RESUMO
Despite extensive analysis of pRB phosphorylation in vitro, how this modification influences development and homeostasis in vivo is unclear. Here, we show that homozygous Rb∆K4 and Rb∆K7 knock-in mice, in which either four or all seven phosphorylation sites in the C-terminal region of pRb, respectively, have been abolished by Ser/Thr-to-Ala substitutions, undergo normal embryogenesis and early development, notwithstanding suppressed phosphorylation of additional upstream sites. Whereas Rb∆K4 mice exhibit telomere attrition but no other abnormalities, Rb∆K7 mice are smaller and display additional hallmarks of premature aging including infertility, kyphosis, and diabetes, indicating an accumulative effect of blocking pRb phosphorylation. Diabetes in Rb∆K7 mice is insulin-sensitive and associated with failure of quiescent pancreatic ß-cells to re-enter the cell cycle in response to mitogens, resulting in induction of DNA damage response (DDR), senescence-associated secretory phenotype (SASP), and reduced pancreatic islet mass and circulating insulin level. Pre-treatment with the epigenetic regulator vitamin C reduces DDR, increases cell cycle re-entry, improves islet morphology, and attenuates diabetes. These results have direct implications for cell cycle regulation, CDK-inhibitor therapeutics, diabetes, and longevity.
Assuntos
Envelhecimento/fisiologia , Ácido Ascórbico/farmacologia , Diabetes Mellitus Experimental/prevenção & controle , Proteína do Retinoblastoma/metabolismo , Animais , Senescência Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina/antagonistas & inibidores , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patologia , Fator de Transcrição E2F1/metabolismo , Desenvolvimento Embrionário/genética , Feminino , Fibroblastos/efeitos dos fármacos , Técnicas de Introdução de Genes , Células Secretoras de Insulina/patologia , Camundongos , Fosforilação , Gravidez , Proteína do Retinoblastoma/genética , Telômero/genéticaRESUMO
Migration is essential for the laminar stratification and connectivity of neurons in the central nervous system. In the retina, photoreceptors (PRs) migrate to positions according to birthdate, with early-born cells localizing to the basal-most side of the outer nuclear layer. It was proposed that apical progenitor mitoses physically drive these basal translocations non-cell autonomously, but direct evidence is lacking, and whether other mechanisms participate is unknown. Here, combining loss- or gain-of-function assays to manipulate cell cycle regulators (Sonic hedgehog, Cdkn1a/p21) with an in vivo lentiviral labelling strategy, we demonstrate that progenitor division is one of two forces driving basal translocation of rod soma. Indeed, replacing Shh activity rescues abnormal rod translocation in retinal explants. Unexpectedly, we show that rod differentiation also promotes rod soma translocation. While outer segment function or formation is dispensable, Crx and SNARE-dependent synaptic function are essential. Thus, both non-cell and cell autonomous mechanisms underpin PR soma sublaminar positioning in the mammalian retina.
Assuntos
Neurossecreção , Células Fotorreceptoras Retinianas Bastonetes , Animais , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Proteínas Hedgehog/metabolismo , Retina/metabolismo , Diferenciação Celular , MamíferosRESUMO
Emerging evidence suggests that intracellular molecules and organelles transfer between cells during embryonic development, tissue homeostasis and disease. We and others recently showed that transplanted and host photoreceptors engage in bidirectional transfer of intracellular material in the recipient retina, a process termed material transfer (MT). We used cell transplantation, advanced tissue imaging approaches, genetic and pharmacologic interventions and primary cell culture to characterize and elucidate the mechanism of MT. We show that MT correlates with donor cell persistence and the accumulation of donor-derived proteins, mitochondria and transcripts in acceptor cells in vivo. MT requires cell contact in vitro and is associated with the formation of stable microtubule-containing protrusions, termed photoreceptor nanotubes (Ph NTs), that connect donor and host cells in vivo and in vitro. Ph NTs mediate GFP transfer between connected cells in vitro. Furthermore, interfering with Ph NT outgrowth by targeting Rho GTPase-dependent actin remodelling inhibits MT in vivo. Collectively, our observations provide evidence for horizontal exchange of intracellular material via nanotube-like connections between neurons in vivo.
