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1.
PLoS Biol ; 19(9): e3001377, 2021 09.
Artículo en Inglés | MEDLINE | ID: mdl-34491983

RESUMEN

Forming an embryo from a zygote poses an apparent conflict for epigenetic regulation. On the one hand, the de novo induction of cell fate identities requires the establishment and subsequent maintenance of epigenetic information to harness developmental gene expression. On the other hand, the embryo depends on cell proliferation, and every round of DNA replication dilutes preexisting histone modifications by incorporation of new unmodified histones into chromatin. Here, we investigated the possible relationship between the propagation of epigenetic information and the developmental cell proliferation during Xenopus embryogenesis. We systemically inhibited cell proliferation during the G1/S transition in gastrula embryos and followed their development until the tadpole stage. Comparing wild-type and cell cycle-arrested embryos, we show that the inhibition of cell proliferation is principally compatible with embryo survival and cellular differentiation. In parallel, we quantified by mass spectrometry the abundance of a large set of histone modification states, which reflects the developmental maturation of the embryonic epigenome. The arrested embryos developed abnormal stage-specific histone modification profiles (HMPs), in which transcriptionally repressive histone marks were overrepresented. Embryos released from the cell cycle block during neurulation reverted toward normality on morphological, molecular, and epigenetic levels. These results suggest that the cell cycle block by HUA alters stage-specific HMPs. We propose that this influence is strong enough to control developmental decisions, specifically in cell populations that switch between resting and proliferating states such as stem cells.


Asunto(s)
Epigénesis Genética , Código de Histonas , Xenopus laevis/embriología , Animales , Afidicolina/farmacología , Ciclo Celular , Proliferación Celular/efectos de los fármacos , Embrión no Mamífero/embriología , Inhibidores Enzimáticos/farmacología , Hidroxiurea/farmacología
2.
PLoS Genet ; 13(5): e1006757, 2017 May.
Artículo en Inglés | MEDLINE | ID: mdl-28498870

RESUMEN

Zygotic gene expression programs control cell differentiation in vertebrate development. In Xenopus, these programs are initiated by local induction of regulatory genes through maternal signaling activities in the wake of zygotic genome activation (ZGA) at the midblastula transition (MBT). These programs lay down the vertebrate body plan through gastrulation and neurulation, and are accompanied by massive changes in chromatin structure, which increasingly constrain cellular plasticity. Here we report on developmental functions for Brahma related gene 1 (Brg1), a key component of embyronic SWI/SNF chromatin remodeling complexes. Carefully controlled, global Brg1 protein depletion in X. tropicalis and X. laevis causes embryonic lethality or developmental arrest from gastrulation on. Transcriptome analysis at late blastula, before development becomes arrested, indicates predominantly a role for Brg1 in transcriptional activation of a limited set of genes involved in pattern specification processes and nervous system development. Mosaic analysis by targeted microinjection defines Brg1 as an essential amplifier of gene expression in dorsal (BCNE/Nieuwkoop Center) and ventral (BMP/Vent) signaling centers. Moreover, Brg1 is required and sufficient for initiating axial patterning in cooperation with maternal Wnt signaling. In search for a common denominator of Brg1 impact on development, we have quantitatively filtered global mRNA fluctuations at MBT. The results indicate that Brg1 is predominantly required for genes with the highest burst of transcriptional activity. Since this group contains many key developmental regulators, we propose Brg1 to be responsible for raising their expression above threshold levels in preparation for embryonic patterning.


Asunto(s)
Adenosina Trifosfatasas/genética , ADN Helicasas/genética , Transcripción Genética , Animales , Blástula/crecimiento & desarrollo , Blástula/metabolismo , Diferenciación Celular/genética , Cromatina/genética , Proteínas Cromosómicas no Histona/genética , ADN Helicasas/biosíntesis , Desarrollo Embrionario/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Herencia Materna/genética , Factores de Transcripción/genética , Vía de Señalización Wnt/genética , Xenopus/genética , Xenopus/crecimiento & desarrollo , Cigoto/crecimiento & desarrollo , Cigoto/metabolismo
3.
PLoS Genet ; 9(1): e1003188, 2013.
Artículo en Inglés | MEDLINE | ID: mdl-23382689

