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1.
Nat Commun ; 10(1): 5115, 2019 11 11.
Artículo en Inglés | MEDLINE | ID: mdl-31712603

RESUMEN

The Epithelial to Mesenchymal Transition (EMT) regulates cell plasticity during embryonic development and in disease. It is dynamically orchestrated by transcription factors (EMT-TFs), including Snail, Zeb, Twist and Prrx, all activated by TGF-ß among other signals. Here we find that Snail1 and Prrx1, which respectively associate with gain or loss of stem-like properties and with bad or good prognosis in cancer patients, are expressed in complementary patterns during vertebrate development and in cancer. We show that this complementarity is established through a feedback loop in which Snail1 directly represses Prrx1, and Prrx1, through direct activation of the miR-15 family, attenuates the expression of Snail1. We also describe how this gene regulatory network can establish a hierarchical temporal expression of Snail1 and Prrx1 during EMT and validate its existence in vitro and in vivo, providing a mechanism to switch and select different EMT programs with important implications in development and disease.


Asunto(s)
Transición Epitelial-Mesenquimal/genética , Redes Reguladoras de Genes , Animales , Línea Celular , Embrión de Pollo , Predisposición Genética a la Enfermedad , Proteínas de Homeodominio , Humanos , Ratones Endogámicos C57BL , MicroARNs/metabolismo , Pronóstico , Regiones Promotoras Genéticas , Factores de Transcripción de la Familia Snail/metabolismo , Pez Cebra/embriología
2.
BMC Mol Biol ; 9: 30, 2008 Mar 26.
Artículo en Inglés | MEDLINE | ID: mdl-18366763

RESUMEN

BACKGROUND: Overexpression of the human DYRK1A gene due to the presence of a third gene copy in trisomy 21 is thought to play a role in the pathogenesis of Down syndrome. The observation of gene dosage effects in transgenic mouse models implies that subtle changes in expression levels can affect the correct function of the DYRK1A gene product. We have therefore characterized the promoter of the human DYRK1A gene in order to study its transcriptional regulation. RESULTS: Transcription start sites of the human DYRK1A gene are distributed over 800 bp within a region previously identified as an unmethylated CpG island. We have identified a new alternative noncoding 5'-exon of the DYRK1A gene which is located 772 bp upstream of the previously described transcription start site. Transcription of the two splicing variants is controlled by non-overlapping promoter regions that can independently drive reporter gene expression. We found no evidence of cell- or tissue-specific promoter usage, but the two promoter regions differed in their activity and their regulation. The sequence upstream of exon 1A (promoter region A) induced about 10-fold higher reporter gene activity than the sequence upstream of exon 1B (promoter region B). Overexpression of the transcription factor E2F1 increased DYRK1A mRNA levels in Saos2 and Phoenix cells and enhanced the activity of promoter region B three- to fourfold. CONCLUSION: The identification of two alternatively spliced transcripts whose transcription is initiated from differentially regulated promoters regions indicates that the expression of the DYRK1A gene is subject to complex control mechanisms. The regulatory effect of E2F1 suggests that DYRK1A may play a role in cell cycle regulation or apoptosis.


Asunto(s)
Factor de Transcripción E2F1/metabolismo , Regiones Promotoras Genéticas/genética , Proteínas Serina-Treonina Quinasas/genética , Proteínas Tirosina Quinasas/genética , Regulación hacia Arriba/genética , Empalme Alternativo/efectos de los fármacos , Secuencia de Bases , Sitios de Unión , Línea Celular Tumoral , Cromatina/metabolismo , Colforsina/farmacología , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Análisis Mutacional de ADN , Bases de Datos de Ácidos Nucleicos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Reporteros , Humanos , Datos de Secuencia Molecular , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Eliminación de Secuencia , Factor de Transcripción Sp1/metabolismo , Sitio de Iniciación de la Transcripción , Regulación hacia Arriba/efectos de los fármacos , Quinasas DyrK
3.
Sci Rep ; 6: 32474, 2016 09 01.
Artículo en Inglés | MEDLINE | ID: mdl-27581768

