RESUMEN
To maintain fertility, male mice re-repress transposable elements (TEs) that were de-silenced in the early gonocytes before their differentiation into spermatogonia. However, the mechanism of TE silencing re-establishment remains unknown. Here, we found that the DNA-binding protein Morc1, in cooperation with the methyltransferase SetDB1, deposits the repressive histone mark H3K9me3 on a large fraction of activated TEs, leading to heterochromatin. Morc1 also triggers DNA methylation, but TEs targeted by Morc1-driven DNA methylation only slightly overlapped with those repressed by Morc1/SetDB1-dependent heterochromatin formation, suggesting that Morc1 silences TEs in two different manners. In contrast, TEs regulated by Morc1 and Miwi2, the nuclear PIWI-family protein, almost overlapped. Miwi2 binds to PIWI-interacting RNAs (piRNAs) that base-pair with TE mRNAs via sequence complementarity, while Morc1 DNA binding is not sequence specific, suggesting that Miwi2 selects its targets, and then, Morc1 acts to repress them with cofactors. A high-ordered mechanism of TE repression in gonocytes has been identified.
Asunto(s)
Heterocromatina , ARN de Interacción con Piwi , Animales , Masculino , Ratones , Proteínas Argonautas/genética , Proteínas Argonautas/metabolismo , Metilación de ADN , Elementos Transponibles de ADN/genética , Drosophila melanogaster/genética , Heterocromatina/genética , Proteínas Nucleares/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismoRESUMEN
Non-small-cell lung carcinomas do not sufficiently respond to cancer chemotherapeutic drugs. Combination effects of cancer chemotherapy drugs (paclitaxel and carboplatin) with nobiletin or powdered Shiikuwasha extract from Citrus depressa were examined by isobologram and combination index analyses. It was demonstrated that the combination generated a synergistic inhibitory effect against the proliferation of the human non-small-cell lung carcinoma cell lines A549 and H460 and that of the two chemotherapy drugs, paclitaxel was responsible for this synergistic effect. Furthermore, the percentage of apoptotic cells was decreased with increasing rates of nobiletin to paclitaxel and carboplatin. These findings were considered to be attributed to the ability of nobiletin to regulate cells in the G1 phase, which escaped cell death initiated by paclitaxel and carboplatin. An antitumor activity assay showed that this combination significantly suppressed the growth of subcutaneous A549 tumor xenografts in nude mice.
Asunto(s)
Antineoplásicos/uso terapéutico , Carboplatino/uso terapéutico , Carcinoma de Pulmón de Células no Pequeñas/tratamiento farmacológico , Citrus/química , Flavonas/uso terapéutico , Neoplasias Pulmonares/tratamiento farmacológico , Paclitaxel/uso terapéutico , Animales , Antineoplásicos/farmacología , Antineoplásicos Fitogénicos/farmacología , Antineoplásicos Fitogénicos/uso terapéutico , Protocolos de Quimioterapia Combinada Antineoplásica , Apoptosis , Carboplatino/farmacología , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Sinergismo Farmacológico , Femenino , Flavonas/farmacología , Humanos , Ratones Endogámicos BALB C , Paclitaxel/farmacología , Fitoterapia , Extractos Vegetales/farmacología , Extractos Vegetales/uso terapéuticoRESUMEN
The Eph family of receptor tyrosine kinases regulates numerous biological processes. To examine the biochemical and developmental contributions of specific structural motifs within Eph receptors, wild-type or mutant forms of the EphA4 receptor were ectopically expressed in developing Xenopus embryos. Wild-type EphA4 and a mutant lacking both the SAM domain and PDZ binding motif were constitutively tyrosine phosphorylated in vivo and catalytically active in vitro. EphA4 induced loss of cell adhesion, ventro-lateral protrusions, and severely expanded posterior structures in Xenopus embryos. Moreover, mutation of a conserved SAM domain tyrosine to phenylalanine (Y928F) enhanced the ability of EphA4 to induce these phenotypes, suggesting that the SAM domain may negatively regulate some aspects of EphA4 activity in Xenopus. Analysis of double mutants revealed that the Y928F EphA4 phenotypes were dependent on kinase activity; juxtamembrane sites of tyrosine phosphorylation and SH2 domain-binding were required for cell dissociation, but not for posterior protrusions. The induction of protrusions and expansion of posterior structures is similar to phenotypic effects observed in Xenopus embryos expressing activated FGFR1. Furthermore, the budding ectopic protrusions induced by EphA4 express FGF-8, FGFR1, and FGFR4a. In addition, antisense morpholino oligonucleotide-mediated loss of FGF-8 expression in vivo substantially reduced the phenotypic effects in EphA4Y928F expressing embryos, suggesting a connection between Eph and FGF signaling.