RESUMEN

Post-translational modifications (PTMs) of histones exert fundamental roles in regulating gene expression. During development, groups of PTMs are constrained by unknown mechanisms into combinatorial patterns, which facilitate transitions from uncommitted embryonic cells into differentiated somatic cell lineages. Repressive histone modifications such as H3K9me3 or H3K27me3 have been investigated in detail, but the role of H4K20me3 in development is currently unknown. Here we show that Xenopus laevis Suv4-20h1 and h2 histone methyltransferases (HMTases) are essential for induction and differentiation of the neuroectoderm. Morpholino-mediated knockdown of the two HMTases leads to a selective and specific downregulation of genes controlling neural induction, thereby effectively blocking differentiation of the neuroectoderm. Global transcriptome analysis supports the notion that these effects arise from the transcriptional deregulation of specific genes rather than widespread, pleiotropic effects. Interestingly, morphant embryos fail to repress the Oct4-related Xenopus gene Oct-25. We validate Oct-25 as a direct target of xSu4-20h enzyme mediated gene repression, showing by chromatin immunoprecipitaton that it is decorated with the H4K20me3 mark downstream of the promoter in normal, but not in double-morphant, embryos. Since knockdown of Oct-25 protein significantly rescues the neural differentiation defect in xSuv4-20h double-morphant embryos, we conclude that the epistatic relationship between Suv4-20h enzymes and Oct-25 controls the transit from pluripotent to differentiation-competent neural cells. Consistent with these results in Xenopus, murine Suv4-20h1/h2 double-knockout embryonic stem (DKO ES) cells exhibit increased Oct4 protein levels before and during EB formation, and reveal a compromised and biased capacity for in vitro differentiation, when compared to normal ES cells. Together, these results suggest a regulatory mechanism, conserved between amphibians and mammals, in which H4K20me3-dependent restriction of specific POU-V genes directs cell fate decisions, when embryonic cells exit the pluripotent state.


Asunto(s)
Diferenciación Celular , N-Metiltransferasa de Histona-Lisina/genética , Placa Neural , Factores del Dominio POU , Proteínas de Xenopus/genética , Xenopus laevis , Animales , Técnicas de Cultivo de Célula , Linaje de la Célula , Cromatina/genética , Metilación de ADN , Embrión no Mamífero , Células Madre Embrionarias/citología , Células Madre Embrionarias/metabolismo , Regulación del Desarrollo de la Expresión Génica , Técnicas de Silenciamiento del Gen , N-Metiltransferasa de Histona-Lisina/metabolismo , Placa Neural/crecimiento & desarrollo , Placa Neural/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Factores del Dominio POU/genética , Factores del Dominio POU/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrollo
4.
Cell Syst ; 11(6): 653-662.e8, 2020 12 16.
Artículo en Inglés | MEDLINE | ID: mdl-33296683

RESUMEN

DNA replication during cell division leads to dilution of histone modifications and can thus affect chromatin-mediated gene regulation, raising the question of how the cell-cycle shapes the histone modification landscape, particularly during embryogenesis. We tackled this problem by manipulating the cell cycle during early Xenopus laevis embryogenesis and analyzing in vivo histone H4K20 methylation kinetics. The global distribution of un-, mono-, di-, and tri-methylated histone H4K20 was measured by mass spectrometry in normal and cell-cycle-arrested embryos over time. Using multi-start maximum likelihood optimization and quantitative model selection, we found that three specific biological methylation rate constants were required to explain the measured H4K20 methylation state kinetics. While demethylation is essential for regulating H4K20 methylation kinetics in non-cycling cells, demethylation is very likely dispensable in rapidly dividing cells of early embryos, suggesting that cell-cycle-mediated dilution of H4K20 methylation is an essential regulatory component for shaping its epigenetic landscape during early development. A record of this paper's transparent peer review process is included in the Supplemental Information.