Assuntos
Células Fotorreceptoras de Vertebrados/metabolismo , Células Fotorreceptoras de Vertebrados/ultraestrutura , Retina/citologia , Actinas/metabolismo , Animais , Transporte Biológico , Sobrevivência Celular , Vesículas Extracelulares , Feminino , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Mitocôndrias/metabolismo , Retina/fisiologia , Retinoblastoma/metabolismo , Retinoblastoma/patologia , Transducina/metabolismo , Proteínas rho de Ligação ao GTP/genética , Proteínas rho de Ligação ao GTP/metabolismoRESUMO
Why are some cell types more prone to transformation than others? In this issue, Xu et al. (2009) show that retinoblastoma cells co-opt several intrinsic features of cone photoreceptors for their survival and growth.
Assuntos
Retinoblastoma/metabolismo , Animais , Proliferação de Células , Sobrevivência Celular , Humanos , Camundongos , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Receptor X Retinoide gama/metabolismoRESUMO
Multipotent retinal progenitor cells (RPCs) generate various cell types in a precise chronological order, but how exactly cone photoreceptor production is restricted to early stages remains unclear. Here, we show that the POU-homeodomain factors Pou2f1/Pou2f2, the homologs of Drosophila temporal identity factors nub/pdm2, regulate the timely production of cones in mice. Forcing sustained expression of Pou2f1 or Pou2f2 in RPCs expands the period of cone production, whereas misexpression in late-stage RPCs triggers ectopic cone production at the expense of late-born fates. Mechanistically, we report that Pou2f1 induces Pou2f2 expression, which binds to a POU motif in the promoter of the rod-inducing factor Nrl to repress its expression. Conversely, conditional inactivation of Pou2f2 in RPCs increases Nrl expression and reduces cone production. Finally, we provide evidence that Pou2f1 is part of a cross-regulatory cascade with the other temporal identity factors Ikzf1 and Casz1. These results uncover Pou2f1/2 as regulators of the temporal window for cone genesis and, given their widespread expression in the nervous system, raise the possibility of a general role in temporal patterning.This article has an associated 'The people behind the papers' interview.
Assuntos
Proteínas do Olho/metabolismo , Fator 1 de Transcrição de Octâmero/metabolismo , Fator 2 de Transcrição de Octâmero/metabolismo , Retina/metabolismo , Células Fotorreceptoras Retinianas Cones/metabolismo , Animais , Drosophila/metabolismo , Feminino , Camundongos , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Células Fotorreceptoras Retinianas Bastonetes/metabolismo , Células-Tronco/metabolismoRESUMO
Until recently, the existence of extracellular kinase activity was questioned. Many proteins of the central nervous system are targeted, but it remains unknown whether, or how, extracellular phosphorylation influences brain development. Here we show that the tyrosine kinase vertebrate lonesome kinase (VLK), which is secreted by projecting retinal ganglion cells, phosphorylates the extracellular protein repulsive guidance molecule b (RGMb) in a dorsal-ventral descending gradient. Silencing of VLK or RGMb causes aberrant axonal branching and severe axon misguidance in the chick optic tectum. Mice harboring RGMb with a point mutation in the phosphorylation site also display aberrant axonal pathfinding. Mechanistic analyses show that VLK-mediated RGMb phosphorylation modulates Wnt3a activity by regulating LRP5 protein gradients. Thus, the secretion of VLK by projecting neurons provides crucial signals for the accurate formation of nervous system circuitry. The dramatic effect of VLK on RGMb and Wnt3a signaling implies that extracellular phosphorylation likely has broad and profound effects on brain development, function and disease.
Assuntos
Orientação de Axônios , Axônios/metabolismo , Animais , Camundongos , Proteínas do Tecido Nervoso/metabolismo , FosforilaçãoRESUMO
BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/µl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.
