Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 47
Filtrar
Más filtros












Base de datos
Intervalo de año de publicación
1.
Biol Pharm Bull ; 47(7): 1338-1344, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-39048355

RESUMEN

The initiation of DNA replication is tightly controlled by the licensing system that loads replicative DNA helicases onto replication origins to form pre-replicative complexes (pre-RCs) once per cell cycle. Cdc10-dependent transcript 1 (Cdt1) plays an essential role in the licensing reaction by recruiting mini-chromosome maintenance (MCM) complexes, which are eukaryotic replicative DNA helicases, to their origins via direct protein-protein interactions. Cdt1 interacts with other pre-RC components, the origin recognition complex, and the cell division cycle 6 (Cdc6) protein; however, the molecular mechanism by which Cdt1 functions in the MCM complex loading process has not been fully elucidated. Here, we analyzed the protein-protein interactions of recombinant Cdt1 and observed that Cdt1 self-associates via the central region of the molecule, which is inhibited by the endogenous licensing inhibitor, geminin. Mutation of two ß-strands of the winged-helix domain in the central region of Cdt1 attenuated its self-association but could still interact with other pre-RC components and DNA similarly to wild-type Cdt1. Moreover, the Cdt1 mutant showed decreased licensing activity in Xenopus egg extracts. Together, these results suggest that the self-association of Cdt1 is crucial for licensing.


Asunto(s)
Proteínas de Ciclo Celular , Geminina , Animales , Geminina/metabolismo , Geminina/genética , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , Replicación del ADN , Proteínas de Xenopus/metabolismo , Proteínas de Xenopus/genética , Xenopus laevis , Dominios Proteicos , Xenopus , Humanos , Proteínas de Unión al ADN
2.
BMC Biol ; 21(1): 265, 2023 11 20.
Artículo en Inglés | MEDLINE | ID: mdl-37981687

RESUMEN

BACKGROUND: Lepidoptera is one of the most species-rich animal groups, with substantial karyotype variations among species due to chromosomal rearrangements. Knowledge of the evolutionary patterns of lepidopteran chromosomes still needs to be improved. RESULTS: Here, we used chromosome-level genome assemblies of 185 lepidopteran insects to reconstruct an ancestral reference genome and proposed a new chromosome nomenclature. Thus, we renamed over 5000 extant chromosomes with this system, revealing the historical events of chromosomal rearrangements and their features. Additionally, our findings indicate that, compared with autosomes, the Z chromosome in Lepidoptera underwent a fast loss of conserved genes, rapid acquisition of lineage-specific genes, and a low rate of gene duplication. Moreover, we presented evidence that all available 67 W chromosomes originated from a common ancestor chromosome, with four neo-W chromosomes identified, including one generated by fusion with an autosome and three derived through horizontal gene transfer. We also detected nearly 4000 inter-chromosomal gene movement events. Notably, Geminin is transferred from the autosome to the Z chromosome. When located on the autosome, Geminin shows female-biased expression, but on the Z chromosome, it exhibits male-biased expression. This contributes to the sexual dimorphism of body size in silkworms. CONCLUSIONS: Our study sheds light on the complex evolutionary history of lepidopteran chromosomes based on ancestral chromosome reconstruction and novel chromosome nomenclature.


Asunto(s)
Evolución Biológica , Lepidópteros , Animales , Femenino , Masculino , Geminina/genética , Genoma , Cromosomas Sexuales/genética , Lepidópteros/genética , Evolución Molecular
3.
FEBS Lett ; 597(7): 985-994, 2023 04.
Artículo en Inglés | MEDLINE | ID: mdl-36905332

RESUMEN

Genome editing with CRISPR-Cas9, particularly for therapeutic purposes, should be accomplished via the homology-directed repair (HDR) pathway, which exhibits greater precision than other pathways. However, one of the issues to be solved is that genome editing efficiency with HDR is generally low. A Streptococcus pyogenes Cas9 (SpyCas9) fusion with human Geminin (Cas9-Gem) reportedly increases HDR efficiency slightly. In contrast, we found that regulation of SpyCas9 activity with an anti-CRISPR protein (AcrIIA4) fused to Chromatin licensing and DNA replication factor 1 (Cdt1) significantly increases HDR efficiency and reduces off-target effects. Here, another anti-CRISPR protein, AcrIIA5, was applied, and the combined use of Cas9-Gem and Anti-CRISPR+Cdt1 showed synergistic enhancement of HDR efficiency. The method may be applicable to various anti-CRISPR/CRISPR-Cas combinations.


