MRT-67307 manufacturer histone demethylase activity is a determinant of XCI timing We next asked whether histone demethylation is critical for the establishment as well as the maintenance of XCI. To address these questions, we tested whether the inhibition of the JmjC 10 / 17 Dynamics of Histone Demethylation in Female 
ESCs histone demethylases can alter the timing of XCI. Female ESCs were culture in the absence or presence of GSK-J4 for 3 hr and then subjected to differentiation by forming EBs concomitant with LIF removal. At day 4 of differentiation, we evaluated the XCI status using Xist RNA fluorescent in situ hybridization coupled with anti-H3K27me3 immunostaining, marks that decorate the entire female inactive X-chromosome. We then evaluated individual cells for the presence of both Xist and H3K27me3 foci. We found that EBs derived from GSK-J4 treated female ESCs show a statistically higher ratio of both Xist and H3K27me3 positive foci, indicating that the JmjC histone demethylases play an important role in XCI timing. Next, we clarified the role of Utx in XCI by first depleting Utx by siRNA and then inducing these ESCs to differentiate using N2B27 serum-free media. The expression levels of Oct4, Tsix, and Xist were measured 1 day after the induction of cellular differentiation. Our results show that the knockdown of Utx results in a significant upregulation of Xist, whereas the expression of Tsix is reduced. These findings indicate that Utx is a negative regulator of XCI during female ESC differentiation. The expression of Oct4 did not show an alteration after knockdown of Utx. Finally, to test the significance of the JmjC histone demethylases for XCI maintenance, we measured the expression of several X-linked genes in GSK-J4 treated female MEFs. We observed an increased expression of Hdac6 after GSK-J4 exposure. In contrast, the other X-linked genes did not alter expression. Previous studies have shown that ectopic Xist expression induces secondary XCI in cis, suggesting that the inhibition of JmjC histone demethylation activates Xist from the Xi. Collectively, these results show that the JmjC histone demethylases influence the timing of XCI in female differentiating ESCs but are dispensable for the maintenance of XCI along the X-chromosome. Discussion The dynamics of XCI/XCR in female ESCs provides an excellent model for epigenetic reprogramming between the pluripotent and differentiated states. XCI occurs during cellular differentiation, while XCR transpires during dedifferentiation towards pluripotency. A previous report has linked the Prdm14 and Tsix to XCR but the underlying mechanism for their action is unknown. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19783938 In this study, we have revealed the pivotal roles of Utx for the expression of Prdm14, Tsix, and Xist in female ESCs. Our qChIP and knockdown studies show that Utx selectively occupies the TSSs of Prdm14 and Tsix, and Xist intron 1 and maintains the expression of these genes. Consistently, AA enhances demethylation of H3K27me3 and activates the expression of Prdm14 and Tsix in the absence of GSK-J4, indicating that AA-induced up-regulation of these genes is H3K27 demethylation-dependent. In addition to these genes, we identified genes down-regulated by GSK-J4 using RNA-Seq, including Utf1, which has been previously PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19786154 reported as an Utx target. We also found that GSK-J4 exposure increases the expression and H3K4me3 levels of Xist, Nodal, and HoxC13. These results are consistent with the recent report from the Helin lab indicating that GSK-J