RESUMEN
Global histone hyperacetylation is suggested to play a critical role for replacement of histones by transition proteins and protamines to compact the genome during spermiogenesis. However, the underlying mechanisms for hyperacetylation-mediated histone replacement remains poorly understood. Here, we report that EPC1 and TIP60, two critical components of the mammalian nucleosome acetyltransferase of H4 (NuA4) complexes, are coexpressed in male germ cells. Strikingly, genetic ablation of either Epc1 or Tip60 disrupts hyperacetylation and impairs histone replacement, in turn causing aberrant spermatid development. Taking these observations together, we reveal an essential role of the NuA4 complexes for histone hyperacetylation and subsequent compaction of the spermatid genome.
Asunto(s)
Histona Acetiltransferasas/metabolismo , Histonas/metabolismo , Proteínas Represoras/metabolismo , Espermátides/crecimiento & desarrollo , Espermatogénesis , Transactivadores/metabolismo , Acetilación , Animales , Células Cultivadas , Regulación del Desarrollo de la Expresión Génica , Técnicas de Inactivación de Genes , Histona Acetiltransferasas/genética , Lisina Acetiltransferasa 5 , Masculino , Ratones , Proteínas Represoras/genética , Espermátides/metabolismo , Transactivadores/genéticaRESUMEN
Krüppel-like factor 3 (KLF3/BKLF), a member of the Krüppel-like factor (KLF) family of transcription factors, is a widely expressed transcriptional repressor with diverse biological roles. Although there is considerable understanding of the molecular mechanisms that allow KLF3 to silence the activity of its target genes, less is known about the signal transduction pathways and post-translational modifications that modulate KLF3 activity in response to physiological stimuli. We observed that KLF3 is modified in a range of different tissues and found that the serine/threonine kinase homeodomain-interacting protein kinase 2 (HIPK2) can both bind and phosphorylate KLF3. Mass spectrometry identified serine 249 as the primary phosphorylation site. Mutation of this site reduces the ability of KLF3 to bind DNA and repress transcription. Furthermore, we also determined that HIPK2 can phosphorylate the KLF3 co-repressor C-terminal binding protein 2 (CtBP2) at serine 428. Finally, we found that phosphorylation of KLF3 and CtBP2 by HIPK2 strengthens the interaction between these two factors and increases transcriptional repression by KLF3. Taken together, our results indicate that HIPK2 potentiates the activity of KLF3.
Asunto(s)
Proteínas Portadoras/fisiología , Proteínas de Unión al ADN/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Fosfoproteínas/metabolismo , Proteínas Serina-Treonina Quinasas/fisiología , Oxidorreductasas de Alcohol , Secuencia de Aminoácidos , Animales , Células COS , Chlorocebus aethiops , Proteínas Co-Represoras , ADN/química , Ensayo de Cambio de Movilidad Electroforética , Factores de Transcripción de Tipo Kruppel/química , Ratones , Datos de Secuencia Molecular , Células 3T3 NIH , Fosforilación , Unión Proteica , Procesamiento Proteico-Postraduccional , Transcripción Genética , Activación TranscripcionalRESUMEN
UNLABELLED: Polycomb-group (PcG) proteins play crucial roles in self-renewal of stem cells by suppressing a host of genes through histone modifications. Identification of the downstream genes of PcG proteins is essential for elucidation of the molecular mechanisms of stem cell self-renewal. However, little is known about the PcG target genes in tissue stem cells. We found that the PcG protein, Ring1B, which regulates expression of various genes through monoubiquitination of histone H2AK119, is essential for expansion of hepatic stem/progenitor cells. In mouse embryos with a conditional knockout of Ring1B, we found that the lack of Ring1B inhibited proliferation and differentiation of hepatic stem/progenitor cells and thereby inhibited hepatic organogenesis. These events were characterized by derepression of cyclin-dependent kinase inhibitors (CDKIs) Cdkn1a and Cdkn2a, known negative regulators of cell proliferation. We conducted clonal culture experiments with hepatic stem/progenitor cells to investigate the individual genetic functions of Ring1B, Cdkn1a, and Cdkn2a. The data showed that the cell-cycle inhibition caused by Ring1B depletion was reversed when Cdkn1a and Cdkn2a were suppressed simultaneously, but not when they were suppressed individually. CONCLUSION: Our results show that expansion of hepatic stem/progenitor cells requires Ring1B-mediated epigenetic silencing of Cdkn1a and Cdkn2a, demonstrating that Ring1B simultaneously regulates multiple CDKIs in tissue stem/progenitor cells.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Células Madre Embrionarias/citología , Hígado/citología , Complejo Represivo Polycomb 1/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Animales , Diferenciación Celular/fisiología , Proliferación Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Epigénesis Genética/fisiología , Femenino , Regulación del Desarrollo de la Expresión Génica/fisiología , Hígado/embriología , Hígado/fisiología , Masculino , Ratones , Ratones Noqueados , Organogénesis/fisiología , Complejo Represivo Polycomb 1/genética , Embarazo , Ubiquitina-Proteína Ligasas/genéticaRESUMEN