RESUMEN

To orchestrate the genomic response to cellular stress signals, p53 recognizes and binds to DNA containing specific and well-characterized p53-responsive elements (REs). Differences in RE sequences can strongly affect the p53 transactivation capacity and occur even between closely related species. Therefore, the identification and characterization of a species-specific p53 Binding sistes (BS) consensus sequence and of the associated target genes may help to provide new insights into the evolution of the p53 regulatory networks across different species. Although p53 functions were studied in a wide range of species, little is known about the p53-mediated transcriptional signature in Danio rerio. Here, we designed and biochemically validated a computational approach to identify novel p53 target genes in Danio rerio genome. Screening all the Danio rerio genome by pattern-matching-based analysis, we found p53 RE-like patterns proximal to 979 annotated Danio rerio genes. Prioritization analysis identified a subset of 134 candidate pattern-related genes, 31 of which have been investigated in further biochemical assays. Our study identified runx1, axin1, traf4a, hspa8, col4a5, necab2, and dnajc9 genes as novel direct p53 targets and 12 additional p53-controlled genes in Danio rerio genome. The proposed combinatorial approach resulted to be highly sensitive and robust for identifying new p53 target genes also in additional animal species.


Asunto(s)
Genoma , Elementos de Respuesta , Transcripción Genética , Proteína p53 Supresora de Tumor/genética , Pez Cebra/genética , Animales , Proteína Axina/genética , Proteína Axina/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas de Unión al Calcio/genética , Proteínas de Unión al Calcio/metabolismo , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Subunidad alfa 2 del Factor de Unión al Sitio Principal/genética , Subunidad alfa 2 del Factor de Unión al Sitio Principal/metabolismo , Evolución Molecular , Regulación de la Expresión Génica , Proteínas del Choque Térmico HSC70/genética , Proteínas del Choque Térmico HSC70/metabolismo , Proteínas del Choque Térmico HSP40/genética , Proteínas del Choque Térmico HSP40/metabolismo , Regiones Promotoras Genéticas , Unión Proteica , Alineación de Secuencia , Transducción de Señal , Factor 4 Asociado a Receptor de TNF/genética , Factor 4 Asociado a Receptor de TNF/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismo
4.
J Cell Sci ; 122(Pt 10): 1574-83, 2009 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-19383720

RESUMEN

Notch signalling is used throughout the animal kingdom to spatially and temporally regulate cell fate, proliferation and differentiation. Its importance is reflected in the dramatic effects produced on both development and health by small variations in the strength of the Notch signal. The Down-syndrome-associated kinase DYRK1A is coexpressed with Notch in various tissues during embryonic development. Here we show that DYRK1A moves to the nuclear transcription compartment where it interacts with the intracellular domain of Notch promoting its phosphorylation in the ankyrin domain and reducing its capacity to sustain transcription. DYRK1A attenuates Notch signalling in neural cells both in culture and in vivo, constituting a novel mechanism capable of modulating different developmental processes that can also contribute to the alterations observed during brain development in animal models of Down syndrome.


Asunto(s)
Síndrome de Down/enzimología , Neocórtex/enzimología , Neuronas/enzimología , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Tirosina Quinasas/metabolismo , Receptor Notch1/metabolismo , Transducción de Señal , Transporte Activo de Núcleo Celular , Animales , Línea Celular , Núcleo Celular/enzimología , Síndrome de Down/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones , Mutación , Neocórtex/embriología , Fosforilación , Proteínas Serina-Treonina Quinasas/genética , Estructura Terciaria de Proteína , Proteínas Tirosina Quinasas/genética , Ratas , Receptor Notch1/genética , Transcripción Genética , Transfección , Quinasas DyrK
5.
Genes Dev ; 16(24): 3173-85, 2002 Dec 15.
Artículo en Inglés | MEDLINE | ID: mdl-12502739

RESUMEN

Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1(-/-) tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1(-/-) tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1(-/-) tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Proto-Oncogénicas/metabolismo , Diente/embriología , Factores de Transcripción/fisiología , Proteínas de Pez Cebra , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Proteínas del Citoesqueleto/metabolismo , Ensayo de Cambio de Movilidad Electroforética , Inducción Embrionaria , Células Epiteliales/fisiología , Factor 4 de Crecimiento de Fibroblastos , Expresión Génica , Proteínas Hedgehog , Factor de Unión 1 al Potenciador Linfoide , Mesodermo/fisiología , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Odontogénesis , Proteínas Recombinantes/metabolismo , Transducción de Señal , Transactivadores/genética , Transactivadores/metabolismo , Proteínas Wnt , beta Catenina
6.
Genes Dev ; 18(22): 2718-23, 2004 Nov 15.
Artículo en Inglés | MEDLINE | ID: mdl-15545629

RESUMEN

Wnt signaling, which is mediated by LEF1/TCF transcription factors, has been placed upstream of the Notch pathway in vertebrate somitogenesis. Here, we examine the molecular basis for this presumed hierarchy and show that a targeted mutation of Lef1, which abrogates LEF1 function and impairs the activity of coexpressed TCF factors, affects the patterning of somites and the expression of components of the Notch pathway. LEF1 was found to bind multiple sites in the Dll1 promoter in vitro and in vivo. Moreover, mutations of LEF1-binding sites in the Dll1 promoter impair expression of a Dll1-LacZ transgene in the presomitic mesoderm. Finally, the induced expression of LEF1-beta-catenin activates the expression of endogenous Dll1 in fibroblastic cells. Thus, Wnt signaling can affect the Notch pathway by a LEF1-mediated regulation of Dll1.


