Abstract
Senescence, a state of stable growth arrest, plays an important role in ageing and age‐related diseases in vivo. Although the INK4/ARF locus is known to be essential for senescence programmes, the key regulators driving p16 and ARF transcription remain largely underexplored. Using siRNA screening for modulators of the p16/pRB and ARF/p53/p21 pathways in deeply senescent human mammary epithelial cells (DS HMECs) and fibroblasts (DS HMFs), we identified EGR2 as a novel regulator of senescence. EGR2 expression is up‐regulated during senescence, and its ablation by siRNA in DS HMECs and HMFs transiently reverses the senescent phenotype. We demonstrate that EGR2 activates the ARF and p16 promoters and directly binds to both the ARF and p16 promoters. Loss of EGR2 down‐regulates p16 levels and increases the pool of p16− p21− ‘reversed’ cells in the population. Moreover, EGR2 overexpression is sufficient to induce senescence. Our data suggest that EGR2 is a direct transcriptional activator of the p16/pRB and ARF/p53/p21 pathways in senescence and a novel marker of senescence.
Highlights
The limited replicative capacity of cultured human cells, resulting in senescence, was first described by Hayflick and Moorhead (1961) and has since been implicated to play an important role during in vivo ageing and age-related diseases (van Deursen, 2014)
There was a small but significant enrichment for early growth response 2 (EGR2) binding sites at the promoters of genes up-regulated in human mammary epithelial cells (HMECs) Deep senescence (DS). Ten of these genes were identified as hits for senescence reversal in the DS HMEC screen, including p16, and nine of these were identified as hits in the human mammary fibroblasts (HMFs) siRNA screen, including the top hit S100A4, suggesting that EGR2 may act as a senescence regulator by activating the expression of these genes
As EGR4 expression is not increased in DS compared to early proliferating (EP) HMECs or HMFs ((GEO: GSE58035, Figure 3g), we suggest that EGR2 may be important for activation of the p16 promoter in epithelial and fibroblast senescence
Summary
The limited replicative capacity of cultured human cells, resulting in senescence, was first described by Hayflick and Moorhead (1961) and has since been implicated to play an important role during in vivo ageing and age-related diseases (van Deursen, 2014). In epithelial cells, it is defined when cultures at p16-dependent stasis undergo no further expansion upon at least two serial passages (Lowe et al, 2015, Methods) In fibroblasts, it is further characterised by additional markers of senescence, most notably the senescence- associated secretory phenotype (SASP) (Coppé et al, 2008; Rodier et al, 2009), accompanied by elevated reactive oxygen species (ROS) levels (Lowe et al, 2015; Passos et al, 2010) and a loss of lamin B1 (Freund et al, 2012). We perform siRNA screens in DS HMECs and human mammary fibroblasts (HMFs) in order to further understand the key regulators upstream of the p16/ pRB and ARF/p53/p21 pathways which drive senescence. We present evidence that early growth response 2 (EGR2) acts as a transcriptional activator of p16 and ARF in senescence and is a novel marker of senescence
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