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1.
Nat Commun ; 11(1): 4159, 2020 08 27.
Artigo em Inglês | MEDLINE | ID: mdl-32855415

RESUMO

The periodic cartilage and smooth muscle structures in mammalian trachea are derived from tracheal mesoderm, and tracheal malformations result in serious respiratory defects in neonates. Here we show that canonical Wnt signaling in mesoderm is critical to confer trachea mesenchymal identity in human and mouse. At the initiation of tracheal development, endoderm begins to express Nkx2.1, and then mesoderm expresses the Tbx4 gene. Loss of ß-catenin in fetal mouse mesoderm causes loss of Tbx4+ tracheal mesoderm and tracheal cartilage agenesis. The mesenchymal Tbx4 expression relies on endodermal Wnt activation and Wnt ligand secretion but is independent of known Nkx2.1-mediated respiratory development, suggesting that bidirectional Wnt signaling between endoderm and mesoderm promotes trachea development. Activating Wnt, Bmp signaling in mouse embryonic stem cell (ESC)-derived lateral plate mesoderm (LPM) generates tracheal mesoderm containing chondrocytes and smooth muscle cells. For human ESC-derived LPM, SHH activation is required along with WNT to generate proper tracheal mesoderm. Together, these findings may contribute to developing applications for human tracheal tissue repair.


Assuntos
Endoderma/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Traqueia/metabolismo , Via de Sinalização Wnt/genética , beta Catenina/genética , Animais , Diferenciação Celular/genética , Células Cultivadas , Endoderma/citologia , Endoderma/embriologia , Células-Tronco Embrionárias Humanas/metabolismo , Humanos , Mesoderma/citologia , Mesoderma/embriologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Células-Tronco Embrionárias Murinas/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fator Nuclear 1 de Tireoide/genética , Fator Nuclear 1 de Tireoide/metabolismo , Traqueia/citologia , Traqueia/embriologia , beta Catenina/metabolismo
2.
Int Heart J ; 61(4): 761-768, 2020 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-32641638

RESUMO

Congenital heart defect (CHD) represents the most common birth deformity, afflicting 1% of all births worldwide, and accounts for substantial morbidity and mortality. Increasing evidence highlights the pivotal roles of genetic etiologies in the pathogenesis of CHD, and pathogenic mutations in multiple genes, including TBX5 encoding a cardiac core transcription factor key to cardiovascular morphogenesis, have been involved in CHD. However, due to pronounced genetic heterogeneity of CHD, the genetic determinants underlying CHD in most cases remain obscure. In this investigation, by sequencing analysis of the coding exons and flanking introns of the TBX5 gene in 198 unrelated patients affected with CHD, a novel heterozygous mutation, NM_000192.3: c.692C>T; p. (Pro231Leu), was identified in an index patient with familial double outlet right ventricle (DORV), ventricular septal defect (VSD), and atrioventricular block (AVB). Genetic analysis of the proband's pedigree showed that the mutation co-segregated with the diseases. The missense mutation, which altered the amino acid conserved evolutionarily, was absent from 266 unrelated healthy subjects. Functional analyses with a dual-luciferase reporter assay system unveiled that the Pro231Leu-mutant TBX5 was associated with significantly reduced transcriptional activity on its target genes MYH6 and NPPA. Furthermore, the mutation disrupted the synergistic transactivation between TBX5 and NKX2-5 as well as GATA4, two other transcription factors causally linked to CHD. This study firstly links TBX5 loss-of-function mutation to familial DORV, VSD, and AVB, which provides novel insight into the mechanism underpinning CHD and AVB, suggesting potential implications for genetic evaluation and individualized treatment of patients affected by CHD and AVB.


Assuntos
Bloqueio Atrioventricular/genética , Cardiopatias Congênitas/genética , Proteínas com Domínio T/genética , Adolescente , Adulto , Animais , Estudos de Casos e Controles , Bovinos , Criança , Pré-Escolar , Cães , Feminino , Humanos , Lactente , Masculino , Camundongos , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Ratos , Adulto Jovem
3.
Proc Natl Acad Sci U S A ; 117(28): 16409-16417, 2020 07 14.
Artigo em Inglês | MEDLINE | ID: mdl-32601185

RESUMO

The polar trophoblast overlays the epiblast in eutherian mammals and, depending on the species, has one of two different fates. It either remains a single-layered, thinning epithelium called "Rauber's layer," which soon disintegrates, or, alternatively, it keeps proliferating, contributing heavily to the population of differentiating, invasive trophoblast cells and, at least in mice, to the induction of gastrulation. While loss of the persistent polar trophoblast in mice leads to reduced induction of gastrulation, we show here that prevention of the loss of the polar trophoblast in cattle results in ectopic domains of the gastrulation marker, BRACHYURY This phenotype, and increased epiblast proliferation, arose when Rauber's layer was maintained for a day longer by countering apoptosis through BCL2 overexpression. This suggests that the disappearance of Rauber's layer is a necessity, presumably to avoid excessive signaling interactions between this layer and the subjacent epiblast. We note that, in all species in which the polar trophoblast persists, including humans and mice, ectopic polar trophoblast signaling is prevented via epiblast cavitation which leads to the (pro)amniotic cavity, whose function is to distance the central epiblast from such signaling interactions.