Asunto(s)
Sistemas CRISPR-Cas , Edición Génica , Humanos , Geminina/genética , Reparación del ADN por Recombinación , Proteínas de Ciclo Celular/genética
4.
Stem Cell Res ; 67: 103031, 2023 03.
Artículo en Inglés | MEDLINE | ID: mdl-36702081

RESUMEN

We previously generated a doxycycline-inducible H2B-mTurq2 reporter in hiPSCs to track cells and study cell division and apoptosis. To improve visualization of cycling cells, we introduced a ubiquitously transcribed mScarletI-Geminin (GMMN) (1-110) into the previously untargeted second AAVS1 allele. Fusion to the N-terminal part of GMNN provided tightly controlled mScarletI expression during the cell cycle. mScarletI fluorescence increased gradually from the S-phase through the M-phase of the cell cycle and was lost at the metaphase-anaphase transition. The resulting hiPSC reporter line generated, which we named ProLiving, is a valuable tool to study cell division and cell cycle characteristics in living hiPSC-derived cells.


Asunto(s)
Células Madre Pluripotentes Inducidas , Geminina/genética , Geminina/metabolismo , Células Madre Pluripotentes Inducidas/metabolismo , Ciclo Celular , División Celular , Proteínas de Ciclo Celular/genética
5.
Cell Rep ; 41(11): 111810, 2022 12 13.
Artículo en Inglés | MEDLINE | ID: mdl-36516767

RESUMEN

Multiciliated ependymal cells and adult neural stem cells are components of the adult neurogenic niche, essential for brain homeostasis. These cells share a common glial cell lineage regulated by the Geminin family members Geminin and GemC1/Mcidas. Ependymal precursors require GemC1/Mcidas expression to massively amplify centrioles and become multiciliated cells. Here, we show that GemC1-dependent differentiation is initiated in actively cycling radial glial cells, in which a DNA damage response, including DNA replication-associated damage and dysfunctional telomeres, is induced, without affecting cell survival. Genotoxic stress is not sufficient by itself to induce ependymal cell differentiation, although the absence of p53 or p21 in progenitors hinders differentiation by maintaining cell division. Activation of the p53-p21 pathway downstream of GemC1 leads to cell-cycle slowdown/arrest, which permits timely onset of ependymal cell differentiation in progenitor cells.


Asunto(s)
Células-Madre Neurales , Proteína p53 Supresora de Tumor , Geminina/genética , Geminina/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Epéndimo/metabolismo , Células Ependimogliales/metabolismo , Células-Madre Neurales/metabolismo , Diferenciación Celular
6.
Pathol Res Pract ; 239: 154170, 2022 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-36257233

RESUMEN

Tumour cells develop by accumulating changes in the genome that result in changes of main cellular processes. Aberrations of basic processes such as replication and chromatin reassembly are particularly important for genomic (in)stability. The aim of this study was to analyse the expression of genes whose products are crucial for the regulation of replication and chromatin reassembly during lymphomagenesis (DNMT1, PCNA, MCM2, CDT1, EZH2, GMNN, EP300). Non-tumour B cells were used as a control, and follicular lymphoma (FL) and the two most common groups of diffuse large B cell lymphoma (DLBCL) samples were used as a model for tumour progression. The results showed that there are significant changes in the expression of the analysed genes in lymphomagenesis, but also that these changes do not display linearity when assessed in relation to the degree of tumour aggression. Additionally, an integrated bioinformatics analysis of the difference in the expression of selected genes between tumour and non-tumour samples, and between tumour samples (FL vs. DLBCL) in five GEO datasets, did not show a consistent pattern of difference among the datasets.


Asunto(s)
Linfoma Folicular , Linfoma de Células B Grandes Difuso , Linfoma no Hodgkin , Humanos , Antígeno Nuclear de Célula en Proliferación , Linfoma no Hodgkin/genética , Linfoma de Células B Grandes Difuso/patología , Linfoma Folicular/patología , Cromatina , Proteínas de Ciclo Celular/genética , Geminina/genética , Proteína Potenciadora del Homólogo Zeste 2/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Proteína p300 Asociada a E1A
7.
J Stomatol Oral Maxillofac Surg ; 123(6): e828-e832, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35217222

RESUMEN

OBJECTIVE: Uncontrolled proliferation and aberrations in cell-cycle progression are fundamental issues in cancer. In this study we aimed to determine and compare deoxyribonucleic acid (DNA) replication licensing factors at the mRNA and protein levels among squamous cell carcinomas (SCCs) of the lip, facial-skin and oral cavity. MATERIALS AND METHODS: A total of 103 lip, oral and face SCCs were immunohistochemically stained with MCM2 (mini-chromosome maintenance 2), geminin, and ki67, and their labeling-indices were calculated. Also, 57 SCCs from the same regions along with their adjacent normal tissues underwent quantitative reverse transcription-polymerase chain reaction analysis. RESULTS: All three proteins were overexpressed in the studied SCCs, but only geminin (P = 0.004) showed significant difference among the three regions, with higher levels in oral SCCs compared to lip (P = 0.005) and skin (P = 0.024) tumors. Geminin expression did not differ between skin- and lip-SCCs (P = 0.822). MCM2/ki67 ratio was higher in oral- compared to skin-neoplasms (P = 0.039), but no difference was found in geminin/ki67 among the SCC-subsites. There were significant differences in MCM2 and geminin mRNA between carcinomatous- and normal-tissues in all tumors, but not among the three locations. CONCLUSION: MCM2 and geminin are involved in the tumorigenesis of lip, face and oral SCC at both mRNA- and protein-levels. Geminin may have a role in the site-specific biologic behavior of SCC. Skin SCCs had the highest proportion of licensed non-proliferating cells, while actively proliferating cells were more prominent in oral tumors. Regarding DNA replication, lip SCCs seem to be closer to skin tumors compared to their oral counterparts.


