RESUMEN
Mcm2, a subunit of the minichromosome maintenance proteins 2-7 (Mcm2-7) helicase best known for its role in DNA replication, contains a histone binding motif that facilitates the transfer of parental histones following DNA replication. Here, we show that Mcm2 is important for the differentiation of mouse embryonic stem (ES) cells. The Mcm2-2A mutation defective in histone binding shows defects in silencing of pluripotent genes and the induction of lineage-specific genes. The defects in the induction of lineage-specific genes in the mutant cells are likely, at least in part, due to reduced binding to Asf1a, a histone chaperone that binds Mcm2 and is important for nucleosome disassembly at bivalent chromatin domains containing repressive H3K27me3 and active H3K4me3 modifications during differentiation. Mcm2 localizes at transcription starting sites and the binding of Mcm2 at gene promoters is disrupted in both Mcm2-2A ES cells and neural precursor cells (NPCs). Reduced Mcm2 binding at bivalent chromatin domains in Mcm2-2A ES cells correlates with decreased chromatin accessibility at corresponding sites in NPCs. Together, our studies reveal a novel function of Mcm2 in ES cell differentiation, likely through manipulating chromatin landscapes at bivalent chromatin domains.
Asunto(s)
Histonas , Células-Madre Neurales , Animales , Ratones , Histonas/metabolismo , Células-Madre Neurales/metabolismo , Cromatina , Ensamble y Desensamble de Cromatina , Diferenciación Celular , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Chaperonas Moleculares/metabolismoRESUMEN
Magnesium chloride and polyamines stabilize DNA and chromatin. Furthermore, they can induce nucleosome aggregation and chromatin condensation in vitro. To determine the effects of elevating the cation concentrations in the nucleus of a living cell, we microinjected various concentrations of mono-, di- and polyvalent cation solutions into the nuclei of mouse embryonic stem (ES) cells and traced their fates. Here, we show that an elevation of either MgCl2, spermidine or spermine concentration in the nucleus exerts a significant effect on mouse ES cells, and can differentiate a certain population of the cells into trophectoderm, a lineage that mouse ES cells do not normally generate, or endoderm. It is hypothesized that the cell differentiation was most probably caused by the condensation of chromatin including the Oct3/4 locus, which was induced by the elevated concentrations of these cations.
Asunto(s)
Endodermo/citología , Cloruro de Magnesio/química , Células Madre Embrionarias de Ratones/citología , Poliaminas/química , Animales , Cationes , Diferenciación Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Cromatina/química , Relación Dosis-Respuesta a Droga , Ratones , Espermidina/química , Espermina/químicaRESUMEN
Differentiation of embryonic stem (ES) cells is a heterogeneous process which is influenced by different parameters, including growth and differentiation factors. The aim of the present study was to investigate the effect of bone morphogenetic protein-4 (BMP4) signaling on differentiation of mouse ES cells to endodermal lineages. For this purpose, differentiation of the ES cells was induced by embryoid body (EB) formation through hanging drop method. During the suspension stage, EBs were treated with BMP4 in a medium containing either fetal bovine serum (FBS) or knockout serum replacement (KoSR). After plating, EBs showed differentiation to a heterogeneous population of specialized cell types. Two weeks after plating, all the experimental groups expressed three germ layer markers and some primitive and definitive endoderm-specific genes. Quantitative real-time PCR analysis showed higher expression levels of Sox17, Pdx1, Cdx2 and Villin mRNAs in the KoSR plus BMP4 condition and higher Gata4 and Afp expression levels in the FBS plus BMP4 condition. Formation of visceral endoderm and derivatives of definitive endoderm was detected in the BMP4 treated EBs. In conclusion, we demonstrated that both BMP4 signaling and serum composition have significant roles in differentiation of mouse ES cells towards endodermal lineages.
RESUMEN
Oxidative stresses induced by reactive oxygen species (ROS) have been shown to be involved in several physiological and pathophysiological processes, such as cell proliferation and differentiation. Steroid hormones can protect cells against apoptosis or induce cell proliferation by several mechanisms. Among androgenic hormones, dihydrotestosterone (DHT) is generated by a 5alpha- reduction of testosterone. Unlike testosterone, DHT cannot be aromatized to estradiol, therefore DHT is considered a pure androgenic steroid. This study was conducted to examine the effect of DHT (10(-7) M) on H(2)O(2) (10(-3) M) -induced injuries in mouse embryonic stem (ES) cells. H(2)O(2) induced ROS generation and increased lipid peroxide formation and DNA fragmentation. These effects of H(2)O(2) were inhibited by pretreatment with DHT. H(2)O(2) also increased the phosphorylation of p38 MAPK, SAPK/JNK and nuclear factor kappa B (NF-kappaB), but DHT blocked these effects. Moreover, H(2)O(2) decreased DNA synthesis and the levels of cell cycle regulatory proteins [cyclin D1, cyclin E, cyclin-dependent kinase (CDK) 2, and CDK 4]. These effects of H(2)O(2) were inhibited by pretreatment with DHT. In conclusion, DHT may partially prevent H(2)O(2)-induced cell injury through inhibition of ROS and ROS-induced activation of p38 MAPK, SAPK/JNK and NF-kappaB in mouse ES cells.