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1.
Nat Commun ; 12(1): 224, 2021 01 11.
Artigo em Inglês | MEDLINE | ID: mdl-33431851

RESUMO

The neural circuitry mediating taste has been mapped out from the periphery to the cortex, but genetic identity of taste-responsive neurons has remained elusive. Here, we describe a population of neurons in the gustatory region of the parabrachial nucleus that express the transcription factor Satb2 and project to taste-associated regions, including the gustatory thalamus and insular cortex. Using calcium imaging in awake, freely licking mice, we show that Satb2 neurons respond to the five basic taste modalities. Optogenetic activation of these neurons enhances taste preferences, whereas chronic inactivation decreases the magnitude of taste preferences in both brief- and long-access taste tests. Simultaneous inactivation of Satb2 and calcitonin gene-related peptide neurons in the PBN abolishes responses to aversive tastes. These data suggest that taste information in the parabrachial nucleus is conveyed by multiple populations of neurons, including both Satb2 and calcitonin gene-related peptide neurons.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neurônios/fisiologia , Núcleos Parabraquiais/fisiologia , Percepção Gustatória/fisiologia , Fatores de Transcrição/metabolismo , Animais , Peptídeo Relacionado com Gene de Calcitonina/metabolismo , Feminino , Proteínas de Fluorescência Verde/metabolismo , Masculino , Camundongos Endogâmicos C57BL , Estimulação Física , Paladar/fisiologia
2.
Am J Hum Genet ; 108(2): 346-356, 2021 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-33513338

RESUMO

Whereas large-scale statistical analyses can robustly identify disease-gene relationships, they do not accurately capture genotype-phenotype correlations or disease mechanisms. We use multiple lines of independent evidence to show that different variant types in a single gene, SATB1, cause clinically overlapping but distinct neurodevelopmental disorders. Clinical evaluation of 42 individuals carrying SATB1 variants identified overt genotype-phenotype relationships, associated with different pathophysiological mechanisms, established by functional assays. Missense variants in the CUT1 and CUT2 DNA-binding domains result in stronger chromatin binding, increased transcriptional repression, and a severe phenotype. In contrast, variants predicted to result in haploinsufficiency are associated with a milder clinical presentation. A similarly mild phenotype is observed for individuals with premature protein truncating variants that escape nonsense-mediated decay, which are transcriptionally active but mislocalized in the cell. Our results suggest that in-depth mutation-specific genotype-phenotype studies are essential to capture full disease complexity and to explain phenotypic variability.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/genética , Mutação , Transtornos do Neurodesenvolvimento/genética , Cromatina/metabolismo , Feminino , Estudos de Associação Genética , Haploinsuficiência , Humanos , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/química , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Modelos Moleculares , Mutação de Sentido Incorreto , Ligação Proteica , Domínios Proteicos , Transcrição Genética
3.
Int J Mol Sci ; 22(1)2020 Dec 23.
Artigo em Inglês | MEDLINE | ID: mdl-33374770

RESUMO

MET-amplified gastric cancer cells are extremely sensitive to MET inhibition in vitro, whereas clinical efficacy of MET inhibitors is disappointing. The compensatory activation of other oncogenic growth factor receptors may serve as an underlying mechanism of resistance. In this study, we analyzed the role of HER receptors, in particular HER3 and its ligand heregulin, in this respect. This also included the chromatin-organizer protein SATB1, as an established regulator of HER expression in other tumor entities. In a panel of MET-amplified gastric carcinoma cell lines, cell growth under anchorage-dependent and independent conditions was studied upon inhibitor treatment or siRNA-mediated knockdown. Expression analyses were performed using RT-qPCR, FACS, and immunoblots. Signal transduction was monitored via antibody arrays and immunoblots. As expected, MET inhibition led to a growth arrest and inhibition of MAPK signaling. Strikingly, however, this was accompanied by a rapid and profound upregulation of the oncogenic receptor HER3. This finding was determined as functionally relevant, since HER3 activation by HRG led to partial MET inhibitor resistance, and MAPK/Akt signaling was even found enhanced upon HRG+MET inhibitor treatment compared to HRG alone. SATB1 was identified as mediator of HER3 upregulation. Concomitantly, SATB1 knockdown prevented upregulation of HER3, thus abrogating the HRG-promoted rescue from MET inhibition. Taken together, our results introduce the combined HER3/MET inhibition as strategy to overcome resistance towards MET inhibitors.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Proteínas Proto-Oncogênicas c-met/antagonistas & inibidores , Receptor ErbB-3/genética , Neoplasias Gástricas/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Ligação à Região de Interação com a Matriz/genética , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-met/metabolismo , Pirazinas/farmacologia , Receptor ErbB-3/metabolismo , Neoplasias Gástricas/genética , Triazóis/farmacologia , Regulação para Cima
4.
Nature ; 583(7817): 625-630, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32669713