Asunto(s)
Puntos de Control del Ciclo Celular/genética , Xenopus laevis/embriología , Animales , Proliferación Celular , Desmetilación , Metilación
5.
Cancer Lett ; 442: 299-309, 2019 02 01.
Artículo en Inglés | MEDLINE | ID: mdl-30445206

RESUMEN

Melanoma is an aggressive cancer with poor prognosis, requiring personalized management of advanced stages and establishment of molecular markers. Melanomas derive from melanocytes, which specifically express tyrosinase, the rate-limiting enzyme of melanin-synthesis. We demonstrate that melanomas with high levels of DNp73, a cancer-specific variant of the p53 family member p73 and driver of melanoma progression show, in contrast to their less-aggressive low-DNp73 counterparts, hypopigmentation in vivo. Mechanistically, reduced melanin-synthesis is mediated by a DNp73-activated IGF1R/PI3K/AKT axis leading to tyrosinase ER-arrest and proteasomal degradation. Tyrosinase loss triggers reactivation of the EMT signaling cascade, a mesenchymal-like cell phenotype and increased invasiveness. DNp73-induced depigmentation, Slug increase and changes in cell motility are recapitulated in neural crest-derived melanophores of Xenopus embryos, underscoring a previously unnoticed physiological role of tyrosinase as EMT inhibitor. This data provides a mechanism of hypopigmentation accompanying cancer progression, which can be exploited in precision diagnosis of patients with melanoma-associated hypopigmentation (MAH), currently seen as a favorable prognostic factor. The DNp73/IGF1R/Slug signature in colorless lesions might aid to clinically discriminate between patients with MAH-associated metastatic disease and those, where MAH is indeed a sign of regression.


Asunto(s)
Transición Epitelial-Mesenquimal , Hipopigmentación/enzimología , Melaninas/metabolismo , Melanocitos/enzimología , Melanoma/enzimología , Monofenol Monooxigenasa/metabolismo , Neoplasias Cutáneas/enzimología , Proteína Tumoral p73/metabolismo , Animales , Línea Celular Tumoral , Movimiento Celular , Humanos , Hipopigmentación/genética , Hipopigmentación/patología , Melanocitos/patología , Melanoma/genética , Melanoma/patología , Ratones , Monofenol Monooxigenasa/genética , Invasividad Neoplásica , Complejo de la Endopetidasa Proteasomal/metabolismo , Proteolisis , Especies Reactivas de Oxígeno/metabolismo , Receptor IGF Tipo 1 , Receptores de Somatomedina/genética , Receptores de Somatomedina/metabolismo , Transducción de Señal , Neoplasias Cutáneas/genética , Neoplasias Cutáneas/patología , Factores de Transcripción de la Familia Snail/genética , Factores de Transcripción de la Familia Snail/metabolismo , Proteína Tumoral p73/genética , Xenopus laevis
6.
Dev Cell ; 51(5): 632-644.e6, 2019 12 02.
Artículo en Inglés | MEDLINE | ID: mdl-31630981

RESUMEN

Gene transcription in eukaryotes is regulated through dynamic interactions of a variety of different proteins with DNA in the context of chromatin. Here, we used mass spectrometry for absolute quantification of the nuclear proteome and methyl marks on selected lysine residues in histone H3 during two stages of Drosophila embryogenesis. These analyses provide comprehensive information about the absolute copy number of several thousand proteins and reveal unexpected relationships between the abundance of histone-modifying and -binding proteins and the chromatin landscape that they generate and interact with. For some histone modifications, the levels in Drosophila embryos are substantially different from those previously reported in tissue culture cells. Genome-wide profiling of H3K27 methylation during developmental progression and in animals with reduced PRC2 levels illustrates how mass spectrometry can be used for quantitatively describing and comparing chromatin states. Together, these data provide a foundation toward a quantitative understanding of gene regulation in Drosophila.