Assuntos
Teste de Ácido Nucleico para COVID-19/métodos , COVID-19/diagnóstico , Kit de Reagentes para Diagnóstico , SARS-CoV-2/isolamento & purificação , Manejo de Espécimes/métodos , Humanos , Nasofaringe/virologia , RNA Viral/isolamento & purificação , Sensibilidade e EspecificidadeRESUMO
The chromatin-remodeling enzyme BRG1 is critical for interferon-gamma (IFN-gamma)-mediated gene induction. Promoter-proximal elements are sufficient to mediate BRG1 dependency at some IFN-gamma targets. In contrast, we show here that at CIITA, which encodes the 'master regulator' of induction of major histocompatibility complex class II, distal elements conferred BRG1 dependency. At the uninduced locus, many sites formed BRG1-independent loops. One loop juxtaposed a far downstream element adjacent to a far upstream site. Notably, BRG1 was recruited to the latter site, which triggered the appearance of a histone 'mark' linked to activation. This subtle change was crucial, as subsequent IFN-gamma-induced recruitment of the transcription factors STAT1, IRF1 and p300, as well as histone modifications, accessibility and additional loops, showed BRG1 dependency. Like BRG1, each remote element was critical for the induction of CIITA expression. Thus, BRG1 regulates CIITA through many interdependent remote enhancers, not through the promoter alone.
Assuntos
Cromatina/genética , DNA Helicases/genética , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica , Proteínas Nucleares/genética , Transativadores/genética , Fatores de Transcrição/genética , Expressão Gênica , Genes Reguladores/genética , Células HeLa , Humanos , Interferon gama/metabolismo , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase , Regiões Promotoras Genéticas , Ativação TranscricionalRESUMO
Transcriptional activators bind DNA and recruit cofactors to modify chromatin. The extent to which these two events are separable is unclear. Here, using a custom ChIP tiling array to map chromatin modifications, we show that interferon-γ-induced DNA binding of signal transducer and activator of transcription 1 (STAT1), typically associated with the transcription factor interferon regulatory factor 1 (IRF1), causes histone acetylation (H3ac, H4ac). In contrast, among IRF1 sites lacking concomitant STAT1 recruitment, only 25% underwent inducible histone acetylation, 31% exhibited constitutive histone acetylation, and 44% had no histone acetylation. These latter "orphan sites" also lacked other activating modifications (e.g. H3K4me1, H3K4me2) and were typically remote from transcription start sites. In these cases the closest gene was typically an IFNγ-inducible locus that did not respond to IFNγ in this setting. Orphan sites were detected in different cell types, suggesting broad relevance. Despite an atypical downstream response (i.e. no histone modifications), IRF1 binding depended on SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily A, member 4 (SMARCA4 or BRG1), as is typical of active IRF1 enhancers. Although SMARCA4 permitted IRF1 access to the orphan sites, there was no corecruitment of the histone acetyltransferases CREB-binding protein (CBP) and p300. Orphan sites were constitutively unacetylated, and several were marked with repressive chromatin modifications (e.g. H3K27me3). In conclusion, although IRF1 can trigger enhanceosome formation independently of STAT1, its ability to do so depends on local chromatin cues.
Assuntos
Histonas/metabolismo , Fator Regulador 1 de Interferon/química , Fator Regulador 1 de Interferon/metabolismo , Acetilação , Sítios de Ligação , Cromatina/metabolismo , Células HeLa , Humanos , Fator de Transcrição STAT1/metabolismoRESUMO
BACKGROUND: STAT1 and IRF1 collaborate to induce interferon-γ (IFNγ) stimulated genes (ISGs), but the extent to which they act alone or together is unclear. The effect of single nucleotide polymorphisms (SNPs) on in vivo binding is also largely unknown. RESULTS: We show that IRF1 binds at proximal or distant ISG sites twice as often as STAT1, increasing to sixfold at the MHC class I locus. STAT1 almost always bound with IRF1, while most IRF1 binding events were isolated. Dual binding sites at remote or proximal enhancers distinguished ISGs that were responsive to IFNγ versus cell-specific resistant ISGs, which showed fewer and mainly single binding events. Surprisingly, inducibility in one cell type predicted ISG-responsiveness in other cells. Several dbSNPs overlapped with STAT1 and IRF1 binding motifs, and we developed methodology to rapidly assess their effects. We show that in silico prediction of SNP effects accurately reflects altered binding both in vitro and in vivo. CONCLUSIONS: These data reveal broad cooperation between STAT1 and IRF1, explain cell type specific differences in ISG-responsiveness, and identify genetic variants that may participate in the pathogenesis of immune disorders.
