The studies reported here demonstrate that endogenous p53 is acetylated at K120 within the DNA binding domain. Rare mutations that convert K120 either to a conservative arginine or to a non-conservative glutamate or methionine residue have been reported in human cancer (Deissler et al., 2004
; Hashimoto et al., 1999
; Hayes et al., 1999
; Leitao et al., 2004
; Meyers et al., 1993
). We show here that acetylation of K120 is catalyzed by the closely related MYST family enzymes hMOF and TIP60 and that K120 acetylation is rapidly induced in cells exposed to genotoxic stress. Interestingly, the tumor-derived K120R mutation both blocks K120 acetylation and inhibits apoptosis induction by p53. However, this mutant retains the ability to promote cell cycle arrest. Consistent with this, loss of K120 acetylation, by direct mutation or depletion of hMOF and TIP60 specifically inhibits the transcription of key pro-apoptotic p53 targets, while non-apoptotic targets are still robustly induced. As a potential mechanism explaining why K120 acetylation is critical for apoptosis but not cell cycle arrest, we demonstrate that the acetyl-K120 form of endogenous p53 selectively accumulates at the promoters of certain pro-apoptotic target genes.
The inability of the K120R mutation to promote apoptosis and induce BAX and PUMA transcription could result from a loss of acetylation potential or from structural changes in the protein. Several lines of evidence support the model in which the K120R defect results primarily from a loss of acetylation potential. First, the K120R mutant retains the ability to both bind and transactivate the target genes p21 and hMDM2 (). Second, depletion of the enzymes responsible for K120 acetylation in response to DNA damage yields a phenotype similar to mutation of K120 to arginine (). Third, the acetyl-K120 form of p53 selectively accumulates at pro-apoptotic promoters (), consistent with it having an active functional role at these genes. Collectively, these findings support the model that the K120R mutation is unable to promote apoptosis due to a lack of K120 acetylation and not due to structural defects.
The overlapping ability of hMOF and TIP60 to acetylate K120 is similar to the overlapping lysine substrates on p53 shared by p300 and CBP as well as PCAF and GCN5. It remains unclear why cells express two enzymes that share the same substrate. One potential explanation is that the two enzymes are redundant so that one can protect against the loss of the other. However, both hMOF and TIP60 appear to contribute independently to K120 acetylation as depletion of either enzyme alone is sufficient to partially reduce K120 acetylation. An alternative explanation may be that distinctions in the type of genotoxic stress or tissue being examined dictate which enzyme catalyzes K120 acetylation. In support of this notion, depletion of TIP60 in the osteosarcoma cell line U2OS and the breast cancer cell line MCF-7 results in a greater reduction of K120 acetylation and pro-apoptotic gene transcription than does hMOF depletion ( and ). However, in the lung cancer cell line H1299, hMOF displays greater catalytic activity over TIP60 in acetylating K120 (). Furthermore, depletion of hMOF in H1299 cells reduces the ability of p53ER to activate BAX expression, where as TIP60 shRNA has a minimal affect ( and data not shown). It should be noted that interpretations drawn from experiments involving TIP60 and hMOF shRNA regarding p53 signaling should consider that both TIP60 and hMOF also participate in the DNA damage signaling pathway at points that lie well upstream of p53 activation and K120 acetylation (Gupta et al., 2005
; Ikura et al., 2000
; Kusch et al., 2004
; Legube et al., 2002
; Sun et al., 2005
). Therefore, further analysis is required to elucidate under which scenarios K120 is acetylated by these enzymes.
Although we have shown that acetylation of K120 is required for the activation of the p53 pro-apoptotic target genes BAX and PUMA, K120 acetylation does not appear to affect all p53 pro-apoptotic target genes. For example, the NOXA gene is still induced by the K120R mutant of p53 in the H1299 cell line and the acetyl-K120 form of endogenous p53 fails to accumulate to significant levels at the NOXA promoter after DNA damage treatment of LNCaP cells (Supp. Figure 4). While the role of NOXA in p53-mediated apoptosis is less universal and more cell-type specific than that of BAX and PUMA (Villunger et al., 2003
), this finding does suggest that not all apoptotic targets have equal requirements for the K120-acetyl form of p53. This differential requirement may reflect differences in the promoter structure and/or cofactors needed for transactivation of the different subsets of target genes.
