Our senescence induction method of choice was replicative ex

Our senescence induction method of choice was replicative exhaustion, although p300 KD was also able to delay the onset of OIS (Figures 7Q and 7R). RS has been implicated in tissues with high turnover such as skin, gut, and blood (Hornsby, 2002) and likely the few mitotic Garcinol receptor in the body, such as adult stem cells (Liu and Rando, 2011). Senescent cells that spuriously appear early in life due to acute stresses not related to replicative exhaustion, serve an anti-tumor role and are cleared by the immune system. In support, OIS enhancers are proximal to SASP genes (Tasdemir et al., 2016) whose expression attracts immune cells for rapid clearance. However, RS cells likely appear later in life, compromise cellular fitness, tissue integrity, and tumor suppression (Campisi, 1997). In support, we found enhancers in RS are proximal to genes related to nucleotide metabolic dysfunction and peroxisomal genes that likely promote oxidative stress and DNA damage (Figures S6A–S6C). A potential negative aspect of our senescence delay strategy, given its primordial role in tumor suppression and wound healing, may be a propensity for cancer. However, a depletion of p300 protein levels (rather than bi-allelic mutation or CRISPR knockout) did not show immortalization at least in the tested cell type (Figures 2A–2C). Furthermore, we detected fewer TIFs in cells harboring p300 hairpin (Figures 2G and 2H). This result is particularly important because p300 mutations have been implicated in cancer where TIFs are more likely (Iyer et al., 2004). Thus, our result demonstrates that a modest depletion of p300, while delaying senescence, does not have a pro-cancer effect, at least not in IMR90 cells. Importantly, cells with p300 depletion eventually establish senescence as assayed by growth cessation (Figures 2A–2C) and SA-β-gal activity (Figures 2I and 2J), also supporting the notion that p300 depletion does not lead to acquisition of cancer characteristics. We conclude, that in combination with safe senolytics, senescence delay by modest depletion of p300 may be a beneficial future therapeutic strategy in treating age-related diseases in the future. In addition to implicated roles in cancer, homozygous mutations in p300 or CBP are early embryonic lethal as are CBP/p300 double heterozygotes suggesting critical roles in development (Iyer et al., 2004). Thus, p300 conforms to the antagonistic pleiotropy theory of aging whereby a single gene controls multiple antagonistic traits, in this case, development and aging (Moskalev et al., 2014). Finally, our findings also provide major mechanistic insights into the role of HATs, histone acetylation, enhancer licensing, and target gene regulation in senescence. We dissected the role of p300 at SEs and contrast it to the paralog CBP, using pooled screening methodology, proteomic, and global genomic measurements. Run-on sequencing mapped active and functional enhancers licensed during senescence establishment. These mechanistic studies lay the foundation for translational discoveries probing the in vivo role of p300 in senescence, tissue, and organismal aging. However, we acknowledge that the distinct roles of p300 and CBP need further exploration, particularly because validation via simultaneous knock out and rescue in IMR90 primary cells was not feasible. Additionally, differences in shRNA-mediated KD efficiency and antibody-based immunoprecipitation may confound our conclusions. A pertinent future question is the basis of altered p300 function in senescent cells, for example, whether post-translational modifications augment p300 catalytic activity in senescence. With commercial availability of inhibitors and potential of genome editing, we anticipate p300 as a novel therapeutic target against aging and age-associated diseases.
STAR★Methods
Acknowledgments We thank Christopher Lord, Katherine Alexander, Digbijay Mahat, Ronen Marmorstein, and Daniel Bose for suggestions related to the screen, PRO-seq, and general information on p300 and CBP. We thank Gabor Egervári for statistical analysis and Raffaella Nativio for discussions. This work was supported by NIH (P01AG031862 to S.L.B., P.D.A., and F.B.J. and CA196539, GM110174, and AI118891 to B.A.G.), CPRIT (R1306 to W.D.), Ted Nash Long Life Foundation (to W.D.), American Heart Association (15POST21230000 to P.S.), and AFAR Irene Diamond Transition Award (DIAMOND 17113 to P.S.). Cell images in Graphical Abstract courtesy of Servier Medical Art, licensed under a Creative Commons Attribution 3.0 Unported License.