Abstract 4845: Control of Notch Signaling by Reversible Acetylation Through the Antagonistic Activities of p300/PCAF and SIRT1 in Endothelial Cells

Abstract

Signaling by Notch receptors is a key cell-cell communication mechanism essential for development and homeostasis of the cardiovascular system. Several critical steps of angiogenic blood vessel growth are coordinated by the Notch pathway and even subtle perturbations in Notch activity can profoundly influence endothelial cell behavior and blood vessel growth, yet very little is known about the dynamics and intrinsic regulation of endothelial Notch signaling. Here, we show that reversible acetylation regulates the amplitude and duration of Notch responses by altering protein turnover of the Notch1 intracellular domain (NICD) in endothelial cells. We report that the histone acetyltransferases (HATs) p300 and PCAF not only acetylate nucleosomal histones at Notch target promoters, but also the NICD itself. Acetylation of NICD induced by p300 and PCAF results in increased NICD stability and enhanced Notch target gene expression. Likewise, blocking histone deacetylase (HDAC) activity using the class III HDAC inhibitor nicotinamid (NAM) leads to a substantial increase in NICD acetylation while class I/II HDAC inhibition has no significant effect suggesting that class III HDACs primarily target NICD for deacetylation. Among the class III HDACs with deacetylase activity (SIRT1, 2, 3, 5), specifically SIRT1 associates with NICD through its catalytic domain and silencing of SIRT1 enhances NICD acetylation. Wild type SIRT1, but not a catalytically inactive mutant catalyzes the deacetylation of NICD in a nicotinamid-dependent manner indicating that SIRT1 acts as a direct NICD deacetylase, which counteracts HAT-induced NICD acetylation. Inactivation of SIRT1 by NAM or RNAi-mediated knock down results in enhanced NICD protein stability by blocking proteasomal degradation. Consistent with these results, knock down of SIRT1 amplifies Notch target gene expression in endothelial cells in response to NICD overexpression or treatment with the Notch ligand Delta-like 4 (Dll4). Taken together, these findings support a model, in which Notch signaling is dynamically regulated by reversible acetylation and suggest that the antagonistic activities of p300/PCAF and SIRT1 act as rheostat to fine-tune endothelial Notch responses.