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1.
Development ; 149(12)2022 06 15.
Artículo en Inglés | MEDLINE | ID: mdl-35608036

RESUMEN

HBXIP, also named LAMTOR5, has been well characterized as a transcriptional co-activator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5, and that depletion of Hbxip compromises the self-renewal of embryonic stem cells (ESCs), with reduced expression of pluripotency genes, reduced cell proliferation and decreased colony-forming capacity. In addition, both Hbxip-/- ESCs and E7.5 embryos displayed defects in ectodermal and mesodermal differentiation. Mechanistically, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, displayed differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/- and p18-/- mice, lacking subunits of the Ragulator complex, also shared similar phenotypes, embryonic lethality and retarded growth around E7-E8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.


Asunto(s)
Desarrollo Embrionario , Células Madre Embrionarias , Animales , Diferenciación Celular/genética , Desarrollo Embrionario/genética , Diana Mecanicista del Complejo 1 de la Rapamicina/genética , Ratones , Transducción de Señal
2.
Nucleic Acids Res ; 49(20): 11596-11613, 2021 11 18.
Artículo en Inglés | MEDLINE | ID: mdl-34723322

RESUMEN

Using the programmable RNA-sequence binding domain of the Pumilio protein, we FLAG-tagged Xist (inactivated X chromosome specific transcript) in live mouse cells. Affinity pulldown coupled to mass spectrometry was employed to identify a list of 138 candidate Xist-binding proteins, from which, Ssb (also known as the lupus autoantigen La) was validated as a protein functionally critical for X chromosome inactivation (XCI). Extensive XCI defects were detected in Ssb knockdown cells, including chromatin compaction, death of female mouse embryonic stem cells during in vitro differentiation and chromosome-wide monoallelic gene expression pattern. Live-cell imaging of Xist RNA reveals the defining XCI defect: Xist cloud formation. Ssb is a ubiquitous and versatile RNA-binding protein with RNA chaperone and RNA helicase activities. Functional dissection of Ssb shows that the RNA chaperone domain plays critical roles in XCI. In Ssb knockdown cells, Xist transcripts are unstable and misfolded. These results show that Ssb is critically involved in XCI, possibly as a protein regulating the in-cell structure of Xist.


Asunto(s)
Pliegue del ARN , ARN Largo no Codificante/química , Proteínas de Unión al ARN/metabolismo , Inactivación del Cromosoma X , Animales , Autoantígenos/química , Autoantígenos/metabolismo , Sitios de Unión , Línea Celular , Ratones , Unión Proteica , ARN Largo no Codificante/metabolismo , Proteínas de Unión al ARN/química , Proteínas de Unión al ARN/genética
3.
J Mol Biol ; 430(17): 2734-2746, 2018 08 17.
Artículo en Inglés | MEDLINE | ID: mdl-29800566

RESUMEN

Xist (inactivated X chromosome specific transcript) is a prototype long noncoding RNA in charge of epigenetic silencing of one X chromosome in each female cell in mammals. In a genetic screen, we identify Mageb3 and its homologs Mageb1 and Mageb2 as genes functionally required for Xist-mediated gene silencing. Mageb1-3 are previously uncharacterized genes belonging to the MAGE (melanoma-associated antigen) gene family. Mageb1-3 are expressed in undifferentiated ES cells and early stages of in vitro differentiation, a critical time window of X chromosome inactivation. Mageb3 showed both cytoplasmic and nuclear localization without enrichment on the inactive X (Xi). Mageb3 interacted with Polycomb group ring finger 3 (Pcgf3), a RING finger protein involved in recruiting Polycomb activities onto Xi. Mageb3 overexpression stabilized Pcgf3 protein. Mageb1-3 gene knockout affected H3K27me3 enrichment and the spreading of gene silencing along Xi. These data suggested that Mageb3 might regulate the recruitment of the Polycomb complex onto Xi and subsequent H3K27me3 modification through Pcgf3. Moreover, the nucleolar enrichment of Mageb3 was diminished when nuclear matrix factor hnRNP U is overexpressed, implying the interaction between Mageb3 and nuclear matrix, which is another possible mechanism for Mageb3 to regulate X chromosome inactivation.


