RESUMEN
Repression of endogenous retroviruses (ERVs) in mammals involves several epigenetic mechanisms. Acute loss of the maintenance methyltransferase Dnmt1 induces widespread DNA demethylation and transcriptional activation of ERVs, including CpG-rich IAP (intracisternal A particle) proviruses. Here, we show that this effect is not due simply to a loss of DNA methylation. Conditional deletions reveal that both Dnmt1 and Np95 are essential for maintenance DNA methylation. However, while IAPs are derepressed in Dnmt1-ablated embryos and embryonic stem cells (ESCs), these ERVs remain silenced when Np95 is deleted alone or in combination with Dnmt1. This paradoxical phenotype results from an ectopic interaction between NP95 and the H3K9 methyltransferase SETDB1. Normally, SETDB1 maintains silencing of IAPs, but in the absence of DNMT1, prolonged binding of NP95 to hemimethylated DNA transiently disrupts SETDB1-dependent H3K9me3 deposition. Thus, our observations reveal an unexpected antagonistic interplay between two repressive pathways involved in retroviral silencing in mammalian cells.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , ADN/metabolismo , Retrovirus Endógenos/metabolismo , N-Metiltransferasa de Histona-Lisina/metabolismo , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/metabolismo , Activación Viral , Animales , Proteínas Potenciadoras de Unión a CCAAT , Diferenciación Celular , ADN (Citosina-5-)-Metiltransferasa 1 , Femenino , Dosificación de Gen , Regulación del Desarrollo de la Expresión Génica , Silenciador del Gen , Genes de Partícula A Intracisternal , Sitios Genéticos , Histonas/metabolismo , Lisina/metabolismo , Ratones , Ratones Noqueados , Modelos Biológicos , Mutación/genética , Proteínas Nucleares/química , Placenta/metabolismo , Embarazo , Unión Proteica , Dominios Proteicos , Trofoblastos/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
The Polycomb group of proteins forms at least two distinct complexes designated the Polycomb repressive complex-1 (PRC1) and PRC2. These complexes cooperate to mediate transcriptional repression of their target genes, including the Hox gene cluster and the Cdkn2a genes. Mammalian Polycomb-like gene Pcl2/Mtf2 is expressed as four different isoforms, and the longest one contains a Tudor domain and two plant homeodomain (PHD) fingers. Pcl2 forms a complex with PRC2 and binds to Hox genes in a PRC2-dependent manner. We show that Pcl2 is a functional component of PRC2 and is required for PRC2-mediated Hox repression. Pcl2, however, exhibits a profound synergistic effect on PRC1-mediated Hox repression, which is not accompanied by major alterations in the local trimethylation of histone H3 at lysine 27 (H3K27me3) or PRC1 deposition. Pcl2 therefore functions in collaboration with both PRC2 and PRC1 to repress Hox gene expression during axial development. Paradoxically, in embryonic fibroblasts, Pcl2 is shown to activate the expression of Cdkn2a and promote cellular senescence, presumably by suppressing the catalytic activity of PRC2 locally. Taken together, we show that Pcl2 differentially regulates Polycomb-mediated repression of Hox and Cdkn2a genes. We therefore propose a novel role for Pcl2 to modify functional engagement of PRC2 and PRC1, which could be modulated by sensing cellular circumstances.
Asunto(s)
Genes Homeobox , Genes p16 , N-Metiltransferasa de Histona-Lisina/metabolismo , Familia de Multigenes , Isoformas de Proteínas/metabolismo , Proteínas Represoras/metabolismo , Animales , Células Cultivadas , Fibroblastos/citología , Fibroblastos/fisiología , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Ratones , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Regiones Promotoras Genéticas , Isoformas de Proteínas/genética , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Represoras/genéticaRESUMEN
The spontaneous dominant mouse mutant, Elbow knee synostosis (Eks), shows elbow and knee joint synosotsis, and premature fusion of cranial sutures. Here we identify a missense mutation in the Fgf9 gene that is responsible for the Eks mutation. Through investigation of the pathogenic mechanisms of joint and suture synostosis in Eks mice, we identify a key molecular mechanism that regulates FGF9 signaling in developing tissues. We show that the Eks mutation prevents homodimerization of the FGF9 protein and that monomeric FGF9 binds to heparin with a lower affinity than dimeric FGF9. These biochemical defects result in increased diffusion of the altered FGF9 protein (FGF9(Eks)) through developing tissues, leading to ectopic FGF9 signaling and repression of joint and suture development. We propose a mechanism in which the range of FGF9 signaling in developing tissues is limited by its ability to homodimerize and its affinity for extracellular matrix heparan sulfate proteoglycans.
