EGCG and Gcn5 conversation assay The DNA sequence encoding the HAT domain of yGCN5 (99\262) was amplified by PCR and was sub\cloned into a pET\28a vector for overexpression. much like those associated with warmth shock and were inversely correlated with the gene expression profiles of aged yeast and aged worms. Through a bioinformatic analysis, we also found that HAT inhibition activated subtelomeric genes in human cell lines. Together, our results suggest that inhibiting the HAT Gcn5 may be an effective means of increasing longevity. gene alone, however, does not increase the lifespan of cells, and homozygous knockout of causes murine embryos to be malformed by E8.5 and to pass away by E11 (Lin et al., 2008), suggesting GCN5 is essential for developmental processes. Gcn5 is known to mediate H3 acetylation at both lysine 9 and 18. The physiological importance of these specific site preferences in the context of aging, however, remains uncertain and requires further study. In this study, we offer novel evidence indicating that the partial inhibition of specific HATs can mediate the rejuvenation of yeast and human cell lines. This increase in lifespan is achieved via disrupting H3 acetylation that is dependent upon Gcn5 and the linked protein Ngg1. Using site\specific mutations, we were able to confirm that Gcn5 preferentially mediates H3 acetylation on K9 and TMS K18 residues and that acetylation of these two sites is usually associated with the observed lifespan extension. We also used low glucose media in order to demonstrate the ability of HAT inhibition to mimic the effects of caloric restriction. Through RNA sequencing, we further decided that HAT inhibitors largely influenced the expression of genes found in subtelomeric domains. In knockdown cell lines, we observed both delayed replicative senescence and decreased markers of aging. 2.?RESULTS 2.1. HAT inhibition increases the lifespan of yeast and human cell lines As the activation of Sir2 has been linked to the prolongation of model organism lifespans (Imai, Armstrong, Kaeberlein, & Guarente, 2000), we wanted to assess whether inhibiting HATs would accomplish a similar effect. We employed two types of microfluidic chips in this study. One is the island chip derived from a previous study (Zhang et al., 2012). The other is a altered U\shape chip (Jo, Liu, Gu, Dang, & Qin, 2015), as shown in Physique S1a. Cells were pumped in using a microfluidic device, and the budding timing across the entirety of the lifespan was continuously monitored for 60h via repeated microscopic imaging. We found that the HAT inhibitors epigallocatechin gallate (EGCG)(Choi et al., 2009), anacardic acid (AA), garcinol (GA), and curcumin all prolonged the replicative lifespan of these cells by 50%, 50%, 33%, and 29%, respectively (Physique?1a,?,b).b). Cell cycle length throughout the entire yeast lifespan was correspondingly reduced, with cells dividing more smoothly following HAT inhibition (Physique?1c,?,d,d, Physique S1bCd). It is important to note that HAT inhibitors not only extend lifespan but also prevent cell cycle extension at the end of life. In yeast, this corresponds to a suppression of the reduction in health at the end of life. As EGCG achieved the most marked lifespan extension, it was used for further experimentation. Epigallocatechin gallate has been experimentally reported to increase the lifespan of worms, (Wagner et al., 2015), and rats (Niu et al., 2013). Previous studies have attributed such extensions to the antioxidant activity of EGCG. As such, we assessed the ability of the strong antioxidant N\acetylcysteine (NAC) (Zafarullah, Li, Sylvester, & Ahmad, 2003), to extend yeast lifespan, revealing that it only mediated a 5% increase in yeast lifespana less dramatic increase than that observed upon EGCG treatment (Physique?1e, Physique S1e). This suggests that other factors beyond antioxidant activity are linked to EGCG\mediated yeast lifespan prolongation. We therefore hypothesized that it facilitates this effect via its HAT\inhibitory activity. Indeed, we found that EGCG treatment was associated with reduced overall Ac\lysine levels (Physique?1f), with a preferential impact TMS on Ac\H3 relative to Ac\H4 expression (Physique?1f). These results indicate that HAT inhibition can mediate a substantial increase TMS in yeast lifespan in a manner potentially linked to H3 histone acetylation. Open in a separate window Physique 1 HAT inhibitors increased the lifespan of both yeast Hgf and human cell lines. (a) HAT inhibitors increased the replicative lifespan of treated cells as compared to wild\type (WT) cells in SD media. (b) EGCG.