Asunto(s)
Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana , Proteínas de la Membrana/metabolismo , Mutación/genética , Proteínas Proto-Oncogénicas/metabolismo , Somitos/fisiología , Factores de Transcripción/fisiología , Animales , Tipificación del Cuerpo , Células Cultivadas , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Humanos , Péptidos y Proteínas de Señalización Intracelular , Operón Lac/fisiología , Factor de Unión 1 al Potenciador Linfoide , Proteínas de la Membrana/genética , Mesodermo/citología , Mesodermo/fisiología , Ratones , Ratones Noqueados , Ratones Transgénicos , Células 3T3 NIH , Regiones Promotoras Genéticas/genética , Proteínas Proto-Oncogénicas/genética , Receptores Notch , Transducción de Señal , Somitos/citología , Transactivadores/genética , Transactivadores/metabolismo , Factores de Transcripción/genética , Proteínas Wnt , beta Catenina , beta-Galactosidasa/metabolismo
7.
Development ; 130(3): 623-33, 2003 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-12490567

RESUMEN

Transcriptional cascades responsible for initiating the formation of vertebrate embryonic structures such as limbs are not well established. Limb formation occurs as a result of interplay between fibroblast growth factor (FGF) and Wnt signaling. What initiates these signaling cascades and thus limb bud outgrowth at defined locations along the anteroposterior axis of the embryo is not known. The T-box transcription factor TBX5 is important for normal heart and limb formation, but its role in early limb development is not well defined. We report that mouse embryos lacking Tbx5 do not form forelimb buds, although the patterning of the lateral plate mesoderm into the limb field is intact. Tbx5 is not essential for an early establishment of forelimb versus hindlimb identity. In the absence of Tbx5, the FGF and Wnt regulatory loops required for limb bud outgrowth are not established, including initiation of Fgf10 expression. Tbx5 directly activates the Fgf10 gene via a conserved binding site, providing a simple and direct mechanism for limb bud initiation. Lef1/Tcf1-dependent Wnt signaling is not essential for initiation of Tbx5 or Fgf10 transcription, but is required in concert with Tbx5 for maintenance of normal levels of Fgf10 expression. We conclude that Tbx5 is not essential for the early establishment of the limb field in the lateral plate mesoderm but is a primary and direct initiator of forelimb bud formation. These data suggest common pathways for the differentiation and growth of embryonic structures downstream of T-box genes.


Asunto(s)
Miembro Anterior/embriología , Proteínas de Dominio T Box/fisiología , Proteínas de Pez Cebra , Animales , Tipificación del Cuerpo/genética , Tipificación del Cuerpo/fisiología , Factor 10 de Crecimiento de Fibroblastos , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/fisiología , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Deformidades Congénitas de las Extremidades/embriología , Deformidades Congénitas de las Extremidades/genética , Ratones , Ratones Noqueados , Modelos Biológicos , Fenotipo , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/fisiología , Transducción de Señal , Proteínas de Dominio T Box/deficiencia , Proteínas de Dominio T Box/genética , Proteínas Wnt
8.
Development ; 129(10): 2541-53, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11973284

