RESUMEN
Coordinating epigenomic inheritance and cell cycle progression is essential for organogenesis. UHRF1 connects these functions during development by facilitating maintenance of DNA methylation and cell cycle progression. Here, we provide evidence resolving the paradoxical phenotype of uhrf1 mutant zebrafish embryos which have activation of pro-proliferative genes and increased number of hepatocytes in S-phase, but the liver fails to grow. We uncover decreased Cdkn2a/b and persistent Cdk4/6 activation as the mechanism driving uhrf1 mutant hepatocytes into S-phase. This induces replication stress, DNA damage and Atr activation. Palbociclib treatment of uhrf1 mutants prevented aberrant S-phase entry, reduced DNA damage, and rescued most cellular and developmental phenotypes, but it did not rescue DNA hypomethylation, transposon expression or the interferon response. Inhibiting Atr reduced DNA replication and increased liver size in uhrf1 mutants, suggesting that Atr activation leads to dormant origin firing and prevents hepatocyte proliferation. Cdkn2a/b was downregulated pro-proliferative genes were also induced in a Cdk4/6 dependent fashion in the liver of dnmt1 mutants, suggesting DNA hypomethylation as a mechanism of Cdk4/6 activation during development. This shows that the developmental defects caused by DNA hypomethylation are attributed to persistent Cdk4/6 activation, DNA replication stress, dormant origin firing and cell cycle inhibition.
Asunto(s)
Proteínas de la Ataxia Telangiectasia Mutada , Quinasa 4 Dependiente de la Ciclina , Quinasa 6 Dependiente de la Ciclina , Metilación de ADN , Hígado , Pez Cebra , Animales , Proteínas de la Ataxia Telangiectasia Mutada/genética , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Ciclo Celular/genética , Puntos de Control del Ciclo Celular/genética , División Celular/genética , Quinasa 4 Dependiente de la Ciclina/genética , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/genética , Quinasa 6 Dependiente de la Ciclina/metabolismo , ADN/metabolismo , Replicación del ADN/genética , Embrión no Mamífero , Hígado/crecimiento & desarrollo , Hígado/metabolismo , Fase S , Pez Cebra/genética , Pez Cebra/metabolismo , Activación Enzimática/genéticaRESUMEN
Acquisition of cellular fate during development is initiated and maintained by well-coordinated patterns of gene expression that are dictated by the epigenetic landscape and genome organization in the nucleus. While the epigenetic marks that mediate developmental gene expression patterns during organogenesis have been well studied, less is known about how epigenetic marks influence nuclear organization during development. This study examines the relationship between nuclear structure, chromatin accessibility, DNA methylation, and gene expression during hepatic outgrowth in zebrafish larvae. We investigate the relationship between these features using mutants that lack DNA methylation. Hepatocyte nuclear morphology was established coincident with hepatocyte differentiation at 80 h post-fertilization (hpf), and nuclear shape and size continued to change until the conclusion of outgrowth and morphogenesis at 120 hpf. Integrating ATAC-Seq analysis with DNA methylation profiling of zebrafish livers at 120 hpf showed that closed and highly methylated chromatin occupies most transposable elements and that open chromatin correlated with gene expression. DNA hypomethylation, due to mutation of genes encoding ubiquitin-like, containing PHD and RING Finger Domains 1 (uhrf1) and DNA methyltransferase (dnmt1), did not block hepatocyte differentiation, but had dramatic effects on nuclear organization. Hepatocytes in uhrf1 mutants have large, deformed nuclei with multiple nucleoli, downregulation of nucleolar genes, and a complete lack of the nuclear lamina. Loss of lamin B2 staining was phenocopied by dnmt1 mutation. Together, these data show that hepatocyte nuclear morphogenesis coincides with organ morphogenesis and outgrowth, and that DNA methylation directs chromatin organization, and, in turn, hepatocyte nuclear shape and size during liver development.
