hibition of H3K27 demethylation activates the demethylase-independent function of Utx Taken together, our data indicate that Utx directly regulates the expression of Prdm14 and Tsix in a demethylase-dependent manner, and suggest that Utx controls Xist via demethylase-independent mechanisms. Ascorbic acid enhances the demethylase activity of Utx and induces its target genes L-ascorbic acid /Vitamin C is a potential activator of -ketoglutarate-dependent oxygenases. Although previous studies reveal that the demethylation of 5-methyl cytosine, histone 3 lysine 9, and histone 3 lysine 36 enhance after AA exposure, it is unknown whether PCI-32765 19784385″ title=View Abstract(s)”>PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 AA regulates H3K27 demethylation. We therefore tested whether AA can facilitate the demethylase activity of Utx. To do this, we overexpressed HEK cells with a C-terminal catalytic domain of UTX protein fused with a nuclear localization signal sequence SV40NLS and evaluated the demethylase activity with or without AA by immunostaining with anti-H3K27me3 antibodies. We found that AA treated cells show a statistically significant reduction in H3K27me3 signal intensity. AA can also enhance demethylation of H3K27me3 using lysates from UTX-CSV40NLS-expressing cells. These results indicate that AA enhances the demethylation of H3K27me3. Next, we treated female ESCs with AA and evaluated the expression levels of the genes tested above. Consistently, the Prdm14 and Tsix levels increase after AA. In contrast, we 5 / 17 Dynamics of Histone Demethylation in Female ESCs Fig 3. Utx binds to the transcriptional start sites of Prdm14, and Tsix, and Xist intron 1 and regulates these genes in ESCs. Female ESCs were subjected to qChIP using anti-Utx antibodies and the primer sets for the TSSs of Oct4, Nanog, Prdm14, Tcl1, Tsix, and Xist; as well as Xist intron 1. The graphs represent the mean fold values of enrichment relative to IgG control from three independent experiments. Error bars show one standard deviation from the mean. Female ESCs were transfected with a control siRNA and two different siRNAs for Utx. The transfectants were subjected to western blot with anti-Utx antibodies 72 hr post transfection. Actin is used as a protein loading control. The graph represents the 
fold change of Utx and Actin proteins. The relative RNA expression was measured by RT-qPCR in the Utx depleted cells. The graph represents the mean values of three independent experiments. Error bars represent one standard deviation from the mean. Student’s t-test was used for statistical analysis. p<0.05; p<0.01. Female ESCs were treated with 10 M of GSK-J4 for 24 hr and then subjected to qChIP using antiUtx antibodies. doi:10.1371/journal.pone.0125626.g003 found an increased expression of Xist in AA treated cells, suggesting an H3K27me3 demethylation-independent mechanism. Indeed, it has been reported that AA treatment induces the global demethylation of 5-methyl cytosine, converting 5mC to 5-hydroxy methyl cytosine in ESCs via a Ten eleven translocated -dependent manner. We evaluated the levels of H3K27me3 and 5hmC at the TSSs of Prdm14, Tsix, and Xist as well as Xist-int1 after AA treatment. The H3K27me3 levels are reduced and the 5hmC levels are increased at all 6 / 17 Dynamics of PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 Histone Demethylation in Female ESCs Fig 4. Ascorbic acid enhances demethylation of H3K27me3 and induces Prdm14, Tsix, and Xist. The catalytic domain of Flag-tagged UTX protein was overexpressed in HEK cells, in the presence or absence of ascorbic acid. The transfectants wer
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