Assuntos
Fator Regulador 1 de Interferon/genética , Interferon gama/imunologia , Polimorfismo de Nucleotídeo Único , Fator de Transcrição STAT1/genética , Elementos Facilitadores Genéticos , Genes MHC Classe I , Células HeLa , Humanos , Fator Regulador 1 de Interferon/imunologia , Fator Regulador 1 de Interferon/metabolismo , Locos Secundários de Histocompatibilidade , Ligação Proteica , Fator de Transcrição STAT1/imunologia , Fator de Transcrição STAT1/metabolismo , Regulação para CimaRESUMO
We showed previously that C57BL/6 congenic mice with an introgressed homozygous 70 cM (125.6 Mb) to 100 cM (179.8 Mb) interval on c1 from the lupus-prone New Zealand Black (NZB) mouse develop high titers of antinuclear Abs and severe glomerulonephritis. Using subcongenic mice, we found that a genetic locus in the 88-96 cM region was associated with altered dendritic cell (DC) function and synergized with T cell functional defects to promote expansion of pathogenic proinflammatory T cell subsets. In this article, we show that the promoter region of the NZB gene encoding the SLAM signaling pathway adapter molecule EWS-activated transcript 2 (EAT-2) is polymorphic, which results in an â¼ 70% reduction in EAT-2 in DC. Silencing of the EAT-2 gene in DC that lacked this polymorphism led to increased production of IL-12 and enhanced differentiation of T cells to a Th1 phenotype in T cell-DC cocultures, reproducing the phenotype observed for DC from congenic mice with the NZB c1 70-100 cM interval. SLAM signaling was shown to inhibit production of IL-12 by CD40L-activated DCs. Consistent with a role for EAT-2 in this inhibition, knockdown of EAT-2 resulted in increased production of IL-12 by CD40-stimulated DC. Assessment of downstream signaling following CD40 cross-linking in the presence or absence of SLAM cross-linking revealed that SLAM coengagement blocked activation of p38 MAPK and JNK signaling pathways in DC, which was reversed in DC with the NZB EAT-2 allele. We conclude that EAT-2 negatively regulates cytokine production in DC downstream of SLAM engagement and that a genetic polymorphism that disturbs this process promotes the development of lupus.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/fisiologia , Anticorpos Antinucleares/imunologia , Células Dendríticas/imunologia , Lúpus Eritematoso Sistêmico/genética , Células Th1/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Anticorpos Antinucleares/genética , Sequência de Bases , Ligante de CD40/metabolismo , Diferenciação Celular/genética , Diferenciação Celular/imunologia , Células Cultivadas , Técnicas de Cocultura , Feminino , Subunidade p35 da Interleucina-12/biossíntese , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Lúpus Eritematoso Sistêmico/imunologia , Ativação Linfocitária/imunologia , Camundongos , Camundongos Endogâmicos NZB , Dados de Sequência Molecular , Interferência de RNA , RNA Interferente Pequeno , Transdução de Sinais/imunologia , Células Th1/citologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
CIITA (or MHC2TA) coordinates constitutive and IFN-γ-induced expression of MHC class II genes. IFN-γ responsiveness of CIITA requires BRG1 (SMARCA4), the ATPase engine of the chromatin remodeling SWI/SNF complex (also called BAF). SWI/SNF is defective in many human cancers, providing a mechanism to explain IFN-γ resistance. BRG1 dependency is mediated through remote elements. Short CIITA reporters lacking these elements respond to IFN-γ, even in BRG1-deficient cells, suggesting that BRG1 counters a remote repressive influence. The nature of this distal repressor is unknown, but it would represent a valuable therapeutic target to reactivate IFN-γ responsiveness in cancer. In this article, we show that the polycomb repressive complex 2 (PRC2) components EZH2 and SUZ12, as well as the associated histone mark H3K27me3, are codetected at interenhancer regions across the CIITA locus. IFN-γ caused a BRG1-dependent reduction in H3K27me3, associated with nucleosome displacement. SUZ12 knockdown restored IFN-γ responsiveness in BRG1-null cells, and it mimicked the ability of BRG1 to induce active histone modifications (H3K27ac, H3K4me) at the -50-kb enhancer. Thus, PRC2 confers BRG1 dependency on the CIITA locus. Our data suggest that, in addition to its known roles in promoting stemness and proliferation, PRC2 may inhibit immune surveillance, and it could be targeted to reactivate CIITA expression in SWI/SNF deficient cancers.
