Then, the cells were washed twice with warm PBS

tudies here on Utx. To elucidate the roles of H3K27 demethylation on the expression of pluripotent genes, we MedChemExpress Tipifarnib treated female undifferentiated ESCs with GSK-J4, a small molecule inhibitor specific for Utx and Jmjd3 H3K27 demethylase catalytic activity. Inhibition of H3K27me3 demethylation following GSK-J4 treatment was confirmed by quantitative chromatin immunoprecipitation using anti-H3K27me3 antibodies at the transcriptional start sites of the pluripotent genes Oct4, Nanog, Prdm14, and Tcl1. Indeed, we observe an increase of H3K27me3 at these loci following GSK-J4 exposure, in particular at the Prdm14 TSS, which shows the highest signal of H3K27me3 in both the control and the GSK-J4 treated ESCs. The expression levels of these genes were measured by reverse-transcription, quantitative PCR. Nanog, Prdm14, and Tcl1 show reduced expression with GSK-J4 treatment although the expression of Oct4 is slightly decreased. Two independent male and female ESCs treated with GSK-J4 confirms the altered gene expression. Taken together, these results suggest that the H3K27 demethylase activity is necessary for the expression of Nanog, Prdm14, and Tcl1. 2 / 17 Dynamics of Histone Demethylation in Female ESCs Fig 1. GSK-J4 treatment results in reduction of Nanog, Prdm14, and Tcl1 expression. RNA expression of Utx and Jmjd3 during the differentiation of female ESCs. ESCs were differentiated by removal of LIF and forming embryoid bodies. RNA was extracted from undifferentiated ESCs ), day 4 and day 8 of differentiation. Relative RNA expression was determined with RT-qPCR. The graph represents mean values of three independent experiments. Error bars show one standard deviation from the mean. Western blot analysis of Utx, Jmjd3, and Oct4 during differentiation. Histone H3 was used a protein loading control. LF2 ESCs were treated with 10 M GSK-J4 for 24 hr and subjected to quantitative chromatin immunoprecipitation using anti-H3K27me3 antibodies and primer sets for the transcriptional start sites of the indicated genes. The graph represents mean values of fold enrichment relative to IgG control from three independent experiments. Error bars represent on standard deviation from the mean. Alteration of Oct4, Nanog, Prdm14, and Tcl1 expression following GSK-J4 treatment. LF2 ESCs were treated by 10 M GSK-J4 for 24 hr and RNA levels were measured by RT-qPCR. The graph represents the mean values of three independent experiments. Error bars show one standard deviation from the mean. doi:10.1371/journal.pone.0125626.g001 GSK-J4 diminishes Tsix and induces Xist expression XCI in the mouse embryo can be faithfully recapitulated ex vivo by inducing the differentiation of female ESCs. Both female X-chromosomes are active in undifferentiated ESCs. During cellular differentiation, Tsix expression extinguishes and Xist is robustly upregulated reflecting the gradual silencing of the entire inactive X-chromosome. Following GSK-J4 exposure, the level of H3K27me3 is increased at the TSSs of Tsix and Xist. We also observe enhanced 3 / 17 Dynamics of Histone Demethylation in Female ESCs Fig 2. GSK-J4 demethylase inhibitor extinguishes Tsix and induces Xist expression. RNA expression levels of Tsix and Xist PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 during the differentiation of female ESCs. RNA was PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 extracted from indicated stages of cells and subjected to RT-qPCR. The graph is representative of mean values of three independent experiments. Error bars represent one standard deviation from the mean. Female ESCs were treate