Histone acetyltransferase p300 induces de novo super-enhancers to drive cellular senescence [DNA-seq]

Cellular senescence is a state of stable proliferative arrest induced by stress and is associated with a pro-inflammatory program. Senescent cells have anti-tumorigenic properties, however, their accumulation during aging contributes to chronic inflammation and age-related diseases. While senescence is associated with profound alterations of the epigenome, a systematic view of epigenetic factors in regulating senescence is lacking. Here, we curated a library of short hairpin RNAs for targeted silencing of all known epigenetic factors and performed a high-throughput loss-of-function screen to identify epigenetic proteins whose downregulation can delay replicative senescence of primary human cells. This screen identified multiple new players regulating senescence, including the histone acetyltransferase p300 that was found to be a primary driver of the senescent phenotype. p300, but not the paralogous CBP, induces a dynamic hyper-acetylated chromatin state in senescence and promotes the formation of active enhancer elements in the non-coding genome, leading to a senescence-specific gene expression program. Our work illustrates a causal role of histone acetyltransferases and acetylation in senescence, and suggests p300 as a potential therapeutic target for senescence and age-related diseases. Overall design: This is a short hairpin RNA screen designed to identify epigenetic regulator genes which limit lifespan and promote replicative senescence in fibroblasts. It operates on the principle that a cell infected with a lentiviral vector expressing an shRNA targeting such a gene should exhibit prolonged lifespan and delayed senescence, outcompeting other cells in a serially passaged culture. Individual plasmids for five short hairpin RNAs per target gene (3,455 hairpins representing 717 epigenetic regulator targets) were randomly assigned to five initial pools denoted A, B, C, D, or E. Each pool included additional control hairpins verified to accelerate (lamin B1) or delay (p53) senescence, as well as a non-targeting control hairpin (NTC). Lentiviral supernatants were generated from these pools and combined into all possible pairwise combinations (AB, AC, AD, etc) to form 10 paired pools that were used to infect 10 plates of IMR90 fibroblasts. Each plate was passaged out, and cells were removed and lysed at PD40 (proliferating control), PD45, PD50, PD55, and PD60 (senescence). Cell lysate from the five time-points was tested for the presence of shRNAs by high-throughput sequencing of genomic DNA to detect the expressing plasmid. Each initial pool (and each plasmid) is thus represented four times in the paired pool strategy. The data were sequenced paired-end over two lanes per sample, resulting in four FASTQ files per sample.