Assuntos
DNA Helicases/genética , Regulação Neoplásica da Expressão Gênica/genética , Neoplasias/genética , Proteínas Nucleares/genética , Complexo Repressor Polycomb 2/genética , Transativadores/genética , Fatores de Transcrição/genética , Western Blotting , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Loci Gênicos , Humanos , Neoplasias/imunologia , Interferência de RNA , Reação em Cadeia da Polimerase em Tempo RealRESUMO
The epigenetic regulator Bmi1 controls proliferation in many organs. Reexpression of cell cycle proteins such as cyclin-dependent kinases (CDKs) is a hallmark of neuronal apoptosis in neurodegenerative diseases. Here we address the potential role of Bmi1 as a key regulator of cell cycle proteins during neuronal apoptosis. We show that several cell cycle proteins are expressed in different models of retinal degeneration and required in the Rd1 photoreceptor death process. Deleting E2f1, a downstream target of CDKs, provided temporary protection in Rd1 mice. Most importantly, genetic ablation of Bmi1 provided extensive photoreceptor survival and improvement of retinal function in Rd1 mice, mediated by a decrease in cell cycle markers and regulators independent of p16(Ink4a) and p19(Arf). These data reveal that Bmi1 controls the cell cycle-related death process, highlighting this pathway as a promising therapeutic target for neuroprotection in retinal dystrophies.
Assuntos
Apoptose/fisiologia , Proteínas de Ciclo Celular/metabolismo , Epigênese Genética/fisiologia , Regulação da Expressão Gênica/fisiologia , Complexo Repressor Polycomb 1/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Degeneração Retiniana/metabolismo , Análise de Variância , Animais , Fator de Transcrição E2F1/metabolismo , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , Técnicas Histológicas , Camundongos , Camundongos Knockout , Microscopia de Fluorescência , Complexo Repressor Polycomb 1/genética , Proteínas Proto-Oncogênicas/genéticaRESUMO
The activating E2f transcription factors (E2f1, E2f2 and E2f3) induce transcription and are widely viewed as essential positive cell cycle regulators. Indeed, they drive cells out of quiescence, and the 'cancer cell cycle' in Rb1 null cells is E2f-dependent. Absence of activating E2fs in flies or mammalian fibroblasts causes cell cycle arrest, but this block is alleviated by removing repressive E2f or the tumour suppressor p53, respectively. Thus, whether activating E2fs are indispensable for normal division is an area of debate. Activating E2fs are also well known pro-apoptotic factors, providing a defence against oncogenesis, yet E2f1 can limit irradiation-induced apoptosis. In flies this occurs through repression of hid (also called Wrinkled; Smac/Diablo in mammals). However, in mammals the mechanism is unclear because Smac/Diablo is induced, not repressed, by E2f1, and in keratinocytes survival is promoted indirectly through induction of DNA repair targets. Thus, a direct pro-survival function for E2f1-3 and/or its relevance beyond irradiation has not been established. To address E2f1-3 function in normal cells in vivo we focused on the mouse retina, which is a relatively simple central nervous system component that can be manipulated genetically without compromising viability and has provided considerable insight into development and cancer. Here we show that unlike fibroblasts, E2f1-3 null retinal progenitor cells or activated Müller glia can divide. We attribute this effect to functional interchangeability with Mycn. However, loss of activating E2fs caused downregulation of the p53 deacetylase Sirt1, p53 hyperacetylation and elevated apoptosis, establishing a novel E2f-Sirt1-p53 survival axis in vivo. Thus, activating E2fs are not universally required for normal mammalian cell division, but have an unexpected pro-survival role in development.