Assuntos
Trofoblastos/citologia , Animais , Apoptose , Bovinos , Diferenciação Celular , Proliferação de Células , Feminino , Proteínas Fetais/genética , Proteínas Fetais/metabolismo , Gastrulação , Camadas Germinativas/embriologia , Camadas Germinativas/metabolismo , Camadas Germinativas/fisiopatologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Trofoblastos/metabolismo
4.
Proc Natl Acad Sci U S A ; 117(31): 18617-18626, 2020 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-32675240

RESUMO

Genome-wide association studies have identified noncoding variants near TBX3 that are associated with PR interval and QRS duration, suggesting that subtle changes in TBX3 expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous Tbx3 mutant (Tbx3 +/-) mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in Tbx3 +/- mutants. Notably, Tbx3 +/- atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near Ryr2, an up-regulated chamber-enriched gene, and in Cacna1g, a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs Cacna1g expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the TBX3 locus.


Assuntos
Nó Atrioventricular , Proteínas com Domínio T , Transcriptoma/genética , Animais , Arritmias Cardíacas , Nó Atrioventricular/metabolismo , Nó Atrioventricular/fisiologia , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Camundongos , Camundongos Transgênicos , Mutação/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/genética , Canal de Liberação de Cálcio do Receptor de Rianodina/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
5.
Nat Neurosci ; 23(6): 741-753, 2020 06.
Artigo em Inglês | MEDLINE | ID: mdl-32393895

RESUMO

During sleep and awake rest, the neocortex generates large-scale slow-wave (SW) activity. Here, we report that the claustrum coordinates neocortical SW generation. We established a transgenic mouse line that enabled the genetic interrogation of a subpopulation of claustral glutamatergic neurons. These neurons received inputs from and sent outputs to widespread neocortical areas. The claustral neuronal firings mostly correlated with cortical SW activity. In vitro optogenetic stimulation of the claustrum induced excitatory postsynaptic responses in most neocortical neurons, but elicited action potentials primarily in inhibitory interneurons. In vivo optogenetic stimulation induced a synchronized down-state featuring prolonged silencing of neural activity in all layers of many cortical areas, followed by a down-to-up state transition. In contrast, genetic ablation of claustral neurons attenuated SW activity in the frontal cortex. These results demonstrate a crucial role of claustral neurons in synchronizing inhibitory interneurons across wide cortical areas for the spatiotemporal coordination of SW activity.


Assuntos
Claustrum/fisiologia , Neocórtex/fisiologia , Sono de Ondas Lentas/fisiologia , Potenciais de Ação/fisiologia , Animais , Potenciais Pós-Sinápticos Excitadores/fisiologia , Interneurônios/fisiologia , Camundongos , Camundongos Transgênicos , Inibição Neural/fisiologia , Neurônios/fisiologia , Optogenética , Proteínas com Domínio T/genética
6.
PLoS One ; 15(5): e0226539, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32413046

RESUMO

A murine model to study the effect of cold-induced stress (CIS) on Chlamydia muridarum genital infection and immune response has been developed in our laboratory. Previous results in the lab show that CIS increases the intensity of chlamydia genital infection, but little is known about the effects and mechanisms of CIS on the differentiation and activities of CD4+ T cell subpopulations and bone marrow-derived dendritic cells (BMDCs). The factors that regulate the production of T helper 1 (Th1) or T helper 2 (Th2) cytokines are not well defined. In this study, we examined whether CIS modulates the expressions of beta-adrenergic receptor (ß-AR), transcription factors, hallmark cytokines of Th1 and Th2, and differentiation of BMDCs during C. muridarum genital infection in the murine model. Our results show that the mRNA level of the beta2-adrenergic receptor (ß2-AR) compared to ß1-AR and ß3-AR was high in the mixed populations of CD4+ T cells and BMDCs. Furthermore, we observed decreased expression of T-bet, low level of Interferon-gamma (IFN-γ) production, increased expression of GATA-3, and Interleukin-4 (IL-4) production in CD4+ T cells of stressed mice. Exposure of BMDCs to Fenoterol, ß2-AR agonist, or ICI118,551, ß2-AR antagonist, revealed significant ß2-AR stimulation or inhibition, respectively, in stressed mice. Moreover, co-culturing of mature BMDCs and naïve CD4+ T cells increased the production of IL-4, IL-10, L-17, and IL-23 cytokines, suggesting that stimulation of ß2-AR leads to the increased production of Th2 cytokines. Overall, our results show for the first time that CIS promotes the switching from a Th1 to Th2 cytokine environment. This was evidenced in the murine stress model by the overexpression of GATA-3 concurrent with elevated IL-4 production, reduced T-bet expression, and IFN-γ secretion.


