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1.
Development ; 144(16): 2925-2939, 2017 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-28698226

RESUMO

Complex cytoplasmic nucleotide-sensing mechanisms can recognize foreign DNA based on a lack of methylation and initiate an immune response to clear the infection. Zebrafish embryos with global DNA hypomethylation caused by mutations in the ubiquitin-like with PHD and ring finger domains 1 (uhrf1) or DNA methyltransferase 1 (dnmt1) genes exhibit a robust interferon induction characteristic of the first line of defense against viral infection. We found that this interferon induction occurred in non-immune cells and examined whether intracellular viral sensing pathways in these cells were the trigger. RNA-seq analysis of uhrf1 and dnmt1 mutants revealed widespread induction of Class I retrotransposons and activation of cytoplasmic DNA viral sensors. Attenuating Sting, phosphorylated Tbk1 and, importantly, blocking reverse transcriptase activity suppressed the expression of interferon genes in uhrf1 mutants. Thus, activation of transposons in cells with global DNA hypomethylation mimics a viral infection by activating cytoplasmic DNA sensors. This suggests that antiviral pathways serve as surveillance of cells that have derepressed intragenomic parasites due to DNA hypomethylation.


Assuntos
Metilação de DNA/fisiologia , Retroelementos/genética , Peixe-Zebra/embriologia , Animais , DNA (Citosina-5-)-Metiltransferase 1 , DNA (Citosina-5-)-Metiltransferases/genética , DNA (Citosina-5-)-Metiltransferases/metabolismo , Metilação de DNA/genética , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Transativadores/genética , Transativadores/metabolismo , Peixe-Zebra/metabolismo , Proteínas de Peixe-Zebra/genética , Proteínas de Peixe-Zebra/metabolismo
2.
Genes Dev ; 24(22): 2517-30, 2010 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-21078818

RESUMO

Wnt ligands signal through ß-catenin and are critically involved in cell fate determination and stem/progenitor self-renewal. Wnts also signal through ß-catenin-independent or noncanonical pathways that regulate crucial events during embryonic development. The mechanism of noncanonical receptor activation and how Wnts trigger canonical as opposed to noncanonical signaling have yet to be elucidated. We demonstrate here that prototype canonical Wnt3a and noncanonical Wnt5a ligands specifically trigger completely unrelated endogenous coreceptors-LRP5/6 and Ror1/2, respectively-through a common mechanism that involves their Wnt-dependent coupling to the Frizzled (Fzd) coreceptor and recruitment of shared components, including dishevelled (Dvl), axin, and glycogen synthase kinase 3 (GSK3). We identify Ror2 Ser 864 as a critical residue phosphorylated by GSK3 and required for noncanonical receptor activation by Wnt5a, analogous to the priming phosphorylation of low-density receptor-related protein 6 (LRP6) in response to Wnt3a. Furthermore, this mechanism is independent of Ror2 receptor Tyr kinase functions. Consistent with this model of Wnt receptor activation, we provide evidence that canonical and noncanonical Wnts exert reciprocal pathway inhibition at the cell surface by competition for Fzd binding. Thus, different Wnts, through their specific coupling and phosphorylation of unrelated coreceptors, activate completely distinct signaling pathways.


Assuntos
Proteínas Wnt/metabolismo , Animais , Linhagem Celular , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Receptores Frizzled/metabolismo , Humanos , Proteínas Relacionadas a Receptor de LDL/metabolismo , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Camundongos , Fosforilação , Receptores Órfãos Semelhantes a Receptor Tirosina Quinase/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Proteína Wnt-5a , Proteína Wnt3 , Proteína Wnt3A
3.
Development ; 137(11): 1907-17, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20460369

RESUMO

Microcephaly affects approximately 1% of the population and is associated with mental retardation, motor defects and, in some cases, seizures. We analyzed the mechanisms underlying brain size determination in a mouse model of human microcephaly. The Hertwig's anemia (an) mutant shows peripheral blood cytopenias, spontaneous aneuploidy and a predisposition to hematopoietic tumors. We found that the an mutation is a genomic inversion of exon 4 of Cdk5rap2, resulting in an in-frame deletion of exon 4 from the mRNA. The finding that CDK5RAP2 human mutations cause microcephaly prompted further analysis of Cdk5rap2(an/an) mice and we demonstrated that these mice exhibit microcephaly comparable to that of the human disease, resulting from striking neurogenic defects that include proliferative and survival defects in neuronal progenitors. Cdk5rap2(an/an) neuronal precursors exit the cell cycle prematurely and many undergo apoptosis. These defects are associated with impaired mitotic progression coupled with abnormal mitotic spindle pole number and mitotic orientation. Our findings suggest that the reduction in brain size observed in humans with mutations in CDK5RAP2 is associated with impaired centrosomal function and with changes in mitotic spindle orientation during progenitor proliferation.