Asunto(s)
Ensamble y Desensamble de Cromatina , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Código de Histonas , Animales , Cromatina/genética , Cromatina/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Proteoma/genética , Proteoma/metabolismo
7.
Genesis ; 46(2): 81-6, 2008 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-18257044

RESUMEN

The restricted expression of XTcf-4 in the anterior midbrain is regulated via an active wnt/beta-catenin pathway (Kunz et al.,2004, Dev Biol 273:390-401). The molecular mechanism of this autoregulatory loop, however, remained elusive. Here we show that the activity of a 1,775 bp promoter fragment containing a consensus Lef/Tcf binding site at position -1,437 to -1,428 is upregulated by activating transcription factors of the Lef/Tcf family. Furthermore, chromatin immunoprecipitation revealed that endogenous beta-catenin is bound to the Lef/Tcf site on the promoter. Thus, regulation of XTcf-4 by canonical wnt-signaling is directly controlled by binding to and activating a consensus Lef/Tcf binding site within its own promoter.


Asunto(s)
Regiones Promotoras Genéticas , Factores de Transcripción TCF/genética , Factores de Transcripción TCF/metabolismo , Animales , Secuencia de Bases , Línea Celular , Inmunoprecipitación de Cromatina , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Proteína 2 Similar al Factor de Transcripción 7 , Transfección , Proteínas Wnt/metabolismo , Proteínas de Xenopus , Xenopus laevis
8.
Mech Dev ; 115(1-2): 79-89, 2002 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-12049769

RESUMEN

The development of skeletal muscle in the vertebrate embryo is controlled by a transcriptional cascade that includes the four myogenic regulatory factors Myf-5, MyoD, Myogenin, and MRF4. The dynamic expression pattern of myf-5 during myogenesis is thought to be consistent with its role during early determination of the myogenic lineage. To study the factors and mechanisms, which regulate myf-5 transcription in Xenopus, we isolated a genomic DNA clone containing 4858 bp of Xmyf-5 5' flanking region. Using a transgenic reporter assay, we show here that this genomic contig is sufficient to recapitulate the dynamic stage- and tissue-specific expression pattern of Xmyf-5 from the gastrula to tail bud stages. For the primary induction of myf-5 transcription, we identify three main regulatory elements, which are responsible for (i) activation in dorsal mesoderm, (ii) activation in ventral mesoderm, and (iii) repression in midline mesoderm, respectively. Their combined activities define the two-winged expression domain of myf-5 in the preinvoluted mesoderm. Repression in midline mesoderm is mediated by a single TCF binding site located in the 5' end of the -4.8 kbp sequence, which binds XTcf-3 protein in vitro. Endogenous Wnt signaling in the lateral mesoderm is required to overcome the long-range repression through this distal TCF site, and to stimulate myf-5 transcription independently from it. The element for ventral mesoderm activation responds to Activin. Together, these results describe a regulatory mosaic of repression and activation, which defines the myf-5 expression profile in the frog gastrula.


Asunto(s)
Secuencia de Consenso , Gástrula/fisiología , Expresión Génica , Proteínas HMGB/metabolismo , Mesodermo/fisiología , Proteínas Musculares/genética , Transactivadores , Factores de Transcripción/metabolismo , Proteínas de Pez Cebra , Activinas/metabolismo , Animales , Animales Modificados Genéticamente , Sitios de Unión , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Receptores Frizzled , Genes Reporteros , Proteínas Fluorescentes Verdes , Proteínas HMGB/genética , Proteínas Luminiscentes/genética , Factor de Unión 1 al Potenciador Linfoide , Factor 5 Regulador Miogénico , Proteínas/genética , Proteínas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Secuencias Reguladoras de Ácidos Nucleicos , Transducción de Señal , Factores de Transcripción TCF , Factor de Transcripción 3 , Proteína 1 Similar al Factor de Transcripción 7 , Factores de Transcripción/genética , Proteínas Wnt , Proteínas de Xenopus , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismo
9.
Gene ; 326: 59-66, 2004 Feb 04.
Artículo en Inglés | MEDLINE | ID: mdl-14729263

RESUMEN

All eukaryotes share a common nuclear infrastructure, in which DNA is packaged into nucleosomal chromatin. Its functional states, in particular the accessibility of the chromatin fiber to trans-acting factors, are determined by two classes of evolutionarily conserved enzymes, i.e. histone modifying enzymes and ATP-driven nucleosome remodeling machines. Browsing the annotated human genome database, we establish here a family of SNF2-like nuclear ATPases, which are the core enzymatic subunits of chromatin remodeling protein complexes. Homologues of those human genes are also to a large extent found in the Xenopus laevis genome, indicating a high degree of sequence conservation of this family among vertebrates. Expression analyses of the ATPase family of proteins reveal stage- and tissue-specific domains of peak RNA expression during early frog embryogenesis. These dynamic expression profiles suggest specific functional requirements for individual members of this family throughout early stages of vertebrate development.