Assuntos
Infecções por Chlamydia/imunologia , Resposta ao Choque Frio , Células Th1/imunologia , Células Th2/imunologia , Agonistas Adrenérgicos beta/farmacologia , Antagonistas Adrenérgicos beta/farmacologia , Animais , Células Cultivadas , Chlamydia muridarum , Células Dendríticas/efeitos dos fármacos , Células Dendríticas/imunologia , Feminino , Fenoterol/farmacologia , Interferon gama/genética , Interferon gama/metabolismo , Interleucinas/genética , Interleucinas/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Propanolaminas/farmacologia , Receptores Adrenérgicos beta/genética , Receptores Adrenérgicos beta/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Células Th1/efeitos dos fármacos , Células Th2/efeitos dos fármacos , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
PLoS Biol ; 18(4): e3000696, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-32275659

RESUMO

It is well known that various developmental signals play diverse roles in hematopoietic stem and progenitor cell (HSPC) production; however, how these signaling pathways are orchestrated remains incompletely understood. Here, we report that Rab5c is essential for HSPC specification by endocytic trafficking of Notch and AKT signaling in zebrafish embryos. Rab5c deficiency leads to defects in HSPC production. Mechanistically, Rab5c regulates hemogenic endothelium (HE) specification by endocytic trafficking of Notch ligands and receptor. We further show that the interaction between Rab5c and Appl1 in the endosome is required for the survival of HE in the ventral wall of the dorsal aorta through AKT signaling. Interestingly, Rab5c overactivation can also lead to defects in HSPC production, which is attributed to excessive endolysosomal trafficking inducing Notch signaling defect. Taken together, our findings establish a previously unrecognized role of Rab5c-mediated endocytic trafficking in HSPC development and provide new insights into how spatiotemporal signals are orchestrated to accurately execute cell fate transition.


Assuntos
Células-Tronco Hematopoéticas/fisiologia , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptores Notch/metabolismo , Proteínas rab5 de Ligação ao GTP/metabolismo , Animais , Animais Geneticamente Modificados , Embrião não Mamífero , Endocitose , Endotélio/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Células HEK293 , Humanos , Receptores Notch/genética , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/genética , Peixe-Zebra/embriologia , Peixe-Zebra/genética , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo , Proteínas rab5 de Ligação ao GTP/química , Proteínas rab5 de Ligação ao GTP/genética
8.
PLoS One ; 15(4): e0232216, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32348326

RESUMO

BACKGROUND: The knowledge of hereditary predisposition has changed our understanding of Pulmonary Arterial Hypertension. Genetic testing has been widely extended and the application of Pulmonary Arterial Hypertension specific gene panels has allowed its inclusion in the diagnostic workup and increase the diagnostic ratio compared to the traditional sequencing techniques. This is particularly important in the differential diagnosis between Pulmonary Arterial Hypertension and Pulmonary Venoocclusive Disease. METHODS: Since November 2011, genetic testing is offered to all patients with idiopathic, hereditable and associated forms of Pulmonary Arterial Hypertension or Pulmonary Venoocclusive Disease included in the Spanish Registry of Pulmonary Arterial Hypertension. Herein, we present the clinical phenotype and prognosis of all Pulmonary Arterial Hypertension patients with disease-associated variants in TBX4. RESULTS: Out of 579 adults and 45 children, we found in eight patients from seven families, disease-causing associated variants in TBX4. All adult patients had a moderate-severe reduction in diffusion capacity. However, we observed a wide spectrum of clinical presentations, including Pulmonary Venoocclusive Disease suspicion, interstitial lung disease, pulmonary vascular abnormalities and congenital heart disease. CONCLUSIONS: Genetic testing is now essential for a correct diagnosis work-up in Pulmonary Arterial Hypertension. TBX4-associated Pulmonary Arterial Hypertension has marked clinical heterogeneity. In this regard, a genetic study is extremely useful to obtain an accurate diagnosis and provide appropriate management.


Assuntos
Hipertensão Pulmonar Primária Familiar/genética , Variação Genética , Proteínas com Domínio T/genética , Adolescente , Adulto , Criança , Pré-Escolar , Códon sem Sentido , Diagnóstico Diferencial , Hipertensão Pulmonar Primária Familiar/diagnóstico , Hipertensão Pulmonar Primária Familiar/diagnóstico por imagem , Feminino , Deleção de Genes , Predisposição Genética para Doença , Humanos , Masculino , Pessoa de Meia-Idade , Mutação de Sentido Incorreto , Linhagem , Fenótipo , Polimorfismo de Nucleotídeo Único , Prognóstico , Pneumopatia Veno-Oclusiva/diagnóstico , Pneumopatia Veno-Oclusiva/genética
9.
BMC Med Genet ; 21(1): 78, 2020 04 15.
Artigo em Inglês | MEDLINE | ID: mdl-32293321