Asunto(s)
Hepatocitos/metabolismo , Transactivadores/metabolismo , Proteínas de Pez Cebra/metabolismo , Animales , Diferenciación Celular/genética , Núcleo Celular/genética , Cromatina/metabolismo , Ensamble y Desensamble de Cromatina/genética , ADN (Citosina-5-)-Metiltransferasa 1/metabolismo , Metilación de ADN/genética , Epigénesis Genética/genética , Expresión Génica/genética , Larva/genética , Hígado/embriología , Hígado/metabolismo , Organogénesis/genética , Transactivadores/genética , Transactivadores/fisiología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/fisiologíaRESUMEN
Activation of transposable elements (TEs) can cause cellular damage. Cytoplasmic nucleic acid sensing pathways evolved to detect pathogens, but can also serve to cull cells with inappropriate TE activation as TEs can be viral mimetics. Epigenetic silencing of TEs is mediated in part by DNA methylation, but it is not clear if TE activation or the immune system contribute to the cellular damage caused by loss of DNA methylation. Here, we provide mechanistic insight into the observation of an activated interferon response in the liver of zebrafish larvae with deletion in critical components of the DNA methylation machinery, uhrf1 and dnmt1. We focus on dissecting the relationship between DNA methylation, TE activation and induction of an immune response through cytoplasmic DNA and double stranded RNA sensing pathways and identify tnfa as a mediator of cell death in the liver of these mutants. Integrated RNAseq and methylome analysis identified LTR transposons as the most upregulated in these mutants and also the most methylated in control larvae, indicating a direct role of DNA methylation in suppressing this TE subclass. RNAseq analysis from these same samples revealed expression signatures of a type-I interferon response and of tnfa activation, mimicking the pattern of gene expression in virally infected cells. CRISPR/Cas9 mediated depletion of the cellular antiviral sensors sting and mavs reduced expression of interferon response genes and tnfa depletion dramatically reduced cell death in uhrf1 mutant livers. This suggests that the antiviral response induced by DNA hypomethylation and TE activation in the liver is mediated by the signaling pathways activated by both cytoplasmic double stranded RNA and DNA and that tnfa mediates cell death as a potential mechanism to eliminate these damaged cells.
Asunto(s)
ADN (Citosina-5-)-Metiltransferasa 1/genética , Elementos Transponibles de ADN , Inmunidad/genética , Hígado/enzimología , Imitación Molecular , Transactivadores/genética , Virus/inmunología , Proteínas de Pez Cebra/genética , Pez Cebra/genética , Proteínas Adaptadoras Transductoras de Señales/genética , Proteínas Adaptadoras Transductoras de Señales/metabolismo , Animales , Animales Modificados Genéticamente , ADN (Citosina-5-)-Metiltransferasa 1/deficiencia , ADN (Citosina-5-)-Metiltransferasa 1/inmunología , Metilación de ADN , Epigénesis Genética , Interacciones Huésped-Patógeno , Hígado/embriología , Hígado/inmunología , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Retroelementos , Transactivadores/deficiencia , Transactivadores/inmunología , Factor de Necrosis Tumoral alfa/genética , Factor de Necrosis Tumoral alfa/metabolismo , Virus/patogenicidad , Pez Cebra/embriología , Pez Cebra/inmunología , Pez Cebra/metabolismo , Proteínas de Pez Cebra/deficiencia , Proteínas de Pez Cebra/inmunología , Proteínas de Pez Cebra/metabolismoRESUMEN
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.
Asunto(s)
Metilación de ADN/fisiología , Retroelementos/genética , Pez Cebra/embriología , Animales , ADN (Citosina-5-)-Metiltransferasa 1 , ADN (Citosina-5-)-Metiltransferasas/genética , ADN (Citosina-5-)-Metiltransferasas/metabolismo , Metilación de ADN/genética , Transducción de Señal/genética , Transducción de Señal/fisiología , Transactivadores/genética , Transactivadores/metabolismo , Pez Cebra/metabolismo , Proteínas de Pez Cebra/genética , Proteínas de Pez Cebra/metabolismoRESUMEN
The dramatic re-organization of the cancer cell nucleus creates telltale morphological features critical for pathological staging of tumors. In addition, the changes to the mutational and epigenetic landscape in cancer cells alter the structure and stability of the genome and directly contribute to malignancy. DNA methylation is one of the best studied epigenetic changes in cancer, as nearly every type of cancer studied shows a loss of DNA methylation spread across most of the genome. This global hypomethylation is accompanied by hypermethylation at distinct loci, and much of the work on DNA methylation in cancer has focused on how local changes contribute to gene expression. However, the emerging picture is that the changes to DNA methylation in cancer cells has little direct effect on gene expression but instead impacts the organization of the genome in the nucleus. Several recent studies that take a broad view of the cancer epigenome find that the most profound changes to the cancer methylome are spread across large segments of the genome, and that the focal changes are reflective of a whole reorganization of epigenome. Hallmarks of nuclear reorganization in cancer are found in the long regions of chromatin marked by histone methylation (LOCKs) and nuclear lamina interactions (LADs). In this review, we focus on a novel perspective that DNA methylation changes in cancer impact the global structure of heterochromatin, LADs and LOCKs, and how these global changes, in turn, contribute to gene expression changes and genomic stability.
