RESUMO
The complex relationship between metabolic disease risk and body fat distribution in humans involves cellular characteristics which are specific to body fat compartments. Here we show depot-specific differences in the stromal vascual fraction of visceral and subcutaneous adipose tissue by performing single-cell RNA sequencing of tissue specimen from obese individuals. We characterize multiple immune cells, endothelial cells, fibroblasts, adipose and hematopoietic stem cell progenitors. Subpopulations of adipose-resident immune cells are metabolically active and associated with metabolic disease status and those include a population of potential dysfunctional CD8+ T cells expressing metallothioneins. We identify multiple types of adipocyte progenitors that are common across depots, including a subtype enriched in individuals with type 2 diabetes. Depot-specific analysis reveals a class of adipocyte progenitors unique to visceral adipose tissue, which shares common features with beige preadipocytes. Our human single-cell transcriptome atlas across fat depots provides a resource to dissect functional genomics of metabolic disease.
Assuntos
Tecido Adiposo/metabolismo , Doenças Metabólicas/metabolismo , Análise de Célula Única/métodos , Adipócitos/metabolismo , Tecido Adiposo/citologia , Adulto , Distribuição da Gordura Corporal , Feminino , Perfilação da Expressão Gênica , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Masculino , Doenças Metabólicas/patologia , Pessoa de Meia-Idade , Obesidade/metabolismoRESUMO
Glioblastoma (GBM) is the most common and deadly malignant brain cancer of glial cell origin, with a median patient survival of less than 20 months. Transcription factors FOXG1 and TLE1 promote GBM propagation by supporting maintenance of brain tumour-initiating cells (BTICs) with stem-like properties. Here, we characterize FOXG1 and TLE1 target genes in GBM patient-derived BTICs using ChIP-Seq and RNA-Seq approaches. These studies identify 150 direct FOXG1 targets, several of which are also TLE1 targets, involved in cell proliferation, differentiation, survival, chemotaxis and angiogenesis. Negative regulators of NOTCH signalling, including CHAC1, are among the transcriptional repression targets of FOXG1:TLE1 complexes, suggesting a crosstalk between FOXG1:TLE1 and NOTCH-mediated pathways in GBM. These results provide previously unavailable insight into the transcriptional programs underlying the tumour-promoting functions of FOXG1:TLE1 in GBM.
Assuntos
Fatores de Transcrição Forkhead/genética , Redes Reguladoras de Genes , Glioblastoma/genética , Glioblastoma/patologia , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Proteínas do Tecido Nervoso/genética , Proteínas Repressoras/genética , Sítios de Ligação , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proteínas Correpressoras , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Genoma Humano , Fator 3-alfa Nuclear de Hepatócito/metabolismo , Humanos , Proteínas do Tecido Nervoso/metabolismo , Ligação Proteica , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Proteínas Repressoras/metabolismo , Reprodutibilidade dos Testes , gama-Glutamilciclotransferase/metabolismoRESUMO
Embryonal tumors with multilayered rosettes (ETMRs) are rare, deadly pediatric brain tumors characterized by high-level amplification of the microRNA cluster C19MC. We performed integrated genetic and epigenetic analyses of 12 ETMR samples and identified, in all cases, C19MC fusions to TTYH1 driving expression of the microRNAs. ETMR tumors, cell lines and xenografts showed a specific DNA methylation pattern distinct from those of other tumors and normal tissues. We detected extreme overexpression of a previously uncharacterized isoform of DNMT3B originating at an alternative promoter that is active only in the first weeks of neural tube development. Transcriptional and immunohistochemical analyses suggest that C19MC-dependent DNMT3B deregulation is mediated by RBL2, a known repressor of DNMT3B. Transfection with individual C19MC microRNAs resulted in DNMT3B upregulation and RBL2 downregulation in cultured cells. Our data suggest a potential oncogenic re-engagement of an early developmental program in ETMR via epigenetic alteration mediated by an embryonic, brain-specific DNMT3B isoform.