RESUMO

BACKGROUND: The protein Kruppel-like factor 13 (KLF13) is a member of the KLF family and has been identified as a cardiac transcription factor that is involved in heart development. However, the relationship between KLF13 variants and CHDs in humans remains largely unknown. The present study aimed to screen the KLF13 variants in CHD patients and genetically analyze the functions of these variants. METHODS: KLF13 variants were sequenced in a cohort of 309 CHD patients and population-matched healthy controls (n = 200) using targeted sequencing. To investigate the effect of variants on the functional properties of the KLF13 protein, the expression and subcellular localization of the protein, as well as the transcriptional activities of downstream genes and physical interactions with other transcription factors, were assessed. RESULTS: Two heterozygous variants, c.487C > T (P163S) and c.467G > A (S156N), were identified in two out of 309 CHD patients with tricuspid valve atresia and transposition of the great arteries, respectively. No variants were found among healthy controls. The variant c.467G > A (S156N) had increased protein expression and enhanced functionality compared with the wild type, without affecting the subcellular localization. The other variant, c.487C > T (P163S), did not show any abnormalities in protein expression or subcellular localization; however, it inhibited the transcriptional activities of downstream target genes and physically interacted with TBX5, another cardiac transcription factor. CONCLUSION: Our results show that the S156N and P163S variants may affect the transcriptional function of KLF13 and physical interaction with TBX5. These results identified KLF13 as a potential genetic risk factor for congenital heart disease.


Assuntos
Proteínas de Ciclo Celular/genética , Cardiopatias Congênitas/genética , Fatores de Transcrição Kruppel-Like/genética , Proteínas Repressoras/genética , Proteínas com Domínio T/genética , Atresia Tricúspide/genética , Criança , Pré-Escolar , Feminino , Regulação da Expressão Gênica/genética , Coração/fisiopatologia , Cardiopatias Congênitas/fisiopatologia , Heterozigoto , Humanos , Lactente , Recém-Nascido , Masculino , Polimorfismo de Nucleotídeo Único/genética , Transposição dos Grandes Vasos/metabolismo , Transposição dos Grandes Vasos/fisiopatologia , Atresia Tricúspide/fisiopatologia
10.
PLoS One ; 15(4): e0227393, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32236096

RESUMO

BACKGROUND: TBX5 is a transcription factor that has an important role in development of heart. TBX5 variants in the region encoding the T-box domain have been shown to cause cardiac defects, such as atrial septal defect or ventricular septal defect, while TBX5 variants have also been identified in a few cardiomyopathy patients and considered causative. We identified a TBX5 variant (c.791G>A, p.Arg264Lys), that is over-represented in cardiomyopathy patients. This variant is located outside of the T-box domain, and its pathogenicity has not been confirmed by functional analyses. OBJECTIVE: To investigate whether the TBX5 R264K is deleterious and could contribute to the pathogenesis of cardiomyopathy. METHODS AND RESULTS: We developed mice expressing Tbx5 R264K. Mice homozygous for this variant displayed compensated dilated cardiomyopathy; mild decreased fractional shortening, dilatation of the left ventricle, left ventricular wall thinning and increased heart weight without major heart structural disorders. There was no difference in activation of the ANF promotor, a transcriptional target of Tbx5, compared to wild-type. However, analysis of RNA isolated from left ventricular samples showed significant increases in the expression of Acta1 in left ventricle with concomitant increases in the protein level of ACTA1. CONCLUSIONS: Mice homozygous for Tbx5 R264K showed compensated dilated cardiomyopathy. Thus, TBX5 R264K may have a significant pathogenic role in some cardiomyopathy patients independently of T-box domain pathway.


Assuntos
Cardiomiopatia Dilatada/genética , Ventrículos do Coração/patologia , Miocárdio Ventricular não Compactado Isolado/genética , Proteínas com Domínio T/genética , Actinas/metabolismo , Animais , Cardiomiopatia Dilatada/diagnóstico , Cardiomiopatia Dilatada/patologia , Criança , Modelos Animais de Doenças , Ecocardiografia , Feminino , Técnicas de Introdução de Genes , Testes Genéticos , Células HEK293 , Ventrículos do Coração/diagnóstico por imagem , Ventrículos do Coração/crescimento & desenvolvimento , Heterozigoto , Humanos , Lactente , Recém-Nascido , Miocárdio Ventricular não Compactado Isolado/diagnóstico , Masculino , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto , Polimorfismo de Nucleotídeo Único
11.
Nat Immunol ; 21(5): 567-577, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32284593

RESUMO

Unprimed mice harbor a substantial population of 'memory-phenotype' CD8+ T cells (CD8-MP cells) that exhibit hallmarks of activation and innate-like functional properties. Due to the lack of faithful markers to distinguish CD8-MP cells from bona fide CD8+ memory T cells, the developmental origins and antigen specificities of CD8-MP cells remain incompletely defined. Using deep T cell antigen receptor (TCR) sequencing, we found that the TCRs expressed by CD8-MP cells are highly recurrent and distinct from the TCRs expressed by naive-phenotype CD8+ T cells. CD8-MP clones exhibited reactivity to widely expressed self-ligands. T cell precursors expressing CD8-MP TCRs showed upregulation of the transcription factor Eomes during maturation in the thymus, prior to induction of the full memory phenotype, which is suggestive of a unique program triggered by recognition of self-ligands. Moreover, CD8-MP cells infiltrate oncogene-driven prostate tumors and express high densities of PD-1, which suggests potential roles in antitumor immunity and the response to immunotherapy.