Asunto(s)
Matriz Extracelular/metabolismo , Factor 9 de Crecimiento de Fibroblastos/metabolismo , Multimerización de Proteína/fisiología , Animales , Secuencia de Bases , Análisis Mutacional de ADN , Difusión , Factor 9 de Crecimiento de Fibroblastos/genética , Proteoglicanos de Heparán Sulfato/metabolismo , Ratones , Ratones Transgénicos , Datos de Secuencia Molecular , Mutación Missense/fisiología , Unión Proteica , Multimerización de Proteína/genética , Transporte de Proteínas/genética , Transporte de Proteínas/fisiología , Homología de Secuencia de Aminoácido , Especificidad por Sustrato , Sinostosis/genética , Distribución TisularRESUMEN
DNA methyltransferase (cytosine-5) 1 (Dnmt1) is the principal enzyme responsible for maintenance of CpG methylation and is essential for the regulation of gene expression, silencing of parasitic DNA elements, genomic imprinting and embryogenesis. Dnmt1 is needed in S phase to methylate newly replicated CpGs occurring opposite methylated ones on the mother strand of the DNA, which is essential for the epigenetic inheritance of methylation patterns in the genome. Despite an intrinsic affinity of Dnmt1 for such hemi-methylated DNA, the molecular mechanisms that ensure the correct loading of Dnmt1 onto newly replicated DNA in vivo are not understood. The Np95 (also known as Uhrf1 and ICBP90) protein binds methylated CpG through its SET and RING finger-associated (SRA) domain. Here we show that localization of mouse Np95 to replicating heterochromatin is dependent on the presence of hemi-methylated DNA. Np95 forms complexes with Dnmt1 and mediates the loading of Dnmt1 to replicating heterochromatic regions. By using Np95-deficient embryonic stem cells and embryos, we show that Np95 is essential in vivo to maintain global and local DNA methylation and to repress transcription of retrotransposons and imprinted genes. The link between hemi-methylated DNA, Np95 and Dnmt1 thus establishes key steps of the mechanism for epigenetic inheritance of DNA methylation.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN , ADN/metabolismo , Epigénesis Genética , Proteínas Nucleares/química , Proteínas Nucleares/metabolismo , Animales , Proteínas Potenciadoras de Unión a CCAAT , Islas de CpG/genética , ADN/química , ADN (Citosina-5-)-Metiltransferasa 1 , Replicación del ADN , Células Madre Embrionarias/metabolismo , Impresión Genómica , Células HeLa , Heterocromatina/genética , Heterocromatina/metabolismo , Humanos , Ratones , Proteínas Nucleares/deficiencia , Proteínas Nucleares/genética , Antígeno Nuclear de Célula en Proliferación/metabolismo , Estructura Terciaria de Proteína , Retroelementos/genética , Transcripción Genética , Ubiquitina-Proteína LigasasRESUMEN
To address the molecular mechanisms underlying Polycomb group (PcG)-mediated repression of Hox gene expression, we have focused on the binding patterns of PcG gene products to the flanking regions of the Hoxb8 gene in expressing and non-expressing tissues. In parallel, we followed the distribution of histone marks of transcriptionally active H3 acetylated on lysine 9 (H3-K9) and methylated on lysine 4 (H3-K4), and of transcriptionally inactive chromatin trimethylated on lysine 27 (H3-K27). Chromatin immunoprecipitation revealed that the association of PcG proteins, and H3-K9 acetylation and H3-K27 trimethylation around Hoxb8 were distinct in tissues expressing and not expressing the gene. We show that developmental changes of these epigenetic marks temporally coincide with the misexpression of Hox genes in PcG mutants. Functional analyses, using mutant alleles impairing the PcG class 2 component Rnf2 or the Suz12 mutation decreasing H3-K27 trimethylation, revealed that interactions between class 1 and class 2 PcG complexes, mediated by trimethylated H3-K27, play decisive roles in the maintenance of Hox gene repression outside their expression domain. Within the expression domains, class 2 PcG complexes appeared to maintain the transcriptionally active status via profound regulation of H3-K9 acetylation. The present study indicates distinct roles for class 2 PcG complexes in transcriptionally repressed and active domains of Hoxb8 gene.
