Role of methyltransferase Set7 in obesity-related endothelial activation: functions beyond histone methylation

Abstract

Abstract Introduction Obesity is associated with environmental changes such as hyperglycemia, hyperinsulinemia, inflammation, and elevated free fatty acids, all of which compromise endothelial function1,2. We have previously reported that in individuals with type 2 diabetes, lysine methyltransferase Set7 is involved in hyperglycemia-induced endothelial damage through monomethylation of histone 3 at lysine 4 (H3K4me1)3. Apart from such histone-driven epigenetic modulation, Set7-induced methylation of non-histone proteins is emerging as a novel mechanism underlying the (dys)regulation of many biological processes4. Purpose To investigate the role of Set7-related endothelial damage in cells exposed to different obesity-associated stimuli and to determine its impact on the protein methylome in these settings. Methods Human aortic endothelial cells (HAECs) were exposed to high concentrations of glucose, insulin, TNFa, or fatty acids (palmitate or oleate). Set7 overexpression, silencing, and inhibition studies were also performed in these cells. Gene and protein expression were assessed by RT-PCR and immunoblotting respectively, whereas production of the reactive oxygen species (ROS) was measured by electron spin resonance spectroscopy. Specific antibodies were used for the enrichment of lysine mono-, di-, and tri-methylated proteins and LC-MS/MS proteomic analysis was carried out for their identification. Results Each obesity model in HAECs increased the expression of nearly all the inflammatory markers tested and, except for TNFa, they also increased the expression of Set7. Significant changes in the global levels of H3K4me1, as well as of methylated proteins were also observed in these cells. Studies overexpressing a mutant form of Set7, which is lacking the SET domain, revealed that Set7 methyltransferase activity is required to define the protein methylome in HAECs. Similar results were obtained after Set7 silencing and inhibition. Despite no differences were observed in the production of ROS when modulating Set7 expression in HAECs, overexpression of an active form of Set7 and its silencing/inhibition resulted in a consistent up- and down-regulation, respectively, of several inflammatory mediators and adhesion molecules. Finally, proteomics analysis identified, for the first time, the protein methylome of endothelial cells, comprising almost 2,700 methylated proteins involved in essential biological processes such as RNA metabolism and cytoskeleton. Conclusions Our results indicate that Set7 may contribute to inflammatory changes in response to obesogenic insults. Moreover, our findings uncover a dual role of methyltransferase Set7 in governing the methylation of both histone and non-histone proteins in endothelial cells. These preliminary findings open a new field of research aiming to further elucidate the molecular mechanisms underlying the modulation of the protein methylome in the endothelium and its role in endothelial damage.