RESUMO

The recent discovery of N6-methyladenine (N6-mA) in mammalian genomes suggests that it may serve as an epigenetic regulatory mechanism1. However, the biological role of N6-mA and the molecular pathways that exert its function remain unclear. Here we show that N6-mA has a key role in changing the epigenetic landscape during cell fate transitions in early development. We found that N6-mA is upregulated during the development of mouse trophoblast stem cells, specifically at regions of stress-induced DNA double helix destabilization (SIDD)2-4. Regions of SIDD are conducive to topological stress-induced unpairing of the double helix and have critical roles in organizing large-scale chromatin structures3,5,6. We show that the presence of N6-mA reduces the in vitro interactions by more than 500-fold between SIDD and SATB1, a crucial chromatin organizer that interacts with SIDD regions. Deposition of N6-mA also antagonizes SATB1 function in vivo by preventing its binding to chromatin. Concordantly, N6-mA functions at the boundaries between euchromatin and heterochromatin to restrict the spread of euchromatin. Repression of SIDD-SATB1 interactions mediated by N6-mA is essential for gene regulation during trophoblast development in cell culture models and in vivo. Overall, our findings demonstrate an unexpected molecular mechanism for N6-mA function via SATB1, and reveal connections between DNA modification, DNA secondary structures and large chromatin domains in early embryonic development.


Assuntos
Adenina/análogos & derivados , DNA/química , DNA/metabolismo , Desenvolvimento Embrionário , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Adenina/metabolismo , Animais , Pareamento de Bases , Desenvolvimento Embrionário/genética , Eucromatina/genética , Eucromatina/metabolismo , Feminino , Humanos , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Células-Tronco/citologia , Células-Tronco/metabolismo , Termodinâmica , Trofoblastos/citologia
5.
Hum Cell ; 33(4): 1155-1164, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32504285

RESUMO

Hepatocellular carcinoma (HCC) remains a lethal cancer type for both males and females. MicroRNAs (miRNAs) contribute to the initiation, development and metastasis of cancer. Although several miRNAs have been identified as drivers or suppressors of HCC, the molecular mechanisms of many miRNAs have not been investigated. Currently, we discovered that miR-4270-5p was a significantly downregulated miRNA in HCC. We revealed that miR-4270-5p overexpression inhibited cell proliferation and invasion of HCC cells. The data manifested that miR-4270-5p directly targeted SATB2, a key regulator of epithelial mesenchymal transition (EMT), in HCC cells and reversed the EMT process. The rescue experiments suggested that SATB2 overexpression reversed the biological function of miR-4270-5p in HCC cells. Clinical data indicated that SATB2 expression was negatively correlated with miR-4270-5p levels in HCC patients. Our findings provided potential targets for prognosis and treatment of patients with HCC.


Assuntos
Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Proliferação de Células/genética , Regulação Neoplásica da Expressão Gênica/genética , Expressão Gênica/genética , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/genética , MicroRNAs/metabolismo , MicroRNAs/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Linhagem Celular Tumoral , Regulação para Baixo/genética , Transição Epitelial-Mesenquimal/genética , Feminino , Humanos , Neoplasias Hepáticas/terapia , Masculino , Terapia de Alvo Molecular
6.
Mol Cell ; 78(5): 876-889.e6, 2020 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-32502422

RESUMO

Many microRNAs (miRNAs) are generated from primary transcripts containing multiple clustered stem-loop structures that are thought to be recognized and cleaved by the Microprocessor complex as independent units. Here, we uncover an unexpected mode of processing of the bicistronic miR-15a-16-1 cluster. We find that the primary miR-15a stem-loop is not processed on its own but that the presence of the neighboring primary miR-16-1 stem-loop on the same transcript can compensate for this deficiency in cis. Using a CRISPR/Cas9 screen, we identify SAFB2 (scaffold attachment factor B2) as an essential co-factor in this miR-16-1-assisted pri-miR-15 cleavage and describe SAFB2 as an accessory protein of the Microprocessor. Notably, SAFB2-mediated cleavage expands to other clustered pri-miRNAs, indicating a general mechanism. Together, our study reveals an unrecognized function of SAFB2 in miRNA processing and suggests a scenario in which SAFB2 enables the binding and processing of suboptimal Microprocessor substrates in clustered primary miRNA transcripts.


Assuntos
Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/metabolismo , Proteínas Associadas à Matriz Nuclear/metabolismo , Receptores Estrogênicos/metabolismo , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Células HEK293 , Humanos , Sequências Repetidas Invertidas/genética , Sequências Repetidas Invertidas/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , MicroRNAs/genética , Proteínas Associadas à Matriz Nuclear/genética , Conformação de Ácido Nucleico , Processamento Pós-Transcricional do RNA/genética , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética
7.
PLoS One ; 15(5): e0232695, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32379794