WIP1 (wild-type p53-induced phosphatase 1) functions as a homeostatic regulator of the ataxia telangiectasia mutated (ATM)-mediated signaling pathway in response to ionizing radiation (IR). Here we identify homeodomain-interacting protein kinase 2 (HIPK2) as a protein kinase that targets WIP1 for phosphorylation and proteasomal degradation. In unstressed cells, WIP1 is constitutively phosphorylated by HIPK2 and maintained at a low level by proteasomal degradation. In response to IR, ATM-dependent AMPKα2-mediated HIPK2 phosphorylation promotes inhibition of WIP1 phosphorylation through dissociation of WIP1 from HIPK2, followed by stabilization of WIP1 for termination of the ATM-mediated double-strand break (DSB) signaling cascade. Notably, HIPK2 depletion impairs IR-induced γ-H2AX foci formation, cell-cycle checkpoint activation, and DNA repair signaling, and the survival rate of hipk2+/- mice upon γ-irradiation is markedly reduced compared to wild-type mice. Taken together, HIPK2 plays a critical role in the initiation of DSB repair signaling by controlling WIP1 levels in response to IR.
Asunto(s)
Proteínas Portadoras/metabolismo , Daño del ADN/efectos de la radiación , Reparación del ADN , Fosfoproteínas Fosfatasas/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Puntos de Control del Ciclo Celular , Línea Celular Tumoral , Daño del ADN/genética , Células HEK293 , Células HeLa , Histonas/metabolismo , Humanos , Ratones , Ratones Transgénicos , Fosforilación , Proteína Fosfatasa 2C , Radiación Ionizante , Transducción de Señal , UbiquitinaciónRESUMEN
Two distinct Polycomb complexes, PRC1 and PRC2, collaborate to maintain epigenetic repression of key developmental loci in embryonic stem cells (ESCs). PRC1 and PRC2 have histone modifying activities, catalyzing mono-ubiquitination of histone H2A (H2AK119u1) and trimethylation of H3 lysine 27 (H3K27me3), respectively. Compared to H3K27me3, localization and the role of H2AK119u1 are not fully understood in ESCs. Here we present genome-wide H2AK119u1 maps in ESCs and identify a group of genes at which H2AK119u1 is deposited in a Ring1-dependent manner. These genes are a distinctive subset of genes with H3K27me3 enrichment and are the central targets of Polycomb silencing that are required to maintain ESC identity. We further show that the H2A ubiquitination activity of PRC1 is dispensable for its target binding and its activity to compact chromatin at Hox loci, but is indispensable for efficient repression of target genes and thereby ESC maintenance. These data demonstrate that multiple effector mechanisms including H2A ubiquitination and chromatin compaction combine to mediate PRC1-dependent repression of genes that are crucial for the maintenance of ESC identity. Utilization of these diverse effector mechanisms might provide a means to maintain a repressive state that is robust yet highly responsive to developmental cues during ES cell self-renewal and differentiation.
Asunto(s)
Histonas , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Ubiquitinación , Animales , Línea Celular , Cromatina/genética , Regulación del Desarrollo de la Expresión Génica , N-Metiltransferasa de Histona-Lisina/metabolismo , Histonas/genética , Histonas/metabolismo , Ratones , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 1/genética , Complejo Represivo Polycomb 1/metabolismo , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Ubiquitinación/genéticaRESUMEN
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
In eukaryotes, Suv39h H3K9 trimethyltransferases are required for pericentric heterochromatin formation and function. In early mouse preimplantation embryos, however, paternal pericentric heterochromatin lacks Suv39h-mediated H3K9me3 and downstream marks. Here we demonstrate Ezh2-independent targeting of maternally provided polycomb repressive complex 1 (PRC1) components to paternal heterochromatin. In Suv39h2 maternally deficient zygotes, PRC1 also associates with maternal heterochromatin lacking H3K9me3, thereby revealing hierarchy between repressive pathways. In Rnf2 maternally deficient zygotes, the PRC1 complex is disrupted, and levels of pericentric major satellite transcripts are increased at the paternal but not the maternal genome. We conclude that in early embryos, Suv39h-mediated H3K9me3 constitutes the dominant maternal transgenerational signal for pericentric heterochromatin formation. In absence of this signal, PRC1 functions as the default repressive back-up mechanism. Parental epigenetic asymmetry, also observed along cleavage chromosomes, is resolved by the end of the 8-cell stage--concurrent with blastomere polarization--marking the end of the maternal-to-embryonic transition.
