and 0.05; **, 0.01. at Ser-15 or Ser-37 residues turned on transcription by binding its p53RE1 or p53RE3 sites during early DDR. p53RE1 uniquely contained three p53-binding half-sites, a structural feature important for transcriptional activation by phosphorylated p53 Ser-15Ser-37. During the later DDR phase, a KAISO-mediated acetylated p53 form (represented by a p53QRQ acetyl-mimic) robustly activated transcription by acting on p53RE1 Rabbit polyclonal to USP53 in which this structural feature is not significant, but it provided MJN110 sufficient KAISO levels to confer a p53 apoptotic code. These results suggest that the critical apoptosis regulator is a p53 target gene that is differently regulated by phosphorylated p53 or acetylated p53, depending on DDR stage. (cyclin-dependent kinase inhibitor 1) and various apoptosis effector genes. KAISO is a critical regulator of p53-mediated apoptosis, under genotoxic stress conditions, in mammalian cells (6). We also showed that by DNA-PK was reported to inhibit p53 interaction with the its repressor, Mdm2 (15). Phosphorylation at Ser-37 is also important for the transcriptional activity of p53 (16). Another important modification of p53, acetylation, also plays important roles in response to various types of MJN110 DNA damage. Acetylation has been shown to increase p53 sequence-specific DNA-binding capacity, through the recruitment of coactivators, and to enhance its stabilization by inhibiting ubiquitination of p53 by MDM2 (17,C20). Specifically, p53 is acetylated at Lys-370, Lys-372, Lys-381, and Lys-382 by p300/CBP and at Lys-320 by p300/CREB-binding proteinCassociated factor (PCAF) (21,C24). Phosphorylation at the p53 N terminus enhances its interaction with the acetyltransferase p300/CBP and acetylates p53. Phosphorylation at N-terminal serines, such as Ser-15, -33, and -37, has been reported to recruit p300/CBP and PCAF to induce p53 acetylation in response to DNA damage (25, 26). Phosphorylation of p53 at Ser-46 by UV-activated HIPK2 facilitates CBP-mediated acetylation of p53 at Lys-382 to promote p53-dependent gene expression (27). These reports suggest that phosphorylation and acetylation of p53 may selectively induce p53 target gene expression and exert p53 functions, such as apoptosis. The gene is absent or mutated in a high proportion of human cancers (28, 29), often leading to expression of a full-length protein that is deficient in certain functions, such as specific DNA binding. Many p53-mutant forms exert a dominant-negative effect, serving to abrogate the ability of WT p53 to inhibit cellular transformation or mediate DNA repair (30). Disruption of p53 functions promotes checkpoint defects, cellular immortalization, genomic instability, and inappropriate survival, allowing the continued proliferation of abnormal cells (31, 32). Activated p53 binds to specific DNA sequences of its target genes after DNA damage, functioning as a homotetramer and binding to p53-response elements; however, mutants of p53, including each of the four hot spots frequently altered in human cancers, fail to bind to the p53-binding consensus dimer (33). The consensus-binding site of p53 contains two copies of a 10-bp motif (5-PuPuPuC(A/T)(T/A)GPyPyPy-3), separated by a 0C21-bp spacer. One copy of the motif was insufficient for site-specific DNA binding (34). Genes activated by p53 include a negative cell cycle regulator, (and expression is dramatically increased by DNA damage only in cells with WT p53 and not in cells with p53 hot spot mutations. Consequently, we investigated how gene expression is controlled by p53 through p53REs in its 5-upstream regulatory and coding regions. During early phases of DDR, p53 phosphorylated at Ser-15 and Ser-37 up-regulates and early DDR genes to activate apoptosis during later phases of DDR. Phosphorylated p53 Ser-15Ser-37 and acetylation-coded (cell death code) (6) p53 differentially regulate expression via MJN110 time differences of their expression and differential reading of its structural features by p53RE1. Results KAISO gene expression is increased only in cells expressing WT p53 and not in the cells with hot spot p53 mutants or lacking p53 Previously, we showed that temporal expression patterns of KAISO and p53 are similar in cells treated with etoposide (6). We further investigated expression patterns of KAISO and p53 in various cell types expressing no p53, WT p53, or hot spot p53 mutations upon treatment with the DNA-damaging agent etoposide. and mRNA levels were increased by 7C16-fold at 3 h of etoposide treatment in HCT116 colorectal cancer p53+/+ cells. However, in HCT116 p53?/?cells or in p53-mutated cancer cells, such as SNU61, Colo320DM, LS1034, and HT-29, neither nor mRNA levels were significantly increased by etoposide (Fig. 1in H1299 lung cancer cells lacking endogenous p53 (Fig. 1and mRNA.