Asunto(s)
Carcinoma de Células Escamosas , Neoplasias Faciales , Neoplasias de los Labios , Neoplasias de la Boca , Humanos , Carcinoma de Células Escamosas/genética , Carcinoma de Células Escamosas/patología , Replicación del ADN , Geminina/genética , Geminina/metabolismo , Inmunohistoquímica , Antígeno Ki-67/genética , Antígeno Ki-67/metabolismo , Componente 2 del Complejo de Mantenimiento de Minicromosoma/genética , Componente 2 del Complejo de Mantenimiento de Minicromosoma/metabolismo , Neoplasias de la Boca/genética , Neoplasias de la Boca/patología , ARN Mensajero/genética , Neoplasias Faciales/genética , Neoplasias Faciales/metabolismo , Neoplasias de los Labios/genética , Neoplasias de los Labios/metabolismo
8.
Exp Cell Res ; 410(1): 112951, 2022 01 01.
Artículo en Inglés | MEDLINE | ID: mdl-34843715

RESUMEN

Endoreplication, known as endocycles or endoreduplication, is a cell cycle variant in which the genomic DNA is re-replicated without mitosis leading to polyploidy. Endoreplication is essential for the development and functioning of the different organs in animals and plants. Deletion of Geminin, a DNA replication licensing inhibitor, causes DNA re-replication or damage. However, the role of Geminin in endoreplication is still unclear. Here, we studied the role of Geminin in the endoreplication of the silk gland cells of silkworms by constructing two transgenic silkworm strains, including BmGeminin1-overexpression and BmGeminin1-RNA interference. Interference of BmGeminin1 led to body weight gain, increased silk gland volume, increased DNA content, and enhanced DNA re-replication activity relative to wild-type Dazao. Meanwhile, overexpression of BmGeminin1 showed an opposite phenotype compared to the BmGem1-RNAi strain. Furthermore, RNA-sequencing of the transgenic strains was carried out to explore how BmGeminin1 regulates DNA re-replication. Our data demonstrated a vital role of Geminin in the regulation of endoreplication in the silk gland of silkworms.


Asunto(s)
Bombyx/genética , Replicación del ADN/genética , Geminina/genética , Seda/genética , Animales , Bombyx/metabolismo , Ciclo Celular/genética , Geminina/antagonistas & inhibidores , Mitosis/genética , Interferencia de ARN , Seda/biosíntesis
9.
Bioengineered ; 12(2): 11482-11489, 2021 12.
Artículo en Inglés | MEDLINE | ID: mdl-34874226

RESUMEN

Low-grade cervical squamous intraepithelial lesion is a precancerous neoplasia that has appreciable probability to evolve into malignancy. To explore the prognostic value of HPV 16/18 genotyping and geminin mRNA quantification in predicting the progressiveness of LSIL. We recruited 212 participants who were negative for intraepithelial lesion or malignancy (NILM 76), low-grade squamous intraepithelial lesion (LSIL 85), high-grade squamous intraepithelial lesion (HSIL 36) and cervical intraepithelial neoplasia grade cervical cancer grade 3, (CIN3 15) patients. Tissues were obtained during excisional treatment. HPV 16/18 genotyping and geminin mRNA qRT-PCR were performed. HPV 16/18 positivity rate and geminin mRNA level were integrated with the clinical parameters into a multivariate logistic model. Area under curve was yielded based on receiver operation curve derived from this multivariate logistic model. Follow-up visits were performed to LSIL patients with progression. HSIL patients have higher HPV 16/18 positivity rate and geminin mRNA levels than LSIL. Among HSIL, CIN3 have higher HPV 16/18 positivity rate and geminin mRNA levels. Multivariate logistic analysis showed that HPV 16/18 positivity and geminin mRNA expression status are independent factors for differentiating HSIL and LSIL. The baseline HPV 16/18 positivity rate and geminin mRNA levels of 18 LSIL patients who developed HSIL are significantly higher than non-progressive LSIL patients. The values examined at follow-up timepoints were also higher than baseline. These results suggest that geminin is implicated in the progression of LSIL and combining HPV 16/18 genotyping and geminin mRNA qRT-PCR could potentially differentiating the progressive LSIL and improve the efficacy of clinical intervention.