Asunto(s)
Factores de Crecimiento de Fibroblastos/metabolismo , Regulación del Desarrollo de la Expresión Génica , Receptor EphA4/fisiología , Secuencias de Aminoácidos , Animales , Western Blotting , Catálisis , Adhesión Celular , Membrana Celular/metabolismo , Clonación Molecular , Factor 8 de Crecimiento de Fibroblastos , Hibridación in Situ , Ratones , Mutación , Fenotipo , Fosforilación , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , ARN/química , ARN/metabolismo , ARN Mensajero/metabolismo , Receptor EphA4/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Tirosina/química , Xenopus , Dominios Homologos srcRESUMEN
In zebrafish embryos, the axons of the posterior trigeminal (Vp) and facial (VII) motoneurons project stereotypically to a small number of target muscles derived from the first and second branchial arches (BA1, BA2). Use of the Islet1 (Isl1)-GFP transgenic line enabled precise real-time observations of the growth cone behaviour of the Vp and VII motoneurons within BA1 and BA2. Screening for N-ethyl-N-nitrosourea-induced mutants identified seven distinct mutations affecting different steps in the axonal pathfinding of these motoneurons. The class 1 mutations caused severe defasciculation and abnormal pathfinding in both Vp and VII motor axons before they reached their target muscles in BA1. The class 2 mutations caused impaired axonal outgrowth of the Vp motoneurons at the BA1-BA2 boundary. The class 3 mutation caused impaired axonal outgrowth of the Vp motoneurons within the target muscles derived from BA1 and BA2. The class 4 mutation caused retraction of the Vp motor axons in BA1 and abnormal invasion of the VII motor axons in BA1 beyond the BA1-BA2 boundary. Time-lapse observations of the class 1 mutant, vermicelli (vmc), which has a defect in the plexin A3 (plxna3) gene, revealed that Plxna3 acts with its ligand Sema3a1 for fasciculation and correct target selection of the Vp and VII motor axons after separation from the common pathways shared with the sensory axons in BA1 and BA2, and for the proper exit and outgrowth of the axons of the primary motoneurons from the spinal cord.
Asunto(s)
Axones/fisiología , Desarrollo Embrionario/genética , Nervio Facial/embriología , Receptores de Superficie Celular/fisiología , Nervio Trigémino/embriología , Proteínas de Pez Cebra/fisiología , Pez Cebra/embriología , Animales , Neuronas Motoras/fisiología , Mutación , Factores de Crecimiento Nervioso , Receptores de Superficie Celular/genética , Semaforinas/fisiología , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
In the developing vertebrate retina, progenitor cells initially proliferate but begin to produce postmitotic neurons when neuronal differentiation occurs. However, the mechanism that determines whether retinal progenitor cells continue to proliferate or exit from the cell cycle and differentiate is largely unknown. Here, we report that histone deacetylase 1 (Hdac1) is required for the switch from proliferation to differentiation in the zebrafish retina. We isolated a zebrafish mutant, ascending and descending (add), in which retinal cells fail to differentiate into neurons and glial cells but instead continue to proliferate. The cloning of the add gene revealed that it encodes Hdac1. Furthermore, the ratio of the number of differentiating cells to that of proliferating cells increases in proportion to Hdac activity, suggesting that Hdac proteins regulate a crucial step of retinal neurogenesis in zebrafish. Canonical Wnt signaling promotes the proliferation of retinal cells in zebrafish, and Notch signaling inhibits neuronal differentiation through the activation of a neurogenic inhibitor, Hairy/Enhancer-of-split (Hes). We found that both the Wnt and Notch/Hes pathways are activated in the add mutant retina. The cell-cycle progression and the upregulation of Hes expression in the add mutant retina can be inhibited by the blockade of Wnt and Notch signaling, respectively. These data suggest that Hdac1 antagonizes these pathways to promote cell-cycle exit and the subsequent neurogenesis in zebrafish retina. Taken together, these data suggest that Hdac1 functions as a dual switch that suppresses both cell-cycle progression and inhibition of neurogenesis in the zebrafish retina.
