Objective-Altering endothelial biology through epigenetic modifiers is an attractive novel concept, which is, however, just in its beginnings. We therefore set out to identify chromatin modifiers important for endothelial gene expression and contributing to angiogenesis. Approach and Results-To identify chromatin modifying enzymes in endothelial cells, histone demethylases were screened by microarray and polymerase chain reaction. The histone 3 lysine 4 demethylase JARID1B was identified as a highly expressed enzyme at the mRNA and protein levels. Knockdown of JARID1B by shRNA in human umbilical vein endothelial cells attenuated cell migration, angiogenic sprouting, and tube formation. Similarly, pharmacological inhibition and overexpression of a catalytic inactive JARID1B mutant reduced the angiogenic capacity of human umbilical vein endothelial cells. To identify the in vivo relevance of JARID1B in the vascular system, Jarid1b knockout mice were studied. As global knockout results in increased mortality and developmental defects, tamoxifen-inducible and endothelial-specific knockout mice were generated. Acute knockout of Jarid1b attenuated retinal angiogenesis and endothelial sprout outgrowth from aortic segments. To identify the underlying mechanism, a microarray experiment was performed, which led to the identification of the antiangiogenic transcription factor HOXA5 to be suppressed by JARID1B. Importantly, downregulation or inhibition of JARID1B, but not of JARID1A and JARID1C, induced HOXA5 expression in human umbilical vein endothelial cells. Consistently, chromatin immunoprecipitation revealed that JARID1B occupies and reduces the histone 3 lysine 4 methylation levels at the HOXA5 promoter, demonstrating a direct function of JARID1B in endothelial HOXA5 gene regulation. Conclusions-JARID1B, by suppressing HOXA5, maintains the endothelial angiogenic capacity in a demethylasedependent manner.
Epigenetic marks critically control gene expression and thus the cellular activity state. The functions of many epigenetic modifiers in the vascular system have not yet been studied. We screened for histone modifiers in endothelial cells and observed a fairly high expression of the histone plant homeodomain finger protein 8 (PHF8). Given its high expression, we hypothesize that this histone demethylase is important for endothelial cell function. Overexpression of PHF8 catalyzed the removal of methyl-groups from histone 3 lysine 9 (H3K9) and H4K20, whereas knockdown of the enzyme increased H3K9 methylation. Knockdown of PHF8 by RNAi also attenuated endothelial proliferation and survival. As a functional readout endothelial migration and tube formation was studied. PHF8 siRNA attenuated the capacity for migration and developing of capillary-like structures. Given the impact of PHF8 on cell cycle genes, endothelial E2F transcription factors were screened, which led to the identification of the gene repressor E2F4 to be controlled by PHF8. Importantly, PHF8 maintains E2F4 but not E2F1 expression in endothelial cells. Consistently, chromatin immunoprecipitation revealed that PHF8 reduces the H3K9me2 level at the E2F4 transcriptional start site, demonstrating a direct function of PHF8 in endothelial E2F4 gene regulation. Conclusion: PHF8 by controlling E2F4 expression maintains endothelial function.
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