Assuntos
Linfócitos T CD8-Positivos/imunologia , Neoplasias da Próstata/imunologia , Receptores de Antígenos de Linfócitos T/genética , Proteínas com Domínio T/metabolismo , Timo/fisiologia , Animais , Autoantígenos/imunologia , Diferenciação Celular , Seleção Clonal Mediada por Antígeno , Células Clonais , Memória Imunológica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Receptor de Morte Celular Programada 1/metabolismo , Proteínas com Domínio T/genética , Regulação para Cima
12.
PLoS Biol ; 18(3): e3000648, 2020 03.
Artigo em Inglês | MEDLINE | ID: mdl-32182234

RESUMO

The memory CD8 T-cell pool must select for clones that bind immunodominant epitopes with high affinity to efficiently counter reinfection. At the same time, it must retain a level of clonal diversity to allow recognition of pathogens with mutated epitopes. How the level of diversity within the memory pool is controlled is unclear, especially in the context of a selective drive for antigen affinity. We find that preservation of clones that bind the activating antigen with low affinity depends on expression of the transcription factor Eomes in the first days after antigen encounter. Eomes is induced at low activating signal strength and directly drives transcription of the prosurvival protein Bcl-2. At higher signal intensity, T-bet is induced which suppresses Bcl-2 and causes a relative survival advantage for cells of low affinity. Clones activated with high-affinity antigen form memory largely independent of Eomes and have a proliferative advantage over clones that bind the same antigen with low affinity. This causes high-affinity clones to prevail in the memory pool, despite their relative survival deficit. Genetic or therapeutic targeting of the Eomes/Bcl-2 axis reduces the clonal diversity of the memory pool, which diminishes its ability to respond to pathogens carrying mutations in immunodominant epitopes. Thus, we demonstrate on a molecular level how sufficient diversity of the memory pool is established in an environment of affinity-based selection.


Assuntos
Apoptose/imunologia , Linfócitos T CD8-Positivos/imunologia , Memória Imunológica , Proteínas com Domínio T/imunologia , Animais , Variação Antigênica/imunologia , Sobrevivência Celular/imunologia , Células Cultivadas , Seleção Clonal Mediada por Antígeno/genética , Seleção Clonal Mediada por Antígeno/imunologia , Regulação da Expressão Gênica/imunologia , Ativação Linfocitária , Camundongos , Células Precursoras de Linfócitos T/imunologia , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/imunologia , Receptores de Antígenos de Linfócitos T/genética , Receptores de Antígenos de Linfócitos T/imunologia , Transdução de Sinais , Proteínas com Domínio T/genética
13.
Proc Natl Acad Sci U S A ; 117(12): 6836-6843, 2020 03 24.
Artigo em Inglês | MEDLINE | ID: mdl-32144139

RESUMO

Visuomotor impairments characterize numerous neurological disorders and neurogenetic syndromes, such as autism spectrum disorder (ASD) and Dravet, Fragile X, Prader-Willi, Turner, and Williams syndromes. Despite recent advances in systems neuroscience, the biological basis underlying visuomotor functional impairments associated with these clinical conditions is poorly understood. In this study, we used neuroimaging connectomic approaches to map the visuomotor integration (VMI) system in the human brain and investigated the topology approximation of the VMI network to the Allen Human Brain Atlas, a whole-brain transcriptome-wide atlas of cortical genetic expression. We found the genetic expression of four genes-TBR1, SCN1A, MAGEL2, and CACNB4-to be prominently associated with visuomotor integrators in the human cortex. TBR1 gene transcripts, an ASD gene whose expression is related to neural development of the cortex and the hippocampus, showed a central spatial allocation within the VMI system. Our findings delineate gene expression traits underlying the VMI system in the human cortex, where specific genes, such as TBR1, are likely to play a central role in its neuronal organization, as well as on specific phenotypes of neurogenetic syndromes.


Assuntos
Canais de Cálcio/genética , Córtex Motor/fisiopatologia , Canal de Sódio Disparado por Voltagem NAV1.1/genética , Transtornos do Neurodesenvolvimento/patologia , Proteínas/genética , Proteínas com Domínio T/genética , Córtex Visual/fisiopatologia , Adulto , Idoso , Transtorno do Espectro Autista/genética , Transtorno do Espectro Autista/patologia , Mapeamento Encefálico , Estudos de Coortes , Epilepsias Mioclônicas/genética , Epilepsias Mioclônicas/patologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Transtornos do Neurodesenvolvimento/genética , Síndrome de Prader-Willi/genética , Síndrome de Prader-Willi/patologia , Desempenho Psicomotor , Percepção Visual
14.
J Pathol ; 251(1): 87-99, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32154590