Assuntos
Apoptose , Fatores de Transcrição E2F/deficiência , Retina/citologia , Retina/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismo , Acetilação , Animais , Divisão Celular , Sobrevivência Celular , Inibidor de Quinase Dependente de Ciclina p21/antagonistas & inibidores , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Fatores de Transcrição E2F/genética , Fatores de Transcrição E2F/metabolismo , Fator de Transcrição E2F1/deficiência , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/deficiência , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F3/deficiência , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Fibroblastos , Camundongos , Camundongos Knockout , Neuroglia/citologia , Neuroglia/metabolismo , Sirtuína 1/metabolismo , Proteína Supressora de Tumor p53/metabolismoRESUMO
In the established model of mammalian cell cycle control, the retinoblastoma protein (Rb) functions to restrict cells from entering S phase by binding and sequestering E2f activators (E2f1, E2f2 and E2f3), which are invariably portrayed as the ultimate effectors of a transcriptional program that commit cells to enter and progress through S phase. Using a panel of tissue-specific cre-transgenic mice and conditional E2f alleles we examined the effects of E2f1, E2f2 and E2f3 triple deficiency in murine embryonic stem cells, embryos and small intestines. We show that in normal dividing progenitor cells E2f1-3 function as transcriptional activators, but contrary to the current view, are dispensable for cell division and instead are necessary for cell survival. In differentiating cells E2f1-3 function in a complex with Rb as repressors to silence E2f targets and facilitate exit from the cell cycle. The inactivation of Rb in differentiating cells resulted in a switch of E2f1-3 from repressors to activators, leading to the superactivation of E2f responsive targets and ectopic cell divisions. Loss of E2f1-3 completely suppressed these phenotypes caused by Rb deficiency. This work contextualizes the activator versus repressor functions of E2f1-3 in vivo, revealing distinct roles in dividing versus differentiating cells and in normal versus cancer-like cell cycles.
Assuntos
Diferenciação Celular , Fatores de Transcrição E2F/metabolismo , Células-Tronco Embrionárias/citologia , Células-Tronco Embrionárias/metabolismo , Regulação da Expressão Gênica , Proteínas Repressoras/metabolismo , Alelos , Animais , Apoptose , Ciclo Celular/genética , Ciclo Celular/fisiologia , Proliferação de Células , Fatores de Transcrição E2F/deficiência , Fatores de Transcrição E2F/genética , Fator de Transcrição E2F1/deficiência , Fator de Transcrição E2F1/genética , Fator de Transcrição E2F1/metabolismo , Fator de Transcrição E2F2/deficiência , Fator de Transcrição E2F2/genética , Fator de Transcrição E2F2/metabolismo , Fator de Transcrição E2F3/deficiência , Fator de Transcrição E2F3/genética , Fator de Transcrição E2F3/metabolismo , Embrião de Mamíferos/citologia , Embrião de Mamíferos/metabolismo , Feminino , Intestino Delgado/citologia , Intestino Delgado/metabolismo , Camundongos , Camundongos Transgênicos , Proteínas Repressoras/deficiência , Proteínas Repressoras/genética , Proteína do Retinoblastoma/deficiência , Proteína do Retinoblastoma/metabolismoRESUMO
BACKGROUND: Despite the disease relevance, understanding of human retinal development lags behind that of other species. We compared the kinetics of gene silencing or induction during ganglion cell development in human and murine retina. RESULTS: Induction of POU4F2 (BRN3B) marks ganglion cell commitment, and we detected this factor in S-phase progenitors that had already silenced Cyclin D1 and VSX2 (CHX10). This feature was conserved in human and mouse retina, and the fraction of Pou4f2+ murine progenitors labeled with a 30 min pulse of BrdU matched the fraction of ganglion cells predicted to be born in a half-hour period. Additional analysis of 18 markers revealed many with conserved kinetics, such as the POU4F2 pattern above, as well as the surprising maintenance of "cell cycle" proteins KI67, PCNA, and MCM6 well after terminal mitosis. However, four proteins (TUBB3, MTAP1B, UCHL1, and RBFOX3) showed considerably delayed induction in human relative to mouse retina, and two proteins (ISL1, CALB2) showed opposite kinetics, appearing on either side of terminal mitosis depending on the species. CONCLUSION: With some notable exceptions, human and murine ganglion cell differentiation show similar kinetics, and the data add weight to prior studies supporting the existence of biased ganglion cell progenitors.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiologia , Diferenciação Celular/fisiologia , Proteínas do Olho/metabolismo , Células Ganglionares da Retina/metabolismo , Células-Tronco/metabolismo , Animais , Humanos , Camundongos , Células Ganglionares da Retina/citologia , Células-Tronco/citologiaRESUMO
The cellular effects of the genetic defects associated with tumorigenesis are context dependent. To better understand the reasons that different cell types require distinct combinations of mutations to form tumours, it is essential to identify and characterize a tumour's 'cell of origin'. Retinoblastoma, a rare childhood cancer of the retina that is caused by RB inactivation, is a good model in which to search for a tumour cell of origin, because retinal development is well understood and the initiating genetic lesion is well characterized. Identifying the cell of origin for this tumour would advance our understanding of how cellular context affects the requirement of specific mutations for cancer initiation and progression.
