Abstract 301: Epigenetic Reprogramming during SS-dependent Differentiation of Mouse Embryonic Stem Cells: Role of Nitric Oxide and Histone Deacetylases.
Introduction and Background: Prior work indicates that shear stress (SS) reprograms mouse embryonic stem cells (ES) to express cardiovascular lineage markers at least in part through the occurrence of epigenetic chromatin modifications. Nitric oxide (NO) is involved in SS signalling in vascular cells and facilitates cardiomyogenesis in ES. In this report, we show the results of experiments performed to assess the molecular mechanism activated by NO in the SS-dependent cardiovascular differentiation of ES. Methods and Results: Exposure of ES to the NO synthase inhibitor S-methyl-thiosourea (SMT), inhibited the SS-dependent expression of cardiovascular markers in ES while treatment with nitric oxide donors anticipated cardiovascular differentiation in ES deprived from leukaemia inhibitory factor (LIF) thus indicating a role for NO in the SS-dependent differentiation of ES cells. The expression of developmentally regulated genes requires the fine tuning of the chromatin condensation/decondensation process in a temporally and spatially-regulated manner, a phenomenon strictly dependent on the activity of chromatin modifying enzymes such as Histone Acetyltransferases (HATs) and Histone Deacetylases (HDACs). Further experiments revealed that, after LIF removal, the direct exposure of ES to NO donors induced the nuclear localization of class II HDACs. Similar results were obtained in adult endothelial cells. Specifically, in this condition, members of the class II HDACs family, namely HDAC-4 and -7, were found shuttling from the cytoplasm to the nucleus of ES cells as early as 1 hour after LIF deprivation. This phenomenon was transient as the class II HDACs returned cytosolic in 6 hours. Intriguingly, NO donors allowed the nuclear retention of these molecules beyond the 6 hours timepoint. This observation was paralleled by a significant reduction of histone H3 and H4 acetylation indicating the presence of a prolonged histone deacetylase activity in the ES nuclei upon NO treatment. Conclusions: These data suggest a direct role of NO in the regulation of class II HDACs function and in the chromatin remodelling of ES cells and provide new insights about the role of NO during the cardiovascular commitment of embryonic stem cells.