Assuntos
Proteínas de Ciclo Celular/genética , Centrossomo/fisiologia , Segregação de Cromossomos/genética , Peptídeos e Proteínas de Sinalização Intracelular/genética , Proteínas do Tecido Nervoso/genética , Anemia/genética , Animais , Sequência de Bases , Proteínas de Ciclo Celular/fisiologia , Segregação de Cromossomos/fisiologia , Primers do DNA/genética , Modelos Animais de Doenças , Éxons , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/deficiência , Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Mutantes , Microcefalia/genética , Microcefalia/patologia , Mitose/genética , Mitose/fisiologia , Proteínas do Tecido Nervoso/deficiência , Proteínas do Tecido Nervoso/fisiologia , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/patologia , Gravidez , RNA Mensageiro/genética , Deleção de Sequência , Células-Tronco/patologia
4.
Biochem J ; 435(1): 175-85, 2011 Apr 01.
Artigo em Inglês | MEDLINE | ID: mdl-21214517

RESUMO

UHRF1 [ubiquitin-like protein, containing PHD (plant homeodomain) and RING finger domains 1] is required for cell cycle progression and epigenetic regulation. In the present study, we show that depleting cancer cells of UHRF1 causes activation of the DNA damage response pathway, cell cycle arrest in G2/M-phase and apoptosis dependent on caspase 8. The DNA damage response in cells depleted of UHRF1 is illustrated by: phosphorylation of histone H2AX on Ser139, phosphorylation of CHK (checkpoint kinase) 2 on Thr68, phosphorylation of CDC25 (cell division control 25) on Ser216 and phosphorylation of CDK1 (cyclin-dependent kinase 1) on Tyr15. Moreover, we find that UHRF1 accumulates at sites of DNA damage suggesting that the cell cycle block in UHRF1-depleted cells is due to an important role in damage repair. The consequence of UHRF1 depletion is apoptosis; cells undergo activation of caspases 8 and 3, and depletion of caspase 8 prevents cell death induced by UHRF1 knockdown. Interestingly, the cell cycle block and apoptosis occurs in p53-containing and -deficient cells. From the present study we conclude that UHRF1 links epigenetic regulation with DNA replication.


Assuntos
Apoptose , Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Dano ao DNA , Fase G2 , Proteínas Estimuladoras de Ligação a CCAAT/genética , Proteína Quinase CDC2/metabolismo , Caspase 3/metabolismo , Caspase 8/genética , Caspase 8/metabolismo , Linhagem Celular Tumoral , Quinase do Ponto de Checagem 2 , Dano ao DNA/efeitos da radiação , Inativação Gênica , Histonas/metabolismo , Humanos , Marcação In Situ das Extremidades Cortadas , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas Serina-Treonina Quinases/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , RNA Interferente Pequeno , Ubiquitina-Proteína Ligases , Raios Ultravioleta/efeitos adversos , Fosfatases cdc25/metabolismo
5.
Birth Defects Res C Embryo Today ; 93(2): 194-203, 2011 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-21671358

RESUMO

Zebrafish embryos are an exceptional system for studying vertebrate development. Historically, studies using zebrafish to uncover key players in developmentally regulated gene expression have entailed detailed analysis of transcription factors. It is now apparent that epigenetic modifications of both DNA and histone tails are equally important in the regulation of gene expression during development. As such, blocking the function of key epigenetic modifiers impairs development, albeit with surprising tissue specificity. For instance, DNA methylation is an important epigenetic mark that is depleted in embryos lacking dnmt1 and uhrf1. These embryos display developmental defects in the eye, liver, pancreas, and larval lethality. Interestingly, human tumors derived from these same organs have aberrant changes in DNA methylation and altered expression of genes that are thought to contribute to formation of these cancers. These observations have provided a mechanistic basis for treating cancer with drugs that block the enzymes that facilitate DNA and histone modifications. Thus, it is important to understand the consequences of targeting these factors in a whole animal. We review the use of zebrafish for probing the genetic, cellular, and physiological response to alterations in the epigenome and highlight exciting data illustrating that epigenetic studies using zebrafish can inform and impact cancer biology.