Asunto(s)
Proteínas de Unión al ADN , Regulación del Desarrollo de la Expresión Génica , Proteínas de Homeodominio/metabolismo , Proteínas Represoras , Transcripción Genética , Animales , Línea Celular , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Embrión de Mamíferos/anatomía & histología , Embrión de Mamíferos/fisiología , Histonas/metabolismo , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Ubiquitina-Proteína LigasasRESUMEN
The Polycomb group (PcG) gene products form multimeric protein complexes and contribute to anterior-posterior (A-P) specification via the transcriptional regulation of Hox cluster genes. The Drosophila polyhomeotic genes and their mammalian orthologues, Phc1, Phc2, and Phc3, encode nuclear proteins that are constituents of evolutionarily conserved protein complexes designated class II PcG complexes. In this study, we describe the generation and phenotypes of Phc2-deficient mice. We show posterior transformations of the axial skeleton and premature senescence of mouse embryonic fibroblasts associated with derepression of Hox cluster genes and Cdkn2a genes, respectively. Synergistic actions of a Phc2 mutation with Phc1 and Rnf110 mutations during A-P specification, coimmunoprecipitation of their products from embryonic extracts, and chromatin immunoprecipitation by anti-Phc2 monoclonal antibodies suggest that Hox repression by Phc2 is mediated through the class II PcG complexes, probably via direct binding to the Hox locus. The genetic interactions further reveal the functional overlap between Phc2 and Phc1 and a strict dose-dependent requirement during A-P specification and embryonic survival. Functional redundancy between Phc2 and Phc1 leads us to hypothesize that the overall level of polyhomeotic orthologues in nuclei is a parameter that is critical in enabling the class II PcG complexes to exert their molecular functions.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación del Desarrollo de la Expresión Génica/fisiología , Genes Homeobox , Proteínas de Homeodominio/antagonistas & inhibidores , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Factores de Transcripción/metabolismo , Animales , Tipificación del Cuerpo/fisiología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Desarrollo Embrionario/fisiología , Proteínas de Homeodominio/biosíntesis , Proteínas de Homeodominio/metabolismo , Ratones , Especificidad de Órganos , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas Represoras/biosíntesis , Factores de Transcripción/genéticaRESUMEN
Polycomb group (PcG) proteins are responsible for the stable repression of homeotic (Hox) genes by forming multimeric protein complexes. We show (1) physical interaction between components of the U2 small nuclear ribonucleoprotein particle (U2 snRNP), including Sf3b1 and PcG proteins Zfp144 and Rnf2; and (2) that Sf3b1 heterozygous mice exhibit skeletal transformations concomitant with ectopic Hox expressions. These alterations are enhanced by Zfp144 mutation but repressed by Mll mutation (a trithorax-group gene). Importantly, the levels of Sf3b1 in PcG complexes were decreased in Sf3b1-heterozygous embryos. These findings suggest that Sf3b1-PcG protein interaction is essential for true PcG-mediated repression of Hox genes.
Asunto(s)
Huesos/embriología , Proteínas de Unión al ADN/metabolismo , Regulación hacia Abajo/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas de Homeodominio/biosíntesis , Fosfoproteínas/metabolismo , Ribonucleoproteína Nuclear Pequeña U2/metabolismo , Animales , Proteínas de Unión al ADN/genética , Regulación hacia Abajo/genética , Regulación del Desarrollo de la Expresión Génica/genética , N-Metiltransferasa de Histona-Lisina , Proteínas de Homeodominio/genética , Ratones , Ratones Noqueados , Mutación , Proteína de la Leucemia Mieloide-Linfoide , Complejo Represivo Polycomb 1 , Unión Proteica/genética , Unión Proteica/fisiología , Proto-Oncogenes/genética , Factores de Empalme de ARN , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína LigasasRESUMEN
The mab-21 gene was first identified because of its requirement for ray identity specification in Caenorhabditis elegans. It is now known to constitute a family of genes that are highly conserved from vertebrates to invertebrates, and two homologues Mab21l1 and Mab21l2 have been identified in many species. Here we describe the generation of Mab21l2-deficient mice, which have defects in eye and body wall formation. The mutant mouse eye has a rudimentary retina, as a result of insufficient invagination of the optic vesicle due to deficient proliferation, causing the absence of lens. The defects in optic vesicle development correlate with reduced expression of Chx10, which is also required for retina development; Rx, Lhx2, and Pax6 expression is not significantly affected. We conclude that Mab21l2 expression is essential for optic vesicle growth and formation of the optic cup, its absence causing reduced expression of Chx10. Mutant mice also display abnormal extrusion of abdominal organs, defects in ventral body wall formation, resulting in death in utero at mid-gestational stage. Our results reveal that Mab21l2 plays crucial roles in retina and in ventral body wall formation.