RESUMO

The oncogene brachyury (TBXT) is a T-box transcription factor that is overexpressed in multiple solid tumors and is associated with tumor aggressiveness and poor patient prognosis. Gliomas comprise the most common and aggressive group of brain tumors, and at the present time the functional and clinical impact of brachyury expression has not been investigated previously in these neoplasms. Brachyury expression (mRNA and protein) was assessed in normal brain (n = 67), glioma tissues (n = 716) and cell lines (n = 42), and further in silico studies were undertaken using genomic databases totaling 3115 samples. Our glioma samples were analyzed for copy number (n = 372), promoter methylation status (n = 170), and mutation status (n = 1569 tissues and n = 52 cell lines) of the brachyury gene. The prognostic impact of brachyury expression was studied in 1524 glioma patient tumors. The functional impact of brachyury on glioma proliferation, viability, and cell death was evaluated both in vitro and in vivo. Brachyury was expressed in the normal brain, and significantly downregulated in glioma tissues. Loss of brachyury was associated with tumor aggressiveness and poor survival in glioma patients. Downregulation of brachyury was not associated with gene deletion, promoter methylation, or inactivating point mutations. Brachyury re-expression in glioma cells was found to decrease glioma tumorigenesis by induction of autophagy. These data strongly suggest that brachyury behaves as a tumor suppressor gene in gliomas by modulating autophagy. It is important to note that brachyury constitutes an independent positive biomarker of patient prognosis. Our findings indicate that the role of brachyury in tumorigenesis may be tissue-dependent and demands additional investigation to guide rational interventions. © 2020 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Assuntos
Neoplasias Encefálicas/patologia , Proteínas Fetais/metabolismo , Regulação Neoplásica da Expressão Gênica , Glioma/genética , Proteínas com Domínio T/metabolismo , Animais , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Linhagem Celular Tumoral , Proteínas Fetais/genética , Genes Supressores de Tumor/fisiologia , Glioma/patologia , Humanos , Camundongos , Prognóstico , Regiões Promotoras Genéticas , Proteínas com Domínio T/genética , Fatores de Transcrição/metabolismo
15.
DNA Cell Biol ; 39(4): 671-682, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-32040341

RESUMO

Comprehensive genomic testing will be required to identify appropriate targets for the precision therapy of breast cancer. Although RNA sequencing (RNA-seq) is an unparalleled platform for this purpose, existing molecular-based prognostic signatures are not optimal for RNA-seq data. In this study, we analyzed RNA-seq datasets to generate a novel prognostic gene signature for breast cancer patients. RNA-seq and clinical datasets from breast cancer patients were obtained from The Cancer Genome Atlas and randomly assigned to training (n = 379) and test (n = 378) cohorts. Using the training cohort, sequential univariate Cox analysis, robust likelihood-based survival analysis, and stepwise multivariable Cox analysis identified a five-gene signature composed of one long noncoding RNA gene and four protein-coding genes. The five-gene signature was then used to dichotomize patients into risk groups and validated using Kaplan-Meier and multivariable Cox analyses. In the full test cohort, the high-risk group had worse overall survival (hazard ratio [HR] = 4.74, 95% confidence interval [CI] = 2.33-9.64, p < 0.0001) and worse relapse-free survival (HR = 2.26, 95% CI = 1.11-4.61, p = 0.024) than the low-risk group. Similarly, overall survival was worse in the high-risk group within nearly all clinically important subsets, including early stage disease (I/II) (HR = 7.87, 95% CI = 3.69-16.77, p < 0.0001), and luminal A (HR = 4.23, 95% CI = 1.11-16.12, p = 0.034), luminal B (HR = 12.79, 95% CI = 2.74-59.69, p = 0.001), and basal (HR = 18.11, 95% CI = 3.21-102.05, p = 0.001) subtypes. Notably, the five-gene signature exhibited superior prognostic performance compared with the Oncotype DX 21-gene signature. This novel five-gene signature may therefore be a powerful prognostic tool for personalized treatment of breast cancer patients as part of an integrated RNA-seq clinical sequencing program.


Assuntos
Biomarcadores Tumorais/genética , Neoplasias da Mama/genética , RNA Longo não Codificante/genética , RNA Mensageiro/genética , Sequência de Bases , Neoplasias da Mama/mortalidade , Classe I de Fosfatidilinositol 3-Quinases/genética , Intervalo Livre de Doença , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Funções Verossimilhança , Pessoa de Meia-Idade , Receptor ErbB-2/metabolismo , Receptores Estrogênicos/metabolismo , Receptores de Progesterona/metabolismo , Análise de Sequência de RNA , Proteínas com Domínio T/genética
16.
Neurobiol Aging ; 89: 83-88, 2020 05.
Artigo em Inglês | MEDLINE | ID: mdl-32007278