Asunto(s)
Adenosina Trifosfatasas/genética , Proteínas de Unión al ADN/genética , Embrión no Mamífero/metabolismo , Perfilación de la Expresión Génica , Proteínas Nucleares , Factores de Transcripción/genética , Xenopus laevis/genética , Adenosina Trifosfatasas/metabolismo , Animales , ADN Helicasas , Proteínas de Unión al ADN/metabolismo , Embrión no Mamífero/enzimología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Hibridación in Situ , Familia de Multigenes/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Factores de Transcripción/metabolismo , Xenopus laevis/embriología
10.
FEBS Lett ; 547(1-3): 1-6, 2003 Jul 17.
Artículo en Inglés | MEDLINE | ID: mdl-12860376

RESUMEN

Though the Wnt/beta-catenin signaling pathway is known to play key roles during Xenopus axis specification, whether it signals exclusively through Lef/Tcf transcription factors in this process remains unclear. To investigate this issue, we generated transgenic frog embryos expressing green fluorescent protein (GFP) driven by a Lef/Tcf-dependent and Wnt/beta-catenin-responsive promoter. This promoter is highly sensitive and even detects maternal beta-catenin activity prior to the large-scale transcription of zygotic genes. Unexpectedly, GFP expression was observed only in some, but not all, known Wnt/beta-catenin-positive territories in Xenopus early development. Furthermore, ubiquitous expression of dominant Lef-1 protein variants from transgenes revealed that zygotic Lef/Tcf activity is required for the ventroposterior development of Xenopus embryos. In summary, our results suggest that endogenous Wnt/beta-catenin activity does not result in obligatory Lef/Tcf-dependent gene activation, and that the ventroposteriorizing activity of zygotic Wnt-8 signaling is mediated by Lef/Tcf proteins.


Asunto(s)
Tipificación del Cuerpo/genética , Proteínas del Citoesqueleto/genética , Proteínas de Unión al ADN/genética , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/genética , Proteínas del Grupo de Alta Movilidad/genética , Proteínas Proto-Oncogénicas/genética , Transactivadores/genética , Factores de Transcripción/genética , Xenopus/embriología , Xenopus/genética , Proteínas de Pez Cebra , Animales , Animales Modificados Genéticamente , Cadherinas/genética , Embrión no Mamífero/fisiología , Genes Reporteros , Genes fos , Proteínas Fluorescentes Verdes , Leucina Zippers , Luciferasas/genética , Proteínas Luminiscentes/genética , Factor de Unión 1 al Potenciador Linfoide , Factor 1 Relacionado con NF-E2 , Proteínas Tirosina Quinasas/genética , Transducción de Señal , Factores de Transcripción TCF , Proteína 1 Similar al Factor de Transcripción 7 , Proteína 2 Similar al Factor de Transcripción 7 , Activación Transcripcional , Proteínas Wnt , Proteínas de Xenopus/genética , Cigoto/fisiología , beta Catenina
11.
Neuroreport ; 14(17): 2163-6, 2003 Dec 02.
Artículo en Inglés | MEDLINE | ID: mdl-14625440

RESUMEN

During vertebrate neural development, inductive signals derived from the roof plate specify dorsal neural cell types of spinal cord as well as adjacent dermomytotome. Noggin has been demonstrated to play a pivotal role in this signaling network but how its expression is regulated remains unclear. To investigate this issue, we identified a 2066 bp Xenopus noggin 5' flanking sequence which recapitulates the roof-plate expression of endogenous gene in transgenic frog tadpoles and we further mapped the roof-plate enhancer to a sequence as short as 79 bp. Our results, therefore, not only shed light on but also provide a powerful tool to the study of neural and somite patterning.