RESUMO

BACKGROUND: The proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells (HBMScs) are modulated by a variety of microRNAs (miRNAs). SATB homeobox 2 (SATB2) is a critical transcription factor that contributes to maintain the balance of bone metabolism. However, it remains unclear how the regulatory relationship between miR-103 and SATB2 on HBMScs proliferation and osteogenic differentiation. METHODS: HBMScs were obtained from Cyagen Biosciences and successful induced osteogenic differentiation. The proliferation abilities of HBMScs after treatment with agomiR-103 and antagomiR-103 were assessed using a cell counting Kit-8 (CCK-8) assay, and osteogenic differentiation was determined using alizarin red S staining and alkaline phosphatase (ALP) activity assay. The expression levels of miR-103, SATB2, and associated osteogenic differentiation biomarkers, including RUNX family transcription factor 2 (RUNX2), bone gamma-carboxyglutamate protein (BGLAP), and secreted phosphoprotein 1 (SPP1), were evaluated using real-time qPCR and Western blot. The regulatory sites of miR-103 on SATB2 were predicted using bioinformatics software and validated using a dual luciferase reporter assay. The underlying mechanism of miR-103 on SATB2-medicated HBMScs proliferation and osteogenic differentiation were confirmed by co-transfection of antagomiR-103 and SATB2 siRNA. RESULTS: The expression of miR-103 in HBMScs after induction of osteogenic differentiation was reduced in a time-dependent way. Overexpression of miR-103 by transfection of agomiR-103 suppressed HBMScs proliferation and osteogenic differentiation, while silencing of miR-103 by antagomiR-103 abolished these inhibitory effects. Consistently, RUNX2, BGLAP and SPP1 mRNA and protein expression were decreased in agomiR-103 treated HBMScs compared with those in agomiR-NC group. Meanwhile, antagomiR-103 upregulated the mRNA and protein expression levels of RUNX2, BGLAP and SPP1 in HBMScs. Further studies revealed that SATB2 was a direct target gene of miR-103. BMSCs transfected with agomiR-103 exhibited significantly downregulated protein expression level of SATB2, whereas knockdown of miR-103 promoted it. Additionally, rescue assays confirmed that silencing of SATB2 partially reversed the effects of antagomiR-103 induced HBMScs proliferation and osteogenic differentiation. CONCLUSIONS: The present results suggested that miR-103 negatively regulates SATB2 to serve an inhibitory role in the proliferation and osteogenic differentiation of HBMScs, which sheds light upon a potential therapeutic target for treating bone-related diseases.


Assuntos
Diferenciação Celular/fisiologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , MicroRNAs/fisiologia , Osteogênese/fisiologia , Fatores de Transcrição/metabolismo , Células da Medula Óssea , Células Cultivadas , Regulação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais
8.
J Cancer Res Ther ; 16(1): 7-12, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32362602

RESUMO

Background: Xist is a long noncoding RNA involved in the X chromosome inactivation in females. It may act as an onco-suppressor gene in hematologic malignancies, and its activity is strongly dependent from SATB1 gene expression. However, its potential role in Hodgkin's disease (HD) onset and progression is unknown. Materials and Methods: Three gene expression microarray datasets were analyzed for the expression of Xist and SATB1 in patients with classical HD, namely, GDS4222 (130 patients and 54,000 gene features), GSE39134 (29 patients and 54,000 features), and E-MEXP-507 (29 patients and 27,648 probes). The first two were oligonucleotide arrays (platform: Affymetrix gene chip HG-U133-Plus2), whereas the latter was a cDNA two-channel array (platform: OncoChip. v2). Summary and time-dependent receiver operating characteristic (ROC) analysis were applied to obtain a summary measure (summary area under the ROC curve [sAUC]) of association between gene expression and unfavorable patient outcome in each probe set. Results: Xist was overexpressed among females in each data set. A slight overexpression was associated with a good prognosis both in males (sAUC = 0.75, 95% confidence interval [CI]: 0.70-0.80) and at a lesser extent, in females (sAUC = 0.64, 95% CI: 0.59-0.69). However, this finding was limited to the analysis of the biggest database (GDS4222). No association was found between Xist and SATB1 expression. Conclusions: A reactivation of Xist might act as an onco-suppressor gene in male patients with HD, which seems independent from SATB1 expression. The possibility that Xist could contribute to the better survival of female patients should also be investigated.


Assuntos
Bases de Dados de Ácidos Nucleicos/estatística & dados numéricos , Perfilação da Expressão Gênica/métodos , Regulação Neoplásica da Expressão Gênica , Genes Supressores de Tumor , Doença de Hodgkin/genética , Doença de Hodgkin/patologia , RNA Longo não Codificante/genética , Adulto , Biologia Computacional/métodos , Progressão da Doença , Feminino , Doença de Hodgkin/metabolismo , Humanos , Masculino , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Pessoa de Meia-Idade , Adulto Jovem
9.
Sci Rep ; 10(1): 8615, 2020 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-32451408