Assuntos
Neoplasias da Retina/genética , Retinoblastoma/genética , Células-Tronco/citologia , Animais , Linhagem Celular Transformada , Linhagem Celular Tumoral , Progressão da Doença , Genes do Retinoblastoma , Humanos , Modelos Biológicos , Mutação , Neoplasias/metabolismo , Retina/metabolismo , Neoplasias da Retina/patologia , Retinoblastoma/patologia , Fatores de TempoRESUMO
Opposite expression and pro- or anti-cancer function of YAP and its paralog TAZ/WWTR1 stratify cancers into binary YAPon and YAPoff classes. These transcriptional coactivators are oncogenic in YAPon cancers. In contrast, YAP/TAZ are silenced epigenetically along with their integrin and extracellular matrix adhesion target genes in neural and neuroendocrine YAPoff cancers (e.g., small cell lung cancer, retinoblastoma). Forced YAP/TAZ expression induces these targets, causing cytostasis in part through Integrin-αV/ß5, independent of the integrin-binding RGD ligand. Other effectors of this anti-cancer YAP function are unknown. Here, using CRISPR screens, we link the Netrin receptor UNC5B to YAP-induced cytostasis in YAPoff cancers. Forced YAP expression induces UNC5B through TEAD DNA-binding partners, as either TEAD1/4-loss or a YAP mutation that disrupts TEAD-binding (S94A) blocks whereas a TEAD-activator fusion (TEAD(DBD)-VP64) promotes UNC5B induction. Ectopic YAP expression also upregulates UNC5B relatives and their netrin ligands in YAPoff cancers. Netrins are considered pro-tumorigenic, but knockout and peptide/decoy-receptor blocking assays reveal that in YAPoff cancers UNC5B and Netrin-1 can cooperate with integrin-αV/ß5 to mediate YAP-induced cytostasis. These data pinpoint an unsuspected Netrin-1/UNC5B/integrin-αV/ß5 axis as a critical effector of YAP tumor suppressor activity.
RESUMO
Regulation of tissue size is a poorly understood process. Mammalian Staufen 2 (Stau2) is a double-stranded mRNA binding protein known to regulate dendrite formation in vitro as well as cell survival and migration in vivo. Three Stau2 isoforms have been identified in the brain of mammals. Here we show that all these Stau2 isoforms are also expressed in the developing eye of chicken embryos. Strikingly, ectopic expression of Stau2 was sufficient to increase eye size, suggesting a novel biological role of Stau2 in eye morphogenesis. Moreover, down regulation of Stau2 in vivo resulted in a small eye. Microphthalmia was not associated with either increased cell death or differentiation but with reduced cell proliferation. Rescue experiments showed that all three Stau2 isoforms present in the developing eye could prevent microphthalmia. Finally, we showed that Stau2 silencing decreased HES-1 and Sox-2 in the developing eye. These data highlight a new biological function for Stau2 and suggest that translation control of specific Stau2-associated transcripts may be a key regulator of tissue size.