Asunto(s)
Regulación hacia Abajo/fisiología , Histona Desacetilasas/metabolismo , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Proteínas de la Membrana/metabolismo , Retina/embriología , Transducción de Señal/genética , Proteínas de Pez Cebra/metabolismo , Pez Cebra/embriología , Secuencia de Aminoácidos , Animales , Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular/fisiología , Proliferación Celular , Ciclina D1/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Regulación hacia Abajo/genética , Inhibidores de Crecimiento/genética , Inhibidores de Crecimiento/fisiología , Histona Desacetilasa 1 , Histona Desacetilasas/genética , Proteínas de la Membrana/antagonistas & inhibidores , Datos de Secuencia Molecular , Mutación , Receptores Notch , Retina/citología , Proteínas Supresoras de Tumor/metabolismo , Proteínas Wnt , Pez Cebra/genética , Proteínas de Pez Cebra/genéticaRESUMEN
Pbx1 is a homeodomain protein that functions in complexes with other homeodomain-containing proteins to regulate gene expression during embryogenesis and oncogenesis. Pbx proteins bind DNA cooperatively as heterodimers or higher order complexes with Meis family members and Hox proteins and are believed to specify cell identity during development. Here, we present evidence that Pbx1, in partnership with Meis1b, can regulate posterior neural markers and neural crest marker genes during Xenopus development. A Xenopus homolog of the Pbx1b homeodomain protein was isolated and shown to be expressed throughout embryogenesis. Xpbx1b expression overlaps with Xmeis1 in several areas, including the lateral neural folds, caudal branchial arch, hindbrain, and optic cup. When ectopically expressed, Xpbx1b can synergize with Xmeis1b to promote posterior neural and neural crest gene expression in ectodermal explants. Further, a physical interaction between these two homeodomain proteins is necessary for induction of these genes in embryonic tissue. In addition, coexpression of Xmeis1b and Xpbx1b leads to a prominent shift in the localization of Xmeis1b from the cytoplasm to the nucleus, suggesting that nuclear transport or retention of Xmeis1b may depend upon Xpbx1b. Finally, expression of a mutant construct in which Xpbx1b protein is fused to the repressor domain from Drosophila Engrailed inhibits posterior neural and neural crest gene expression. These data indicate that Xpbx1b and its partner, Xmeis1b, function in a transcriptional activation complex during hindbrain and neural crest development.
Asunto(s)
Proteínas de Unión al ADN/fisiología , Proteínas de Homeodominio/fisiología , Proteínas de Neoplasias/fisiología , Cresta Neural/metabolismo , Proteínas Proto-Oncogénicas/fisiología , Proteínas de Xenopus/fisiología , Xenopus/embriología , Secuencia de Aminoácidos , Animales , Northern Blotting , Western Blotting , ADN Complementario/metabolismo , Proteínas de Unión al ADN/biosíntesis , Dimerización , Biblioteca de Genes , Proteínas de Homeodominio/biosíntesis , Inmunohistoquímica , Hibridación in Situ , Datos de Secuencia Molecular , Proteína 1 del Sitio de Integración Viral Ecotrópica Mieloide , Proteínas de Neoplasias/biosíntesis , Factor de Transcripción 1 de la Leucemia de Células Pre-B , Pruebas de Precipitina , Unión Proteica , Estructura Terciaria de Proteína , Proteínas Proto-Oncogénicas/biosíntesis , ARN/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Tiempo , Transcripción Genética , Proteínas de Xenopus/biosíntesisRESUMEN
Bioactive phospholipids, which include sphingosine-1-phosphate, lysophosphatidic acid, ceramide and their derivatives regulate a wide variety of cellular functions in culture such as proliferation, apoptosis and differentiation. The availability of these lipids and their products is regulated by the lipid phosphate phosphatases (LPPs). Here we show that mouse embryos deficient for LPP3 fail to form a chorio-allantoic placenta and yolk sac vasculature. A subset of embryos also show a shortening of the anterior-posterior axis and frequent duplication of axial structures that are strikingly similar to the phenotypes associated with axin deficiency, a critical regulator of Wnt signaling. Loss of LPP3 results in a marked increase in beta-catenin-mediated TCF transcription, whereas elevated levels of LPP3 inhibit beta-catenin-mediated TCF transcription. LPP3 also inhibits axis duplication and leads to mild ventralization in Xenopus embryo development. Although LPP3 null fibroblasts show altered levels of bioactive phospholipids, consistent with loss of LPP3 phosphatase activity, mutant forms of LPP3, specifically lacking phosphatase activity, were able to inhibit beta-catenin-mediated TCF transcription and also suppress axis duplication, although not as effectively as intact LPP3. These results reveal that LPP3 is essential to formation of the chorio-allantoic placenta and extra-embryonic vasculature. LPP3 also mediates gastrulation and axis formation, probably by influencing the canonical Wnt signaling pathway. The exact biochemical roles of LPP3 phosphatase activity and its undefined effect on beta-catenin-mediated TCF transcription remain to be determined.