Assuntos
Metilação de DNA , Desenvolvimento Embrionário/fisiologia , Epigênese Genética/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Fígado/embriologia , Modelos Animais , Neoplasias/fisiopatologia , Peixe-Zebra , Acetilação , Animais , Histonas/metabolismo , Fígado/fisiopatologia , Metilação , Neoplasias/genética
6.
Cancer Cell ; 25(2): 196-209, 2014 Feb 10.
Artigo em Inglês | MEDLINE | ID: mdl-24486181

RESUMO

Ubiquitin-like with PHD and RING finger domains 1 (UHRF1) is an essential regulator of DNA methylation that is highly expressed in many cancers. Here, we use transgenic zebrafish, cultured cells, and human tumors to demonstrate that UHRF1 is an oncogene. UHRF1 overexpression in zebrafish hepatocytes destabilizes and delocalizes Dnmt1 and causes DNA hypomethylation and Tp53-mediated senescence. Hepatocellular carcinoma (HCC) emerges when senescence is bypassed. tp53 mutation both alleviates senescence and accelerates tumor onset. Human HCCs recapitulate this paradigm, as UHRF1 overexpression defines a subclass of aggressive HCCs characterized by genomic instability, TP53 mutation, and abrogation of the TP53-mediated senescence program. We propose that UHRF1 overexpression is a mechanism underlying DNA hypomethylation in cancer cells and that senescence is a primary means of restricting tumorigenesis due to epigenetic disruption.


Assuntos
Proteínas Estimuladoras de Ligação a CCAAT/metabolismo , Carcinoma Hepatocelular/patologia , Metilação de DNA , Neoplasias Hepáticas/patologia , Animais , Proteínas Estimuladoras de Ligação a CCAAT/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/mortalidade , Células Cultivadas , Senescência Celular , Estudos de Coortes , Biologia Computacional , Hepatócitos/citologia , Hepatócitos/metabolismo , Humanos , Immunoblotting , Fígado/metabolismo , Fígado/patologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/metabolismo , Mutação/genética , Prognóstico , Taxa de Sobrevida , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/metabolismo , Ubiquitina-Proteína Ligases , Peixe-Zebra
7.
Neural Dev ; 6: 3, 2011 Jan 07.
Artigo em Inglês | MEDLINE | ID: mdl-21214893

RESUMO

BACKGROUND: The dorsal thalamus acts as a gateway and modulator for information going to and from the cerebral cortex. This activity requires the formation of reciprocal topographic axon connections between thalamus and cortex. The axons grow along a complex multistep pathway, making sharp turns, crossing expression boundaries, and encountering intermediate targets. However, the cellular and molecular components mediating these steps remain poorly understood. RESULTS: To further elucidate the development of the thalamocortical system, we first created a thalamocortical axon reporter line to use as a genetic tool for sensitive analysis of mutant mouse phenotypes. The TCA-tau-lacZ reporter mouse shows specific, robust, and reproducible labeling of thalamocortical axons (TCAs), but not the overlapping corticothalamic axons, during development. Moreover, it readily reveals TCA pathfinding abnormalities in known cortical mutants such as reeler. Next, we performed an unbiased screen for genes involved in thalamocortical development using random mutagenesis with the TCA reporter. Six independent mutant lines show aberrant TCA phenotypes at different steps of the pathway. These include ventral misrouting, overfasciculation, stalling at the corticostriatal boundary, and invasion of ectopic cortical cell clusters. An outcross breeding strategy coupled with a genomic panel of single nucleotide polymorphisms facilitated genetic mapping with small numbers of mutant mice. We mapped a ventral misrouting mutant to the Emx2 gene, and discovered that some TCAs extend to the olfactory bulbs in this mutant. Mapping data suggest that other lines carry mutations in genes not previously known for roles in thalamocortical development. CONCLUSIONS: These data demonstrate the feasibility of a forward genetic approach to understanding mammalian brain morphogenesis and wiring. A robust axonal reporter enabled sensitive analysis of a specific axon tract inside the mouse brain, identifying mutant phenotypes at multiple steps of the pathway, and revealing a new aspect of the Emx2 mutant. The phenotypes highlight vulnerable choice points and latent tendencies of TCAs, and will lead to a refined understanding of the elements and interactions required to form the thalamocortical system.


Assuntos
Axônios/fisiologia , Córtex Cerebral , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Mutação/genética , Fenótipo , Tálamo , Fatores de Transcrição/genética , Alquilantes/farmacologia , Animais , Padronização Corporal/efeitos dos fármacos , Padronização Corporal/genética , Moléculas de Adesão Celular Neuronais/genética , Moléculas de Adesão Celular Neuronais/metabolismo , Córtex Cerebral/embriologia , Córtex Cerebral/crescimento & desenvolvimento , Córtex Cerebral/metabolismo , Embrião de Mamíferos , Etilnitrosoureia/farmacologia , Proteínas da Matriz Extracelular/genética , Proteínas da Matriz Extracelular/metabolismo , Feminino , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Testes Genéticos/métodos , Óperon Lac/genética , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Vias Neurais/anormalidades , Vias Neurais/embriologia , Vias Neurais/crescimento & desenvolvimento , Proteína Reelina , Serina Endopeptidases/genética , Serina Endopeptidases/metabolismo , Tálamo/embriologia , Tálamo/crescimento & desenvolvimento , Tálamo/metabolismo , beta-Galactosidase/metabolismo
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