Asunto(s)
Células del Asta Posterior/embriología , Regiones Promotoras Genéticas/fisiología , Proteínas/genética , Somitos/fisiología , Transcripción Genética/genética , Proteínas de Xenopus/genética , Animales , Animales Modificados Genéticamente , Proteínas Portadoras , Femenino , Proteínas de Xenopus/fisiología , Xenopus laevis
12.
Cardiovasc Res ; 97(3): 454-63, 2013 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-23241315

RESUMEN

AIMS: Future cardiac repair strategies will require a profound understanding of the principles underlying cardiovascular differentiation. Owing to its extracorporal and rapid development, Xenopus laevis provides an ideal experimental system to address these issues in vivo. Whereas mammalian MesP1 is currently regarded as the earliest marker for the cardiovascular system, several MesP1-related factors from Xenopus-mespa, mespb, and mespo-have been assigned only to somitogenesis so far. We, therefore, analysed these genes comparatively for potential contributions to cardiogenesis. METHODS AND RESULTS: RNA in situ hybridizations revealed a novel anterior expression domain exclusively occupied by mespa during gastrulation, which precedes the prospective heart field. Correspondingly, when overexpressed mespa most strongly induced cardiac markers in vivo as well as ex vivo. Transference to murine embryonic stem (ES) cells and subsequent FACS analyses for Flk-1 and Troponin I confirmed the high potential of mespa as a cardiac inducer. In vivo, Morpholino-based knockdown of mespa protein led to a dramatic loss of pro-cardiac and sarcomeric markers, which could be rescued either by mespa itself or human MesP1, but neither by mespb nor mespo. Epistatic analysis positioned mespa upstream of mespo and mespb, and revealed positive autoregulation for mespa at the time of its induction. CONCLUSIONS: Our findings contribute to the understanding of conserved events initiating vertebrate cardiogenesis. We identify mespa as functional amphibian homologue of mammalian MesP1. These results will enable the dissection of cardiac specification from the very beginning in the highly versatile Xenopus system.


Asunto(s)
Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/fisiología , Diferenciación Celular/fisiología , Corazón/embriología , Miocitos Cardíacos/citología , Proteínas de Xenopus/fisiología , Xenopus laevis/embriología , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/genética , Células Cultivadas , Células Madre Embrionarias/citología , Células Madre Embrionarias/fisiología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Corazón/fisiología , Ratones , Modelos Animales , Morfolinos/farmacología , Miocitos Cardíacos/fisiología , Proteínas de Xenopus/genética , Xenopus laevis/fisiología
13.
PLoS One ; 7(5): e36136, 2012.
Artículo en Inglés | MEDLINE | ID: mdl-22590521

RESUMEN

Maternal Wnt/ß-Catenin signaling establishes a program of dorsal-specific gene expression required for axial patterning in Xenopus. We previously reported that a subset of dorsally expressed genes depends not only on Wnt/ß-Catenin stimulation, but also on a MyD88-dependent Toll-like receptor/IL1-receptor (TLR/IL1-R) signaling pathway. Here we show that these two signal transduction cascades converge in the nucleus to coactivate gene transcription in blastulae through a direct interaction between ß-Catenin and NF-κB proteins. A transdominant inhibitor of NF-κB, ΔNIκBα, phenocopies loss of MyD88 protein function, implicating Rel/NF-κB proteins as selective activators of dorsal-specific gene expression. Sensitive axis formation assays in the embryo demonstrate that dorsalization by Wnt/ß-Catenin requires NF-κB protein activity, and vice versa. Xenopus nodal-related 3 (Xnr3) is one of the genes with dual ß-Catenin/NF-κB input, and a proximal NF-κB consensus site contributes to the regional activity of its promoter. We demonstrate in vitro binding of Xenopus ß-Catenin to several XRel proteins. This interaction is observed in vivo upon Wnt-stimulation. Finally, we show that a synthetic luciferase reporter gene responds to both endogenous and exogenous ß-Catenin levels in an NF-κB motif dependent manner. These results suggest that ß-Catenin acts as a transcriptional co-activator of NF-κB-dependent transcription in frog primary embryonic cells.


