RESUMEN
Lineage specification of the three germ layers occurs during early embryogenesis and is critical for normal development. The nucleosome remodeling and deacetylase (NuRD) complex is a repressive chromatin modifier that plays a role in lineage commitment. However, the role of chromodomain helicase DNA-binding protein 4 (CHD4), one of the core subunits of the NuRD complex, in neural lineage commitment is poorly understood. Here, we report that the CHD4/NuRD complex plays a critical role in neural differentiation of mouse embryonic stem cells (ESCs). We found that RNAi-mediated Chd4 knockdown suppresses neural differentiation, as did knockdown of methyl-CpG-binding domain protein Mbd3, another NuRD subunit. Chd4 and Mbd3 knockdowns similarly affected changes in global gene expression during neural differentiation and up-regulated several mesendodermal genes. However, inhibition of mesendodermal genes by knocking out the master regulators of mesendodermal lineages, Brachyury and Eomes, through a CRISPR/Cas9 approach could not restore the impaired neural differentiation caused by the Chd4 knockdown, suggesting that CHD4 controls neural differentiation by not repressing other lineage differentiation processes. Notably, Chd4 knockdown increased the acetylation levels of p53, resulting in increased protein levels of p53. Double knockdown of Chd4 and p53 restored the neural differentiation rate. Furthermore, overexpression of BCL2, a downstream factor of p53, partially rescued the impaired neural differentiation caused by the Chd4 knockdown. Our findings reveal that the CHD4/NuRD complex regulates neural differentiation of ESCs by down-regulating p53.
Asunto(s)
Diferenciación Celular , ADN Helicasas/metabolismo , Regulación hacia Abajo , Neuronas/metabolismo , Nucleosomas/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , Animales , Línea Celular , ADN Helicasas/genética , Técnicas de Silenciamiento del Gen , Ratones , Células Madre Embrionarias de Ratones , Neuronas/citología , Nucleosomas/genética , Proteínas Proto-Oncogénicas c-bcl-2/biosíntesis , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteína p53 Supresora de Tumor/genéticaRESUMEN
The Activin/Nodal/TGF-ß signaling pathway plays a major role in maintaining mouse epiblast stem cells (EpiSCs). The EpiSC-maintaining medium, which contains Activin A and bFGF, induces differentiation of mouse embryonic stem cells (ESCs) to EpiSCs. Here, we show that Activin A also has an ability to efficiently propagate ESCs without differentiation to EpiSCs when combined with a MEK inhibitor PD0325901. ESCs cultured in Activin+PD retained high-level expression of naive pluripotency-related transcription factors. Genomewide analysis showed that the gene expression profile of ESCs cultured in Activin+PD resembles that of ESCs cultured in 2i. ESCs cultured in Activin+PD also showed features common to the naive pluripotency of ESCs, including the preferential usage of the Oct4 distal enhancer and the self-renewal response to Wnt pathway activation. Our finding shows a role of Activin/Nodal/TGF-ß signaling in stabilizing self-renewal gene regulatory networks in ESCs.
Asunto(s)
Activinas/farmacología , Sistema de Señalización de MAP Quinasas/efectos de los fármacos , Células Madre Embrionarias de Ratones/efectos de los fármacos , Células Madre Embrionarias de Ratones/metabolismo , Activinas/química , Animales , Diferenciación Celular/fisiología , Proliferación Celular/fisiología , Células Cultivadas , Estratos Germinativos/citología , Estratos Germinativos/metabolismo , Células HEK293 , Humanos , Sistema de Señalización de MAP Quinasas/fisiología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/antagonistas & inhibidores , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Células Madre Embrionarias de Ratones/citología , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/efectos de los fármacos , Células Madre Pluripotentes/metabolismo , Factores de Transcripción/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Vía de Señalización WntRESUMEN
The discreteness of cell fates is an inherent and fundamental feature of multicellular organisms. Here we show that cross-antagonistic mechanisms of actions of MyoD and PPARγ, which are the master regulators of muscle and adipose differentiation, respectively, confer robustness to the integrity of cell differentiation. Simultaneous expression of MyoD and PPARγ in mesenchymal stem/stromal cells led to the generation of a mixture of multinucleated myotubes and lipid-filled adipocytes. Interestingly, hybrid cells (i.e., lipid-filled myotubes) were not generated, suggesting that these differentiation programs are mutually exclusive. Mechanistically, although exogenously expressed MyoD was rapidly degraded in adipocytes through ubiquitin-proteasome pathways, exogenously expressed PPARγ was not downregulated in myotubes. In PPARγ-expressing myotubes, PPARγ-dependent histone hyperacetylation was inhibited in a subset of adipogenic gene loci, including that of C/EBPα, an essential effector of PPARγ. Thus, the cross-repressive interactions between MyoD- and PPARγ-induced differentiation programs ensure discrete cell-fate decisions.