Asunto(s)
Embrión de Mamíferos/metabolismo , Desarrollo Embrionario/fisiología , Impresión Genómica , Heterocromatina/fisiología , Metiltransferasas/fisiología , Ratones/embriología , Proteínas Represoras/fisiología , Animales , Apoptosis/fisiología , Blastómeros/metabolismo , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/fisiología , Embrión de Mamíferos/ultraestructura , Proteína Potenciadora del Homólogo Zeste 2 , Femenino , Técnica del Anticuerpo Fluorescente , Heterocromatina/ultraestructura , N-Metiltransferasa de Histona-Lisina/genética , N-Metiltransferasa de Histona-Lisina/fisiología , Integrasas/metabolismo , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Oocitos/metabolismo , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Proteínas/genética , Proteínas/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transcripción Genética , Transgenes/fisiología , Ubiquitina-Proteína LigasasRESUMEN
Macrophages have a wide variety of activities and it is largely unknown how the diverse phenotypes of macrophages contribute to pathological conditions in the different types of tissue injury in vivo. In this study we established a novel animal model of acute respiratory distress syndrome caused by the dysfunction of alveolar epithelial type II (AE2) cells and examined the roles of alveolar macrophages in the acute lung injury. The human diphtheria toxin (DT) receptor (DTR), heparin-binding epidermal growth factor-like growth factor (HB-EGF), was expressed under the control of the lysozyme M (LysM) gene promoter in the mice. When DT was administrated to the mice they suffered from acute lung injury and died within 4 days. Immunohistochemical examination revealed that AE2 cells as well as alveolar macrophages were deleted via apoptosis in the mice treated with DT. Consistent with the deletion of AE2 cells, the amount of surfactant proteins in bronchoalveolar lavage fluid was greatly reduced in the DT-treated transgenic mice. When bone marrow from wild-type mice was transplanted into irradiated LysM-DTR mice, the alveolar macrophages became resistant to DT but the mice still suffered from acute lung injury by DT administration. Compared with the mice in which both AE2 cells and macrophages were deleted by DT administration, the DT-treated LysM-DTR mice with DT-resistant macrophages showed less severe lung injury with a reduced amount of hepatocyte growth factor in bronchoalveolar lavage fluid. These results indicate that macrophages play a protective role in noninflammatory lung injury caused by the selective ablation of AE2 cells.
Asunto(s)
Macrófagos Alveolares/fisiología , Alveolos Pulmonares/patología , Síndrome de Dificultad Respiratoria/prevención & control , Animales , Citocinas/biosíntesis , Toxina Diftérica/toxicidad , Factor de Crecimiento Similar a EGF de Unión a Heparina , Factor de Crecimiento de Hepatocito/biosíntesis , Humanos , Receptores de Hialuranos/fisiología , Etiquetado Corte-Fin in Situ , Péptidos y Proteínas de Señalización Intercelular , Pulmón/efectos de los fármacos , Pulmón/patología , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Muramidasa/genética , Receptores de Superficie Celular/fisiología , Síndrome de Dificultad Respiratoria/etiologíaRESUMEN
Regulation of histone methylation is critical for proper gene expression and chromosome function. Suppressor of Zeste 12 (SUZ12) is a requisite member of the EED/EZH2 histone methyltransferase complexes, and is required for full activity of these complexes in vitro. In mammals and flies, SUZ12/Su(z)12 is necessary for trimethylation of histone H3 on lysine 27 (H3K27me3) on facultative heterochromatin. However, Su(z)12 is unique among Polycomb Group Proteins in that Su(z)12 mutant flies exhibit gross defects in position effect variegation, suggesting a role for Su(z)12 in constitutive heterochromatin formation. We investigated the role of Suz12 in constitutive heterochromatin and discovered that Suz12 is required for histone H3 lysine 9 tri-methylation (H3K9me3) in differentiated but not undifferentiated mouse embryonic stem cells. Knockdown of SUZ12 in human cells caused a reduction in H3K27me3 and H3K9me3, and altered the distribution of HP1 alpha. In contrast, EZH2 knockdown caused loss of H3K27me3 but not H3K9me3, indicating that SUZ12 regulates H3-K9 methylation in an EZH2-independent fashion. This work uncovers a role for SUZ12 in H3-K9 methylation.
