If applicable with a repeated measure approach, Bonferroni post hoc test was performed

4 can inhibit both the H3K27 and H3K4 demethylases in cell culture. However, depletion of Utx results in reduced expression of Xist, Nodal, and HoxC13, suggesting that Utx activates these genes in an H3K27me3-independent manner. Taken together, our results show that JW 55 site GSK-J4 alters the gene expression by interfering with both H3K27me3 and H3K4me3 demethylation. Two different JmjC family members, Kdm2 and Kdm5, demethylate H3K4me3. The Kdm2 family consists of Kdm2a and Kdm2b and can also demethylate H3K36me2. Previous studies reveal that Kdm2b binds to GC-rich promoters across the entire genome in male ESCs and regulates PRC1 complex PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19784385 recruitment. The Kdm5 family includes Kdm5a, Kdm5b, Kdm5c, and Kdm5d and is specific for the demethylation of H3K4. 11 / 17 Dynamics of Histone Demethylation in Female ESCs Fig 7. JmjC histone demethylases influence the timing of XCI in female ESCs. Xist RNA FISH combined with anti-H3K27me3 immunofluorescence. Female ESCs were cultured in the presence or absence of 10 M GSK-J4 for 3 hr and then subjected to the induction of differentiation to day 4 EBs as described above. Quantification of the Xist/H3K27me3 foci positive cells. The graph represents the percentage of Xist + H3K27me3 dual-stained, positive foci cells versus the dual-stained negative cells. The standard deviations are represented as error bars from three independent experiments. The Fisher’s exact test was used for the statistical analysis. “n” indicates the total number of counted cells. p<0.01. Depletion of Utx induces Xist expression in differentiating female ESCs. Female ESCs were transfected with two independent Utx siRNAs and 12 / 17 Dynamics of Histone Demethylation in Female ESCs plated onto undifferentiated cellular conditions. Then, the media was changed to N2B27 and a second siRNA transfection was performed 24 hr later. The cells were cultured an additional 24 hr, harvested, and subjected to RT-qPCR. The graph represents the means from three independent experiments with the standard deviations as error bars. The Student's t-test was used for the statistical analysis. p<0.05; p<0.01; n.s. = not significant. The expression of X-linked genes upon GSK-J4 treatment in female MEFs. Female MEFs were treated with GSK-J4 for 24 hr, harvested, and subjected to RT-qPCR. The graph represents the means from three independent experiments with the standard deviations shown as error bars. Working models for the Utx H3K27 demethylase. Utx maintains the expression of a subset of genes such as Prmd14 and Tsix by demethylating H3K27me3. These genes are repressed by GSK-J4. Ascorbic acid can enhance the demethylase activity PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19785045 of Utx, activating Prdm14 and Tsix GSK-J4 can also inhibit H3K4me3 demethylases to induce gene expression of genes such as Xist. Utx enriches its target by GSK-J4 via unknown mechanisms, which may activate the demethylase-independent functions of Utx. doi:10.1371/journal.pone.0125626.g007 We investigated the publically available ChIP-Seq database for Kdm2a, Kdm2b, and Kdm5c binding to see whether their occupancy correlates with the altered gene expression we observed following GSK-J4 exposure. Indeed, 16 of 160 up-regulated genes and only 1 of 29 down-regulated genes following GSK-J4 exposure have significant peaks of Kdm5c. We did not observe a correlation between gene expression alteration and the occupancy of Kdm2a or Kdm2b. This may reflect the possibility that GSK-J4 mainly contributes to the inhibition of the Kdm5 rather than Kdm2