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1.
Stem Cells ; 37(6): 743-753, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-30801858

RESUMEN

The scaffold protein Symplekin (Sympk) is involved in cytoplasmic RNA polyadenylation, transcriptional modulation, and the regulation of epithelial differentiation and proliferation via tight junctions. It is highly expressed in embryonic stem cells (ESCs), in which its role remains unknown. In this study, we found Sympk overexpression in mouse ESCs significantly increased colony formation, and Sympk deletion via CRISPR/Cas9 decreased colony formation. Sympk promoted ESC growth and its overexpression sustained ESC pluripotency, as assessed by teratoma and chimeric mouse formation. Genomic stability was preserved in these cells after long-term passage. The domain of unknown function 3453 (DUF3453) in Sympk was required for its interaction with the key pluripotent factor Oct4, and its depletion led to impaired colony formation. Sympk activated proliferation-related genes and suppressed differentiation-related genes. Our results indicate that Sympk interacts with Oct4 to promote self-renewal and pluripotency in ESCs and preserves genome integrity; accordingly, it has potential value for stem cell therapies. Stem Cells 2019;37:743-753.


Asunto(s)
Proteínas del Citoesqueleto/genética , Regulación del Desarrollo de la Expresión Génica , Genoma , Proteínas de la Membrana/genética , Células Madre Embrionarias de Ratones/metabolismo , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/genética , Células Madre Pluripotentes/metabolismo , Animales , Sistemas CRISPR-Cas , Diferenciación Celular , Línea Celular , Proliferación Celular , Proteínas del Citoesqueleto/deficiencia , Eliminación de Gen , Perfilación de la Expresión Génica , Genes Reporteros , Inestabilidad Genómica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas de la Membrana/deficiencia , Ratones , Células Madre Embrionarias de Ratones/citología , Proteínas Nucleares/deficiencia , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Células Madre Pluripotentes/citología , Transducción de Señal , Teratoma/genética , Teratoma/metabolismo , Teratoma/patología , Uniones Estrechas/metabolismo
2.
J Cell Sci ; 129(14): 2767-77, 2016 07 15.
Artículo en Inglés | MEDLINE | ID: mdl-27235421

RESUMEN

Skeletal myogenesis is a multistep process in which basic helix-loop-helix (bHLH) transcription factors, such as MyoD (also known as MyoD1), bind to E-boxes and activate downstream genes. Ccndbp1 is a HLH protein that lacks a DNA-binding region, and its function in skeletal myogenesis is currently unknown. We generated Ccndbp1-null mice by using CRISPR-Cas9. Notably, in Ccndbp1-null mice, the cross sectional area of the skeletal tibialis anterior muscle was smaller, and muscle regeneration ability and grip strength were impaired, compared with those of wild type. This phenotype resembled that of myofiber hypotrophy in some human myopathies or amyoplasia. Ccndbp1 expression was upregulated during C2C12 myogenesis. Ccndbp1 overexpression promoted myogenesis, whereas knockdown of Ccndbp1 inhibited myogenic differentiation. Co-transfection of Ccndbp1 with MyoD and/or E47 (encoded by TCF3) significantly enhanced E-box-dependent transcription. Furthermore, Ccndbp1 physically associated with MyoD but not E47. These data suggest that Ccndbp1 regulates muscle differentiation by interacting with MyoD and enhancing its binding to target genes. Our study newly identifies Ccndbp1 as a positive modulator of skeletal myogenic differentiation in vivo and in vitro, providing new insights in order to decipher the complex network involved in skeletal myogenic development and related diseases.


Asunto(s)
Desarrollo de Músculos , Músculo Esquelético/metabolismo , Factores de Transcripción/metabolismo , Animales , Cardiotoxinas/toxicidad , Diferenciación Celular/efectos de los fármacos , Línea Celular , Elementos E-Box/genética , Técnicas de Silenciamiento del Gen , Hipertrofia , Ratones Noqueados , Desarrollo de Músculos/efectos de los fármacos , Fibras Musculares Esqueléticas/efectos de los fármacos , Fibras Musculares Esqueléticas/patología , Músculo Esquelético/efectos de los fármacos , Proteína MioD/metabolismo , Fenotipo , Unión Proteica/efectos de los fármacos , Regeneración/efectos de los fármacos , Factores de Transcripción/deficiencia , Activación Transcripcional/efectos de los fármacos , Activación Transcripcional/genética , Regulación hacia Arriba/efectos de los fármacos
4.
Nat Cell Biol ; 25(7): 1004-1016, 2023 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-37322289