Asunto(s)
Tipificación del Cuerpo/fisiología , Núcleo Celular/metabolismo , FN-kappa B/metabolismo , Elementos de Respuesta/fisiología , Transcripción Genética/fisiología , Vía de Señalización Wnt/fisiología , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Antígenos de Diferenciación/genética , Antígenos de Diferenciación/metabolismo , Blástula/citología , Blástula/embriología , Núcleo Celular/genética , FN-kappa B/genética , Receptores Inmunológicos/genética , Receptores Inmunológicos/metabolismo , Factor de Crecimiento Transformador beta , Proteínas Wnt/genética , Proteínas Wnt/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis , beta Catenina/genética , beta Catenina/metabolismo
14.
PLoS One ; 6(7): e22548, 2011.
Artículo en Inglés | MEDLINE | ID: mdl-21814581

RESUMEN

Vertebrate embryos are derived from a transitory pool of pluripotent cells. By the process of embryonic induction, these precursor cells are assigned to specific fates and differentiation programs. Histone post-translational modifications are thought to play a key role in the establishment and maintenance of stable gene expression patterns underlying these processes. While on gene level histone modifications are known to change during differentiation, very little is known about the quantitative fluctuations in bulk histone modifications during development. To investigate this issue we analysed histones isolated from four different developmental stages of Xenopus laevis by mass spectrometry. In toto, we quantified 59 modification states on core histones H3 and H4 from blastula to tadpole stages. During this developmental period, we observed in general an increase in the unmodified states, and a shift from histone modifications associated with transcriptional activity to transcriptionally repressive histone marks. We also compared these naturally occurring patterns with the histone modifications of murine ES cells, detecting large differences in the methylation patterns of histone H3 lysines 27 and 36 between pluripotent ES cells and pluripotent cells from Xenopus blastulae. By combining all detected modification transitions we could cluster their patterns according to their embryonic origin, defining specific histone modification profiles (HMPs) for each developmental stage. To our knowledge, this data set represents the first compendium of covalent histone modifications and their quantitative flux during normogenesis in a vertebrate model organism. The HMPs indicate a stepwise maturation of the embryonic epigenome, which may be causal to the progressing restriction of cellular potency during development.


Asunto(s)
Embrión no Mamífero/metabolismo , Células Madre Embrionarias/metabolismo , Epigenómica , Histonas/química , Xenopus laevis/embriología , Xenopus laevis/genética , Animales , Blástula/citología , Blástula/metabolismo , Western Blotting , Diferenciación Celular , Cromatografía Liquida , Embrión no Mamífero/citología , Perfilación de la Expresión Génica , Histonas/metabolismo , Lisina/química , Lisina/genética , Metilación , Ratones , Ratones Endogámicos ICR , Procesamiento Proteico-Postraduccional , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción
15.
Genes Dev ; 21(8): 973-83, 2007 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-17438000

RESUMEN

Experiments in Xenopus have illustrated the importance of extracellular morphogens for embryonic gene regulation in vertebrates. Much less is known about how induction leads to the correct positioning of boundaries; for example, between germ layers. Here we report that the neuroectoderm/mesoderm boundary is controlled by the chromatin remodeling ATPase CHD4/Mi-2beta. Gain and loss of CHD4 function experiments shifted this boundary along the animal-vegetal axis at gastrulation, leading to excess mesoderm formation at the expense of neuroectoderm, or vice versa. This phenotype results from specific alterations in gene transcription, notably of the neural-promoting gene Sip1 and the mesodermal regulatory gene Xbra. We show that CHD4 suppresses Sip1 transcription by direct binding to the 5' end of the Sip1 gene body. Furthermore, we demonstrate that CHD4 and Sip1 expression levels determine the "ON" threshold for Nodal-dependent but not for eFGF-dependent induction of Xbra transcription. The CHD4/Sip1 epistasis thus constitutes a regulatory module, which balances mesoderm and neuroectoderm formation.