As discussed above, the finding that the K120R mutation occurs in human cancer suggests that acetylation by MYST family proteins may be critical to the tumor suppressor function of p53. Consistent with this, hMOF levels are frequently decreased in human tumor samples (S. Rea and A. Akhtar, personal communication). A role for TIP60 in tumorigenesis has also been suggested by recent studies (Kim et al., 2005
; Squatrito et al., 2006
). It is therefore tempting to speculate that loss of K120 acetylation is partially responsible for the roles played by hMOF or TIP60 in cancer. Furthermore, a role for TIP60 in the UV-induced transcription of several p53 targets was recently reported (Tyteca et al., 2006
The next series of challenges that are presented by the identification of this pathway relate to understanding precisely why K120 acetylation is required for p53-mediated transcription of apoptotic target genes, BAX and PUMA. It is possible that the presence of an acetyl group on K120 alters the interaction this residue makes with DNA. This is supported by structural studies suggesting that K120 indeed participates in dictating the preference of p53 for distinct classes of binding sites (Kitayner et al., 2006
). However, the finding that the non-acetylated K120R mutant retains significant binding to the PUMA promoter and exhibits only a partial defect at the BAX promoter suggests that a direct effect on DNA binding may not explain the critical role of this residue (). More likely is a model in which increased transcription of pro-apoptotic targets results from the ability of the acetyl-K120 form of p53 to recruit essential transcriptional cofactors that modify nucleosomal histones, stabilize the p53-DNA interaction or otherwise augment mRNA synthesis. For example, the ASPP family of proteins (ASPP1 and ASPP2) preferentially localize to p53 pro-apoptotic promoters, such as BAX (Samuels-Lev et al., 2001
), similar to the acetyl-K120 form of p53. Furthermore, the ASPP proteins specifically enhance the binding of p53 to the BAX promoter and consequently the apoptotic function of p53 (Samuels-Lev et al., 2001
). Therefore the modest defect in p53 K120R binding to the BAX promoter may result from its inability to bind cofactors that enhance p53-DNA interactions, such as the ASPP proteins. Finally, although the ASPP proteins do not directly interact with the L1 loop, it has been proposed that the L1 loop influences the binding of ASPP2 to p53 (Friedler et al., 2005
). Thus, acetylation of K120 may result in enhanced recruitment of the ASPP proteins or other essential cofactors to pro-apoptotic promoters.
Previous studies have identified other post-translational modifications of p53 that are specifically associated with the induction of apoptosis, but not cell cycle arrest. These modifications include the phosphorylation of serines 20 and 46 and the acetylation of lysine 373 (Bulavin et al., 1999
; Jack et al., 2002
; Knights et al., 2006
; Oda et al., 2000
). While the role of these modifications in apoptosis induction remains unclear (Thompson et al., 2004
), there may be some interplay between them and K120 acetylation. This type of crosstalk between distinct post-translational modifications occurs on histones (Sun and Allis, 2002
) and has been suggested to occur on p53 as well (Sakaguchi et al., 1998
). Current efforts are aimed at determining whether the acetylation of K120 requires or influences other p53 post-translational modifications.
The acetylation of lysine 120 within the DNA binding domain of p53 is distinct from most other sites of p53 modification in that it is disrupted by mutation in human cancer. Also unusual is that this naturally occurring p53 mutation selectively affects the ability of p53 to induce apoptosis without blocking cell cycle arrest. Similarly, the MYST family of enzymes that catalyze K120 acetylation are implicated in the DNA damage response pathway and appear to be deregulated in cancer as well. Considered with the finding that the acetyl-K120 form of p53 selectively accumulates at key pro-apoptotic promoters, these studies define a pathway of p53 regulation that likely participates in determining whether the cellular response to DNA damage elicits cell cycle arrest or apoptosis. Further analysis will be required to gain a complete understanding of the contribution made by the genetic lesions in this pathway to human cancer.