Assuntos
Neoplasias Encefálicas/genética , Cromossomos Humanos Par 19 , DNA (Citosina-5-)-Metiltransferases/genética , Proteínas de Membrana/genética , MicroRNAs/genética , Neoplasias Embrionárias de Células Germinativas/genética , Pré-Escolar , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA , Regulação Neoplásica da Expressão Gênica , Fusão Gênica , Humanos , Masculino , Isoformas de Proteínas , Proteína p130 Retinoblastoma-Like/genética , Ensaios Antitumorais Modelo de Xenoenxerto , DNA Metiltransferase 3BRESUMO
The reelin signaling pathway regulates nervous system function after birth, in addition to its role in regulating neuronal positioning during embryogenesis. The receptor-dependent, reelin-induced tyrosine phosphorylation of the Dab1 docking protein is an established prerequisite for biological responses to this ligand. Here we show that the inactivation of a conditional Dab1 allele reduces process complexity in correctly positioned neurons in the CA1 region of the mouse hippocampus after birth. Reelin stimulation of cultured hippocampal neurons enhances dendritogenesis by approximately twofold and in a manner dependent on Src family kinases. This enhancement is blocked by reducing expression of Crk family proteins, adaptor molecules that interact with Dab1 in a tyrosine phosphorylation-dependent manner. Retrovirally expressed inhibitory RNAs used to reduce Crk and CrkL expression did not block BDNF-enhanced dendritogenesis or influence axonogenesis. Together, this demonstrates that the Crk family proteins are important downstream components of the reelin signaling pathway in the regulation of postnatal hippocampal dendritogenesis.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Moléculas de Adesão Celular Neuronais/fisiologia , Diferenciação Celular/fisiologia , Dendritos/metabolismo , Proteínas da Matriz Extracelular/fisiologia , Hipocampo/metabolismo , Proteínas do Tecido Nervoso/fisiologia , Proteínas Nucleares/metabolismo , Proteínas Proto-Oncogênicas c-crk/metabolismo , Serina Endopeptidases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Forma Celular/fisiologia , Células Cultivadas , Dendritos/ultraestrutura , Regulação para Baixo/genética , Hipocampo/embriologia , Hipocampo/crescimento & desenvolvimento , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Nucleares/genética , Fosforilação , Proteínas Proto-Oncogênicas c-crk/genética , Células Piramidais/citologia , Células Piramidais/metabolismo , RNA Interferente Pequeno , Proteína Reelina , Transdução de Sinais/fisiologia , Coloração pela Prata , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
Reelin-induced Dab1 tyrosine phosphorylation has been implicated in the regulation of neuronal positioning during brain development. The downstream consequences of Dab1 tyrosine phosphorylation are not fully understood, however. Here we identify CrkII, CrkL and Dock1 in complexes bound to tyrosine-phosphorylated Dab1, through mass spectrometry. The CrkII-Dab1 interaction requires tyrosine phosphorylation of Dab1 at residues 220 or 232 and is promoted by Reelin treatment of embryonic forebrain neurons. Unlike other CrkII binding proteins, such as paxillin and p130Cas, expression of Dab1 interfered with CrkII-dependent cell migration of Nara Bladder Tumor II (NBT-II) cells, in a tyrosine phosphorylation-site dependent manner. Overexpression of CrkIIGFP rescued the migration of these cells, suggesting that Dab1 makes Crk a limiting factor for migration. The Dock1-Dab1 association is indirect and requires CrkII. In organisms such as Drosophila melanogaster and Caenorhabditis elegans, signaling complexes, which contain Crk and Dock1 family members are conserved and act through Rac. We show that a rough-eye phenotype in Drosophila caused by exogenous expression of tyrosine-phosphorylated mouse Dab1RFP is partially rescued by a loss-of-function mutation in myoblast city, a Dock1-like gene in Drosophila. We propose a model that tyrosine-phosphorylated Dab1 engages the conserved Crk-Dock1-Rac signaling cassette, but when bound to Dab1 this signaling complex does not support migration.