RESUMO

Psychotic symptoms are a common and debilitating feature of Alzheimer's disease (AD) and are associated with a more rapid course of decline. Current evidence from postmortem and neuroimaging studies implicates frontal, temporal, and parietal lobes, with reported disruptions in monoaminergic pathways. However, the molecular mechanisms underlying this remain unclear. In the present study, we investigated methylomic variation associated with AD psychosis in 3 key brain regions implicated in the etiology of psychosis (prefrontal cortex, entorhinal cortex, and superior temporal gyrus) in postmortem brain samples from 29 AD donors with psychosis and 18 matched AD donors without psychosis. We identified psychosis-associated methylomic changes in a number of loci, with these genes being enriched in known schizophrenia-associated genetic and epigenetic variants. One of these known loci resided in the AS3MT gene-previously implicated in schizophrenia in a large GWAS meta-analysis. We used bisulfite-pyrosequencing to confirm hypomethylation across 4 neighboring CpG sites in the ASM3T gene. Finally, our regional analysis nominated multiple CpG sites in TBX15 and WT1, which are genes that have been previously implicated in AD. Thus one potential implication from our study is whether psychosis-associated variation drives reported associations in AD case-control studies.


Assuntos
Doença de Alzheimer/genética , Doença de Alzheimer/metabolismo , Córtex Cerebral/metabolismo , Metilação de DNA/genética , Transtornos Psicóticos/genética , Doença de Alzheimer/complicações , Monoaminas Biogênicas/metabolismo , Ilhas de CpG/genética , Epigênese Genética , Variação Genética/genética , Humanos , Metiltransferases/genética , Transtornos Psicóticos/etiologia , Esquizofrenia/etiologia , Esquizofrenia/genética , Proteínas com Domínio T/genética , Proteínas WT1/genética
17.
FASEB J ; 34(2): 1970-1982, 2020 02.
Artigo em Inglês | MEDLINE | ID: mdl-31909538

RESUMO

Osterix is a critical transcription factor of mesenchymal stem cell fate, where its loss or loss of Wnt signaling diverts differentiation to a chondrocytic lineage. Intervertebral disc (IVD) degeneration activates the differentiation of prehypertrophic chondrocyte-like cells and inactivates Wnt signaling, but its interactive role with osterix is unclear. First, compared to young-adult (5 mo), mechanical compression of old (18 mo) IVD induced greater IVD degeneration. Aging (5 vs 12 mo) and/or compression reduced the transcription of osterix and notochordal marker T by 40-75%. Compression elevated the transcription of hypertrophic chondrocyte marker MMP13 and pre-osterix transcription factor RUNX2, but less so in 12 mo IVD. Next, using an Ai9/td reporter and immunohistochemical staining, annulus fibrosus and nucleus pulposus cells of young-adult IVD expressed osterix, but aging and compression reduced its expression. Lastly, in vivo LRP5-deficiency in osterix-expressing cells inactivated Wnt signaling in the nucleus pulposus by 95%, degenerated the IVD to levels similar to aging and compression, reduced the biomechanical properties by 45-70%, and reduced the transcription of osterix, notochordal markers and chondrocytic markers by 60-80%. Overall, these data indicate that age-related inactivation of Wnt signaling in osterix-expressing cells may limit regeneration by depleting the progenitors and attenuating the expansion of chondrocyte-like cells.


Assuntos
Envelhecimento/metabolismo , Condrócitos/metabolismo , Condrogênese , Subunidade alfa 1 de Fator de Ligação ao Core/biossíntese , Proteínas Fetais/biossíntese , Degeneração do Disco Intervertebral/metabolismo , Fator de Transcrição Sp7/biossíntese , Proteínas com Domínio T/biossíntese , Envelhecimento/genética , Envelhecimento/patologia , Animais , Antígenos de Diferenciação/biossíntese , Antígenos de Diferenciação/genética , Condrócitos/patologia , Subunidade alfa 1 de Fator de Ligação ao Core/genética , Proteínas Fetais/genética , Regulação da Expressão Gênica , Degeneração do Disco Intervertebral/genética , Degeneração do Disco Intervertebral/patologia , Camundongos , Camundongos Transgênicos , Fator de Transcrição Sp7/genética , Proteínas com Domínio T/genética
18.
DNA Cell Biol ; 39(2): 289-298, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31916853

RESUMO

TBX3 reprograms cardiac myocytes into cells that possess sinoatrial node phenotype, but no specific funny current (If) was detected. We explore whether overexpression of TBX3 alone or combined with HCN2 can reprogram human-induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs) into pacemaker-like cells. HiPSC-CMs were transfected with TBX3 and/or HCN2 in this study. Expression analysis showed that overexpression of TBX3 induces a reduced reduction expression profile of working cardiomyocytes into that of pacemaker cells. Reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and electrophysiological analyses showed a reduced expression of connexins subunits (CX40, CX43), the sodium current (SCN5A, INa), the inward rectified potassium channels (Kir2.1, IK1), and an increased expression of connexins subunits (CX30.2, CX45). No If was detected. The reduction of IK1 resulted in a more depolarized maximum diastolic potential together with an expression of If (generated by HCN2), which they work in synergy to generate spontaneous diastolic depolarization that was the most typical characteristic of pacemaker cells. In conclusion, overexpression of TBX3 and HCN2 could reprogram hiPSC-CMs into pacemaker-like cells. The ability to enable diastolic depolarization formation provides a new strategy for the construction of a biological pacemaker.