RESUMO

The Special AT-rich sequence binding protein 1 (SATB1) is a genome organizer protein that controls gene expression of numerous genes by regulating chromatin architecture and targeting chromatin-remodeling/-modifying enzymes onto specific chromatin regions. SATB1 is overexpressed in various tumors. In head and neck squamous cell carcinoma (HNSCC), SATB1 upregulation is correlated with TNM classification, metastasis, poor prognosis and reduced overall survival. In this paper, we comprehensively analyze cellular and molecular effects of SATB1 in a large set of primary cell lines from primary HNSCC or metastases, using RNAi-mediated knockdown in vitro and, therapeutically, in tumor xenograft mouse models in vivo. In a series of 15 cell lines, major differences in SATB1 levels are observed. In various 2-D and 3-D assays, growth inhibition upon efficient siRNA-mediated SATB1 knockdown depends on the cell line rather than initial SATB1 levels. Inhibitory effects are found to be based on cell cycle deceleration, apoptosis induction, decreased HER3 and Heregulin A&B expression, and effects on EMT genes. In vivo, systemic treatment of tumor xenograft-bearing mice with siRNAs formulated in polymeric nanoparticles inhibits tumor growth of two HNSCC xenograft models, resulting from therapeutic SATB1 reduction and concomitant decrease of proliferation and induction of apoptosis. In conclusion, SATB1 represents a promising target in HNSCC, affecting crucial cellular processes and molecular pathways.


Assuntos
Neoplasias de Cabeça e Pescoço/patologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/patologia , Animais , Apoptose , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Neoplasias de Cabeça e Pescoço/tratamento farmacológico , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/antagonistas & inibidores , Proteínas de Ligação à Região de Interação com a Matriz/genética , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Nanopartículas/química , Estadiamento de Neoplasias , Neuregulina-1/genética , Neuregulina-1/metabolismo , Interferência de RNA , RNA Interferente Pequeno/química , RNA Interferente Pequeno/metabolismo , RNA Interferente Pequeno/uso terapêutico , Receptor ErbB-3/genética , Receptor ErbB-3/metabolismo , Carcinoma de Células Escamosas de Cabeça e Pescoço/tratamento farmacológico , Carcinoma de Células Escamosas de Cabeça e Pescoço/metabolismo , Transplante Heterólogo , Regulação para Cima
10.
Nucleic Acids Res ; 48(11): 5873-5890, 2020 06 19.
Artigo em Inglês | MEDLINE | ID: mdl-32392347

RESUMO

The chromatin organizer SATB1 is highly enriched in thymocytes and is essential for T-cell development. Although SATB1 regulates a large number of genes important for T-cell development, the mechanism(s) regulating expression of SATB1 during this process remain elusive. Using chromatin immune precipitation-seq-based occupancy profiles of H3K4me3 and H3Kme1 at Satb1 gene locus, we predicted four different alternative promoters of Satb1 in mouse thymocytes and characterized them. The expression of Satb1 transcript variants with distinct 5' UTRs occurs in a stage-specific manner during T-cell development and is dependent on TCR signaling. The observed discrepancy between the expression levels of SATB1 mRNA and protein in developing thymocytes can be explained by the differential translatability of Satb1 transcript variants as confirmed by polysome profiling and in vitro translation assay. We show that Satb1 alternative promoters exhibit lineage-specific chromatin accessibility during T-cell development from progenitors. Furthermore, TCF1 regulates the Satb1 P2 promoter switch during CD4SP development, via direct binding to the Satb1 P2 promoter. CD4SP T cells from TCF1 KO mice exhibit downregulation of P2 transcript variant expression as well as low levels of SATB1 protein. Collectively, these results provide unequivocal evidence toward alternative promoter switch-mediated developmental stage-specific regulation of SATB1 in thymocytes.


Assuntos
Cromatina/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Regiões Promotoras Genéticas , Receptores de Antígenos de Linfócitos T/metabolismo , Linfócitos T/citologia , Linfócitos T/metabolismo , Animais , Linfócitos T CD4-Positivos/metabolismo , Diferenciação Celular , Linhagem da Célula , Cromatina/genética , Fator 1-alfa Nuclear de Hepatócito/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Biossíntese de Proteínas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Timócitos/citologia , Timócitos/metabolismo
11.
Proc Natl Acad Sci U S A ; 117(19): 10554-10564, 2020 05 12.
Artigo em Inglês | MEDLINE | ID: mdl-32312821

RESUMO

A unique combination of transcription factor expression and projection neuron identity demarcates each layer of the cerebral cortex. During mouse and human cortical development, the transcription factor CTIP2 specifies neurons that project subcerebrally, while SATB2 specifies neuronal projections via the corpus callosum, a large axon tract connecting the two neocortical hemispheres that emerged exclusively in eutherian mammals. Marsupials comprise the sister taxon of eutherians but do not have a corpus callosum; their intercortical commissural neurons instead project via the anterior commissure, similar to egg-laying monotreme mammals. It remains unknown whether divergent transcriptional networks underlie these cortical wiring differences. Here, we combine birth-dating analysis, retrograde tracing, gene overexpression and knockdown, and axonal quantification to compare the functions of CTIP2 and SATB2 in neocortical development, between the eutherian mouse and the marsupial fat-tailed dunnart. We demonstrate a striking degree of structural and functional homology, whereby CTIP2 or SATB2 of either species is sufficient to promote a subcerebral or commissural fate, respectively. Remarkably, we reveal a substantial delay in the onset of developmental SATB2 expression in mice as compared to the equivalent stage in dunnarts, with premature SATB2 overexpression in mice to match that of dunnarts resulting in a marsupial-like projection fate via the anterior commissure. Our results suggest that small alterations in the timing of regulatory gene expression may underlie interspecies differences in neuronal projection fate specification.


