How tumour suppressor p53 bifurcates cell cycle arrest and apoptosis and executes these distinct pathways is not clearly understood. We show that BAX and PUMA promoters harbour an identical MAR element and are transcriptional targets of SMAR1. On mild DNA damage, SMAR1 selectively represses BAX and PUMA through binding to the MAR independently of inducing p53 deacetylation through HDAC1. This generates an anti-apoptotic response leading to cell cycle arrest. Importantly, knockdown of SMAR1 induces apoptosis, which is abrogated in the absence of p53. Conversely, apoptotic DNA damage results in increased size and number of promyelocytic leukaemia (PML) nuclear bodies with consequent sequestration of SMAR1. This facilitates p53 acetylation and restricts SMAR1 binding to BAX and PUMA MAR leading to apoptosis. Thus, our study establishes MAR as a damage responsive cis element and SMAR1-PML crosstalk as a switch that modulates the decision between cell cycle arrest and apoptosis in response to DNA damage.

The tumor suppressor p53 plays a critical role in maintaining genomic stability. In response to genotoxic stress, p53 levels increase and induce cell-cycle arrest, senescence, or apoptosis, thereby preventing replication of damaged DNA. In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that directly interacts with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome. Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Because of the important role of MDM2 in modulating p53 function, it is critical to understand how MDM2 levels are regulated. Here we show that the F-box protein FBXO31, a candidate tumor suppressor encoded in 16q24.3 for which there is loss of heterozygosity in various solid tumors, is responsible for promoting MDM2 degradation. Following genotoxic stress, FBXO31 is phosphorylated by the DNA damage serine/ threonine kinase ATM, resulting in increased levels of FBXO31. FBXO31 then interacts with and directs the degradation of MDM2, which is dependent on phosphorylation of MDM2 by ATM. FBXO31-mediated loss of MDM2 leads to elevated levels of p53, resulting in growth arrest. In cells depleted of FBXO31, MDM2 is not degraded and p53 levels do not increase following genotoxic stress. Thus, FBXO31 is essential for the classic robust increase in p53 levels following DNA damage.O ne of the most fundamental and extensively studied anticancer mechanisms is the large increase in the levels of the tumor suppressor p53 that occurs following DNA damage (reviewed in ref. 1). The increased p53 then mediates growth arrest and/or apoptosis. The importance of this anticancer mechanism is highlighted by the mutation or functional inactivation of the p53 gene in more than 50% of human cancers (2, 3).In unstressed cells, p53 is maintained at a low level. The major negative regulator of p53 is MDM2, an E3 ubiquitin ligase that interacts directly with p53 and promotes its polyubiquitination, leading to the subsequent destruction of p53 by the 26S proteasome (reviewed in ref. 4). Following DNA damage, MDM2 is degraded rapidly, resulting in increased p53 stability. Originally it was proposed that MDM2 degradation was caused by auto-ubiquitination; however, subsequent experiments showed that the E3 ubiquitin ligase activity of MDM2 is not required for its degradation (5).We originally identified the F-box protein FBXO31 in an RNAi screen as one of 17 factors required for oncogenic BRAF to induce senescence in primary human cells (6). F-box proteins are best known for their role as the substrate-recognition components of the SKP1/CUL1/F-box protein (SCF) class of E3 ubiquitin ligases (7). The F-box motif is responsible for the ability of F-box proteins to interact with the SCF complex and to promote ubiquitination of their targets (8).One of the other genes we isolated in our original RNAi screen was p53 (6), raising the possibility that FBXO31 and p53 function in a...

We previously identified 28 cofactors through which a RAS oncoprotein directs transcriptional silencing of Fas and other tumor suppressor genes (TSGs). Here we performed RNAi-based epistasis experiments and found that RAS-directed silencing occurs through a highly ordered pathway that is initiated by binding of ZFP354B, a sequencespecific DNA-binding protein, and culminates in recruitment of the DNA methyltransferase DNMT1. RNAi and pharmacological inhibition experiments reveal that silencing requires continuous function of RAS and its cofactors and can be rapidly reversed, which may have therapeutic implications for reactivation of silenced TSGs in RAS-positive cancers.