The histone methyltransferase MLL is an upstream regulator of endothelial-cell sprout formation

Posttranslational histone modification by acetylation or methylation regulates gene expression. Here, we investigated the role of the histone lysine methyltransferase MILL for angiogenic functions in human umbilical vein endothelial cells. Suppression of MILL expression by sIRNA or incubation with the pharmacologic methyltransferase inhibitor 5'-deoxy-5'-(methylthio)adenosine significantly decreased endothelial-cell migration and capillary sprout formation, indicating that methyltransferase activity is required for proangiogenic endothelial-cell functions. Because the expression of homeodomain transcription factors (Hox) is regulated by MILL, we elucidated the role of Hox gene expression. MILL silencing was associated with reduced mRNA and protein expression of HoxA9 and HoxD3, whereas HoxB3, HoxB4, HoxB5, and Hox139 were not altered. Overexpression of HoxA9 or HoxD3 partially compensated for impaired migration in MILL sRNA-transfected endothelial cells, suggesting that HoxA9 and HoxD3 both contribute to MLL-dependent migration. As a potential underlying mechanism, MILL siRNA down-regulated mRNA and protein levels of the HoxA9-dependent axon guidance factor EphB4. In contrast, MILL knockdown effects on capillary sprouting were not rescued by HoxA9 or HoxD3 overexpression, indicating that MILL affects additional targets required for 3-dimensional sprout formation. We conclude that MILL regulates endothelial-cell migration via HoxA9 and EphB4, whereas sprout formation requires MLL-dependent signals beyond HoxA9 and HoxD3.