RESUMEN

Proper repair of DNA damage lesions is essential to maintaining genome integrity and preventing the development of human diseases, including cancer. Increasing evidence suggests the importance of the nuclear envelope in the spatial regulation of DNA repair, although the mechanisms of such regulatory processes remain poorly defined. Through a genome-wide synthetic viability screen for PARP-inhibitor resistance using an inducible CRISPR-Cas9 platform and BRCA1-deficient breast cancer cells, we identified a transmembrane nuclease (renamed NUMEN) that could facilitate compartmentalized and non-homologous end joining-dependent repair of double-stranded DNA breaks at the nuclear periphery. Collectively, our data demonstrate that NUMEN generates short 5' overhangs through its endonuclease and 3'→5' exonuclease activities, promotes the repair of DNA lesions-including heterochromatic lamina-associated domain breaks as well as deprotected telomeres-and functions as a downstream effector of DNA-dependent protein kinase catalytic subunit. These findings underline the role of NUMEN as a key player in DNA repair pathway choice and genome-stability maintenance, and have implications for ongoing research into the development and treatment of genome instability disorders.


Asunto(s)
Roturas del ADN de Doble Cadena , Reparación del ADN , Humanos , Reparación del ADN/genética , Proteínas de Unión al ADN/metabolismo , Reparación del ADN por Unión de Extremidades , Endonucleasas/genética
5.
Bioact Mater ; 10: 32-47, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34901527

RESUMEN

Regardless of the advancement of synthetic bone substitutes, allograft-derived bone substitutes still dominate in the orthopaedic circle in the treatments of bone diseases. Nevertheless, the stringent devitalization process jeopardizes their osseointegration with host bone and therefore prone to long-term failure. Hence, improving osseointegration and transplantation efficiency remains important. The alteration of bone tissue microenvironment (TME) to facilitate osseointegration has been generally recognized. However, the concept of exerting metal ionic cue in bone TME without compromising the mechanical properties of bone allograft is challenging. To address this concern, an interfacial tissue microenvironment with magnesium cationc cue was tailored onto the gamma-irradiated allograft bone using a customized magnesium-plasma surface treatment. The formation of the Mg cationic cue enriched interfacial tissue microenvironment on allograft bone was verified by the scanning ion-selective electrode technique. The cellular activities of human TERT-immortalized mesenchymal stem cells on the Mg-enriched grafts were notably upregulated. In the animal test, superior osseointegration between Mg-enriched graft and host bone was found, whereas poor integration was observed in the gamma-irradiated controls at 28 days post-operation. Furthermore, the bony in-growth appeared on magnesium-enriched allograft bone was significant higher. The mechanism possibly correlates to the up-regulation of integrin receptors in mesenchymal stem cells under modified bone TME that directly orchestrate the initial cell attachment and osteogenic differentiation of mesenchymal stem cells. Lastly, our findings demonstrate the significance of magnesium cation modified bone allograft that can potentially translate to various orthopaedic procedures requiring bone augmentation.

6.
Stem Cell Res Ther ; 11(1): 314, 2020 07 23.
Artículo en Inglés | MEDLINE | ID: mdl-32703285

RESUMEN

BACKGROUND: Embryonic stem cells (ESCs) are important source of clinical stem cells for therapy, so dissecting the functional gene regulatory network involved in their self-renewal and proliferation is an urgent task. We previously reported that Ddx56 interacts with the core transcriptional factor Oct4 by mass spectrometry analysis in ESCs. However, the exact function of Ddx56 in ESCs remains unclear. METHODS: We investigated the role of Ddx56 in mouse ESCs (mESCs) through both gain- and loss-of-function strategies. The effect of Ddx56 on mESCs was determined based on morphological changes, involvement in the network of pluripotency markers (Nanog, Oct4, Sox2), and altered lineage marker expression. In addition, the role of Ddx56 in mESCs was evaluated by polysome fractionation, qRT-PCR, and co-immunoprecipitation (co-IP). Finally, RNA sequencing was applied to explore potential network regulation by Ddx56 in mESCs. RESULT: We found that Ddx56 participated in ribosome assembly, as knockout or RNAi knockdown of Ddx56 led to ribosome dysfunction and cell lethality. Surprisingly, exogenous expression of C-terminal domain truncated Ddx56 (Ddx56 ΔC-ter) did not affect ribosome assembly, but decreased mESC proliferation by downregulation of proliferation-related genes and cell cycle changing. In terms of mechanism, Ddx56 interacted with the Oct4 and Sox2 complex by binding to Sox2, whereas Ddx56 ΔC-ter showed weaker interaction with Sox2 and led to retardation of mESC proliferation. CONCLUSIONS: Ddx56 maintains ESC proliferation by conventional regulation of ribosome assembly and interaction with the Oct4 and Sox2 complex.