Asunto(s)
Adenosina Trifosfatasas/metabolismo , Ectodermo/metabolismo , Inducción Embrionaria , Mesodermo/metabolismo , Proteínas de Xenopus/metabolismo , Xenopus laevis/embriología , Activinas/genética , Animales , Relación Dosis-Respuesta a Droga , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Represoras/metabolismo , Proteínas de Dominio T Box/genética , Proteínas de Xenopus/genética , Xenopus laevis/metabolismo
16.
Dev Dyn ; 235(4): 949-57, 2006 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-16493693

RESUMEN

In Drosophila, the Toll/Dorsal pathway triggers the nuclear entry of the Rel protein Dorsal, which controls dorsoventral patterning in early embryogenesis and plays an important role in innate immunity of the adult fly. In vertebrates, the homologous Toll/IL-1 receptor signaling pathway directs the nuclear localization of Rel/NF-kappaB complexes, which activate genes involved in proliferation, apoptosis, and immune response. Recently, first evidence has been reported for the activity of vertebrate Rel proteins and a Toll-like signaling pathway in the dorsoventral patterning process of Xenopus laevis embryos. Given the evolutionary divergence of the fly and frog model organisms, these findings raise the question, to what extent the effector functions of this pathway have been conserved? Here, we report the ability of two Xenopus Rel proteins to partially substitute for several, but not all, functions of the Dorsal protein in Drosophila embryos. Our results suggest the interaction between Rel proteins and their cytoplasmic inhibitors as an important interface of evolutionary adaptation.


Asunto(s)
Tipificación del Cuerpo , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Proteínas Nucleares/metabolismo , Fosfoproteínas/metabolismo , Factor de Transcripción ReIB/metabolismo , Factores de Transcripción/metabolismo , Animales , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrión no Mamífero , Gástrula , Genes de Insecto , Inmunohistoquímica , Microinyecciones , FN-kappa B/metabolismo , Proteínas Nucleares/genética , Fosfoproteínas/genética , Filogenia , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Interleucina-1/genética , Receptores de Interleucina-1/metabolismo , Transducción de Señal , Factores de Tiempo , Receptores Toll-Like/genética , Receptores Toll-Like/metabolismo , Factor de Transcripción ReIB/genética , Factores de Transcripción/genética , Xenopus laevis/embriología , Xenopus laevis/genética , Xenopus laevis/metabolismo
17.
Cell ; 123(7): 1178-9, 2005 Dec 29.
Artículo en Inglés | MEDLINE | ID: mdl-16377556

RESUMEN

Linker histones of the H1 family are among the most abundant components of chromatin. In this issue of Cell, the effect of H1 downregulation on gene expression is examined. Although a 50% reduction of histone H1 levels in embryonic stem cells affects chromatin structure globally, the expression of very few genes is altered. Intriguingly, this study reveals a new link between H1 and DNA methylation.


Asunto(s)
Metilación de ADN , Regulación de la Expresión Génica/fisiología , Histonas/fisiología , Animales , Humanos
18.
Eur J Biochem ; 269(9): 2294-9, 2002 May.
Artículo en Inglés | MEDLINE | ID: mdl-11985611

RESUMEN

During the development of multicellular organisms, both transient and stable gene expression patterns have to be established in a precisely orchestrated sequence. Evidence from diverse model organisms indicates that this epigenetic program involves not only transcription factors, but also the local structure, composition, and modification of chromatin, which define and maintain the accessibility and transcriptional competence of the nucleosomal DNA template. A paradigm for the interdependence of development and chromatin is constituted by the mechanisms controlling the specification and differentiation of the skeletal muscle cell lineage in vertebrates, which is the topic of this review.


Asunto(s)
Cromatina/metabolismo , Músculo Esquelético/embriología , Acetilación , Animales , Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico , Diferenciación Celular , Metilación de ADN , Proteínas de Unión al ADN/fisiología , Regulación del Desarrollo de la Expresión Génica , Histonas/metabolismo , Humanos , Proteína MioD/fisiología , Factores de Transcripción/fisiología , Transcripción Genética
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