Abstract
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.
Highlights
An intact endothelial barrier is essential for vascular function
To uncover a potential function of PHF8 in the endothelium, we first analyzed its expression in different cell types. qRT-PCR revealed that the expression of PHF8 mRNA in human umbilical vein endothelial cells (HUVECs) and microvascular endothelial cells (HMECs) is higher than that of the closest family member JHDM1D (Fig 1A)
A comparison between the cytoplasmic and nuclear cellular fraction revealed that the expression of PHF8 is restricted to the nucleus, which is suggestive for a potential function as histone demethylase in endothelial cells (Fig 1C)
Summary
An intact endothelial barrier is essential for vascular function. It prevents vessel occlusion and controls vascular permeability. Endothelial cells locally proliferate, enlarge and migrate to restore an intact vascular surface. Endothelial cell death (apoptosis), dysfunction or senescence has been implicated in the pathogenesis of numerous vascular diseases such as atherosclerosis, thrombosis and vascular leakage [1,2,3]. Epigenetic control of gene expression by histone modification is a central mechanism determining cell-fate and cell-phenotype maintenance [4].
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