Assuntos
Corpo Caloso/metabolismo , Eutérios/genética , Marsupiais/genética , Animais , Axônios/metabolismo , Evolução Biológica , Encéfalo/metabolismo , Córtex Cerebral/metabolismo , Corpo Caloso/fisiologia , Proteínas de Ligação a DNA/metabolismo , Evolução Molecular , Regulação da Expressão Gênica no Desenvolvimento/genética , Redes Reguladoras de Genes/genética , Humanos , Mamíferos/genética , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Vias Neurais/fisiologia , Neurônios/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
12.
Nucleic Acids Res ; 48(8): 4551-4561, 2020 05 07.
Artigo em Inglês | MEDLINE | ID: mdl-32187371

RESUMO

Phosphorothioate modification is commonly introduced into therapeutic oligonucleotides, typically as a racemic mixture in which either of the two non-bridging phosphate oxygens is replaced by sulfur, which frequently increases affinities with proteins. Here, we used isothermal titration calorimetry and X-ray crystallography to investigate the thermodynamic and structural properties of the interaction between the primary DNA-binding domain (CUTr1) of transcription factor SATB1 and dodecamer DNAs with racemic phosphorothioate modifications at the six sites known to contact CUTr1 directly. For both the modified and unmodified DNAs, the binding reactions were enthalpy-driven at a moderate salt concentration (50 mM NaCl), while being entropy-driven at higher salt concentrations with reduced affinities. The phosphorothioate modifications lowered this susceptibility to salt, resulting in a significantly enhanced affinity at a higher salt concentration (200 mM NaCl), although only some DNA molecular species remained interacting with CUTr1. This was explained by unequal populations of the two diastereomers in the crystal structure of the complex of CUTr1 and the phosphorothioate-modified DNA. The preferred diastereomer formed more hydrogen bonds with the oxygen atoms and/or more hydrophobic contacts with the sulfur atoms than the other, revealing the origins of the enhanced affinity.


Assuntos
DNA/química , Proteínas de Ligação à Região de Interação com a Matriz/química , Oligonucleotídeos Fosforotioatos/química , Cristalografia por Raios X , DNA/metabolismo , Ligação de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Modelos Moleculares , Domínios Proteicos , Estereoisomerismo , Termodinâmica
13.
Nat Commun ; 11(1): 1562, 2020 03 26.
Artigo em Inglês | MEDLINE | ID: mdl-32218434

RESUMO

CCL5 is a unique chemokine with distinct stage and cell-type specificities for regulating inflammation, but how these specificities are achieved and how CCL5 modulates immune responses is not well understood. Here we identify two stage-specific enhancers: the proximal enhancer mediates the constitutive CCL5 expression during the steady state, while the distal enhancer located 1.35 Mb from the promoter induces CCL5 expression in activated cells. Both enhancers are antagonized by RUNX/CBFß complexes, and SATB1 further mediates the long-distance interaction of the distal enhancer with the promoter. Deletion of the proximal enhancer decreases CCL5 expression and augments the cytotoxic activity of tissue-resident T and NK cells, which coincides with reduced melanoma metastasis in mouse models. By contrast, increased CCL5 expression resulting from RUNX3 mutation is associated with more tumor metastasis in the lung. Collectively, our results suggest that RUNX3-mediated CCL5 repression is critical for modulating anti-tumor immunity.


Assuntos
Quimiocina CCL5/genética , Subunidades alfa de Fatores de Ligação ao Core/metabolismo , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Imunidade , Animais , Antígenos CD/metabolismo , Subunidade beta de Fator de Ligação ao Core/metabolismo , Homeostase/genética , Imunidade/genética , Ativação Linfocitária/imunologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Melanoma Experimental/genética , Melanoma Experimental/imunologia , Melanoma Experimental/patologia , Camundongos Endogâmicos C57BL , Camundongos Transgênicos
14.
Nat Cell Biol ; 22(2): 187-199, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31932738

RESUMO

Traditionally viewed as an autodigestive pathway, autophagy also facilitates cellular secretion; however, the mechanisms underlying these processes remain unclear. Here, we demonstrate that components of the autophagy machinery specify secretion within extracellular vesicles (EVs). Using a proximity-dependent biotinylation proteomics strategy, we identify 200 putative targets of LC3-dependent secretion. This secretome consists of a highly interconnected network enriched in RNA-binding proteins (RBPs) and EV cargoes. Proteomic and RNA profiling of EVs identifies diverse RBPs and small non-coding RNAs requiring the LC3-conjugation machinery for packaging and secretion. Focusing on two RBPs, heterogeneous nuclear ribonucleoprotein K (HNRNPK) and scaffold-attachment factor B (SAFB), we demonstrate that these proteins interact with LC3 and are secreted within EVs enriched with lipidated LC3. Furthermore, their secretion requires the LC3-conjugation machinery, neutral sphingomyelinase 2 (nSMase2) and LC3-dependent recruitment of factor associated with nSMase2 activity (FAN). Hence, the LC3-conjugation pathway controls EV cargo loading and secretion.