Asunto(s)
Células Madre Embrionarias de Ratones , Factor 3 de Transcripción de Unión a Octámeros , Animales , Diferenciación Celular , Proliferación Celular , Células Madre Embrionarias/metabolismo , Ratones , Células Madre Embrionarias de Ratones/metabolismo , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/metabolismo , Interferencia de ARN , Ribosomas/genética , Ribosomas/metabolismo
7.
Protein Cell ; 11(9): 641-660, 2020 09.
Artículo en Inglés | MEDLINE | ID: mdl-32458346

RESUMEN

In mammalian cells, long noncoding RNAs (lncRNAs) form complexes with proteins to execute various biological functions such as gene transcription, RNA processing and other signaling activities. However, methods to track endogenous lncRNA dynamics in live cells and screen for lncRNA interacting proteins are limited. Here, we report the development of CERTIS (CRISPR-mediated Endogenous lncRNA Tracking and Immunoprecipitation System) to visualize and isolate endogenous lncRNA, by precisely inserting a 24-repeat MS2 tag into the distal end of lncRNA locus through the CRISPR/Cas9 technology. In this study, we show that CERTIS effectively labeled the paraspeckle lncRNA NEAT1 without disturbing its physiological properties and could monitor the endogenous expression variation of NEAT1. In addition, CERTIS displayed superior performance on both short- and long-term tracking of NEAT1 dynamics in live cells. We found that NEAT1 and paraspeckles were sensitive to topoisomerase I specific inhibitors. Moreover, RNA Immunoprecipitation (RIP) of the MS2-tagged NEAT1 lncRNA successfully revealed several new protein components of paraspeckle. Our results support CERTIS as a tool suitable to track both spatial and temporal lncRNA regulation in live cells as well as study the lncRNA-protein interactomes.


Asunto(s)
Sistemas CRISPR-Cas , Rastreo Celular , Perfilación de la Expresión Génica , Técnicas de Sustitución del Gen , Proteómica , ARN Largo no Codificante/metabolismo , Células HEK293 , Humanos , ARN Largo no Codificante/genética
9.
Biomaterials ; 149: 29-40, 2017 Dec.
Artículo en Inglés | MEDLINE | ID: mdl-28988062

RESUMEN

Cancer stem cells (CSCs) are believed to have a critical role in tumorigenesis, metastasis, therapeutic resistance or recurrence. Therefore, strategies designed to specifically target and eliminate CSCs have become one of the most promising and desirable ways for tumor treatment. Osteosarcoma stem cells (OSCs), the CSCs in osteosarcoma (OS), are critically associated with OS progression. Here, we show that single-walled carbon nanotubes (SWCNTs), including unmodified SWCNT (SWCNT-Raw) and SWCNT-COOH, have the ability to specifically inhibit the process of TGFß1-induced OS cells dedifferentiation, prevent the stem cell phenotypes acquisition in OS cells and reduce the OSC viability under conditions which mimic the OS microenvironment. Concurrently, SWCNT treatment significantly down-regulates the expression of OSC markers in OS, and markedly reduces the tumor microvessel density and tumor growth. Furthermore, we found that SWCNT could suppress the TGFß1-induced activation of TGFß type I receptor and downstream signaling, which are key for the OSC formation and maintenance. Our results reveal an unexpected function of SWCNT in negative modulation of OSCs, and provide significant implications for the potential CSCs-targeted therapeutic applications of SWCNT.


Asunto(s)
Neoplasias Óseas/tratamiento farmacológico , Nanotubos de Carbono/química , Células Madre Neoplásicas/efectos de los fármacos , Osteosarcoma/tratamiento farmacológico , Factor de Crecimiento Transformador beta1/metabolismo , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , Apoptosis , Neoplasias Óseas/irrigación sanguínea , Neoplasias Óseas/metabolismo , Neoplasias Óseas/patología , Desdiferenciación Celular , Línea Celular Tumoral , Proliferación Celular , Regulación hacia Abajo , Femenino , Humanos , Ratones Endogámicos BALB C , Células Madre Neoplásicas/metabolismo , Células Madre Neoplásicas/patología , Osteosarcoma/irrigación sanguínea , Osteosarcoma/metabolismo , Osteosarcoma/patología , Tamaño de la Partícula , Transducción de Señal , Microambiente Tumoral
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