Abstract 822: The histone Methyltransferase MLL is an Upstream Regulator of Endothelial Cell Angiogenic Functions
Posttranslational histone modification by acetylation, phosphorylation, or methylation is involved in the regulation of gene expression. Among homeodomain transcription factors, transcriptional activation of HoxA9, which is critical for endothelial cell differentiation and migration, specifically depends on the presence of the histone H3 lysine K4 methyltransferase MLL. Here, we investigated the role of MLL for angiogenic functions in human umbilical vein endothelial cells. Downregulation of MLL in endothelial cells was associated with reduced mRNA and protein expression of HoxA9 and HoxD3, whereas HoxB4, HoxB5, and HoxB9 were not changed. In parallel, MLL-siRNA impaired endothelial cell migration in a scratched-wound assay (0.73±0.07 vs. scrambled siRNA: 1.12±0.11 mm, p<0.05), and almost completely abolished three-dimensional capillary sprout formation from endothelial cell spheroid cultures into a collagen matrix (33±14 μm cumulative sprout length vs. 436±61 μm with scrambled siRNA, p<0.01). The specific histone H3 lysine K4 methyltransferase inhibitor 5′-deoxy-5′-(methylthio)adenosine (MTA) dose-dependently inhibited migration by 77±15% (5 mM, p<0.05) as well as sprout formation by 97±2% (p<0.05), indicating that the methyltransferase activity of MLL is required for its pro-angiogenic function. Indeed, chromatin immunoprecipitation confirmed the presence of histone H3 lysine K4 methylation within the HoxA9 promoter region in endothelial cells. In co-transfection experiments, simultaneous overexpression of HoxA9 compensated for the decrease in scratched-wound migration in MLL-siRNA-transfected endothelial cells (91±7% vs. MLL-siRNA / mock: 60±8% of scrambled / mock-transfected cells, p<0.05), suggesting that HoxA9 mediates MLL-dependent migration. As a potentially underlying mechanism, MLL-siRNA down-regulated the axon guidance molecule EphB4, a transcriptional target of HoxA9, at both, the mRNA (by 55±9%, p<0.05) and protein level. These data reveal a novel upstream pathway for the regulation of angiogenic functions in endothelial cells, in which MLL-dependent signals critically modulate migration via HoxA9 and EphB4.