Assuntos
Autofagossomos/metabolismo , Autofagia/genética , Vesículas Extracelulares/metabolismo , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Ligação a RNA/genética , Proteínas Adaptadoras de Transporte Vesicular/deficiência , Proteínas Adaptadoras de Transporte Vesicular/genética , Animais , Autofagossomos/química , Proteína 7 Relacionada à Autofagia/deficiência , Proteína 7 Relacionada à Autofagia/genética , Proteínas Relacionadas à Autofagia/deficiência , Proteínas Relacionadas à Autofagia/genética , Transporte Biológico , Biotinilação , Vesículas Extracelulares/química , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Células HEK293 , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/genética , Ribonucleoproteínas Nucleares Heterogêneas Grupo K/metabolismo , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Lisossomos/química , Lisossomos/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Proteínas Associadas aos Microtúbulos/metabolismo , Proteínas Associadas à Matriz Nuclear/genética , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteômica/métodos , Células RAW 264.7 , Pequeno RNA não Traduzido/genética , Pequeno RNA não Traduzido/metabolismo , Proteínas de Ligação a RNA/classificação , Proteínas de Ligação a RNA/metabolismo , Receptores Estrogênicos/genética , Receptores Estrogênicos/metabolismo , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo
15.
Life Sci ; 241: 117113, 2020 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-31805288

RESUMO

An oncogenic role, p21-activated kinase 5 (PAK5), has proven as a significant mediator for many cellular progression, which is expressed highly in human organs such as lung, liver, kidney, blood vessels endothelial cells and inflammatory cells. PAK5 was primitively detected in the cerebrum and accelerated the filopodia formation in neurocytes. It can reverse the effect of Rho and adjust its activity to mediate maintenance and development of nerve axon by binding with Cdc42-GTP. Moreover, PAK5 has been suggested to mediate protean, multitudinous and inscrutable functions in cancer. Currently, many researches indicated that PAK5 was dysregulated in ovarian cancer, cervical cancer, melanoma, osteosarcoma, renal carcinoma, breast cancer, gastric cancer and so on, which was involved in cell proliferation, apoptosis, migration and invasion. This review focuses the latest knowledge on the structure, expression, signalling pathway of PAK5, emphasizing its function in cancer.


Assuntos
Neoplasias/metabolismo , Quinases Ativadas por p21/genética , Quinases Ativadas por p21/metabolismo , Epigênese Genética , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neoplasias/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Quinases Ativadas por p21/química
16.
Histopathology ; 76(2): 251-264, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31233624

RESUMO

AIMS: Special AT-rich sequence-binding protein 2 (SATB2) is a transcriptional regulator with critical roles in brain, craniofacial and skeletal development. It has emerged as a key marker of lower gastrointestinal (GI) tract columnar epithelial and osteoblastic differentiation. Transcription factor immunohistochemistry is useful in assigning site of origin in well-differentiated neuroendocrine tumours (NETs), and has had a limited role in poorly differentiated neuroendocrine carcinomas (NECs). This study sought to evaluate the role of SATB2 in assigning site of origin in neuroendocrine epithelial neoplasms. METHODS AND RESULTS: Tissue microarrays were constructed from the following: 317 NETs (37 thyroid, 46 lung, 16 stomach, 12 duodenum, 70 pancreas, 106 jejunoileum, 24 appendix, and six rectosigmoid), 44 phaeochromocytomas/paragangliomas, and 79 NECs (29 Merkel cell, 30 lung, and 20 extrapulmonary visceral); nine appendiceal and 19 rectal NETs were examined in whole sections. SATB2 immunohistochemistry was scored for extent (%) and intensity (0-3+), with an H-score being calculated. SATB2 was expressed by 96% of rectosigmoid NETs, 79% of appendiceal NETs, and only 7% of other well-differentiated neoplasms (P < 0.0001). Expression in lower GI tract NETs (median H-score of 255) was stronger than in other positive tumours (median H-score of 7) (P < 0.0001). Any SATB2 expression was 86% sensitive/93% specific for lower GI tract origin. SATB2 was expressed by 79% of Merkel cell carcinomas (median H-score of 300), 33% of lung NECs (median H-score of 23), and 60% of extrapulmonary visceral NECs (median H-score of 110), with stronger expression in Merkel cell carcinoma (P < 0.001). At an H-score cutoff of ≥150, SATB2 was 69% sensitive/90% specific for Merkel cell carcinoma. CONCLUSIONS: SATB2 is frequently and strongly expressed by lower GI tract NETs; we have adopted it as our rectal NET marker. Relatively frequent and strong expression in Merkel cell carcinoma may have value in assigning NEC site of origin.