Asunto(s)
Geminina/genética , Papillomavirus Humano 16/genética , Papillomavirus Humano 18/genética , Lesiones Intraepiteliales Escamosas/genética , Displasia del Cuello del Útero/genética , Neoplasias del Cuello Uterino/genética , Adulto , Anciano , Línea Celular Tumoral , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Geminina/metabolismo , Regulación Neoplásica de la Expresión Génica , Genotipo , Humanos , Modelos Logísticos , Persona de Mediana Edad , Análisis Multivariante , Pronóstico , ARN Mensajero/genética , Curva ROC , Lesiones Intraepiteliales Escamosas/patología , Neoplasias del Cuello Uterino/patología , Displasia del Cuello del Útero/patología
10.
Nat Commun ; 12(1): 5779, 2021 10 01.
Artículo en Inglés | MEDLINE | ID: mdl-34599168

RESUMEN

Geminin and its binding partner Cdt1 are essential for the regulation of DNA replication. Here we show that the CULLIN3 E3 ubiquitin ligase adaptor protein SPOP binds Geminin at endogenous level and regulates DNA replication. SPOP promotes K27-linked non-degradative poly-ubiquitination of Geminin at lysine residues 100 and 127. This poly-ubiquitination of Geminin prevents DNA replication over-firing by indirectly blocking the association of Cdt1 with the MCM protein complex, an interaction required for DNA unwinding and replication. SPOP is frequently mutated in certain human cancer types and implicated in tumorigenesis. We show that cancer-associated SPOP mutations impair Geminin K27-linked poly-ubiquitination and induce replication origin over-firing and re-replication. The replication stress caused by SPOP mutations triggers replication catastrophe and cell death upon ATR inhibition. Our results reveal a tumor suppressor role of SPOP in preventing DNA replication over-firing and genome instability and suggest that SPOP-mutated tumors may be susceptible to ATR inhibitor therapy.


Asunto(s)
Geminina/metabolismo , Proteínas Nucleares/genética , Proteínas Represoras/genética , Complejos de Ubiquitina-Proteína Ligasa/genética , Animales , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Replicación del ADN/genética , Replicación del ADN/fisiología , Geminina/genética , Humanos , Masculino , Ratones , Ratones SCID , Proteínas de Mantenimiento de Minicromosoma/genética , Proteínas de Mantenimiento de Minicromosoma/metabolismo , Mutación/genética , Ubiquitinación/genética , Ubiquitinación/fisiología
11.
Elife ; 102021 05 19.
Artículo en Inglés | MEDLINE | ID: mdl-34009124

RESUMEN

To study disease development, an inventory of an organ's cell types and understanding of physiologic function is paramount. Here, we performed single-cell RNA-sequencing to examine heterogeneity of murine pancreatic duct cells, pancreatobiliary cells, and intrapancreatic bile duct cells. We describe an epithelial-mesenchymal transitory axis in our three pancreatic duct subpopulations and identify osteopontin as a regulator of this fate decision as well as human duct cell dedifferentiation. Our results further identify functional heterogeneity within pancreatic duct subpopulations by elucidating a role for geminin in accumulation of DNA damage in the setting of chronic pancreatitis. Our findings implicate diverse functional roles for subpopulations of pancreatic duct cells in maintenance of duct cell identity and disease progression and establish a comprehensive road map of murine pancreatic duct cell, pancreatobiliary cell, and intrapancreatic bile duct cell homeostasis.


Asunto(s)
Perfilación de la Expresión Génica , Heterogeneidad Genética , Conductos Pancreáticos/citología , Análisis de la Célula Individual , Transcriptoma , Animales , Línea Celular , Separación Celular , Daño del ADN , Bases de Datos Genéticas , Modelos Animales de Enfermedad , Transición Epitelial-Mesenquimal , Femenino , Geminina/genética , Geminina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Humanos , Ratones Endogámicos C57BL , Ratones Transgénicos , Morfogénesis , Osteopontina/genética , Osteopontina/metabolismo , Conductos Pancreáticos/metabolismo , Pancreatitis Crónica/genética , Pancreatitis Crónica/metabolismo , Pancreatitis Crónica/patología , Fenotipo , RNA-Seq
12.
PLoS Genet ; 16(8): e1008988, 2020 08.
Artículo en Inglés | MEDLINE | ID: mdl-32841231

RESUMEN

Achieving complete and precise genome duplication requires that each genomic segment be replicated only once per cell division cycle. Protecting large eukaryotic genomes from re-replication requires an overlapping set of molecular mechanisms that prevent the first DNA replication step, the DNA loading of MCM helicase complexes to license replication origins, after S phase begins. Previous reports have defined many such origin licensing inhibition mechanisms, but the temporal relationships among them are not clear, particularly with respect to preventing re-replication in G2 and M phases. Using a combination of mutagenesis, biochemistry, and single cell analyses in human cells, we define a new mechanism that prevents re-replication through hyperphosphorylation of the essential MCM loading protein, Cdt1. We demonstrate that Cyclin A/CDK1 can hyperphosphorylate Cdt1 to inhibit MCM re-loading in G2 phase. The mechanism of inhibition is to block Cdt1 binding to MCM independently of other known Cdt1 inactivation mechanisms such as Cdt1 degradation during S phase or Geminin binding. Moreover, our findings suggest that Cdt1 dephosphorylation at the mitosis-to-G1 phase transition re-activates Cdt1. We propose that multiple distinct, non-redundant licensing inhibition mechanisms act in a series of sequential relays through each cell cycle phase to ensure precise genome duplication.