Assuntos
Diferenciação Celular/genética , Canais Disparados por Nucleotídeos Cíclicos Ativados por Hiperpolarização/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Canais de Potássio/genética , Proteínas com Domínio T/genética , Potenciais de Ação/fisiologia , Relógios Biológicos/genética , Humanos , Miócitos Cardíacos/metabolismo
19.
RNA ; 26(4): 481-491, 2020 04.
Artigo em Inglês | MEDLINE | ID: mdl-31953255

RESUMO

Long noncoding RNAs (lncRNAs) have emerged as critical epigenetic regulators and play important roles in cardiac development and congenital heart disease. In a previous study, we identified a novel lncRNA, Ppp1r1b, with expression highly correlated with myogenesis. However, the molecular mechanism that underlies Ppp1r1b-lncRNA function in myogenic regulation is unknown. By silencing Ppp1r1b-lncRNA, mouse C2C12 and human skeletal myoblasts failed to develop fully differentiated myotubes. Myogenic differentiation was also impaired in PPP1R1B-lncRNA deficient human-induced pluripotent stem cell-derived cardiomyocytes (hiPSCs-CMs). The expression of myogenic transcription factors, including MyoD, Myogenin, and Tbx5, as well as sarcomere proteins, was significantly suppressed in Ppp1r1b-lncRNA inhibited myoblast cells and neonatal mouse heart. Histone modification analysis revealed increased H3K27 tri-methylation at MyoD1 and Myogenin promoters in GapmeR treated C2C12 cells. Furthermore, Ppp1r1b-lncRNA was found to bind to Ezh2, and chromatin isolation by RNA purification (ChIRP) assay revealed enriched interaction of Ppp1r1b-lncRNA with Myod1 and Tbx5 promoters, suggesting that Ppp1r1b-lncRNA induces transcription of myogenic transcription factors by interacting with the polycomb repressive complex 2 (PRC2) at the chromatin interface. Correspondingly, the silencing of Ppp1r1b-lncRNA increased EZH2 binding at promoter regions of myogenic transcription factors. Therefore, our results suggest that Ppp1r1b-lncRNA promotes myogenic differentiation through competing for PRC2 binding with chromatin of myogenic master regulators during heart and skeletal muscle development.


Assuntos
Fosfoproteína 32 Regulada por cAMP e Dopamina/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular , Músculo Esquelético/metabolismo , Miócitos Cardíacos/metabolismo , Complexo Repressor Polycomb 2/metabolismo , RNA Longo não Codificante/genética , Animais , Linhagem Celular , Fosfoproteína 32 Regulada por cAMP e Dopamina/metabolismo , Proteína Potenciadora do Homólogo 2 de Zeste/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Inativação Gênica , Código das Histonas , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteína MyoD/genética , Proteína MyoD/metabolismo , Miogenina/genética , Miogenina/metabolismo , RNA Longo não Codificante/metabolismo , Proteínas com Domínio T/genética , Proteínas com Domínio T/metabolismo
20.
Gene ; 726: 144223, 2020 Feb 05.
Artigo em Inglês | MEDLINE | ID: mdl-31669645

RESUMO

TBX3, a member of the ancient and evolutionary conserved T-box transcription factor family, is a critical developmental regulator of several structures including the heart, mammary glands, limbs and lungs. Indeed, mutations in the human TBX3 lead to ulnar mammary syndrome which is characterized by several clinical malformations including hypoplasia of the mammary and apocrine glands, defects of the upper limb, areola, dental structures, heart and genitalia. In contrast, TBX3 has no known function in adult tissues but is frequently overexpressed in a wide range of epithelial and mesenchymal derived cancers. This overexpression greatly impacts several hallmarks of cancer including bypass of senescence, apoptosis and anoikis, promotion of proliferation, tumour formation, angiogenesis, invasion and metastatic capabilities as well as cancer stem cell expansion. The debilitating consequences of having too little or too much TBX3 suggest that its expression levels need to be tightly regulated. While we have a reasonable understanding of the mutations that result in low levels of functional TBX3 during development, very little is known about the factors responsible for the overexpression of TBX3 in cancer. Furthermore, given the plethora of oncogenic processes that TBX3 impacts, it must be regulating several target genes but to date only a few have been identified and characterised. Interestingly, while there is compelling evidence to support oncogenic roles for TBX3, a few studies have indicated that it may also have tumour suppressor functions in certain contexts. Together, the diverse functional elasticity of TBX3 in development and cancer is thought to involve, in part, the protein partners that it interacts with and this area of research has recently received some attention. This review provides an insight into the significance of TBX3 in development and cancer and identifies research gaps that need to be explored to shed more light on this transcription factor.


Assuntos
Doença/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas com Domínio T/genética , Animais , Transformação Celular Neoplásica/genética , Transformação Celular Neoplásica/patologia , Humanos , Fatores de Transcrição/genética
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