Assuntos
Biomarcadores Tumorais/metabolismo , Carcinoma de Célula de Merkel/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neoplasias Epiteliais e Glandulares/metabolismo , Tumores Neuroendócrinos/metabolismo , Neoplasias Retais/metabolismo , Fatores de Transcrição/metabolismo , Carcinoma de Célula de Merkel/diagnóstico , Carcinoma de Célula de Merkel/patologia , Carcinoma Neuroendócrino/diagnóstico , Carcinoma Neuroendócrino/metabolismo , Carcinoma Neuroendócrino/patologia , Diferenciação Celular , Estudos de Coortes , Diagnóstico Diferencial , Humanos , Imuno-Histoquímica , Iowa , Trato Gastrointestinal Inferior/metabolismo , Trato Gastrointestinal Inferior/patologia , Neoplasias Epiteliais e Glandulares/diagnóstico , Neoplasias Epiteliais e Glandulares/patologia , Tumores Neuroendócrinos/diagnóstico , Tumores Neuroendócrinos/patologia , Neoplasias Retais/diagnóstico , Neoplasias Retais/patologia , Análise Serial de Tecidos
17.
BMC Biol ; 17(1): 104, 2019 12 13.
Artigo em Inglês | MEDLINE | ID: mdl-31830989

RESUMO

BACKGROUND: Polarity is necessary for epithelial cells to perform distinct functions at their apical and basal surfaces. Oral epithelial cell-derived ameloblasts at secretory stage (SABs) synthesize large amounts of enamel matrix proteins (EMPs), largely amelogenins. EMPs are unidirectionally secreted into the enamel space through their apical cytoplasmic protrusions, or Tomes' processes (TPs), to guide the enamel formation. Little is known about the transcriptional regulation underlying the establishment of cell polarity and unidirectional secretion of SABs. RESULTS: The higher-order chromatin architecture of eukaryotic genome plays important roles in cell- and stage-specific transcriptional programming. A genome organizer, special AT-rich sequence-binding protein 1 (SATB1), was discovered to be significantly upregulated in ameloblasts compared to oral epithelial cells using a whole-transcript microarray analysis. The Satb1-/- mice possessed deformed ameloblasts and a thin layer of hypomineralized and non-prismatic enamel. Remarkably, Satb1-/- ameloblasts at the secretory stage lost many morphological characteristics found at the apical surface of wild-type (wt) SABs, including the loss of Tomes' processes, defective inter-ameloblastic adhesion, and filamentous actin architecture. As expected, the secretory function of Satb1-/- SABs was compromised as amelogenins were largely retained in cells. We found the expression of epidermal growth factor receptor pathway substrate 8 (Eps8), a known regulator for actin filament assembly and small intestinal epithelial cytoplasmic protrusion formation, to be SATB1 dependent. In contrast to wt SABs, EPS8 could not be detected at the apical surface of Satb1-/- SABs. Eps8 expression was greatly reduced in small intestinal epithelial cells in Satb1-/- mice as well, displaying defective intestinal microvilli. CONCLUSIONS: Our data show that SATB1 is essential for establishing secretory ameloblast cell polarity and for EMP secretion. In line with the deformed apical architecture, amelogenin transport to the apical secretory front and secretion into enamel space were impeded in Satb1-/- SABs resulting in a massive cytoplasmic accumulation of amelogenins and a thin layer of hypomineralized enamel. Our studies strongly suggest that SATB1-dependent Eps8 expression plays a critical role in cytoplasmic protrusion formation in both SABs and in small intestines. This study demonstrates the role of SATB1 in the regulation of amelogenesis and the potential application of SATB1 in ameloblast/enamel regeneration.


Assuntos
Ameloblastos/fisiologia , Amelogênese , Polaridade Celular , Esmalte Dentário/crescimento & desenvolvimento , Proteínas de Ligação à Região de Interação com a Matriz/genética , Animais , Diferenciação Celular , Humanos , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos
18.
Elife ; 82019 12 23.
Artigo em Inglês | MEDLINE | ID: mdl-31868578

RESUMO

The syndromic autism spectrum disorder (ASD) Timothy syndrome (TS) is caused by a point mutation in the alternatively spliced exon 8A of the calcium channel Cav1.2. Using mouse brain and human induced pluripotent stem cells (iPSCs), we provide evidence that the TS mutation prevents a normal developmental switch in Cav1.2 exon utilization, resulting in persistent expression of gain-of-function mutant channels during neuronal differentiation. In iPSC models, the TS mutation reduces the abundance of SATB2-expressing cortical projection neurons, leading to excess CTIP2+ neurons. We show that expression of TS-Cav1.2 channels in the embryonic mouse cortex recapitulates these differentiation defects in a calcium-dependent manner and that in utero Cav1.2 gain-and-loss of function reciprocally regulates the abundance of these neuronal populations. Our findings support the idea that disruption of developmentally regulated calcium channel splicing patterns instructively alters differentiation in the developing cortex, providing important in vivo insights into the pathophysiology of a syndromic ASD.