Asunto(s)
Replicación del ADN/genética , Genoma Humano/genética , Origen de Réplica/genética , Duplicaciones Segmentarias en el Genoma/genética , Proteína Quinasa CDC2/genética , Proteínas de Ciclo Celular/genética , Ciclina A/genética , Fase G2/genética , Geminina/genética , Genes Duplicados/genética , Células HEK293 , Humanos , Proteínas de Mantenimiento de Minicromosoma/genética , Fosforilación/genética , Fase S/genética
13.
Dev Biol ; 464(1): 11-23, 2020 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-32450229

RESUMEN

Development of the complex structure of the vertebrate limb requires carefully orchestrated interactions between multiple regulatory pathways and proteins. Among these, precise regulation of 5' Hox transcription factor expression is essential for proper limb bud patterning and elaboration of distinct limb skeletal elements. Here, we identified Geminin (Gmnn) as a novel regulator of this process. A conditional model of Gmnn deficiency resulted in loss or severe reduction of forelimb skeletal elements, while both the forelimb autopod and hindlimb were unaffected. 5' Hox gene expression expanded into more proximal and anterior regions of the embryonic forelimb buds in this Gmnn-deficient model. A second conditional model of Gmnn deficiency instead caused a similar but less severe reduction of hindlimb skeletal elements and hindlimb polydactyly, while not affecting the forelimb. An ectopic posterior SHH signaling center was evident in the anterior hindlimb bud of Gmnn-deficient embryos in this model. This center ectopically expressed Hoxd13, the HOXD13 target Shh, and the SHH target Ptch1, while these mutant hindlimb buds also had reduced levels of the cleaved, repressor form of GLI3, a SHH pathway antagonist. Together, this work delineates a new role for Gmnn in modulating Hox expression to pattern the vertebrate limb.


Asunto(s)
Embrión de Mamíferos/embriología , Geminina/metabolismo , Regulación del Desarrollo de la Expresión Génica , Miembro Posterior/embriología , Proteínas de Homeodominio/metabolismo , Factores de Transcripción/metabolismo , Animales , Embrión de Mamíferos/citología , Geminina/genética , Proteínas Hedgehog/genética , Proteínas Hedgehog/metabolismo , Miembro Posterior/citología , Proteínas de Homeodominio/genética , Ratones , Ratones Transgénicos , Receptor Patched-1/genética , Receptor Patched-1/metabolismo , Factores de Transcripción/genética
14.
Biosci Rep ; 39(2)2019 02 28.
Artículo en Inglés | MEDLINE | ID: mdl-30710064

RESUMEN

Geminin is an inhibitor of DNA replication licensing and cell cycle. Our previous study demonstrates that Geminin plays an important role in regulating phenotypic diversity and growth of vascular smooth cells (VSMCs). Ubiquitin-like with PHD and RING Finger domains 1 (UHRF1) is an epigenetic coordinator, whose RING domain confers intrinsic E3 ligase activity, mediating the ubiquitination of several proteins and the protein-protein interaction. Aberrant expression of UHRF1 was related to aggressiveness of multiple human malignancies, where knockdown of UHRF1 led to decreased proliferation of cancer cells. However, it is unclear whether proper UHRF1 function is involved in aberrant proliferation and phenotypic switching of VSMCs via altering Geminin protein levels. In present study, in UHRF1-overexpressing A10 cells, 3H-thymidine and 5-ethynyl-20-deoxyuridine (EdU) and CCK8 were used to examine the proliferation of VSMCs. RT-PCR and Western blot analyses were performed to investigate whether UHRF1-mediated effects were achieved by altering Geminin expression in VSMCs. RNA-seq analysis was performed to dissect related mechanisms or signaling pathways of these effects. The results of in vitro experiments suggested that UHRF1 prompted proliferation and cell cycle of VSMCs via the down-regulation of Geminin protein levels with no change in Geminin mRNA expression. Besides, PI3K-Akt signaling pathway was increased upon UHRF1 up-regulation. Our study demonstrated that overexpressing UHRF1 was involved in VSMCs proliferation through reducing inhibitory Geminin protein levels to promote cell cycle as well as activating PI3K-Akt signaling. This may provide key knowledge for the development of better strategies to prevent diseases related to VSMCs abnormal proliferation.