Asunto(s)
Proteínas Cromosómicas no Histona/metabolismo , Metilación de ADN , Histonas/metabolismo , Proteínas Represoras/fisiología , Animales , Proteínas Portadoras/fisiología , Homólogo de la Proteína Chromobox 5 , Proteínas de Unión al ADN/fisiología , Proteína Potenciadora del Homólogo Zeste 2 , Heterocromatina/metabolismo , N-Metiltransferasa de Histona-Lisina , Humanos , Lisina/metabolismo , Ratones , Proteínas de Neoplasias , Proteínas Nucleares/fisiología , Complejo Represivo Polycomb 2 , Proteínas/fisiología , Distribución Tisular , Factores de Transcripción/fisiologíaRESUMEN
The product of the Scmh1 gene, a mammalian homolog of Drosophila Sex comb on midleg, is a constituent of the mammalian Polycomb repressive complexes 1 (Prc1). We have identified Scmh1 as an indispensable component of the Prc1. During progression through pachytene, Scmh1 was shown to be excluded from the XY body at late pachytene, together with other Prc1 components such as Phc1, Phc2, Rnf110 (Pcgf2), Bmi1 and Cbx2. We have identified the role of Scmh1 in mediating the survival of late pachytene spermatocytes. Apoptotic elimination of Scmh1(-/-) spermatocytes is accompanied by the preceding failure of several specific chromatin modifications at the XY body, whereas synapsis of homologous autosomes is not affected. It is therefore suggested that Scmh1 is involved in regulating the sequential changes in chromatin modifications at the XY chromatin domain of the pachytene spermatocytes. Restoration of defects in Scmh1(-/-) spermatocytes by Phc2 mutation indicates that Scmh1 exerts its molecular functions via its interaction with Prc1. Therefore, for the first time, we are able to indicate a functional involvement of Prc1 during the meiotic prophase of male germ cells and a regulatory role of Scmh1 for Prc1, which involves sex chromosomes.
Asunto(s)
Proteínas Represoras/metabolismo , Espermatocitos/metabolismo , Animales , Apoptosis , Secuencia de Bases , Cartilla de ADN/genética , Histonas/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Mutación , Complejo Represivo Polycomb 1 , Proteínas del Grupo Polycomb , Proteínas Represoras/genética , Espermatocitos/citología , Espermatogénesis/genética , Espermatogénesis/fisiología , Fracciones Subcelulares/metabolismo , Cromosoma X/metabolismo , Cromosoma Y/metabolismoRESUMEN
The vertebrate Polycomb Group (PcG) genes encode proteins that form large multimeric and chromatin-associated complexes implicated in the stable repression of developmentally essential genes. Rnf110 and Phc2 are shown to be components of mammalian PcG multimeric complexes in HeLa cells. Here we report defects in Peyer's patch (PP) development in Rnf110 mutant mice, which is synergically exaggerated by Phc2 mutation. PP development involves a series of inductive interactions and subsequent differentiation and proliferation between lymphoid and mesenchymal cells in late gestational stage. Rnf110 and Phc2 mutations impair development of PP anlagen by affecting proliferation of lymphoid lineage cells populated in PP anlagen in gene-dosage dependent manner. We suggest that PcG complexes may act to mediate certain inductive signals maybe through IL-7Ralpha to allow sufficient proliferation of lymphoid inducer cells during PP organogenesis.
Asunto(s)
Linfocitos/citología , Ganglios Linfáticos Agregados/crecimiento & desarrollo , Ganglios Linfáticos Agregados/inmunología , Proteínas Represoras/fisiología , Animales , Proliferación Celular , Células Cultivadas , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Femenino , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Mucosa Intestinal/metabolismo , Intestinos/embriología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Endogámicos C57BL , Mutación , Ganglios Linfáticos Agregados/embriología , Complejo Represivo Polycomb 1 , Complejo Represivo Polycomb 2 , Proteínas del Grupo Polycomb , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
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
Polycomb group proteins are essential for early development in metazoans, but their contributions to human development are not well understood. We have mapped the Polycomb Repressive Complex 2 (PRC2) subunit SUZ12 across the entire nonrepeat portion of the genome in human embryonic stem (ES) cells. We found that SUZ12 is distributed across large portions of over two hundred genes encoding key developmental regulators. These genes are occupied by nucleosomes trimethylated at histone H3K27, are transcriptionally repressed, and contain some of the most highly conserved noncoding elements in the genome. We found that PRC2 target genes are preferentially activated during ES cell differentiation and that the ES cell regulators OCT4, SOX2, and NANOG cooccupy a significant subset of these genes. These results indicate that PRC2 occupies a special set of developmental genes in ES cells that must be repressed to maintain pluripotency and that are poised for activation during ES cell differentiation.
