Interest in the non coding
Interest in the non-coding genome has recently surfaced with accelerated emphasis in the past few years (Li et al., 2016). Transcription factors (TFs) bind to enhancers and mediate RNA initiation from distal transcriptional start sites (TSS) of genes. Epigenome sequencing of human and mouse during development has revealed an enormous number of enhancers, which provide exquisite tuning of gene regulation (Atlasi and Stunnenberg, 2017, Rada-Iglesias et al., 2011). Enhancers are activated by relaxation of compact chromatin, which occurs by a poorly understood sequence of events. Histone modifying SMER 3 synthesis mediate deposition of marks such as H3K4me1 or H3K27ac, and bidirectional RNAs are transcribed by RNA polymerase II, which together, with additional known and unknown mechanisms, culminate in looping to the target promoter and initiation of mRNA transcription over gene bodies (Hnisz et al., 2013).
The functional importance of enhancers has been implicated by their mutations in diseases. For example, ENCODE sequencing of DNA revealed an unanticipated large number of mutations in enhancers acquired in disease states (Birney et al., 2007). Sequencing of human genomes from cancer-derived tissues reveal that key enhancers tend to harbor mutations that disrupt binding of TFs (Hnisz et al., 2013). While the role of enhancers has been demonstrated in development and cancer, there is limited understanding of enhancer biology in senescence and aging. Enhancers are abundantly decorated with histone acetylation such as H3K27ac. Histone acetylation has long been implicated in yeast and Drosophila aging with an overall model wherein histone hypoacetylation prolongs lifespan by promoting autophagy, while suppressing oxidative stress and necrosis (Peleg et al., 2016) through (1) inhibition of acetyl-CoA producing enzymes, (2) spermidine supplementation, or (3) inactivation of histone acetyltransferases (HATs). However, the role of histone acetylation in cellular senescence is not clearly understood.
In this work, we systematically screened epigenetic proteins to discover potential roles in RS, with the ultimate goal of identifying “druggable” targets and pathways to ameliorate age-related disease. We focused on the HAT p300, for further investigation given the importance of acetylation in aging and the important role of p300 at enhancers (Vo and Goodman, 2001). Our results suggest that p300 serves a specific role in senescence and has the potential to be a novel anti-aging target.
Discussion Our results provide evidence that the HAT p300 is a primary driver of the RS phenotype linked to its ability to license new SEs. New senescence-activated SEs are enriched in H3K4me1 and multiple histone acetyl marks (Figures 4 and S5A), actively transcribe eRNAs (Figures 5D and 5H), gain p300 binding (Figure 7), and direct senescence-specific gene expression (Figure 5F, left). Importantly, depletion of p300 alone was sufficient to downregulate senescence genes (Figure 5F, middle and right) and extend RLS (Figures 2A–2C and 2F). We additionally demonstrate non-redundancy of the role of p300 in senescence as depletion of its paralog CBP, is unable to phenocopy the senescence delay in both RS and OIS models (Figures 7K–7R). We also confirm differential recruitment of p300 and CBP at licensed enhancers with a predominant bias toward p300-mediated regulation (Figure 7A). To our knowledge, this is the first evidence of a unique functional role for p300 despite previous speculation based on distinct occupancy and unique disease profiles of p300 and CBP (Vo and Goodman, 2001). We discovered the role of p300 in senescence in an unbiased shRNA screen focused on epigenetic factors. A major advantage of sub-library screens is the higher representation of individual shRNAs compared to whole genome libraries (Kampmann et al., 2015). This increased representation tremendously improves the signal to noise ratio in screens and increases the sensitivity of assays to distinguish true candidates from false-positives. Additionally, sub-libraries minimize the scale of an experiment, which assists technical execution. Screens of focused libraries with genes of defined function can thus lead to actionable secondary assays. This was evident in successful identification of several candidate genes from our screen that were independently validated (Figures 2 and S2).