Asunto(s)
Proteínas Potenciadoras de Unión a CCAAT/genética , Proliferación Celular , Geminina/genética , Músculo Liso Vascular/citología , Ubiquitina-Proteína Ligasas/genética , Ciclo Celular , Línea Celular , Humanos , Músculo Liso Vascular/metabolismo , Regulación hacia Arriba
15.
J Virol ; 93(5)2019 03 01.
Artículo en Inglés | MEDLINE | ID: mdl-30541837

RESUMEN

Kaposi's sarcoma-associated herpesvirus (KSHV) is a human oncogenic virus, which maintains the persistent infection of the host by intermittently reactivating from latently infected cells to produce viral progenies. While it is established that the replication and transcription activator (RTA) viral transcription factor is required for the induction of lytic viral genes for KSHV lytic reactivation, it is still unknown to what extent RTA alters the host transcriptome to promote KSHV lytic cycle and viral pathogenesis. To address this question, we performed a comprehensive time course transcriptome analysis during KSHV reactivation in B-cell lymphoma cells and determined RTA-binding sites on both the viral and host genomes, which resulted in the identification of the core RTA-induced host genes (core RIGs). We found that the majority of RTA-binding sites at core RIGs contained the canonical RBP-Jκ-binding DNA motif. Subsequently, we demonstrated the vital role of the Notch signaling transcription factor RBP-Jκ for RTA-driven rapid host gene induction, which is consistent with RBP-Jκ being essential for KSHV lytic reactivation. Importantly, many of the core RIGs encode plasma membrane proteins and key regulators of signaling pathways and cell death; however, their contribution to the lytic cycle is largely unknown. We show that the cell cycle and chromatin regulator geminin and the plasma membrane protein gamma-glutamyltransferase 6, two of the core RIGs, are required for efficient KSHV reactivation and virus production. Our results indicate that host genes that RTA rapidly and directly induces can be pivotal for driving the KSHV lytic cycle.IMPORTANCE The lytic cycle of KSHV is involved not only in the dissemination of the virus but also viral oncogenesis, in which the effect of RTA on the host transcriptome is still unclear. Using genomics approaches, we identified a core set of host genes which are rapidly and directly induced by RTA in the early phase of KSHV lytic reactivation. We found that RTA does not need viral cofactors but requires its host cofactor RBP-Jκ for inducing many of its core RIGs. Importantly, we show a critical role for two of the core RIGs in efficient lytic reactivation and replication, highlighting their significance in the KSHV lytic cycle. We propose that the unbiased identification of RTA-induced host genes can uncover potential therapeutic targets for inhibiting KSHV replication and viral pathogenesis.


Asunto(s)
Herpesvirus Humano 8/genética , Proteínas Inmediatas-Precoces/genética , Proteína de Unión a la Señal Recombinante J de las Inmunoglobulinas/metabolismo , Transactivadores/genética , Activación Viral/genética , Línea Celular Tumoral , Geminina/genética , Geminina/metabolismo , Perfilación de la Expresión Génica , Regulación Viral de la Expresión Génica/genética , Células HEK293 , Herpesvirus Humano 8/metabolismo , Herpesvirus Humano 8/patogenicidad , Humanos , Interferencia de ARN , ARN Interferente Pequeño/genética , Latencia del Virus/genética , gamma-Glutamiltransferasa/genética , gamma-Glutamiltransferasa/metabolismo
16.
Stem Cells Transl Med ; 7(11): 792-805, 2018 11.
Artículo en Inglés | MEDLINE | ID: mdl-30272835

RESUMEN

Cell transplantation holds considerable promise for end-stage liver diseases but identifying a suitable, transplantable cell type has been problematic. Here, we describe a novel type of mesenchymal stem cells (MSCs) from human adipose tissue. These cells are different from previously reported MSCs, they are in the euchromatin state with epigenetic multipotency, and express pluripotent markers MYC, KLF4, and GMNN. Most of the genes associated with germ layer specification are modified by H3K4me3 or co-modified by H3K4me3 and H3K27me3. We named this new type of MSCs as adult multipotent adipose-derived stem cells (M-ADSCs). Using a four-step nonviral system, M-ADSCs can be efficiently Induced into hepatocyte like cells with expression of hepatocyte markers, drug metabolizing enzymes and transporters, and the other basic functional properties including albumin (ALB) secretion, glycogen storage, detoxification, low-density lipoprotein intake, and lipids accumulation. In vivo both M-ADSCs-derived hepatoblasts and hepatocytes could form vascularized liver-like tissue, secrete ALB and express metabolic enzymes. Single-cell RNA-seq was used to investigate the important stages in this conversion. M-ADSCs could be converted to a functionally multipotent state during the preinduction stage without undergoing reprogramming process. Our findings provide important insights into mechanisms underlying cell development and conversion. Stem Cells Translational Medicine 2018;7:792-805.


Asunto(s)
Geminina/metabolismo , Hepatocitos/metabolismo , Factores de Transcripción de Tipo Kruppel/metabolismo , Células Madre Mesenquimatosas/metabolismo , Proteínas Proto-Oncogénicas c-myc/metabolismo , Tejido Adiposo/citología , Animales , Biomarcadores/metabolismo , Diferenciación Celular , Geminina/genética , Factor Nuclear 4 del Hepatocito/genética , Factor Nuclear 4 del Hepatocito/metabolismo , Hepatocitos/citología , Humanos , Factor 4 Similar a Kruppel , Factores de Transcripción de Tipo Kruppel/genética , Hígado/metabolismo , Células Madre Mesenquimatosas/citología , Ratones , Ratones Desnudos , Proteínas Proto-Oncogénicas c-myc/genética , ARN/química , ARN/aislamiento & purificación , ARN/metabolismo , Análisis de Secuencia de ARN , Análisis de la Célula Individual , Transcriptoma , alfa-Fetoproteínas/genética , alfa-Fetoproteínas/metabolismo
17.
Biomed Res Int ; 2018: 6543196, 2018.
Artículo en Inglés | MEDLINE | ID: mdl-29984243