RESUMEN
Most growth factor receptor tyrosine kinases (RTKs) signal through similar intracellular pathways, but they often have divergent biological effects. Therefore, elucidating the mechanism of channeling the intracellular effect of RTK stimulation to facilitate specific biological responses represents a fundamental biological challenge. Lens epithelial cells express numerous RTKs with the ability to initiate the phosphorylation (activation) of Erk1/2 and PI3-K/Akt signaling. However, only Fgfr stimulation leads to lens fiber cell differentiation in the developing mammalian embryo. Additionally, within the lens, only Fgfrs activate the signal transduction molecule Frs2α. Loss of Frs2α in the lens significantly increases apoptosis and decreases phosphorylation of both Erk1/2 and Akt. Also, Frs2α deficiency decreases the expression of several proteins characteristic of lens fiber cell differentiation, including Prox1, p57(KIP2), aquaporin 0 and ß-crystallins. Although not normally expressed in the lens, the RTK TrkC phosphorylates Frs2α in response to binding the ligand NT3. Transgenic lens epithelial cells expressing both TrkC and NT3 exhibit several features characteristic of lens fiber cells. These include elongation, increased Erk1/2 and Akt phosphorylation, and the expression of ß-crystallins. All these characteristics of NT3-TrkC transgenic lens epithelial cells depend on Frs2α. Therefore, tyrosine phosphorylation of Frs2α mediates Fgfr-dependent lens cell survival and provides a mechanistic basis for the unique fiber-differentiating capacity of Fgfs on mammalian lens epithelial cells.
Asunto(s)
Diferenciación Celular/genética , Factores de Crecimiento de Fibroblastos/fisiología , Cristalino/embriología , Proteínas de la Membrana/fisiología , Animales , Animales Recién Nacidos , Diferenciación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Supervivencia Celular/genética , Sinergismo Farmacológico , Embrión de Mamíferos , Factores de Crecimiento de Fibroblastos/farmacología , Cristalino/crecimiento & desarrollo , Cristalino/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Ratones , Ratones Transgénicos , Morfogénesis/efectos de los fármacos , Morfogénesis/genética , Morfogénesis/fisiología , Ratas , Ratas Wistar , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/genética , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/metabolismo , Receptor Tipo 2 de Factor de Crecimiento de Fibroblastos/fisiología , Receptor trkC/genética , Receptor trkC/metabolismo , Receptor trkC/fisiología , Regulación hacia Arriba/genéticaRESUMEN
Previous studies suggested that FGF signaling is important for lens formation. However, the times at which FGFs act to promote lens formation, the FGFs that are involved, the cells that secrete them and the mechanisms by which FGF signaling may promote lens formation are not known. We found that transcripts encoding several FGF ligands and the four classical FGF receptors are detectable in the lens-forming ectoderm at the time of lens induction. Conditional deletion of Fgfr1 and Fgfr2 from this tissue resulted in the formation of small lens rudiments that soon degenerated. Lens placodes lacking Fgfr1 and 2 were thinner than in wild-type embryos. Deletion of Fgfr2 increased cell death from the initiation of placode formation and concurrent deletion of Fgfr1 enhanced this phenotype. Fgfr1/2 conditional knockout placode cells expressed lower levels of proteins known to be regulated by FGF receptor signaling, but proteins known to be important for lens formation were present at normal levels in the remaining placode cells, including the transcription factors Pax6, Sox2 and FoxE3 and the lens-preferred protein αA-crystallin. Previous studies identified a genetic interaction between BMP and FGF signaling in lens formation and conditional deletion of Bmpr1a caused increased cell death in the lens placode, resulting in the formation of smaller lenses. In the present study, conditional deletion of both Bmpr1a and Fgfr2 increased cell death beyond that seen in Fgfr2(CKO) placodes and prevented lens formation. These results suggest that the primary role of autocrine or paracrine FGF signaling is to provide essential survival signals to lens placode cells. Because apoptosis was already increased at the onset of placode formation in Fgfr1/2 conditional knockout placode cells, FGF signaling was functionally absent during the period of lens induction by the optic vesicle. Since the expression of proteins required for lens formation was not altered in the knockout placode cells, we can conclude that FGF signaling from the optic vesicle is not required for lens induction.