Assuntos
Processamento Alternativo/fisiologia , Transtorno do Espectro Autista/metabolismo , Canais de Cálcio/metabolismo , Diferenciação Celular/fisiologia , Animais , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Transtorno Autístico , Encéfalo/embriologia , Encéfalo/crescimento & desenvolvimento , Cálcio , Canais de Cálcio/genética , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/metabolismo , Éxons , Regulação da Expressão Gênica no Desenvolvimento , Humanos , Células-Tronco Pluripotentes Induzidas/metabolismo , Síndrome do QT Longo , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Camundongos , Modelos Animais , Mutação , Neurogênese , Neurônios/citologia , Neurônios/metabolismo , Processamento de RNA , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Sindactilia , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo
19.
BMC Res Notes ; 12(1): 770, 2019 Nov 26.
Artigo em Inglês | MEDLINE | ID: mdl-31771640

RESUMO

OBJECTIVES: It is challenging to distinguish between primary ovarian mucinous tumors and metastatic mucinous neoplasms from the lower gastrointestinal tract, including appendiceal tumors. A combination of PAX8 and SATB2 immunohistochemical stains can be used as a diagnostic tool to distinguish between these cases. RESULTS: Immunostaining for SATB2, PAX8, CK7, CK20 and CDX2 was performed on 50 ovarian mucinous neoplasms (OMN) (39 cystadenomas, 4 borderline and 7 adenocarcinomas), 63 mucinous colorectal carcinoma (CRC), and 9 appendiceal mucinous neoplasms (AMN) [8 low grade appendiceal mucinous neoplasms (LAMN) and 1 adenocarcinoma]. PAX8 was positive in 32% of OMN and negative in all CRC and AMN cases. SATB2 was expressed in 2.0% of OMN, 77.8% of AMN, and 49.2% of CRC cases. CK7 was positive in 78.0% of OMN, 33.3% of AMN, and 9.5% of CRC cases. CK20 was expressed in 24.0% of OMN, 88.9% of OMN, and 87.3% of CRC cases. CDX2 was positive in 14.0% of OMN, 100% of AMN, and 90.5% of CRC cases. PAX8 can differentiate between OMN and AMN with high specificity but low sensitivity. CDX2 is the most sensitive marker for CRC and AMN, whereas SATB2 has better specificity.


Assuntos
Adenocarcinoma Mucinoso/diagnóstico , Neoplasias do Apêndice/diagnóstico , Neoplasias do Colo/diagnóstico , Cistadenoma Mucinoso/diagnóstico , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , Neoplasias Ovarianas/diagnóstico , Fator de Transcrição PAX8/metabolismo , Fatores de Transcrição/metabolismo , Adenocarcinoma Mucinoso/metabolismo , Neoplasias do Apêndice/metabolismo , Biomarcadores Tumorais/genética , Biomarcadores Tumorais/metabolismo , Fator de Transcrição CDX2/genética , Fator de Transcrição CDX2/metabolismo , Neoplasias do Colo/metabolismo , Cistadenoma Mucinoso/metabolismo , Diagnóstico Diferencial , Feminino , Humanos , Imuno-Histoquímica , Queratina-20/genética , Queratina-20/metabolismo , Proteínas de Ligação à Região de Interação com a Matriz/genética , Neoplasias Ovarianas/metabolismo , Fator de Transcrição PAX8/genética , Fatores de Transcrição/genética
20.
Dis Markers ; 2019: 6315936, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31737131

RESUMO

Gastric cancer (GC) is currently recognized as one of the most common and fatal tumor worldwide. The identification of novel biomarkers in relation to clinical information as well as extending the knowledge on a multiple crosstalk between various oncogenic pathways implicated in GC carcinogenesis seems pivotal to limit the disease-associated mortality. Therefore, we assessed the expression of HER2, NF-κB, and SATB1 in a total of 104 gastric adenocarcinomas and 30 normal gastric samples and correlated the expression patterns with each other and with some clinicopathological variables. Protein expression was examined by immunohistochemistry (IHC) on tissue microarrays (TMAs), and fluorescence in situ hybridization (FISH) was employed to detect HER2 amplification. In the studied group, HER2 and SATB1 were found to be overexpressed in gastric cancer tissue in comparison to normal gastric mucosa. The expression status of the former protein was seen to differ according to some clinicopathological features, but without statistical significance, whereas the expression of the latter was not importantly associated with any of them. In turn, the NF-κB protein level was significantly related to the presence of lymph node metastasis. HER2 expression was not significantly correlated with that of other proteins, but a positive correlation was found between the expression of SATB1 and NF-κB. Further studies with a larger group of patients combined with in vitro mechanistic experiments are required to fully elucidate the role and relationship of HER2, NF-κB, and SATB1 expression in gastric cancer progression. However, to the best of our knowledge, this study is the first look at a simultaneous evaluation of these three markers in the samples of gastric cancer patients.


Assuntos
Adenocarcinoma/patologia , Proteínas de Ligação à Região de Interação com a Matriz/metabolismo , NF-kappa B/metabolismo , Receptor ErbB-2/metabolismo , Neoplasias Gástricas/patologia , Adenocarcinoma/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/metabolismo , Feminino , Mucosa Gástrica/metabolismo , Mucosa Gástrica/patologia , Humanos , Imuno-Histoquímica , Hibridização in Situ Fluorescente , Metástase Linfática , Masculino , Pessoa de Meia-Idade , Receptor ErbB-2/genética , Neoplasias Gástricas/metabolismo
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