RESUMEN

During somitogenesis, Fgf8 maintains the predifferentiation stage of presomitic mesoderm (PSM) cells and its retraction gives a cue for somite formation. Delta/Notch initiates the expression of oscillation genes in the tail bud and subsequently contributes to somite formation in a periodic way. Whether there exists a critical factor coordinating Fgf8 and Notch signaling pathways is largely unknown. Here, we demonstrate that the loss of function of geminin gave rise to narrower somites as a result of derepressed Fgf8 gradient in the PSM and tail bud. Furthermore, in geminin morphants, the somite boundary could not form properly but the oscillation of cyclic genes was normal, displaying the blurry somitic boundary and disturbed somite polarity along the AP axis. In mechanism, these manifestations were mediated by the disrupted association of the geminin/Brg1 complex with intron 3 of mib1. The latter interaction was found to positively regulate mib1 transcription, Notch activity, and sequential somite segmentation during somitogenesis. In addition, geminin was also shown to regulate the expression of deltaD in mib1-independent way. Collectively, our data for the first time demonstrate that geminin regulates Fgf8 and Notch signaling to regulate somite segmentation during somitogenesis.


Asunto(s)
Geminina/fisiología , Receptores Notch/fisiología , Somitos/efectos de los fármacos , Desarrollo Embrionario , Factor 8 de Crecimiento de Fibroblastos/fisiología , Geminina/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo , Transducción de Señal
18.
J Pathol ; 246(2): 134-140, 2018 10.
Artículo en Inglés | MEDLINE | ID: mdl-29952003

RESUMEN

Geminin, a DNA replication licensing inhibitor, ensures faithful DNA replication in vertebrates. Several studies have shown that geminin depletion in vitro results in rereplication and DNA damage, whereas increased expression of geminin has been observed in human cancers. However, conditional inactivation of geminin during embryogenesis has not revealed any detectable DNA replication defects. In order to examine its role in vivo, we conditionally inactivated geminin in the murine colon and lung, and assessed chemically induced carcinogenesis. We show here that mice lacking geminin develop a significantly higher number of tumors and bear a larger tumor burden than sham-treated controls in urethane-induced lung and azoxymethane/dextran sodium sulfate-induced colon carcinogenesis. Survival is also significantly reduced in mice lacking geminin during lung carcinogenesis. A significant increase in the total number and grade of lesions (hyperplasias, adenomas, and carcinomas) was also confirmed by hematoxylin and eosin staining. Moreover, increased genomic aberrations, identified by increased ATR and γH2AX expression, was detected with immunohistochemistry analysis. In addition, we analyzed geminin expression in human colon cancer, and found increased expression, as well as a positive correlation with ATM/ATR levels and a non-monotonic association with γH2AX. Taken together, our data demonstrate that geminin acts as a tumor suppressor by safeguarding genome stability, whereas its overexpression is also associated with genomic instability. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.


Asunto(s)
Adenoma/genética , Carcinoma/genética , Neoplasias del Colon/genética , Geminina/genética , Genes Supresores de Tumor , Inestabilidad Genómica , Neoplasias Pulmonares/genética , Adenoma/inducido químicamente , Adenoma/metabolismo , Adenoma/patología , Animales , Proteínas de la Ataxia Telangiectasia Mutada/metabolismo , Azoximetano , Carcinoma/inducido químicamente , Carcinoma/metabolismo , Carcinoma/patología , Neoplasias del Colon/inducido químicamente , Neoplasias del Colon/metabolismo , Neoplasias del Colon/patología , Sulfato de Dextran , Modelos Animales de Enfermedad , Geminina/deficiencia , Geminina/metabolismo , Predisposición Genética a la Enfermedad , Histonas/metabolismo , Neoplasias Pulmonares/inducido químicamente , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Ratones Endogámicos C57BL , Ratones Noqueados , Fenotipo , Fosforilación , Uretano
19.
Chromosoma ; 127(2): 151-174, 2018 06.
Artículo en Inglés | MEDLINE | ID: mdl-29243212

