eleven Translocation (TET) 2, which is frequently mutated in myeloid malignancies. The siRNA-mediated knockdown of this enzyme in primary monocytes prevented active DNA demethylation, suggesting that TET2 is essential for the proper execution of this process in human monocytes. CONCLUSIONS: The work described here provides definite evidence that TET2-mediated conversion of 5-methylcytosine to 5-hydroxymethylcytosine initiates targeted, active DNA demethylation in a mature postmitotic myeloid cell type.
Assuntos
Citosina/análogos & derivados , Proteínas de Ligação a DNA/metabolismo , Células Dendríticas/metabolismo , Monócitos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , 5-Metilcitosina/análogos & derivados , Diferenciação Celular , Citosina/metabolismo , Metilação de DNA , Proteínas de Ligação a DNA/genética , Dioxigenases , Epigênese Genética , Técnicas de Silenciamento de Genes , Humanos , Espectrometria de Massas , Proteínas Proto-Oncogênicas/genéticaRESUMO
Systemic concentrations of interleukin-6 (IL-6) are elevated in patients with liver cirrhosis, and impaired hepatic uptake of IL-6 was suggested to contribute to higher levels in these patients. To test this hypothesis IL-6 was measured in portal venous serum (PVS), hepatic venous serum (HVS) and systemic venous serum (SVS) of 41 patients with liver cirrhosis and four patients with normal liver function. IL-6 was higher in PVS than HVS of all blood donors and about 43% of portal vein derived IL-6 was extracted by the healthy liver, and 6.3% by the cirrhotic liver demonstrating markedly impaired removal of IL-6 by the latter. Whereas in patients with CHILD-PUGH stage A IL-6 in HVS was almost 25% lower than in PVS, in patients with CHILD-PUGH stage C IL-6 was similarly abundant in the two blood compartments. Ascites is a common complication in cirrhotic patients and was associated with higher IL-6 levels in all blood compartments without significant differences in hepatic excretion. Hepatic venous pressure gradient did not correlate with the degree of hepatic IL-6 removal excluding hepatic shunting as the principal cause of impaired IL-6 uptake. Furthermore, patients with alcoholic liver cirrhosis had higher IL-6 in all blood compartments than patients with cryptogenic liver cirrhosis. Aetiology of liver cirrhosis did not affect hepatic removal rate indicating higher IL-6 synthesis in patients with alcoholic liver cirrhosis. In summary, the current data provide evidence that impaired hepatic removal of IL-6 is explained by hepatic shunting and liver dysfunction in patients with liver cirrhosis partly explaining higher systemic levels.
Assuntos
Interleucina-6/sangue , Cirrose Hepática/sangue , Cirrose Hepática/fisiopatologia , Fígado/irrigação sanguínea , Fígado/fisiopatologia , Adulto , Idoso , Antropometria , Ascite/sangue , Ascite/complicações , Ascite/fisiopatologia , Estudos de Casos e Controles , Demografia , Feminino , Humanos , Cirrose Hepática/complicações , Testes de Função Hepática , Masculino , Pessoa de Meia-Idade , Veia Porta/fisiopatologiaRESUMO
Chemerin is an adipokine whose systemic concentration and adipose tissue expression is increased in obesity. Chemerin is highly abundant in adipocytes, yet the molecular mechanisms mediating its further induction in obesity have not been clarified. Adipocyte hypertrophy contributes to dysregulated adipokine synthesis, and we hypothesized that excess loading with free fatty acids (FFA) stimulates chemerin synthesis. Chemerin was expressed in mature adipocytes, and differentiation of 3T3-L1 cells in the presence of FFA further increased its level. TNF and IL-6 were induced by FFA, but concentrations were too low to up-regulate chemerin. Sterol regulatory element-binding protein 2 (SREBP2) was activated in these cells, indicative for cholesterol shortage. Suppression of cholesterol synthesis by lovastatin led to activation of SREBP2 and increased chemerin, and supplementation with mevalonate reversed this effect. Knockdown of SREBP2 reduced basal and FFA-induced chemerin. EMSA confirmed binding of 3T3-L1 adipocyte nuclear proteins to a SREBP site in the chemerin promotor. SREBP2 was activated and chemerin was induced in adipose tissue of mice fed a high-fat diet, and higher systemic levels seem to be derived from adipocytes. Lipopolysaccharide-mediated elevation of chemerin was similarly effective as induction by FFA, indicating that both mechanisms are equally important. Chemokine-like receptor 1 was not altered by the incubations mentioned above, and higher expression in fat of mice fed a high-fat diet may reflect increased number of adipose tissue-resident macrophages in obesity. In conclusion, the current data show that adipocyte hypertrophy and chronic inflammation are equally important in inducing chemerin synthesis.