Asunto(s)
Proteínas Portadoras/metabolismo , Regulación del Desarrollo de la Expresión Génica , Células Madre/fisiología , Animales , Proteínas Portadoras/genética , Células Cultivadas , Perfilación de la Expresión Génica , Humanos , Complejos Multiproteicos , Proteínas de Neoplasias , Proteínas Nucleares , Análisis de Secuencia por Matrices de Oligonucleótidos , Complejo Represivo Polycomb 2 , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , ARN Polimerasa II/genética , ARN Polimerasa II/metabolismo , Transducción de Señal/fisiología , Células Madre/citología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Transcripción GenéticaRESUMEN
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
To investigate the biological significance of a longevity mutation found in daf-2 of Caenorhabditis elegans, we generated a homologous murine model by replacing Pro-1195 of insulin receptors with Leu using a targeted knock-in strategy. Homozygous mice died in the neonatal stage from diabetic ketoacidosis, whereas heterozygous mice showed the suppressed kinase activity of the insulin receptor but grew normally without spontaneously developing diabetes during adulthood. We examined heterozygous insulin receptor mutant mice for longevity phenotypes. Under 80% oxygen, mutant female mice survived 33.3% longer than wild-type female mice, whereas mutant male mice survived 18.2% longer than wild-type male mice. These results suggested that mutant mice acquired more resistance to oxidative stress, but the benefit of the longevity mutation was more pronounced in females than males. Manganese superoxide dismutase activity in mutant mice was significantly upregulated, suggesting that the suppressed insulin signaling leads to an enhanced antioxidant defense. To analyze the molecular basis of the gender difference, we administered estrogen to mutant mice. It was found that the survival of mice under 80% oxygen was extended when they were administered estradiol. In contrast, mutant and wild-type female mice showed shortened survivals when their ovaries were removed. The influence of estrogen is remarkable in mutant mice compared with wild-type mice, suggesting that estrogen modulates insulin signaling in mutant mice. Furthermore, we showed additional extension of survival under oxidative conditions when their diet was restricted. Collectively, we show that three distinct signals; insulin, estrogen, and dietary signals work in independent and cooperative ways to enhance the resistance to oxidative stress in mice.
Asunto(s)
Estrógenos/metabolismo , Insulina/metabolismo , Longevidad/fisiología , Estrés Oxidativo/fisiología , Receptor de Insulina/genética , Animales , Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/genética , Femenino , Masculino , Ratones , Mutación , Receptor de Insulina/metabolismo , Superóxido Dismutasa/metabolismo , Regulación hacia ArribaRESUMEN
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 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.
Asunto(s)
Regulación del Desarrollo de la Expresión Génica/fisiología , Proteínas Represoras/genética , Alelos , Animales , Cromatina/fisiología , Cruzamientos Genéticos , Cartilla de ADN , Proteínas de Unión al ADN/genética , Glutatión Transferasa/genética , Homocigoto , Ratones , Ratones Noqueados , Fenotipo , Complejo Represivo Polycomb 1 , Reacción en Cadena de la Polimerasa , Biosíntesis de Proteínas , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Represoras/metabolismo , Transcripción Genética , Dedos de ZincRESUMEN
Manganese superoxide dismutase (MnSOD) is the enzyme that converts toxic O(2)(-) to H(2)O(2) in mitochondria. Previous reports showed that a deficiency of MnSOD in mice was neonatal lethal. Therefore, a model mouse was not available for the analysis of the pathological role of O(2)(-) injuries in adult tissues. To explore an adult-type model mouse, we designed tissue-specific MnSOD conditional knockout mice using a Cre-loxp system. First, we crossbred MnSOD flox mice with transgenic mice expressing Cre recombinase under the control of the chicken actin promoter (CAG). We confirmed that CAG MnSOD knockout mice were completely deficient in MnSOD and died as neonates, validating the use of the Cre-loxp system. Next, we generated liver-specific MnSOD-deficient mice by crossbreeding with Alb-Cre transgenic mice. MnSOD activity and protein were both significantly downregulated in the liver of liver-specific MnSOD knockout mice. However, no obvious morphological abnormality was observed in the liver when biochemical alterations such as lipid peroxidation were not detectable, suggesting a redundant or less important physiological role for MnSOD in the liver than previously thought. In the present study, we successfully generated tissue-specific MnSOD conditional knockout mice that would provide a useful tool for the analysis of various age-associated diseases such as diabetes mellitus, Parkinson's disease, stroke, and heart disease, when crossbred with tissue-specific transgenic Cre mice.