The Role of p300-Mediated Chromatin Acetylation in Senescence Induction




Director of the Epigenetics Program; Professor of Cell and Developmental Biology

Project Summary

My project’s broad goal is to understand how post-translational histone acetylation affects cellular senescence and tissue and organismal aging. Histones are basic proteins that are bound to DNA, forming chromatin; chemical modifications such as acetylation, methylation, and phosphorylation on histones’ tails and globular domains profoundly affect gene expression. These modifications are heritable but also reversible, making them promising targets for therapeutic intervention. The Berger Lab previously identified p300, a histone acetyltransferase, as a co-activator protein whose reduction delays replicative senescence of fetal lung fibroblasts IMR90. Senescence, a stress-induced state in which cells no longer proliferate, is closely linked to aging; it has been shown to drive age-related phenotypes, including the increased incidence of age-related diseases like cancer, cardiovascular disease, and neurodegeneration. Targeted elimination of senescent cells can extend lifespan and healthspan in mice. We ultimately aim to reverse age-related decline and disease by targeting chromatin factors that mediate senescence.

During the award period, one focus of my project was delineating the distinction between p300 and CBP, which are members of the same class of co-activator proteins and are largely known to serve redundant functions. However, studies in our lab identified p300 but not CBP as a factor that regulates senescence. It is still debated whether they have different effects, so I investigated cells that lacked one or the other. To do this, I knocked down p300 and CBP in IMR90 cells using shRNA. I then passaged the cells to senescence. This time course allowed me to compare the number of population doublings before senescence between the p300- and CBP-knockdowns, and showed that there was a significant difference. I confirmed that depletion of p300 but not CBP delays senescence, thus establishing p300 is the primary driver of the senescence phenotype. Western blots and RT-qPCR were performed to confirm that the knockdowns had been successful, were stable, and only targeted one factor. During this process, I optimized a protocol for preparing nuclear extracts for western blots; as p300 and CBP are both very large proteins, previous methods had caused degradation and provided inconclusive results. I have also used novel techniques such as CRISPR, to knockout p300 in cells, and PRO-seq, to look at nascent transcription.

Through my research experience, I have learned numerous cellular and molecular biology practices, both fundamental and advanced, which will be applicable to my intended research career. I have also participated in weekly lab meetings and monthly journal club, allowing me to learn about the projects of other members of the lab and exciting new literature. My research project has contributed greatly to my educational experience by allowing me to connect theories learned in class to hands-on experiments. My previous coursework in both biology and chemistry provided me with the background necessary to understand how and why I used certain assays, and my research experience will give me a deeper understanding in my future courses. I am very grateful for this opportunity to conduct research as an undergraduate, and excited to continue this project throughout the school year.

The Role of p300-Mediated Chromatin Acetylation in Senescence Induction