br Results br Discussion Here we describe

Results
Discussion Here we describe the role for histone demethylases KDM4A and KDM4C in the epigenetic regulation of mESC differentiation to endothelial cells. We showed that KDM4A and KDM4C targeted the tri-methyl histone 3 lysine 9 (H3K9me3), a known repressive marker of gene regulation (Black et al., 2012; Berry and Janknecht 2013) at Flk1 and VE-cadherin promoters. Demethylation of H3K9me3 enabled de-repression at the promoters and thereby induced expression of Flk1 and VE-cadherin genes, the lineage markers of endothelial orexin receptor (Kohler et al., 2013; Park et al., 2013). Differentiation of ESCs into the mesodermal progenitors and then into endothelial cells involves the orderly expression of developmental transcription factors such as Er71 (Kataoka et al., 2011; Lee et al., 2008), upregulation of the VEGF receptor Flk1 (Kohler et al., 2013; Park et al., 2013), and expression of endothelial cell-specific proteins such as VE-cadherin, which allow the cells to form stable adherens junctions and functional vascular networks (Park et al., 2013). In the present study, we demonstrated that KDM4A and KDM4C were essential epigenetic regulators of mESC differentiation to endothelial cells in mice and also contributed to vascular development in the zebrafish. KDM4A and KDM4C functioned independently through histone demethylation at the Flk1 and VE-cadherin promoters, respectively, to induce the endothelial cell lineage. Interestingly, we found that Kdm4c knockdown also suppressed Flk1 expression despite there being no interaction between KDM4C and histones at the Flk1 promoter, therefore suggesting that KDM4C targets additional genes regulating Flk1 expression. Our study examined the role histone demethylases in the endothelial differentiation of ESCs. Using a screen for 28 histone demethylases in FLK1/VE-cad-double-positive endothelial cells, we observed that mRNA and protein expression of Kdm4a and Kdm4c were markedly increased during endothelial differentiation. Importantly, the levels of KDM4A and KDM4C increased at day 6 of differentiation to the same extent seen in adult endothelial cells, suggesting that ESC-derived endothelial cells resembled mature endothelial cells in their histone demethylation signature. Although we did not examine human ESC differentiation to endothelial cells, their emergence may similarly involve KDM4A and KDM4C since histone demethylases are evolutionarily conserved (Berry and Janknecht, 2013; Black et al., 2012; Hou and Yu, 2010; Ye et al., 2012). To buttress the evidence that binding of KDM4A and KDM4C to Flk1 and VE-cadherin promoters reduced tri-methylation of H3K9me3, we compared enrichment of H3K9me3 at the Flk1 and VE-cadherin promoters in mESCs relative to the differentiated endothelial cells. In these studies, we sorted FLK1/VE-cadherin-double-positive cells and determined H3K9me3 methylation. The results clearly demonstrated that methylation was markedly reduced in differentiated cells as compared with undifferentiated cells. In addition, we knocked down Kdm4a or Kdm4c and determined enrichment of H3K9me3 at the Flk1 and VE-cadherin promoters and observed persistent methylation of the Flk1 promoter following Kdm4a knockdown but not following Kdm4c knockdown (data not shown). These results together show the critical role of KDM4A and KDM4C in regulating demethylation at level of Flk1 and VE-cadherin promoters and point to these demethylases as central to differentiation of ESCs to endothelial cells. ESC differentiation into endothelial cells requires the presence of the growth factors BMP-4, bFGF, and VEGF (Blancas et al., 2008), but it is unknown whether endothelial differentiation induced in this manner requires epigenetic regulation. Our data showed that BMP-4 induced the expression of KDM4A within 24 hr, consistent with the central role of KDM4A upregulation during the critical early phase of endothelial lineage commitment. We also observed that KDM4A bound to histones on Flk1 promoter and that Flk1 expression increased 24 hr after the induction of KDM4A. Since Flk1 enables mesodermal progenitors to commit to endothelial lineage fate (Park et al., 2013; Yamashita et al., 2000), our results suggest that an orchestrated differentiation program initiated by BMP-4 is responsible for mediating the upregulation of KDM4A. Interestingly, KDM4C expression was not dependent on KDM4A, as evident by the finding that KDM4A knockdown did not affect the expression of KDM4C. We also observed that KDM4C expression was maximal at 72 hr and induced the expression of VE-cadherin on day 5. Thus, it appears that KDM4A initiated the earlier mesodermal differentiation, whereas KDM4C participates in specifying differentiation of cells toward endothelial cells. A model suggested by our observations is that once FLK1 is expressed through KDM4A, the newly generated FLK1-positive cells in response to VEGF signaling induce expression of KDM4C to mediate VE-cadherin expression and thereby the endothelial lineage commitment.