Asunto(s)
Factores de Crecimiento de Fibroblastos/fisiología , Cristalino/embriología , Transducción de Señal/fisiología , Animales , Receptores de Proteínas Morfogenéticas Óseas/fisiología , Ectodermo/química , Proteínas del Ojo/fisiología , Factores de Crecimiento de Fibroblastos/análisis , Mutación de Línea Germinal , Proteínas de Homeodominio/fisiología , Ratones , Factor de Transcripción PAX6 , Factores de Transcripción Paired Box/fisiología , Receptores de Factores de Crecimiento de Fibroblastos/análisis , Proteínas Represoras/fisiologíaRESUMEN
Growth factor signaling, mediated via receptor tyrosine kinases (RTKs), needs to be tightly regulated in many developmental systems to ensure a physiologically appropriate biological outcome. At one level this regulation may involve spatially and temporally ordered patterns of expression of specific RTK signaling antagonists, such as Sef (similar expression to fgfs). Growth factors, notably FGFs, play important roles in development of the vertebrate ocular lens. FGF induces lens cell proliferation and differentiation at progressively higher concentrations and there is compelling evidence that a gradient of FGF signaling in the eye determines lens polarity and growth patterns. We have recently identified the presence of Sef in the lens, with strongest expression in the epithelial cells. Given the important role for FGFs in lens developmental biology, we employed transgenic mouse strategies to determine if Sef could be involved in regulating lens cell behaviour. Over-expressing Sef specifically in the lens of transgenic mice led to impaired lens and eye development that resulted in microphthalmia. Sef inhibited primary lens fiber cell elongation and differentiation, as well as increased apoptosis, consistent with a block in FGFR-mediated signaling during lens morphogenesis. These results are consistent with growth factor antagonists, such as Sef, being important negative regulators of growth factor signaling. Moreover, the lens provides a useful paradigm as to how opposing gradients of a growth factor and its antagonist could work together to determine and stabilise tissue patterning during development and growth.
Asunto(s)
Diferenciación Celular , Embrión de Mamíferos/citología , Cristalino/citología , Proteínas de la Membrana/fisiología , Animales , Apoptosis , Western Blotting , Embrión de Mamíferos/metabolismo , Células Epiteliales/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/genética , Factores de Crecimiento de Fibroblastos/metabolismo , Humanos , Etiquetado Corte-Fin in Situ , Cristalino/metabolismo , Masculino , Ratones , Ratones Transgénicos , Microftalmía/metabolismo , Microftalmía/patología , Regiones Promotoras Genéticas , ARN Mensajero/genética , Receptores de Factores de Crecimiento de Fibroblastos/fisiología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Cadena A de alfa-Cristalina/genéticaRESUMEN
The vertebrate lens provides an excellent model to study the mechanisms that regulate terminal differentiation. Although fibroblast growth factors (FGFs) are thought to be important for lens cell differentiation, it is unclear which FGF receptors mediate these processes during different stages of lens development. Deletion of three FGF receptors (Fgfr1-3) early in lens development demonstrated that expression of only a single allele of Fgfr2 or Fgfr3 was sufficient for grossly normal lens development, while mice possessing only a single Fgfr1 allele developed cataracts and microphthalmia. Profound defects were observed in lenses lacking all three Fgfrs. These included lack of fiber cell elongation, abnormal proliferation in prospective lens fiber cells, reduced expression of the cell cycle inhibitors p27(kip1) and p57(kip2), increased apoptosis and aberrant or reduced expression of Prox1, Pax6, c-Maf, E-cadherin and alpha-, beta- and gamma-crystallins. Therefore, while signaling by FGF receptors is essential for lens fiber differentiation, different FGF receptors function redundantly.