Asunto(s)
Antígenos de Neoplasias/metabolismo , Embrión de Mamíferos/metabolismo , Fibroblastos/metabolismo , Silenciador del Gen , Genoma , Proteínas de Neoplasias/metabolismo , Interferencia de ARN , Inactivación del Cromosoma X , Animales , Antígenos de Neoplasias/genética , Núcleo Celular , Embrión de Mamíferos/citología , Fibroblastos/citología , Ribonucleoproteína Heterogénea-Nuclear Grupo U/genética , Ribonucleoproteína Heterogénea-Nuclear Grupo U/metabolismo , Masculino , Ratones , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas del Grupo Polycomb/genética , Proteínas del Grupo Polycomb/metabolismo , Fracciones Subcelulares , Transcripción Genética
4.
Sci Rep ; 6: 26804, 2016 05 26.
Artículo en Inglés | MEDLINE | ID: mdl-27226076

RESUMEN

The COP9 signalosome has been implicated in pluripotency maintenance of human embryonic stem cells. Yet, the mechanism for the COP9 signalosome to regulate pluripotency remains elusive. Through knocking down individual COP9 subunits, we demonstrate that Cops2, but not the whole COP9 signalosome, is essential for pluripotency maintenance in mouse embryonic stem cells. Down-regulation of Cops2 leads to reduced expression of pluripotency genes, slower proliferation rate, G2/M cell cycle arrest, and compromised embryoid differentiation of embryonic stem cells. Cops2 also facilitates somatic cell reprogramming. We further show that Cops2 binds to Nanog protein and prevent the degradation of Nanog by proteasome. Moreover, Cops2 functions as transcriptional corepressor to facilitate pluripotency maintenance. Altogether, our data reveal the essential role and novel mechanisms of Cops2 in pluripotency maintenance.


Asunto(s)
Complejo del Señalosoma COP9/metabolismo , Células Madre Embrionarias/citología , Regulación del Desarrollo de la Expresión Génica/fisiología , Proteína Homeótica Nanog/metabolismo , Proteínas Nucleares/fisiología , Factores de Transcripción/fisiología , Transcripción Genética/genética , Secuencia de Aminoácidos , Animales , Complejo del Señalosoma COP9/antagonistas & inhibidores , Complejo del Señalosoma COP9/genética , Complejo del Señalosoma COP9/fisiología , Autorrenovación de las Células , Técnicas de Reprogramación Celular , Cuerpos Embrioides , Células Madre Embrionarias/metabolismo , Técnicas de Silenciamiento del Gen , Ratones , Proteína Homeótica Nanog/genética , Proteínas Nucleares/antagonistas & inhibidores , Proteínas Nucleares/genética , Factor 3 de Transcripción de Unión a Octámeros/antagonistas & inhibidores , Factor 3 de Transcripción de Unión a Octámeros/genética , Factor 3 de Transcripción de Unión a Octámeros/fisiología , Estabilidad Proteica , Proteolisis , Interferencia de ARN , ARN Interferente Pequeño/genética , Factores de Transcripción SOXC/antagonistas & inhibidores , Factores de Transcripción SOXC/genética , Factores de Transcripción SOXC/fisiología , Ovinos/genética , Factores de Transcripción/antagonistas & inhibidores , Factores de Transcripción/genética
5.
Biotechniques ; 56(4): 198-201, 2014.
Artículo en Inglés | MEDLINE | ID: mdl-24724846

RESUMEN

Removing an antibiotic resistance gene allows the same antibiotic to be re-used in the next round of genetic manipulation. Here we applied the CRISPR/Cas system to disrupt the puromycin resistance gene in an engineered mouse embryonic stem cell line and then re-used puromycin selection in the resulting cells to establish stable reporter cell lines. With the CRISPR/Cas system, pre-engineered sequences, such as loxP or FRT, are not required. Thus, this technique can be used to disrupt antibiotic resistance genes that cannot be removed by the Cre-loxP and Flp-FRT systems.


Asunto(s)
Antibacterianos/farmacología , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Farmacorresistencia Microbiana/genética , Vectores Genéticos/genética , Animales , Sistemas CRISPR-Cas , Línea Celular , Supervivencia Celular/efectos de los fármacos , Células Madre Embrionarias , Técnicas Genéticas , Ratones , Puromicina/farmacología , Recombinación Genética/genética
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