Asunto(s)
Abdomen/anomalías , Embrión de Mamíferos/anatomía & histología , Proteínas del Ojo/metabolismo , Ojo/embriología , Morfogénesis , Retina/embriología , Animales , Proteína Morfogenética Ósea 4 , Proteínas Morfogenéticas Óseas/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión de Mamíferos/fisiología , Ojo/anatomía & histología , Ojo/crecimiento & desarrollo , Proteínas del Ojo/genética , Regulación del Desarrollo de la Expresión Génica , Humanos , Hibridación in Situ , Péptidos y Proteínas de Señalización Intracelular , Ratones , Ratones Noqueados , Retina/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Mammalian Polycomb group (PcG) proteins are known to function during the maintenance of spatially restricted expression of Hox cluster genes and cellular proliferation. To understand the molecular basis of PcG functions, it is important to identify the components of mammalian PcG complexes. We isolated mouse YAF2 as a protein that interacts with Ring1B, a known constituent of mammalian PcG complexes. We show that the murine YAF2 locus generates two different transcripts, mYAF2-a and mYAF2-b by alternative splicing of the third exons which encode two YAF2 isoforms of 179 and conceptual 60 amino acids, respectively. At least five exons encoding mYAF2 transcripts are mapped on chromosome 15E3 region. Expression of mYAF2 mRNA was observed in both pre- and postimplantation embryos. In mid-gestation embryos, mYAF2 expression is strongly seen in the region close to the surface ectoderm. Finally, biochemical evidence and colocalization studies in tissue culture cells suggest that the product of the mYAF2 gene is involved in PcG complexes together with Ring1B and/or Ring1A.
Asunto(s)
Empalme Alternativo , Proteínas de Unión al ADN/genética , Embrión de Mamíferos/metabolismo , Proteínas Musculares , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Línea Celular Tumoral , Mapeo Cromosómico , ADN/química , ADN/genética , ADN/aislamiento & purificación , ADN Complementario/química , ADN Complementario/genética , ADN Complementario/aislamiento & purificación , Proteínas de Unión al ADN/metabolismo , Desarrollo Embrionario/genética , Exones , Femenino , Regulación del Desarrollo de la Expresión Génica , Proteínas Fluorescentes Verdes , Humanos , Intrones , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Masculino , Ratones , Datos de Secuencia Molecular , Complejo Represivo Polycomb 1 , Embarazo , Unión Proteica , Isoformas de Proteínas/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/genética , Proteínas Represoras/metabolismo , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Homología de Secuencia de Ácido Nucleico , Factores de Tiempo , Transcripción Genética , Técnicas del Sistema de Dos Híbridos , Ubiquitina-Proteína LigasasRESUMEN
The Foxl1 gene, which encodes a winged helix transcriptional regulator, is expressed in the mesenchymal layer of developing and mature gastrointestinal tract. Foxl1-deficient mice exhibit various defects not only in the epithelial layer of the gastrointestinal tract but also in gut-associated lymphoid tissues. In the small intestine of Foxl1-deficient mice, the formation of Peyer's patches is affected, particularly in the caudal region. This alteration is shown to be due to the delayed formation of Peyer's patches organizing centers as revealed by the expressions of VCAM1 and IL-7 receptor alpha-chain at 17.5 days postcoitus. Peyer's patch defects are concordant with the significantly decreased expression of Lymphotoxin beta-receptor in the caudal region of fetal intestine. Foxl1 is suggested to regulate the responsiveness of fetal intestinal mesenchymal cells to inductive signals mediated by Lymphotoxins during Peyer's patch organogenesis. In addition, constitutive outgrowth of colonic patches due to defects in radioresistant stromal components of colonic patches are seen in Foxl1-deficient mice. Because of the functional similarities of hypertrophic colonic patches to those seen in hapten-induced experimental colitis, this hypertrophy is suggested to involve Lymphotoxin beta-receptor signaling. Together, the data suggest that Foxl1 might be involved in cellular responses of gut-associated lymphoid tissues dependent upon the Lymphotoxins/Lymphotoxin beta-receptor axis.