RESUMEN

To ensure that the genetic material is accurately passed down to daughter cells during mitosis, dividing cells must duplicate their chromosomes and centrosomes once and only once per cell cycle. The same key steps-licensing, duplication, and segregation-control both the chromosome and the centrosome cycle, which must occur in concert to safeguard genome integrity. Aberrations in genome content or centrosome numbers lead to genomic instability and are linked to tumorigenesis. Such aberrations, however, can also be part of the normal life cycle of specific cell types. Multiciliated cells best exemplify the deviation from a normal centrosome cycle. They are post-mitotic cells which massively amplify their centrioles, bypassing the rule for once-per-cell-cycle centriole duplication. Hundreds of centrioles dock to the apical cell surface and generate motile cilia, whose concerted movement ensures fluid flow across epithelia. The early steps that control the generation of multiciliated cells have lately started to be elucidated. Geminin and the vertebrate-specific GemC1 and McIdas are distantly related coiled-coil proteins, initially identified as cell cycle regulators associated with the chromosome cycle. Geminin is required to ensure once-per-cell-cycle genome replication, while McIdas and GemC1 bind to Geminin and are implicated in DNA replication control. Recent findings highlight Geminin family members as early regulators of multiciliogenesis. GemC1 and McIdas specify the multiciliate cell fate by forming complexes with the E2F4/5 transcription factors to switch on a gene expression program leading to centriole amplification and cilia formation. Positive and negative interactions among Geminin family members may link cell cycle control to centriole amplification and multiciliogenesis, acting close to the point of transition from proliferation to differentiation. We review key steps of centrosome duplication and amplification, present the role of Geminin family members in the centrosome and chromosome cycle, and discuss links with disease.


Asunto(s)
Centriolos/metabolismo , Cilios/metabolismo , Geminina/genética , Genoma , Mitosis , Animales , Carcinogénesis/genética , Carcinogénesis/metabolismo , Carcinogénesis/patología , Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centriolos/ultraestructura , Cilios/ultraestructura , Replicación del ADN , Enanismo/genética , Enanismo/metabolismo , Enanismo/patología , Factor de Transcripción E2F4/genética , Factor de Transcripción E2F4/metabolismo , Factor de Transcripción E2F5/genética , Factor de Transcripción E2F5/metabolismo , Geminina/metabolismo , Regulación de la Expresión Génica , Inestabilidad Genómica , Humanos , Proteínas Nucleares/genética , Proteínas Nucleares/metabolismo , Unión Proteica , Transducción de Señal , Factores de Transcripción
20.
mBio ; 8(6)2017 11 07.
Artículo en Inglés | MEDLINE | ID: mdl-29114028

RESUMEN

The cellular response to viral infection is usually studied at the level of cell populations. Currently, it remains an open question whether and to what extent cell-to-cell variability impacts the course of infection. Here we address this by dynamic proteomics-imaging and tracking 400 yellow fluorescent protein (YFP)-tagged host proteins in individual cells infected by herpes simplex virus 1. By quantifying time-lapse fluorescence imaging, we analyze how cell-to-cell variability impacts gene expression from the viral genome. We identify two proteins, RFX7 and geminin, whose levels at the time of infection correlate with successful initiation of gene expression. These proteins are cell cycle markers, and we find that the position in the cell cycle at the time of infection (along with the cell motility and local cell density) can reasonably predict in which individual cells gene expression from the viral genome will commence. We find that the onset of cell division dramatically impacts the progress of infection, with 70% of dividing cells showing no additional gene expression after mitosis. Last, we identify four host proteins that are specifically modulated in infected cells, of which only one has been previously recognized. SUMO2 and RPAP3 levels are rapidly reduced, while SLTM and YTHDC1 are redistributed to form nuclear foci. These modulations are dependent on the expression of ICP0, as shown by infection with two mutant viruses that lack ICP0. Taken together, our results provide experimental validation for the long-held notion that the success of infection is dependent on the state of the host cell at the time of infection.IMPORTANCE High-throughput assays have revolutionized many fields in biology, both by allowing a more global understanding of biological processes and by deciphering rare events in subpopulations. Here we use such an assay, dynamic proteomics, to study viral infection at the single-cell level. We follow tens of thousands of individual cells infected by herpes simplex virus using fluorescence live imaging. Our results link the state of a cell at the time of virus infection with its probability to successfully initiate gene expression from the viral genome. Further, we identified three cellular proteins that were previously unknown to respond to viral infection. We conclude that dynamic proteomics provides a powerful tool to study single-cell differences during viral infection.


Asunto(s)
Herpesvirus Humano 1/fisiología , Interacciones Huésped-Patógeno , Proteómica , Proteínas Reguladoras de la Apoptosis , Proteínas Portadoras/metabolismo , Ciclo Celular , Línea Celular Tumoral , Geminina/genética , Geminina/metabolismo , Regulación de la Expresión Génica , Genoma Viral , Herpesvirus Humano 1/genética , Herpesvirus Humano 1/metabolismo , Humanos , Proteínas Inmediatas-Precoces/genética , Proteínas Inmediatas-Precoces/metabolismo , Mitosis , Mutación , Imagen Óptica , Análisis de la Célula Individual/métodos , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/genética , Proteínas Modificadoras Pequeñas Relacionadas con Ubiquitina/metabolismo , Biología de Sistemas , Ubiquitina-Proteína Ligasas/genética , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Virales/genética , Proteínas Virales/metabolismo , Replicación Viral
SELECCIÓN DE REFERENCIAS
DETALLE DE LA BÚSQUEDA
...