RESUMEN

X-linked and autosomal forms of anhidrotic ectodermal dysplasia syndromes (HED) are characterized by deficient development of several ectodermal organs, including hair, teeth and exocrine glands. The recent cloning of the genes that underlie these syndromes, ectodysplasin (ED1) and the ectodysplasin A receptor (EDAR), and their identification as a novel TNF ligand-receptor pair suggested a role for TNF signaling in embryonic morphogenesis. In the mouse, the genes of the spontaneous mutations Tabby (Ta) and downless (dl) were identified as homologs of ED1 and EDAR, respectively. To gain insight into the function of this signaling pathway in development of skin and hair follicles, we analyzed the expression and regulation of Eda and Edar in wild type as well as Tabby and Lef1 mutant mouse embryos. We show that Eda and Edar expression is confined to the ectoderm and occurs in a pattern that suggests a role of ectodysplasin/Edar signaling in the interactions between the ectodermal compartments and the formation and function of hair placodes. By using skin explant cultures, we further show that this signaling pathway is intimately associated with interactions between the epithelial and mesenchymal tissues. We also find that Ta mutants lack completely the placodes of the first developing tylotrich hairs, and that they do not show patterned expression of placodal genes, including Bmp4, Lef1, Shh, Ptch and Edar, and the genes for beta-catenin and activin A. Finally, we identified activin as a mesenchymal signal that stimulates Edar expression and WNT as a signal that induces Eda expression, suggesting a hierarchy of distinct signaling pathways in the development of skin and hair follicles. In conclusion, we suggest that Eda and Edar are associated with the onset of ectodermal patterning and that ectodysplasin/edar signaling also regulates the morphogenesis of hair follicles.


Asunto(s)
Proteínas de Peces/metabolismo , Folículo Piloso/embriología , Proteínas de la Membrana/metabolismo , Transducción de Señal , Transactivadores , Factor de Necrosis Tumoral alfa/metabolismo , Activinas/genética , Activinas/metabolismo , Animales , Proteínas del Citoesqueleto/genética , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Ectodermo/metabolismo , Ectodisplasinas , Receptor Edar , Células Epidérmicas , Proteínas de Peces/genética , Folículo Piloso/metabolismo , Subunidades beta de Inhibinas/genética , Subunidades beta de Inhibinas/metabolismo , Factor de Unión 1 al Potenciador Linfoide , Proteínas de la Membrana/genética , Mesodermo/metabolismo , Ratones , Ratones Mutantes , Mutación , Técnicas de Cultivo de Órganos , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Proteínas Wnt , beta Catenina
9.
Development ; 131(21): 5469-80, 2004 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-15469977

RESUMEN

Here, we present evidence that Lrp6, a coreceptor for Wnt ligands, is required for the normal formation of somites and bones. By positional cloning, we demonstrate that a novel spontaneous mutation ringelschwanz (rs) in the mouse is caused by a point mutation in Lrp6, leading to an amino acid substitution of tryptophan for the evolutionarily conserved residue arginine at codon 886 (R886W). We show that rs is a hypomorphic Lrp6 allele by a genetic complementation test with Lrp6-null mice, and that the mutated protein cannot efficiently transduce signals through the Wnt/beta-catenin pathway. Homozygous rs mice, many of which are remarkably viable, exhibit a combination of multiple Wnt-deficient phenotypes, including dysmorphologies of the axial skeleton, digits and the neural tube. The establishment of the anteroposterior somite compartments, the epithelialization of nascent somites, and the formation of segment borders are disturbed in rs mutants, leading to a characteristic form of vertebral malformations, similar to dysmorphologies in individuals suffering from spondylocostal dysostosis. Marker expression study suggests that Lrp6 is required for the crosstalk between the Wnt and notch-delta signaling pathways during somitogenesis. Furthermore, the Lrp6 dysfunction in rs leads to delayed ossification at birth and to a low bone mass phenotype in adults. Together, we propose that Lrp6 is one of the key genetic components for the pathogenesis of vertebral segmentation defects and of osteoporosis in humans.


Asunto(s)
Anomalías Musculoesqueléticas/metabolismo , Mutación/genética , Osteogénesis/genética , Receptores de LDL/metabolismo , Somitos/citología , Somitos/metabolismo , Envejecimiento/fisiología , Alelos , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Tipificación del Cuerpo/genética , Polaridad Celular , Proteínas del Citoesqueleto/metabolismo , Proteínas de Unión al ADN/deficiencia , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/anomalías , Embrión de Mamíferos/citología , Embrión de Mamíferos/embriología , Embrión de Mamíferos/metabolismo , Fibroblastos , Proteína-6 Relacionada a Receptor de Lipoproteína de Baja Densidad , Región Lumbosacra/anomalías , Región Lumbosacra/embriología , Ratones , Ratones Noqueados , Datos de Secuencia Molecular , Anomalías Musculoesqueléticas/genética , Anomalías Musculoesqueléticas/patología , Fenotipo , Proteínas Proto-Oncogénicas/metabolismo , Receptores de LDL/química , Receptores de LDL/genética , Alineación de Secuencia , Transducción de Señal , Somitos/química , Transactivadores/metabolismo , Proteínas Wnt , beta Catenina
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