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Sistema Digestivo/embriología , Sistema Digestivo/metabolismo , Tejido Linfoide/embriología , Tejido Linfoide/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Animales , Proteínas de Unión al ADN/deficiencia , Sistema Digestivo/crecimiento & desarrollo , Factores de Transcripción Forkhead , Regulación del Desarrollo de la Expresión Génica , Tejido Linfoide/crecimiento & desarrollo , Receptor beta de Linfotoxina , Mesodermo/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Ganglios Linfáticos Agregados/embriología , Ganglios Linfáticos Agregados/crecimiento & desarrollo , Ganglios Linfáticos Agregados/metabolismo , Receptores de Interleucina-7/metabolismo , Receptores del Factor de Necrosis Tumoral/metabolismo , Transducción de Señal , Factores de Transcripción/deficiencia , Molécula 1 de Adhesión Celular Vascular/metabolismoRESUMEN
The mab-21 gene was first identified because of its requirement for ray identity specification in Caenorhabditis elegans. It is now known to constitute a family of genes that are highly conserved from vertebrates to invertebrates, and two homologs, Mab21l1 and Mab21l2, have been identified in many species. We describe the generation of Mab21l1-deficient mice with defects in eye and preputial gland formation. The mutant mouse eye has a rudimentary lens resulting from insufficient invagination of the lens placode caused by deficient proliferation. Chimera analyses suggest that the lens placode is affected in a cell-autonomous manner, although Mab21l1 is expressed in both the lens placode and the optic vesicle. The defects in lens placode development correlate with delayed and insufficient expression of Foxe3, which is also required for lens development, while Maf, Sox2, Six3 and PAX6 levels are not significantly affected. Significant reduction of Mab21l1 expression in the optic vesicle and overlying surface ectoderm in Sey homozygotes indicates that Mab21l1 expression in the developing eye is dependent upon the functions of Pax6 gene products. We conclude that Mab21l1 expression dependent on PAX6 is essential for lens placode growth and for formation of the lens vesicle; lack of Mab21l1 expression causes reduced expression of Foxe3 in a cell-autonomous manner.
Asunto(s)
Proteínas de Homeodominio/metabolismo , Cristalino/embriología , Animales , Ectodermo/metabolismo , Proteínas del Ojo , Factores de Transcripción Forkhead , Proteínas de Homeodominio/genética , Cristalino/anomalías , Ratones , Ratones Transgénicos , Mutación , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box , Proteínas Represoras , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
The products of the Polycomb group of genes form complexes that maintain the state of transcriptional repression of several genes with relevance to development and in cell proliferation. We have identified Ring1B, the product of the Ring1B gene (Rnf2 - Mouse Genome Informatics), by means of its interaction with the Polycomb group protein Mel18. We describe biochemical and genetic studies directed to understand the biological role of Ring1B. Immunoprecipitation studies indicate that Ring1B form part of protein complexes containing the products of other Polycomb group genes, such as Rae28/Mph1 and M33, and that this complexes associate to chromosomal DNA. We have generated a mouse line bearing a hypomorphic Ring1B allele, which shows posterior homeotic transformations of the axial skeleton and a mild derepression of some Hox genes (Hoxb4, Hoxb6 and Hoxb8) in cells anterior to their normal boundaries of expression in the mesodermal compartment. By contrast, the overexpression of Ring1B in chick embryos results in the repression of Hoxb9 expression in the neural tube. These results, together with the genetic interactions observed in compound Ring1B/Mel18 mutant mice, are consistent with a role for Ring1B in the regulation of Hox gene expression by Polycomb group complexes.